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2 STEMCELL Technologies Inc. TABLE OF CONTENTS
Reprogram Differentiate
4 Reprogramming 24 Differentiation 4 ReproRNA™-OKSGM 24 STEMdiff™ Overview 5 ReproTeSR™ 5 Erythroid Progenitor Reprogramming Kit 25 Mesoderm Differentiation + 5 CD34 Progenitor Reprogramming Kit 25 Mesoderm Differentiation Pathways 6 TeSR™-E7™ 26 STEMdiff™ Mesoderm Induction Medium 26 STEMdiff™ Mesenchymal Progenitor Kit Maintain 27 STEMdiff™ Endothelial Differentiation Kit 28 STEMdiff™ Hematopoietic System 7 Maintenance 31 STEMdiff™ Cardiomyocyte System 7 TeSR™ Overview 32 STEMdiff™ Myogenic System 8 mTeSR™ Plus 33 STEMdiff™ Kidney Organoid Kit 10 TeSR™-AOF 12 mTeSR™1 34 Ectoderm Differentiation 12 TeSR™-E8™ 34 Ectoderm Differentiation Pathways 35 STEMdiff™ Neural System 13 Scale-Up 37 STEMdiff™ Cerebral Organoid Kit 13 mTeSR™3D 38 STEMdiff™ Dorsal and Ventral Forebrain Organoid Kits 13 TeSR™-E8™3D 39 BrainPhys™ Neuronal Medium 39 NeuroFluor™ NeuO 14 Naïve Induction and Maintenance 40 STEMdiff™ Neural Crest Differentiation Kit 14 RSeT™ Feeder-Free 41 STEMdiff™ Microglia Culture System 15 NaïveCult™ 15 hPSC Naïve State qPCR Array 42 Endoderm Differentiation 42 Endoderm Differentiation Pathways 16 Matrices 43 STEMdiff™ Definitive Endoderm Kit 16 Vitronectin XF™ & CellAdhere™ Laminin-521 43 hPSC-Derived Endoderm qPCR Array 44 STEMdiff™ Intestinal Organoid Kit 17 Dissociation Reagents 45 STEMdiff™ Pancreatic Progenitor Kit 17 ReLeSR™ 46 STEMdiff™ Lung Progenitor Kit 17 Gentle Cell Dissociation Reagent 47 STEMdiff™ Branching Lung Organoid Kit 17 ACCUTASE™ & Dispase 48 Flexible User-Directed Differentiation Genome Edit 48 STEMdiff™ APEL™2 & APEL™2-LI 18 Genome Editing 48 TeSR™-E5 & TeSR™-E6 18 CloneR™ 19 ArciTect™ Accessory Products
Characterize 49 Small Molecules & Cytokines 50 AggreWell™ Plates 20 Cell Quality Characterization 51 Antibodies 20 hPSC Genetic Analysis Kit 51 GloCell™ Fixable Viability Dyes 21 STEMdiff™ Trilineage Differentiation Kit 52 Annexin V Dyes 21 hPSC Trilineage Differentiation qPCR Array 53 Product Listing Cryopreserve 22 Cryopreservation 58 References 22 mFreSR™ & FreSR™-S 22 CryoStor® CS10 23 ThawSTAR® CFT2
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Reprogramming ReproRNA™-OKSGM Generate iPS Cells Using a Non-Integrating Reprogramming Vector
ReproRNA™-OKSGM is a single-stranded RNA replicon vector that contains five reprogramming factors: OCT4, KLF4, SOX2, GLIS1, and c-MYC, as well as a puromycin-resistance gene. This RNA vector reprograms human somatic cells, such as fibroblasts, into induced pluripotent stem (iPS) cells with high efficiency and only requires a single transfection. As shown in Figure 1, using ReproRNA™-OKSGM with ReproTeSR™ reprogramming medium allows for iPS cell colony generation under feeder-free conditions with similar reprogramming efficiency to feeder- based systems. ReproRNA™-derived human iPS cell colonies also express markers of undifferentiated cells and retain a normal karyotype. Subsequently, human iPS cells generated with ReproRNA™-OKSGM can be maintained in a TeSR™ maintenance medium and further differentiated into cells of all three germ layers.
Adult Human Dermal Fibroblasts
(%) Why Use ReproRNA™-OKSGM?
NON-VIRAL. Non-integrating vector system.
SELF-REPLICATING VECTOR. Only a single transfection is required.
TM ALL-IN-ONE. Vector contains all reprogramming factors.
HIGHLY EFFICIENT. Comparable fibroblast 1 Figure 1. ReproRNA™-OKSGM Vector Efficiently Reprograms reprogramming efficiency to Sendai virus. Fibroblasts
Human dermal fibroblasts were transfected with the ReproRNA™-OKSGM vector and reprogrammed under feeder-dependent (standard KOSR-containing human embryonic stem (ES) cell medium on inactivated mouse embryonic fibroblasts Learn more at (iMEFs)) or feeder‑free conditions (ReproTeSR™ on Corning®Matrigel®). www.stemcell.com/ReproRNA-OKSGM The efficiency of reprogramming fibroblasts with ReproRNA™-OKSGM and ReproTeSR™ is comparable to that reported with Sendai virus.1 (n ≥ 6; data shown are mean ± SD)
Somatic Cells Mesenchymal – Epithelial iPS Cell Colony (e.g. Fibroblasts) Transition Reprogramming with ReproRNA™-OKSGM
Plate Transfect somatic cells Pick cells with ReproRNA™ colonies
Day
0 1 6 8 15 20 - 28
+ Puromycin + Puromycin + B18R
+ B18R Culture Somatic Cell TeSR™ Growth Medium ReproTeSR™ ReproTeSR™ Medium Medium Medium*
*mTeSR™1, mTeSR™ Plus, TeSR™-E8™, or TeSR™-AOF
Figure 2. Schematic for Reprogramming with ReproRNA™-OKSGM
Somatic cells are transfected with ReproRNA™-OKSGM at day 0 and cultured in Growth Medium (containing puromycin). After 5 days of puromycin selection post-transfection, cells are cultured in ReproTeSR™ for the remainder of the reprogramming induction phase until iPS cell colonies emerge. Recombinant B18R Protein is also added during the first 2 weeks after transfection to inhibit the interferon response and increase cell viability. Typically, by day 20, iPS cell colonies are large enough to be isolated and propagated in a TeSR™ maintenance medium.
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4 STEMCELL Technologies Inc. ReproTeSR™ Reproducibly Generate High Numbers of Integrated Sets of Tools for Human iPS Cell Colonies Reprogramming Human Blood Cells
Erythroid Progenitor CD34+ Progenitor ReproTeSR™ is a complete, defined, xeno-free, and feeder‑free Reprogramming Kit Reprogramming Kit reprogramming medium optimized for the generation of human iPS cells. Use ReproTeSR™ during the induction phase of reprogramming to produce more iPS cell colonies than with traditional KOSR-containing human ES cell media. Human iPS cell colonies generated with ReproTeSR™ express undifferentiated • Enrich cells with • Enrich cells with RosetteSep™ cell markers and exhibit more defined borders, compact RosetteSep™ and and SepMate™ morphology, and reduced differentiation. SepMate™ • Isolate CD34+ cells ReproTeSR™ was optimized for reprogramming blood cells • No isolation step required with EasySep™* and seamlessly integrates with RosetteSep™, SepMate™, • Expand erythroid cells with • Expand CD34+ cells with EasySep™, and StemSpan™ products for isolating and StemSpan™ SFEM II + Erythroid StemSpan™ SFEM II + CD34+ expanding hematopoietic cells. It can also be used to reprogram Expansion Supplement Expansion Supplement other somatic cell types and can be paired with ReproRNA™- • Reprogram cells with • Reprogram cells with OKSGM for reprogramming fibroblasts. iPS cells generated ReproTeSR™ ReproTeSR™ with ReproTeSR™ can be subsequently cultured in a TeSR™ maintenance medium and differentiated with the STEMdiff™ *EasySep™ magnet is not included with the CD34+ Progenitor Reprogramming Kit and must suite of products to cells of all three lineages. Purchase be purchased separately. ReproTeSR™ individually or as part of the Erythroid or CD34+ Progenitor Reprogramming Kits.
Blood Cells Attached Blood Cells Pre-iPS Cell Colony iPS Cell Colony (e.g. CD34+ Cells)
ReproTeSR™ Blood Reprogramming Timeline
Transfect or transduce blood cells Add Pick iPS cell colonies and with reprogramming factors ReproTeSR™ maintain in TeSR™ medium
Day
0 3 5 7 21 - 28 Full-medium change daily
Culture ReproTeSR™ Medium StemSpan™ + Supplement ReproTeSR™ TeSR™ Medium* StemSpan™ + Supplement
*mTeSR™1, mTeSR™ Plus, TeSR™-E8™, or TeSR™-AOF
Figure 3. Schematic of ReproTeSR™ Blood Reprogramming Timeline
ReproTeSR™ is used during the entire induction phase of reprogramming (days 3 to 21). On days 3 and 5, ReproTeSR™ is added to StemSpan™ growth medium (in a fed-batch manner) to facilitate attachment of transfected cells. Attached cells are further cultured in ReproTeSR™ with daily full media changes until putative iPS cell colonies emerge (days 21 to 28). iPS cell colonies can then be isolated and propagated in a TeSR™ maintenance medium.
ReproTeSR™ StemSpan™ + Supplement Learn more at www.stemcell.com/ReproTeSR 3
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TeSR™-E7™ Generate iPS Cells from Fibroblasts Without Why Use TeSR™-E7™? Feeders or Animal Components IDENTIFY AND SELECT COLONIES EASILY. TeSR™-E7™ is a defined, animal component-free (ACF) Pre-screened components ensure high-quality colony reprogramming culture medium optimized for the generation of morphology for improved manual selection. human iPS cells without the use of feeders. It is based on the E7 RAPID SUBCLONING. Reduced differentiation and formulation published by the laboratory of Dr. James Thomson.2 fibroblast growth enables rapid establishment of TeSR™-E7™ is specifically formulated to limit fibroblast overgrowth, homogeneous human iPS cell cultures. resulting in colonies with easily recognizable ES cell-like morphology. REPRODUCIBLE EFFICIENCY. Feeder-free, defined formulation facilitates reproducibly efficient human iPS A B cell generation.
Learn more at www.stemcell.com/TeSR-E7
Figure 4. Primary iPS Cell Colonies Derived in TeSR™-E7™ Have Defined Borders and Reduced Differentiation
(A) TeSR™-E7™ yields easily recognizable iPS cell colonies with defined borders. (B) Unqualified components can result in colonies that have poorly defined edges and higher levels of differentiation. Representative colonies from adult human fibroblasts reprogrammed with episomal vectors containing OCT4, SOX2, KLF4, and c-MYC are shown.
Somatic Cells Mesenchymal - Pre-iPS Cell iPS Cell Established iPS (e.g. Fibroblasts) Epithelial Transition Colony Colony Cell Line
Reprogramming Timeline
Transfect or transduce somatic cells Change medium Pick iPS cell colonies and with reprogramming factors to TeSR™-E7™ maintain in TeSR™ medium
Day
0 3 25 - 30
Culture Medium Somatic Cell Medium TeSR™-E7™ TeSR™ Medium*
*mTeSR™1, mTeSR™ Plus, TeSR™-E8™, or TeSR™-AOF
Figure 5. Schematic of Reprogramming Timeline
TeSR™-E7™ can be used during the entire induction phase of reprogramming (days 3 to 25+). Following reprogramming, iPS cell colonies can be isolated and propagated in feeder-free maintenance systems (e.g. TeSR™ media on Corning® Matrigel® or Vitronectin XF™ matrices). TeSR™ = TeSR™ family of maintenance media.
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6 STEMCELL Technologies Inc. Maintenance Media Overview Maintaining high-quality human pluripotent stem cells (hPSCs), including embryonic stem (ES) and induced pluripotent stem (iPS) cells, is critical for success in hPSC research. The TeSR™ family of feeder-free maintenance media is manufactured using rigorously pre-screened materials to ensure the highest levels of batch-to-batch consistency and experimental reproducibility. These media are based on published formulations2-5 from the laboratory of Dr. James Thomson and are tailored to suit your specific needs.
Select the Right Maintenance Medium for Your Research cGMP Media Minimal Media Minimize Risk in Your Cell Therapy Just the Basics Development TeSR™-E8™ ™ TeSR -AOF • Contains only the 8 most critical components required for 2,5 • No animal-derived raw materials to the secondary level of hPSC maintenance manufacturing • Animal component-free (ACF) formulation with no animal- • Supports high-quality culture morphology, robust attachment, derived raw materials to the primary level of manufacturing and cell expansion • Low-protein when compared to conventional feeder-free • Stabilized FGF2 supports high cell quality while allowing for maintenance media alternate feeding schedules Suspension Culture Versatility for Routine Maintenance and Expansion Scale Up Cell Production ™ mTeSR™ Plus mTeSR 3D • Based on mTeSR™1, optimized for hPSC scale-up • Enhanced buffering and stabilized FGF2 support superior culture morphology and cell growth while allowing for • Fed-batch culture system for a simplified workflow 9 alternate feeding schedules • Scale up to 1 x 10 high-quality, undifferentiated hPSCs in as few as 2 - 3 weeks • Enables heightened single-cell survival when used with CloneR™ TeSR™-E8™3D ™ mTeSR 1 • Based on TeSR™-E8™, optimized for hPSC scale-up in low protein, ACF conditions • Used to maintain thousands of ES and iPS cell lines in over 60 countries for more than 10 years • Fed-batch culture system for a simplified workflow 9 • Scale up to 1 x 10 high-quality, undifferentiated hPSCs in as • Contains pre-screened BSA to stabilize medium, aid in lipid/nutrient transport, and protect cultures from cellular few as 2 - 3 weeks toxins and stresses3
Why Use mTeSR™ and the TeSR™ Media Family?
FEEDER-FREE. Feeder-free hPSC culture minimizes variability by limiting the presence of undefined components and immunogenic material.
PROVEN. mTeSR™ is the most widely published medium for hPSC culture with > 1500 peer-reviewed publications.
OPTIMIZED. The same base formulation in each medium allows for establishment of a continuous TeSR™ media-based workflow.
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mTeSR™ Plus Enjoy Flexible Feeding Schedules and Enhanced Why Use mTeSR™ Plus? Growth Characteristics with Stabilized, cGMP- STABILIZED. Enhanced buffering and stabilized Grade Medium components, including FGF2, support cell quality while allowing for alternate feeding schedules. mTeSR™ Plus is based on the formula of mTeSR™1, the most- ENHANCED GROWTH. Supports superior culture published feeder-free hPSC maintenance medium, and allows for morphology and cell growth characteristics. true culture versatility while supporting high-quality maintenance and expansion of hPSCs. EFFICIENT CLONING. Enables heightened single-cell survival when used with CloneR™. To enhance cell quality attributes, critical medium components— including FGF2—have been stabilized and medium pH is more STREAMLINED. Fully compatible with established consistent. As a result, this medium supports higher cell numbers genome editing and differentiation protocols. with daily feeding, while maintaining consistent quality during REGULATED. Manufactured under relevant cGMPs with restricted feeding schedules. enhanced documentation, including a viral-safe profile to mTeSR™ Plus is now manufactured under relevant cGMPs with the support cell therapy research. same high-quality critical raw materials as the original, non-cGMP product. With the added benefits of enhanced critical raw material traceability, processes, and quality control validations, mTeSR™ Plus mTeSR™ 1 100 mTeSR™ Plus
enables a seamless transition from basic research to drug and cell =0 Competitor S
therapy development. oT et mTeSR™ Plus is compatible with a variety of culture matrices,
including Corning® Matrigel® hESC-Qualified Matrix and elativ mTeSR™ 1 gr 50 Vitronectin XF™. 100 mTeSR™ Plus =0 Competitor S
Each lot of mTeSR™ Plus 5X Supplement is used to prepare oT remainin complete mTeSR™ Plus medium and is then performance-tested in et a culture assay using hPSCs. GF elativ %F % FGF Remaining Relative to t=0
gr 0 50 020406080 Time (Hours) mTeSR™ 1 Time (hours) 100 mTeSR™ Plus remainin
=0 Figure 7. mTeSR™ Plus Maintains Consistent Levels of FGF2 Competitor S GF oT 7.5 Throughout a Weekend-Free Protocol et %F FGF2 0levels remain high in mTeSR™ Plus when kept at 37°C over a 72-hour time 020406080 period. Measured by ELISA. elativ Time (hours) gr pH
pH 50 7.0 Maintain hPSCs on Your Own Schedule remainin GF Skip 2 days = Double feed %F 6.05 01×10 6 2×10 6 3×10 6 020406080 Skip 1 day = Regular feed NumberTime of (hours) Cells AfterAfter 72 Hourshours
Figure 6. mTeSR™ Plus Maintains Optimal pH Levels Throughout The possibilities are endless. Use your regular schedule, or try a Weekend-Free Protocol something new to free up your days.
The pH of spent medium from hPSCs cultured in mTeSR™ Plus is higher than that PASSAGING MON TUE WED THU FRI SAT SUN of hPSCs cultured in mTeSR™1 and another flexible-feeding medium at similar FREQUENCY cell densities. pH and cell numbers were measured after a 72-hour period without 7d P F F F F F F repeat feeding. Range of cell numbers shown represent different densities that would be observed throughout a typical passage. Cell numbers are from one well of a 7d P F F F 2F x x repeat 6-well plate. 6d P x 2F x x F repeat
5d P F 2F x x repeat
3d/4d P F x P 2F x x repeat
Fill Out Your Own
P = Passage; F = Single Feed; 2F = Double Feed 8 STEMCELL Technologies Inc. H1 WLS-1C mTeSR™ Plus mTeSR™1 mTeSR™
Figure 8. Normal Human ES and iPS Cell Morphology Is Observed in mTeSR™ Plus Cultures
Images depict undifferentiated human ES (H1) and iPS (WLS-1C) cells cultured on Corning® Matrigel® matrix in mTeSR™1 with daily feeds or in mTeSR™ Plus with restricted feeds. Cells retain the prominent nucleoli and high nuclear-to-cytoplasmic ratio characteristic of this cell type after 10 passages. Densely packed cells and multi‑layering are prominent when cells are ready to be passaged.
4×10 6 mTeSR™1 Daily Feeding
3×10 6 Restricted Feeding mTeSR™ Plus
Cells Daily Feeding of 2×10 6 Restricted Feeding Number Number of Cells 1×10 6 Division Time (Hours) Division Time
0 02468 Cell Line DaysDays in in Culture Figure 10. hPSCs Maintained in mTeSR™ Plus Demonstrate Figure 9. mTeSR™ Plus Supports Higher Cell Numbers, Even with Equivalent Cell Division Times to Cells Maintained in mTeSR™1 Restricted Feeding Human ES and iPS cells (H9, WLS-1C) cultured in mTeSR™1 or mTeSR™ Plus were Growth curves were obtained for human ES (H9) cells cultured in mTeSR™1 or dissociated to single cells and seeded at 20,000 cells/cm2 on Matrigel®-coated mTeSR™ Plus on Corning® Matrigel® matrix over 7 days with either daily feeds plates. The cells were imaged every 20 minutes on an IncuCyte ZOOM® for three or restricted feeds. Growth curves were determined by seeding 20,000 cells per days with a medium exchange the day after seeding. Individual cell division times well of a 6-well plate as aggregates and counting the cell numbers each day in were determined using single cell tracking. Data points include first, second, and duplicate wells. third cell divisions.
Learn more at www.stemcell.com/mTeSRPlus
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Minimize Risk in Your Cell Therapy Development Our Commitment to Quality and Good Manufacturing Practices
Choosing a cell and tissue culture medium is a critical step in ensuring the quality and performance of your cultures. For cell therapy manufacturers, this choice can have additional impacts on the safety of your cell therapy. Minimize risk by using animal origin-free TeSR™- AOF as part of a high-compliance maintenance workflow that also includes dissociation reagents (Gentle Cell Dissociation Reagent, ReLeSR™), matrices (CellAdhere™ Laminin-521), and cryopreservation reagents (CryoStor® CS10). For more information on how we can support your regulatory needs, including navigating requirements for using TeSR™-AOF in your cell therapy applications, visit www.stemcell.com/regulatory-support or contact your local STEMCELL representative.
TeSR™-AOF Maintain hPSCs for Cell Therapy Development Using cGMP-Grade, Animal Origin-Free, Stabilized Media
Reduce risk and obtain greater numbers of higher quality cells for your human pluripotent stem cell (hPSC)-derived cell therapy development with TeSR™-AOF, manufactured under relevant cGMPs. With no raw materials of animal or human origin to the secondary level of manufacturing, enjoy more straightforward traceability and enhanced viral safety compared to media that are only animal origin- free to the primary level of manufacturing. Use TeSR™-AOF to consistently culture viral-safe, high-quality hPSCs on a schedule that works for you—with whatever cell lines you choose. To enhance cell quality attributes, particularly during restricted feeds, Figure 12. hPSCs Maintained in TeSR™-AOF with Daily and critical medium components have been stabilized, including FGF2 Restricted Feed Schedules Exhibit Comparable Colony Morphology (also known as basic FGF [bFGF]). As a result, TeSR™-AOF allows for both daily and restricted feeding schedules while maintaining cell hPSCs were maintained on Vitronectin XF™ for five passages. Phase-contrast images were taken on day 7 after seeding. For restricted feeds, hPSCs were fed quality and performance. with a double volume (4 mL) of medium on day 2 after passage, followed by TeSR™-AOF is compatible with a variety of culture matrices, including two consecutive skipped days of feeds, with a final single-volume feed (2 mL) on Corning® Matrigel® hESC-Qualified Matrix, Vitronectin XF™, and day 5, prior to passaging on day 6 or 7. hPSCs maintained in TeSR™-AOF exhibit hPSC-like morphology, forming densely packed, round colonies with smooth edge CellAdhere™ Laminin-521. morphology.
B
Figure 11. Native bFGF Levels Are Stabilized at 37°C in TeSR™-AOF TeSR™-AOF and TeSR™-E8™ were incubated at 37°C for 24, 48, and 72 hours. Figure 13. hPSCs Maintained in TeSR™-AOF with Daily and FGF2 levels were measured by Meso Scale Discovery (MSD) immunoassay; data Restricted Feed Schedules Have Comparable Expansion Rates was normalized to t = 0 levels for TeSR™-E8™ and TeSR™-AOF, respectively. FGF2 levels in TeSR™-AOF remain at 36.7 ± 5.61% of t = 0 levels at 72 hours when hPSCs were maintained on Vitronectin XF™ for five passages. At the end of ® incubated at 37°C. Data representative of n = 3 biological replicates ± SD. each passage, cell counts were obtained using the Nucleocounter NC-200™ ChemoMetec automated cell counter to count DAPI-stained nuclei. The log2 transformed cumulative fold expansion was plotted against time in culture (days). 10 STEMCELL Technologies Inc. Why Use TeSR™-AOF?
ANIMAL ORIGIN-FREE. Minimize risk in your ancillary materials by selecting a medium with no animal raw materials to the secondary level of manufacturing.
CONSISTENT CELL GROWTH. Supports high-quality colony morphology, robust attachment, and cell expansion.
REGULATED. Manufactured under relevant cGMPs with enhanced documentation to support cell and gene Figure 14. hPSCs Cultured in TeSR™-AOF with Restricted Feeding therapy research. Maintain Demonstrate Classic hPSC Colony Morphology STABILIZED. Stabilized components, including FGF2, hPSCs maintained in TeSR™-AOF were passaged as aggregates with ReLeSR™ support high cell quality while allowing for alternate passaging reagent every 6 - 7 days for greater than 10 passages. hPSCs maintained in TeSR™-AOF exhibit hPSC-like morphology, forming densely packed, round feeding schedules.. colonies with smooth edge morphology. Homogeneous cell morphology characteristic of hPSCs are observed, including large nucleoli and scant cytoplasm.
A B A B
C Figure 16. hPSCs Cultured in TeSR™-AOF Express Markers of the Undifferentiated State and Differentiate to the Three Germ Layers
(A) hPSCs maintained in TeSR™-AOF exhibit high levels of TRA-1-60 and OCT4 by flow cytometry at passage 5 and 10. Across n = 6 cell lines, the average TRA-1-60 expression was 93.4 ± 3.37 %, and percent OCT4-positive cells was 98.3 ± 1.55%. Data shown represent an average of passage 5 and 10 flow results for each cell line. MG = Matrigel®; VN = Vitronectin XF™. (B) Efficient differentiation to the three germ layers was demonstrated in one hES and one hiPS cell line maintained for > 5 passages in TeSR™-AOF. Cultures were processed for flow cytometry and assessed for CXCR4+/SOX17+ cells on day 5 following differentiation using the STEMdiff™ Definitive Endoderm Kit. Cultures were Figure 15. hPSCs Maintained in TeSR™-AOF Have Improved processed for flow cytometry and assessed for Brachyury (T)+/OCT4- cells on day Attachment and Higher Overall Expansion Compared to Low- 5 following differentiation in STEMdiff™ Mesoderm Induction Medium. Cultures were processed for flow cytometry and assessed for PAX6+/Nestin+ cells on day 7 Protein Medium following monolayer differentiation using STEMdiff™ Neural Induction Medium. (A) hPSCs cultured in TeSR™-AOF demonstrate a higher plating efficiency compared to hPSCs maintained in low-protein medium (TeSR™-E8™). Plating efficiency is calculated by seeding a known number of aggregates and comparing to the number of established colonies on day 7. (B) hPSCs maintained in Learn more at www.stemcell.com/TeSR-AOF TeSR™-AOF exhibit a higher average fold expansion per passage compared to TeSR™-E8™. (C) hPSCs cultured in TeSR™-AOF demonstrate consistent expansion and minimal cell-line-to-cell-line variability between ES and iPS cell lines assessed. Cumulative fold expansion was measured from passage 1 to 5. Data represented as mean plating efficiency or fold expansion across 10 passages ± SD. MG = Matrigel®; VN = Vitronectin XF™. WWW.STEMCELL.COM 11 HUMAN PLURIPOTENT STEM CELLS
mTeSR™1 TeSR™-E8™ Maintain hPSCs in cGMP-Grade, Feeder-Free Maintain hPSCs in Animal Component-Free, hPSC Medium Feeder-Free hPSC Medium
mTeSR™1 is a serum-free and complete cell culture medium that TeSR™-E8™ is based on the E8 formulation2,5 developed by the has been used to successfully maintain thousands of hPSC lines, laboratory of Dr. James Thomson, the lead research group behind with established protocols for applications ranging from gene the design of mTeSR™1. TeSR™-E8™ contains only the most editing and bioreactor expansion to lineage-specific differentiation. critical components required for maintenance of hPSCs, providing mTeSR™1 is designed for use in cell therapy research applications, a simpler medium for hPSC culture. This medium can be used with manufactured following the recommendations of USP <1043> on Vitronectin XF™ for a completely xeno-free system. ancillary materials, and available for use under an approved US FDA Investigational New Drug (IND) application.
Why Use TeSR™-E8™? Why Use mTeSR™1? ANIMAL COMPONENT-FREE. Serum-free, cutting- edge formulation. PROVEN. The most highly validated serum-free hPSC LOW PROTEIN. Minimal formulation contains only medium, backed by a decade of data. mTeSR™1 has the most essential components required for hPSC been used in over 1500 peer-reviewed publications with maintenance. established protocols for a variety of applications.
ROBUST. Contains pre-screened and quality-controlled components.
cGMP. Ensures the highest quality and consistency for A reproducible results.
A 500 µm 25 µm
H1 B
500 µm 150 µm
B 500 µm 25 µm
WLS-1C Figure 18. Normal Human ES and iPS Cell Morphology Is Observed in TeSR™-E8™ Cultures
500 µm 150 µm Undifferentiated (A) human ES (H9) and (B) human iPS (WLS-1C) cells cultured on Corning® Matrigel® in TeSR™-E8™ retain the prominent nucleoli and high nuclear-to-cytoplasmic ratio, characteristic of this cell type. Densely packed cells and Figure 17. Normal Human ES and iPS Cell Morphology Is Observed multilayering are prominent when cells are ready to be passaged. in mTeSR™1 Cultures
Undifferentiated (A) H1 human ES and (B) WLS-1C human iPS cells cultured on Corning® Matrigel® matrix in mTeSR™1 retain the prominent nucleoli and high Learn more at www.stemcell.com/TeSR-E8 nuclear-to-cytoplasmic ratio characteristic of this cell type after 10 passages. Densely packed cells and multilayering are prominent when cells are ready to be passaged.
Learn more at www.stemcell.com/mTeSR1
12 STEMCELL Technologies Inc. Scale-Up Why Use Suspension Culture? Expand Large Numbers of hPSCs in 3D OPTIMIZED. Part of the TeSR™ family, mTeSR™3D, and Suspension culture of hPSCs as 3D aggregates provides a TeSR™-E8™3D are optimized for hPSC suspension culture. convenient method to produce large numbers of high-quality, STREAMLINED WORKFLOW. Fed-batch strategy provides undifferentiated hPSCs with reduced labor and costs. a simplified culture system. hPSCs expanded in the TeSR™ family suspension culture systems SIMPLIFIED CULTURE SYSTEM. Serum-free culture have robust growth, maintain high expression of pluripotent stem system with no microcarriers or external matrix required. cell markers, and retain trilineage differentiation ability. SCALE-UP. Easily produce up to 1 x 109 hPSCs in as few as 2 - 3 weeks. mTeSR™3D VERSATILE. Compatible with a variety of suspension culture vessels. Based on mTeSR™1, mTeSR™3D is optimized for the expansion and scale-up of hPSCs. It is optimized as a fed-batch culture system, in COST-EFFECTIVE. Significant cost savings in both media which required nutrients are added daily, eliminating the need for and labor. daily medium exchanges.
A B TeSR™-E8™3D TeSR™-E8™3D is a low protein, animal component-free medium based on TeSR™-E8™. The system contains only the most critical components for hPSCs, providing a simpler culture medium for robust, large-scale hPSC expansion. It uses a fed-batch feeding strategy that replenishes nutrients daily while reducing labor and costs.
Figure 19. Morphology of hPSC Aggregates Cultured in mTeSR™3D
Characteristic morphology of suspension-cultured hPSC aggregates includes: approximately spherical shape, edges that are clear but not perfectly smooth, and a mottled or pock-marked appearance. Aggregates should be approximately 350 - 400 μm by the end of the passage. Shown are (A) human ES cell line H7 and (B) human iPS cell line STiPS-F016.
Isotype Control Sample Figure 21. Morphology of hPSC Aggregates Cultured in TeSR™-E8™3D Characteristic morphology of suspension-cultured hPSC aggregates includes: approximately spherical shape, edges that are clear but not perfectly smooth, and a mottled or pock-marked appearance. Aggregates should be approximately 350 - 400 μm by the end of the passage. Shown are human ES cell line H1 cultured FITC FITC in TeSR™-E8™3D.
2019-03-18_at_03-56-45pm.fcsIsotype H1 1CControl OCT4.FCS (D07) 2019-03-18_at_03-56-45pm.fcsSample H1 1C OCT4.FCS (G07) 5 5 10 10 OCT4+ OCT4+ 4 4 10 1.00% 10 96.04%
3 FSC FSC 10 3 10
2 10 2 Figure 20. OCT4 Expression of hPSCs Cultured in mTeSR™3D 10
FITC 1 FITC 10 1 10 GRN-B-HLin GRN-B-HLin hPSCs expanded in mTeSR™3D maintain expression of pluripotent stem cell 0 10 0 10 markers. Shown are representative plots of OCT4 expression after 7 passages in -1 10 -1 mTeSR™3D. 10 0 25000 50000 75000 100000 0 25000 50000 75000 100000 FSC-HLinFSC FSC-HLinFSC
Figure 22. OCT4 Expression of hPSCs Cultured in TeSR™-E8™3D Learn more at www.stemcell.com/mTeSR3D hPSCs expanded in TeSR™-E8™3D maintain expression of pluripotent stem cell markers. Shown are representative plots of OCT4 expression after 10 passages in TeSR™-E8™3D.
Learn more at www.stemcell.com/TeSR-E8-3D
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Naïve Induction and Maintenance
RSeT™ Feeder-Free Medium Maintain Feeder-Free Naïve-Like hPSCs in Defined Medium
RSeT™ Feeder-Free is a serum-free medium that reverts primed hPSCs and maintains cells in a naïve-like state without the need of bFGF or feeder cells. RSeT™ Feeder-Free Medium produces robust cultures with a naïve-like morphology and increased expression of key naïve- associated transcripts. This improved formulation enables efficient reversion to a naïve-like state as early as passage 1, without the variability and burden associated with using feeder cells.
A B 10 Why Use RSeT™ Feeder-Free 5 Medium?
0 CONSISTENT. Feeder-free, serum-free formulation with of Fold Change 2 Relative to TBP
Log pre-screened components to ensure batch-to-batch
100 µm -5 Normalized to Primed Sample consistency. KLF2 KLF4 KLF17 OCT4 SOX2 STELLA NANOG DNMT1 TFCP2L1 DNMT3L DNMT3ADNMT3B TRANSGENE-FREE. No exogenous genes required for Figure 23. hPSCs Maintained in RSeT™ Feeder-Free Are Reverted reversion to naïve-like state. to a Naïve-Like State and Express High Levels of Naïve-Associated Genes EFFICIENT. Reversion to naïve-like state as early as passage 1. (A) A representative image of hPSCs that reverted to a naïve-like state after being cultured in RSeT™ Feeder-Free Medium for 1 passage. During reversion, EASY-TO-USE. Simplified formulation and colonies change from a flat morphology to a domed morphology characteristic of straightforward reversion protocol. naïve-state hPSCs. (B) Expression of naïve-associated genes (KLF2, KLF4, KLF17, TFCP2L1, STELLA, and DNMT3L) in hPSCs that were reverted to a naïve-like state in RSeT™ Feeder-Free Medium. Expression levels were measured by qPCR and normalized to levels in primed hPSCs.
Primed Human ES/iPS Cells Reversion to Naïve-Like State Naïve-Like Human ES/iPS Cells
Reversion of Primed to Naïve-Like hPSCs with RSeT™ Feeder-Free
Plate aggregates on Corning® Passage every Matrigel®-coated plates Switch medium Passage 3 - 6 days
Day
0 1 5 8+
Culture TeSR™ TeSR™ Medium RSeT™ Feeder-Free Medium RSeT™ Feeder-Free Medium Medium Medium*
*mTeSR™1, mTeSR™ Plus, or TeSR™-E8™
Figure 24. Schematic of Reversion of Primed to Naïve-Like hPSCs with RSeT™ Feeder-Free
Primed hPSCs are plated as aggregates in a TeSR™ medium (mTeSR™ Plus, mTeSR™1, or TeSR™-E8™). On day 1, TeSR™ medium is replaced with RSeT™ Feeder-Free Medium, and the medium is exchanged every other day. By day 4 or 5, the colonies are generally large enough to be passaged. During the initial culture in RSeT™ Feeder-Free Medium, colonies expand and begin to adopt a tightly packed, highly domed morphology characteristic of naïve-like stem cells with smooth and refractive colony edges as early as passage 1. Developed under license from the Weizman Institute of Science.6
Learn more at www.stemcell.com/RSeT-FeederFree 5
14 STEMCELL Technologies Inc. NaïveCult™ Achieve Serum- and Transgene-Free Induction Why Use NaïveCult™? and Expansion of Naïve Reset hPSCs NAÏVE. Maintains hPSCs with high expression of naïve-associated genes. NaïveCult™ is a serum-free media system that generates transgene-free reset naïve hPSCs from primed hPSCs and allows ROBUST. Works consistently across multiple human ES for their continual maintenance. NaïveCult™ contains pre- and iPS cell lines. screened quality components to work consistently across multiple TRANSGENE-FREE. No exogenous genes required for human embryonic stem (ES) and induced pluripotent stem (iPS) reversion to the naïve state. cell lines.
A B A B
15 20
15 10
10 5
5
of Fold Change 0 of Fold Change 2 2
Relative to TBP Relative to TBP 0 Log Log -5 -5 Normalized to Primed Sample Normalized to Primed Sample KLF2KLF4 KLF2KLF4 KLF17TBX3 OCT4 SOX2 KLF17TBX3 OCT4 SOX2 STELLA DNMT1 STELLA DNMT1 TFCP2L1 DNMT3L NANOG DNMT3B TFCP2L1 DNMT3L NANOG DNMT3B Figure 25. Human ES and iPS Cells Cultured in NaïveCult™ Show DNMT3A DNMT3A Characteristic Morphology of Naïve-State hPSCs Figure 26. hPSCs Cultured in the NaïveCult™ Media System Representative images of human (A) H9 ES cells at passage 7 and (B) WLS-1C Express High Levels of Factors Associated with Naïve hPSCs7-9 iPS cells at passage 9 that were reverted to a naïve state using the NaïveCult™ Human (A) H9 ES cells and (B) WLS-1C iPS cells were reverted using the Induction Kit and subsequently cultured in NaïveCult™ Expansion Medium. During NaïveCult™ Induction Kit and maintained in NaïveCult™ Expansion Medium. reversion, colonies change from a flat morphology to a tightly packed and uniformly Expression levels were measured by quantitative PCR (qPCR) and normalized to domed morphologyReversion with refractive edgesof Primedcharacteristic of naïve-stateto Naïve-Like hPSCs.7-9 hPSCs in NaïveCult™ levels in primed hPSCs.
Switch to Induction Seed primed hPSCs Medium 1 supplemented Switch Dissociate hPSCs Switch Switch as single cells on iMEFs with an HDACi medium into single cells medium medium
Day
0 1 4 11 Passage 3 Passage 4 - 8
Passage every 3 - 4 Passage every 3 - 4 Passage every 3 - 4 days onto iMEFs days onto iMEFs days onto iMEFs
Culture TeSR™ Expansion Induction Medium 1 Induction Medium 2 Induction Medium 3 Medium Medium* Medium
NaïveCult™ NaïveCult™ Induction Kit Expansion Medium
Note: From Day 0 onward, culture under hypoxic conditions (5% O2, 5% CO2). Perform full medium changes daily. *mTeSR™1, mTeSR™ Plus, or TeSR™-E8™
Figure 27. Schematic of Reversion of Primed to Naïve-Like hPSCs in NaïveCult™ 6 Primed hPSCs are plated on irradiated mouse embryonic fibroblasts (iMEFs) and treated with ROCK inhibitor (10 μM Y-27632) for 24 hours in hypoxic conditions. Background differentiation will decrease between passage 3 and 8. At this time, cells can be transferred into NaïveCult™ Expansion Medium for long-term maintenance and expansion. Developed under license from Cambridge Enterprises.7
Learn more at www.stemcell.com/NaiveCult
hPSC Naïve State qPCR Array The hPSC Naïve State qPCR Array provides a validated 90-gene assay to characterize the state of hPSCs in the spectrum from naïve to primed pluripotency. Data analysis is streamlined with our flexible online app (www.stemcell.com/qPCRanalysis).
Learn more at www.stemcell.com/naive-array
WWW.STEMCELL.COM 15 HUMAN PLURIPOTENT STEM CELLS
Matrices Cell culture matrices support the growth and differentiation of human pluripotent stem cells (hPSCs), including human embryonic stem (ES) and induced pluripotent stem (iPS) cells, by mimicking the in vivo extracellular matrix. When used with TeSR™ maintenance media, they provide a robust culture system for cell maintenance under feeder-free conditions.
Vitronectin XF™ Grow and Differentiate hPSCs Under Serum-Free, Why Use Vitronectin XF™ and Feeder-Free Conditions CellAdhere™ Laminin-521?
Vitronectin XF™, developed and manufactured by Nucleus Biologics, DEFINED. Decrease sources of variability in your is a defined, xeno-free cell culture matrix that supports the growth experiment with a recombinant human protein matrix. and differentiation of hPSCs. Use with TeSR™2 or TeSR™-E8™ media COMPATIBLE. Use with any TeSR™ family medium to for a completely xeno-free culture system and complete control over maintain hPSCs. the culture environment, resulting in more consistent cell populations and reproducible results in downstream applications. Human ES and XENO-FREE. Create a completely xeno-free system iPS cells cultured on Vitronectin XF™ retain pluripotency and normal when used with TeSR™2, TeSR™-E8™, or TeSR™-AOF. colony morphology, without the need for an adaptation step. Pair with Gentle Cell Dissociation Reagent (GCDR) or ReLeSR™ when passaging to maintain high-quality cultures.
H9 ESH9 H9Cells ESES Cells WLS-1CWLS-1CWLS-1C iPS Cells iPS Cells CellAdhere™ Laminin-521 Maintain hPSCs Long-Term in Feeder-Free Conditions
CellAdhere™ Laminin-521 is a defined and xeno-free cell culture
matrix that supports the growth and differentiation of ES and iPS cells under feeder-free conditions. Laminin-521 is expressed and secreted by hPSCs in the inner cell mass of the embryo and can therefore be used to create a biologically relevant hPSC culture environment in vitro. For consistent cell populations and reproducible results in downstream applications, use CellAdhere™ Laminin-521 with TeSR™ maintenance media to provide a defined culture substrate for cell maintenance. Compared to other matrices, CellAdhere™ Laminin-521 increases single-cell attachment and survival compared to other matrices and does not require the addition of apoptotic inhibitors during plating. NOTE: While it is possible to passage hPSCs as single cells, this practice may lead to genetic aberrations in the culture. If passaging Figure 28. Human ES and iPS Cells Exhibit Normal Morphology When Cultured on Vitronectin XF™ Cell Culture Matrix in TeSR™-E8™ as single cells, check the karyotype frequently.
Undifferentiated human ES (H9) and iPS (WLS-1C) cell cultures exhibit normal morphology when cultured on Vitronectin XF™. Colonies are round, tightly packed Learn more at www.stemcell.com/Laminin-521 and multilayered, with a high nucleus-to-cytoplasm ratio. Cells were transferred directly from Matrigel® hESC-Qualified Matrix without an adaptation step.
Note: Colonies grown in TeSR™-E8™ have a more condensed and round morphology when grown on Vitronectin XF™ matrix, compared to colonies grown on Matrigel® hESC-Qualified Matrix, which are more diffuse and irregularly shaped.
Learn more at www.stemcell.com/Vitronectin-XF
16 STEMCELL Technologies Inc. Dissociation Reagents ReLeSR™ and Gentle Cell Dissociation Reagent Why Use ReLeSR™ and GCDR? Passage hPSCs, Enzyme-Free SCALABLE. Compatible with culture processes involving closed vessels. ReLeSR™ selectively detaches undifferentiated cells from hPSC cultures, eliminating manual selection and scraping. Passaging CHEMICALLY DEFINED. Enzyme-free formulation hPSCs with ReLeSR™ enables the easy generation of optimally minimizes variability. sized aggregates while eliminating the hassle and variability of EASY-TO-USE. Easily generates optimally sized manual selection. By removing the need for scraping, ReLeSR™ aggregates without manual scraping. more readily enables the use of culture flasks and other closed COMPATIBLE. Use with any TeSR™ family medium, vessels, thus facilitating culture scale-up and automation. Vitronectin XF™ and Corning® Matrigel®. Gentle Cell Dissociation Reagent (GCDR) is an enzyme-free reagent suitable for the dissociation of hPSCs into cell aggregates for routine passaging or into a single-cell suspension. Learn more at www.stemcell.com/ReLeSR ReLeSR™ and GDCR are both manufactured under relevant cGMPs and can be used as part of a high-compliance hPSC maintenance workflow, such as with TeSR™-AOF medium and CellAdhere™ Learn more at www.stemcell.com/GCDR Laminin-521.
Pre-Treatment Post-Treatment
A B ACCUTASE™ For Creation of Single-Cell Suspensions
ACCUTASE™ is a cell detachment solution of proteolytic and
collagenolytic enzymes, and is useful for the routine detachment of
200 µm 200 µm cells from standard tissue culture plasticware and adhesion‑coated plasticware. ACCUTASE™ does not contain mammalian or After Shaking/Tapping bacterial‑derived products. C D
Dispase For Enzymatic Dissociation 200 µm 200 µm Dispase is a commonly used enzyme preparation recommended for Figure 29. ReLeSR™ Selectively Detaches Undifferentiated Cells passaging hPSCs maintained in feeder-free conditions on ® ® from hPSC Cultures Without Manual Selection and Generates Corning Matrigel . Optimally Sized Aggregates
(A) An hPSC culture ready for passaging. Note the presence of differentiated cells at the edge of the undifferentiated hPSC colony. (B) Following incubation with ReLeSR™, the undifferentiated hPSC colony starts to lift off of the cultureware. The differentiated cells remain attached to the cultureware. (C) Following shaking/tapping of the cultureware, the undifferentiated cells completely lift off of the cultureware. (D) The undifferentiated hPSC colony is broken up into optimally sized aggregates for replating.
WWW.STEMCELL.COM 17 HUMAN PLURIPOTENT STEM CELLS
Genome Editing CloneR™ Enhance the Cloning Efficiency and Why Use CloneR™? Single-Cell Survival of hPSCs EFFICIENT. Increased single-cell survival at low and clonal Genome editing of human pluripotent stem cells (hPSCs) relies densities. heavily on the survival of single cells to establish clonal lines. EASY-TO-USE. No adaptation to single-cell passaging CloneR™ is a serum-free supplement formulated for enhancing required. the cloning efficiency and single-cell survival of hPSCs, especially FLEXIBLE. Compatible with TeSR™ maintenance media under clonal and low-density seeding conditions. Designed for use and your choice of cell culture matrix. in feeder-free culture systems, this flexible supplement is compatible with TeSR™ maintenance media, a range of cell culture matrices, ROBUST. Increased cloning efficiency across multiple and cell lines. Unlike current methods, CloneR™ enables the robust human ES and iPS cell lines. generation of clonal hPSC lines without single-cell adaptation, thus minimizing the risk of acquiring genetic abnormalities.
50 50 A B C D ROCK Inhibitor (Y-27632) CloneR™
) 40 40 2
30 30
A B 20 20
10 10 Percent Cloning Efficiency with Cloning Efficiency Percent Density Seeding (25 cells/cm Single-Cell Deposition (1 cell/well) Single-Cell Percent Cloning Efficiency with Low with Low Cloning Efficiency Percent 0 0
H1 H7 H1 H7 H1 H7 H1 H7 H1 H7 H1 H7
WLS-1C WLS-1C WLS-1C WLS-1C WLS-1C WLS-1C Figure 30. CloneR™ Increases the Cloning Efficiency STiPS-M001 STiPS-M001 STiPS-M001 STiPS-M001 STiPS-M001 STiPS-M001
of hPSCs at Low Seeding Densities mTeSRTM1 TeSRTM-E8TM (A-D) hPSCs plated in TeSR™ medium supplemented with CloneR™ demonstrated significantly increased cloning efficiencies compared to cells plated in TeSR™ containing ROCK inhibitor (10 μM Y-27632). (A,B) Representative images of alkaline phosphatase-stained colonies at day 7 in individual wells of a 12-well plate. Human H1 ES cells were seeded at clonal density (100 cells/well, 25 cells/cm2) in (top) mTeSR™1 or (bottom) mTeSR™ Plus and supplemented with (A) ROCK inhibitor or (B) CloneR™ on Vitronectin XF™ cell culture matrix. Cells were hPSCseeded (C) at clonalSingle-Cell density (25 cells/cm 2)Cloning in mTeSR™1 or TeSR™-E8™ with andCloneR™ (D) by single-cell deposition using FACS (seeded at 1 cell/well) in mTeSR™1.
Seed single cells at clonal Full-medium Pick colonies and Long-term density in TeSR™ medium change expand in TeSR™ maintenance in supplemented with CloneR™ Switch medium medium TeSR™ medium
Day
-1 0 2 4 10 14 Full-medium change daily Full-medium change daily
CloneR™
Culture Medium mTeSR™ Plus, mTeSR™1, TeSR™-E8™, or TeSR™-AOF
Matrices Vitronectin XF™ or CellAdhere™ Laminin-521 or Corning® Matrigel®
Culture Vessel Well Plates 10-cm Dish or 96-Well Plate Well Plates
Figure 31. Schematic for hPSC Single-Cell Cloning with CloneR™ 7
On day 0, hPSCs are seeded as single cells at clonal density (e.g. 25 cells/cm2) or sorted at 1 cell per well in 96-well plates in a TeSR™ medium supplemented with CloneR™. On day 2, the cells are fed with TeSR™ medium containing CloneR™ supplement. From day 4, cells are maintained in TeSR™ medium without CloneR™. Colonies are ready to be picked between days 10 - 14. Clonal cell lines can be maintained long-term in a TeSR™ maintenance medium.
Learn more at www.stemcell.com/CloneR
18 STEMCELL Technologies Inc. ArciTect™ Genome Edit hPSCs with a CRISPR-Cas9 System Why Use ArciTect™?
The ArciTect™ product family is a ribonucleoprotein (RNP)-based EFFICIENT. Maximize delivery and expression in difficult- CRISPR-Cas9 genome editing system for hPSCs. From cell culture to-manipulate cell types by using RNP complexes. and single-cell survival to experimental design, designing guide EASY-TO-USE. Simplify genome editing with an RNAs, detection, and validation of editing efficiency, the ArciTect™ integrated Guide RNA Design Tool and cell-type-specific toolkit contains qualified solutions for every step in the hPSC genome protocols. editing workflow. Refer to the Technical Bulletin: Genome Editing of Human Pluripotent Stem Cells (Document #27084) for a protocol RAPID. Get to your results faster with ready-to-use optimized and validated for use with ArciTect™. purified Cas9 proteins and synthetic guide RNAs. PRECISE. Minimize potential off-target cutting with timely degradation of the RNP complex. A B ) 100 * ) 8 ells
Cells 80 6 eC 60 Efficiency Efficiency Viable Viabl 4 of 40 of - + P P 2
GF 20 BF Knockout Knock-in (% (% 0 0 A A P A A P N N rR RN o E RN o E g N rR g s s N trac : :trac NA NA rR rR c c
Figure 32. Efficient Genetic Knockout and Knock-In in Human Pluripotent Stem Cells Using the ArciTect™ CRISPR-Cas9 System
1C-eGFP hPSC lines were cultured in mTeSR™1 (Catalog #85850) supplemented with CloneR™ (#05888) for 24 hours after electroporation with CRISPR-Cas9 RNP complexes containing ArciTect™ Cas9 Nuclease and either ArciTect™ crRNA:tracrRNA duplexes or sgRNA targeting GFP, co-delivered with ssODN encoding nucleotides to convert GFP to BFP. (A) Knockout (% GFP- cells) and (B) knock-in (% BFP+ cells) efficiency were measured by flow cytometry 3 days after electroporation; n = 3. Control samples were not electroporated (no EP). Error bars represent standard deviation.
Learn more at www.ArciTect.com
WWW.STEMCELL.COM 19 HUMAN PLURIPOTENT STEM CELLS
Cell Quality Characterization hPSC Genetic Analysis Kit Detect Most Karyotypic Abnormalities in Human Why Use the hPSC Genetic Analysis ES and iPS Cells Using qPCR Kit?
The hPSC Genetic Analysis Kit contains primer/probe mixes to TARGETED. Designed to detect the majority of detect the majority of karyotypic abnormalities reported in human karyotypic abnormalities observed in human pluripotent embryonic stem (ES) and induced pluripotent stem (iPS) cells. This stem cell (hPSC) cultures. qPCR-based kit enables the genetic screening of multiple human ES RAPID. From cells in culture to results within one day. and iPS cell lines in a rapid and cost-effective manner, and includes COST-EFFECTIVE. Low cost per sample enables more enough material to analyze 20 individual samples in triplicate. Our frequent screening of multiple samples. online hPSC Genetic Analysis Tool (www.stemcell.com/geneticanalysisapp) is designed to help with CONVENIENT. Online hPSC Genetic Analysis Tool for data analysis and interpretation: simply input qPCR data and the streamlined data analysis and interpretation. tool will perform statistical analyses, assist with data interpretation, and provide visual representation of the data.
A B A B Copy Number Copy Number
1q 4p 8q 10p 12p 17q 18q 20q Xp 1q 4p 8q 10p 12p 17q 18q 20q Xp
Figure 33. The hPSC Genetic Analysis Kit Identifies Chromosome Figure 35. The hPSC Genetic Analysis Kit Identifies Chromosome 1 12 Trisomy Duplication via Unbalanced Translocation
Chromosome 12 trisomy in WLS-1C human iPS cell line is (A) detected using the Unbalanced rearrangement of chromosome 1 in the WLS-1C human iPS cell line, hPSC Genetic Analysis Kit and (B) confirmed by G-banding. in which an extra copy of the long (q) arm of chromosome 1 translocated to the short arm (p) of chromosome 21, was (A) detected using the hPSC Genetic Analysis Kit and (B) confirmed by G-banding.
A B C Copy Number Copy Number
Figure 34. The hPSC Genetic Analysis Kit Identifies Chromosome 20q11.21 Duplication
Chromosome 20q duplication in WLS-1C human iPS cell line is (A) detected using the hPSC Genetic Analysis Kit, (B) undetected by G-banding, and (C) confirmed by fluorescent in situ hybridization using probes for 20p11 (green) and 20q11.21 (red). Figure 36. The hPSC Genetic Analysis Kit Identifies Abnormalities in Cultures with Approximately 30% Mosaicism
Genetically normal WLS-1C human iPS cells were mixed in the indicated ratios Learn more at with WLS-1C human iPS cells containing a chromosome 20q duplication. Cultures www.stemcell.com/GeneticAnalysisKit with approximately 30% genetically abnormal cells exhibit a significantly enriched population of 20q11.21 duplication (orange bars).
20 STEMCELL Technologies Inc. STEMdiff™ Trilineage Differentiation Kit STEMdiff™ Trilineage Differentiation Kit
Validate Pluripotency with Directed Differentiation d te rm m rentia rm e er ffe d d The STEMdiff™ Trilineage Differentiation Kit provides a simple cell culture assay to functionally and reproducibly validate the ability of ode so o s Ectoderm Mesoderm Endoderm ct e nd human ES and iPS cells to differentiate to the three germ layers. This kit includes reagents and protocols to perform parallel in vitro directed Undi E M E EB differentiation experiments for each germ layer, clearly establishing trilineage differentiation potential within one week. Clear, quantitative 5 1 assay results evaluated by immunocytochemistry, flow cytometry, or transcriptome analysis make the STEMdiff™ Trilineage Differentiation Kit MT3 hPS C a valuable tool for establishing the pluripotency of human ES and iPS cell lines. 1 S 2 ed t T 2 rm m rentia rm e er ES ffe d d ode so o s ct e nd T 2 Undi E M E EB ™ Why Use the STEMdiff Trilineage oder m
t Differentiation Kit? c 1 5 1 Figure 2. Flow cytometry analysis shows efficient differentiation to all E 2 MT3 three germ layers R hPS C 1 ROBUST. Reproducible directed differentiation to all ES5 S 2 H9 cells were maintained in mTeSR™1, differentiated in vitro using the STEMdiff™ three germ layers across multiple pluripotent cell lines. M1 Trilineage Differentiation Kit and subjected to flow cytometry analysis. This T T 2 representativeCLEAR. experiment Easy-to-interpret shows efficient assay differentiation results. as evidenced by 1 ES m the double-positive expression pattern of lineage-specific markers for each R T 2 DEFINED. Complete, defined cell culture media. germ layer. T oder m t 2
c 1 Mesode r E EFFICIENT. Standardized, one-week protocol. 2 R R M 1 ES5 R4 M1 T T 4 S )
Ectoderm Mesoderm Endoderm 2 1 Ectoderm Mesoderm Endoderm S 1 m R ER1 100
T m E MES r
2 e 2 d s Mesode r l R l 0 T 4 M 1 T End o C e R4 E e 0
T v i E T 1 t 4 S i s Gene Expression (Log ) 2 o 40 S 1 14 S P
ER1 % Positive Cells % Positive % m E MES 20 r
e 2 d Figure 4. Molecular analysis of cultures differentiated with the T 4 0 T STEMdiff™ Trilineage Differentiation Kit show strong separation of End o PAX6 T SOX17 1 E lineage-specific markers E T 1 Figure The38. TheSTEMdiff™ STEMdiff™ Trilineage Trilineage Differentiation Differentiation Kit promotes Kit Promotes H9 cells were maintained in mTeSR™1 and subsequently differentiated in vitro Gene Expression (Log Figure 3. 14 S efficientEfficient differentiation Differentiation to all three to germAll Three layers Germ Layers using either directed differentiation with the STEMdiff™ Trilineage Differentiation Kit or spontaneous differentiation in embryoid bodies (EBs) using a 10-day HumanPluripotent pluripotent stem cellsstem (both cells iPS (bothand ES iPS cells and represented) ES cells were represented) maintained werein Figure 37. Molecular Analysis of Cultures Differentiated with the protocol in serum-containing medium. Undifferentiated cells, differentiated maintainedmTeSR™1, in differentiated mTeSR™1, usingdifferentiated the STEMdiff™ using Trilineage the STEMdiff™ Differentiation Trilineage Kit, and STEMdiff™ Trilineage Differentiation Kit Shows Strong Separation ectoderm, mesoderm and endoderm cells from the directed differentiation kit Differentiationsubjected to Kit flow and cytometry subjected analysis to flow (n cytometry= 13 biological analysis replicates (N = including13 biological 5 and EBs were then subjected to a microarray-based transcriptome analysis of Lineage-Specific Markers replicatesdistinct including cell lines). 5 The distinct markers cell usedlines). for The flow markers cytometry used for for eachflow germcytometry layer arefor to evaluate expression levels of key germ layer markers. Cells differentiated H9 cells were maintained in mTeSR™1 and subsequently differentitated in vitro eachlisted germ below layer the are x-axis. listed below the X-axis. using the STEMdiff™ Trilineage Differentiation Kit showed clear upregulation of using either directed differentiation with the STEMdiff™ Trilineage Differentiation appropriate germ layer-specific markers, whereas the same cells differentiated Kit or spontaneous differentiation in embryoid bodies (EBs) using a 10-day spontaneously in EBs did not show significant upregulation of mesoderm or protocol in serum-containing medium. Undifferentiated cells, differentiated hPSC Trilineage Differentiation endoderm makers. ectoderm, mesoderm, and endoderm cells from the directed differentiation kit and EBs were then subjected to a microarray-based transcriptome analysis to qPCR Array evaluate expression levels of key germ layer markers. Cells differentiated using the STEMdiff™ Trilineage Differentiation Kit showed clear upregulation of appropriate The hPSC Trilineage Differentiation qPCR Array provides a germ layer-specific markers, whereas the same cells differentiated spontaneously validated 90-gene assay to assess gene expression associated in EBs did not show significant upregulation of mesoderm or endoderm markers. with undifferentiated hPSCs or their derivatives undergoing the Forearly a complete stages of list differentiation, of related products, plus housekeeping including specialized controls and cell a culture and storage media, matrices, antibodies, cytokines and small Learn more at www.stemcell.com/TrilineageKit molecules,synthetic visitDNA www.stemcell.com/hPSCworkflow positive control. Data analysis is streamlined or contact with us at [email protected]. our flexible online app (www.stemcell.com/qPCRanalysis). Copyright © 2016 by STEMCELL Technologies Inc. All rights reserved including graphics and images. STEMCELL Technologies & Design, STEMCELL Shield Design, Scientists Helping Scientists, Learn more at www.stemcell.com/trilineage-array and STEMdiff are trademarks of STEMCELL Technologies Canada Inc. mTeSR is a trademark of WARF. All other trademarks are the property of their respective holders. While STEMCELL has made all reasonable efforts to ensure that the information provided by STEMCELL and its suppliers is correct, it makes no warranties or representations as to the accuracy or completeness of such information. WWW.STEMCELL.COM 21
STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485. PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED. HUMAN PLURIPOTENT STEM CELLS
Cryopreservation
Increase Post-Thaw Recovery and Viability with Serum-Free Cryopreservation Media Why Use FreSR™ Media?
™ ™ COMPATIBLE. Optimized for cells cultured in TeSR™ mFreSR and FreSR -S family media.
Conventional methods for cryopreservation of human pluripotent HIGH VIABILITY. Cells have higher post-thaw recovery stem cells (hPSCs) use fetal bovine serum, introducing an undefined and maintain high viability. component into the culture system. FreSR™ cryopreservation ROBUST. hPSCs retain undifferentiated cell markers and media are defined, serum-free, and optimized for use with cells expansion capability. cultured with TeSR™ maintenance media. Cells stored in FreSR™ media have higher recovery and maintain higher viability post- thaw than cells frozen via conventional methods using serum.10-13 mFreSR™ serum‑free medium is optimized for cryopreservation of hPSCs as aggregates. FreSR™-S animal component-free media is Learn more at www.stemcell.com/mFreSR optimized for cryopreservation of cells in single-cell suspension and provides faster post-thaw recovery of hPSC cultures compared with Learn more at www.stemcell.com/FreSR-S conventional freezing methods.
A B
CryoStor® CS10
Storage and cryopreservation of cells and tissues are important parts of the workflow for biological research. CryoStor® CS10 is animal component-free, cGMP-manufactured with USP Figure 39. hPSCs Frozen and Thawed as Single Cells with grade components, and designed to maintain high viability and FreSR™-S Display a Normal Karyotype maximize hPSC cell recovery after long-term storage. CryoStor® Karyograms of WLS-4D1 iPS cells that were frozen and thawed as single CS10 contains 10% dimethyl sulfoxide (DMSO) and provides a cells using FreSR™-S. (A) Thawed cells were seeded into culture containing safe and protective environment for cells and tissues during the TeSR™-E8™ medium and 10 µM Y-27632 and maintained as aggregates for freezing, storage, and thawing processes. five passages. (B) hPSCs were also subjected to a second freeze-thaw cycle as single cells with FreSR™-S and cultured for five passages as aggregates prior to collection of karyotype data.
Why Use CryoStor® CS10? A B ANIMAL COMPONENT-FREE. Chemically defined TM TM formulation.
REGULATORY COMPLIANCE. cGMP-manufactured with USP grade components; US FDA Drug Master File. % Viability % Recovery CONVENIENT. Ready-to-use and available in several size options. Figure 40. High Viability and Recovery of Cells Stored in FreSR™-S
hPSCs cryopreserved as single cells using FreSR™-S have (A) higher post‑thaw recovery (number of cells recovered per number of cells frozen) and (B) maintain higher viability (number of live cells per total number of cells) compared with competitor medium. All data values are plotted as percentages (n = 18, Learn more at www.stemcell.com/CryoStor-CS10 p < 0.0001 for each).
22 STEMCELL Technologies Inc. ThawSTAR® CFT2 Automated Thawing System Why Use ThawSTAR® CFT2? Standardize Your Thawing Performance SAFE. Reduce the risk of contamination by using Through Automation ThawSTAR® CFT2 as part of a sterile process inside a biosafety cabinet. Increase confidence in your cell thawing workflow by using RELIABLE. Achieve consistent, controlled cell thawing the ThawSTAR® CFT2 Automated Thawing System to ensure profiles. sample sterility and consistent thawing performance. ThawSTAR® CFT2—a sensor-based, water-free instrument—reduces the risk of QUALITY CONTROL. Document instrument performance contamination and delivers cell thawing profiles similar to those of and generate an audit trail with ThawSTAR® CFT2 a water bath. Using this convenient and compact system, you can confirmation vials. thaw your cells directly in the biosafety cabinet, in ~2.5 minutes. Built for quality control, ThawSTAR® CFT2 easily integrates into cell thawing processes within research and clinical settings requiring a higher level of compliance. You can document instrument Learn more at performance and create an audit trail by using ThawSTAR® CFT2 www.stemcell.com/ThawSTAR-CFT2 confirmation vials before and after each thaw session. Completing the system, the portable ThawSTAR® CFT2 Transporter protects your cells from transient warming events during handling and transport of frozen vials from long-term storage to the downstream thawing step.
A B
Figure 41. Frozen hPSCs Thawed Using the ThawSTAR® CFT2 Automated Thawing System Show High Recovery and Viability hPSCs cryopreserved in CryoStor® CS10 (Catalog #07930) at a concentration of 1 x 106 cells/vial were retrieved from liquid nitrogen, one week after storage. When thawed using the ThawSTAR® CFT2 Automated Thawing System or a water bath, (A) the mean live cell recovery was 9.05 x 105 vs. 9.35 x 105 cells, respectively and (B) the mean viability was 83.04% vs. 82.93%, respectively. The hPSCs were from 3 different cell lines (M001, 1C, and H9) and were tested in triplicates. Cell recovery and viability was assessed using a Nucleoview™ counter.
WWW.STEMCELL.COM 23 HUMAN PLURIPOTENT STEM CELLS
Differentiation STEMdiff™ Pluripotent Stem Cell Why Use STEMdiff™? Differentiation Media REPRODUCIBLE. Reduce experimental variability with When working with hPSCs, inconsistent differentiation is formulations optimized under rigorous quality controls. a common challenge, even with detailed and established EFFICIENT. Successfully differentiate across multiple ES differentiation protocols.14,15 Use STEMdiff™ to reproducibly and iPS cell lines. differentiate from multiple human embryonic stem (ES) and induced pluripotent stem (iPS) cell lines to cell types and organoids COMPATIBLE. Use with hPSCs cultured with TeSR™ originating from all three embryonic germ layers. For gene- maintenance media. edited or patient-derived hPSC lines, these optimized media and EASY-TO-USE. Standardize your differentiation to all protocols enable the generation of a variety of cell types with the three germ layers with simplified kit formats and detailed same genotype. The STEMdiff™ family of products is part of our protocols. complete system of reagents for hPSC culture and is compatible VERSATILE. Generate and bank progenitor cell types with TeSR™ maintenance media, including mTeSR™1 and for experimental flexibility, or as a reliable cell source for mTeSR™ Plus. customized downstream differentiation. Explore the following pages for tools to support neural, hematopoietic, immune, cardiac, renal, intestinal, pulmonary, pancreatic, and customizable cell and organoid differentiation.
Learn more at www.STEMdiff.com
24 STEMCELL Technologies Inc. Mesoderm Differentiation Pathways
Flexible Products for Differentiation OCT4 TRA-1-60 SSEA-3 • STEMdiff™ APEL™2 Human Pluripotent Stem Cells (hPSCs) • STEMdiff™ APEL™2-LI • TeSR™-E6 • TeSR™-E5 T • Cytokines Early Mesoderm • Small Molecules STEMdiff™ Mesoderm Induction Medium
Intermediate Lateral Plate Mesoderm Paraxial Mesoderm
Urogenital Kidney
STEMdiff™ Kidney Organoid Kit
Hemogenic Cardiac Endothelium Mesoderm CD146 CD73 Mesenchymal Stem/ CD105 Progenitor Cells
™ CD34 CD34 STEMdiff Mesenchymal CD34 CD45 CD31 cTnT Progenitor Kit CD144 CD43 Hematopoietic CD144 Cardiomyocytes Progenitors Endothelial Cells Smooth Muscle Cells
STEMdiff™ STEMdiff™ STEMdiff™ Hematopoietic Kit Endothelial System Cardiomyocyte System
GYPA CD71 Monocyte/ Lymphoid Erythroblasts Macrophage Progenitors Adipocytes Chondrocytes Osteoblasts Progenitors
STEMdiff™ MesenCult™ MesenCult™-ACF MesenCult™ Erythroid Kit Adipogenic Chondrogenic Osteogenic Differentiation Differentiation Differentiation Kit (Human) Kit Medium Kit
Microglia Monocytes Osteoclasts T Cells NK Cells
™ ™ ™ STEMdiff™ Microglia STEMdiff STEMdiff STEMdiff Differentiation Kit Monocyte Kit T Cell Kit NK Cell Kit STEMdiff™ Microglia Maturation Kit
WWW.STEMCELL.COM 25 HUMAN PLURIPOTENT STEM CELLS
STEMdiff™ Mesoderm STEMdiff™ Mesenchymal Induction Medium Progenitor Kit Differentiate to Early Mesoderm, Xeno-Free Derive Functional Mesenchymal Progenitor Cells
STEMdiff™ Mesoderm Induction Medium (MIM) is a defined, The STEMdiff™ Mesenchymal Progenitor Kit is optimized for the xeno-free medium for generation of early mesoderm cells from efficient and reproducible derivation of mesenchymal progenitor human embryonic stem (ES) and induced pluripotent stem (iPS) cells (MPCs) from human ES or iPS cells. This kit contains animal cells. Protocols for mesodermal differentiation can be difficult and component-free (ACF) induction medium, expansion medium, and inconsistent; therefore, use the short and simple STEMdiff™ MIM attachment substrate for the derivation and expansion of MPCs. monolayer protocol to differentiate your human pluripotent stem It uses a simple monolayer protocol to generate MPCs under cells (hPSCs). feeder‑free conditions in three weeks. Human ES or iPS cell-derived MPCs are capable of long-term expansion (Figure 43). The derived STEMdiff™ MIM produces a cell population enriched for early MPCs are characterized by strong expression of cell-surface markers mesoderm, as indicated by positive expression of Brachyury (T), CD73, CD90, and CD105, and lack expression of CD45. MIXL1, and NCAM markers (Figure 42).
1012 1011 ™ 10 Why Use STEMdiff MIM? 10 109
XENO-FREE. Defined formulation for mesoderm 108 induction. 107 106 RAPID. Induction of mesoderm after only 2 - 4 days of 105 differentiation. 104 103 EFFICIENT. Reproducible differentiation of multiple Cumulative Count Cell (Log) 102 human ES and iPS cell lines. P0 P1 P2 P3 P4 P5 P6 P7 P8 MULTIPOTENT. Generates early mesoderm cells that are Passage Number capable of differentiation to multiple downstream cell Figure 43. hPSC-Derived MPCs Generated Using the STEMdiff™ types. Mesenchymal Progenitor Kit Exhibit a High Rate of Cell Expansion in MesenCult™-ACF Plus Medium
The average cell expansion of human MPCs generated from hPSCs using the STEMdiff™ Mesenchymal Progenitor Kit. Error bars represent standard error of A B mean (SEM; n = 5).
™
% Positive Cells Why Use the STEMdiff Relative Expression T OCT4 SOX17 Mesenchymal Progenitor Kit?
Figure 42. STEMdiff™ MIM Efficiently Generates a Homogenous CONSISTENT. Serum-free and ACF formulation. Population of Early Mesoderm Cells ROBUST. Efficient and reproducible generation of MPCs (A) Data showing marker expression characteristic of early mesoderm (positive from multiple human ES or iPS cell lines. Brachyury (T) expression and negative OCT4 and SOX17 expression) on day 5 of the protocol. Data expressed as a mean percentage of cells expressing each marker FAST. Rapid derivation of MPCs in three weeks. ± SD, n = 33 (T, OCT4); n = 5 (SOX17). (B) Expression of undifferentiated cell FUNCTIONAL. Generates MPCs capable of markers (OCT4, SOX2, NANOG) and early mesoderm markers (T, MIXL1, NCAM), long-term expansion and differentiation to adipocytes, measured by qPCR and normalized to levels in undifferentiated cells; n = 2. osteoblasts, and chondrocytes.
Learn more at www.stemcell.com/STEMdiff-MIM Learn more at www.stemcell.com/STEMdiff-MPC
26 STEMCELL Technologies Inc. STEMdiff™ Endothelial Differentiation Kit Efficiently Differentiate hPSCs to Endothelial Cells
The STEMdiff™ Endothelial Differentiation Kit includes attachment substrate, animal component-free (ACF) endothelial induction medium, and endothelial expansion medium. It is optimized for differentiating hPSCs to endothelial-like cells on Corning® Matrigel®. This kit is designed to be used immediately after early mesoderm induction with STEMdiff™ Mesoderm Induction Medium.
A B Why Use the STEMdiff™ Endothelial Differentiation Kit?
EFFICIENT. Generate endothelial cells with no cell enrichment or sorting step required.
RAPID. Expand hPSC-derived endothelial cells faster than Figure 44. STEMdiff™ Endothelial Differentiation Kit Generates in FBS-containing media. Functional hPSC-Derived Endothelial Cells
(A) Endothelial cells generated from hPSCs (F016 cell line) using the STEMdiff™ Endothelial Differentiation Kit take up acetylated LDL when plated at 10,000 cells/cm2. (B) Cells are able to form tubular networks in vitro in a tube formation assay when plated at 20,000 cells/well in a 96-well plate for 24 hrs.
Figure 45. A Representative Flow Cytometric Analysis of Endothelial Marker Expression in hPSC-Derived Endothelial Cells hPSC (H9 cell line)-derived endothelial cells were obtained at Day 7 using STEMdiff™ Endothelial Induction Medium. Greater than 85% of the cells were CD34+ and had high levels of CD31 and CD144 expression. With subsequent passages (up to passage 5), the proportion of cells expressing endothelial markers (CD34, CD31, and CD144) increased.
Learn more at www.stemcell.com/endo-diff
WWW.STEMCELL.COM 27 HUMAN PLURIPOTENT STEM CELLS
STEMdiff™ Hematopoietic System Generate Hematopoietic Progenitor Cells, Immune Cells, and Blood Cells
The STEMdiff™ Hematopoietic Kit consists of serum-free basal medium and supplements designed for the generation of hematopoietic progenitor cells (HPCs). Optimized for a standardized, 12-day differentiation protocol, this kit supports robust differentiation of hPSCs into HPCs that can be identified by the expression of CD34 and CD45, and by the ability to form hematopoietic colonies of multiple lineages in colony- forming unit (CFU) assays with MethoCult™ medium. The resulting HPCs may be used for downstream assays or quantified in a CFU assay with MethoCult™ SF H4636 medium, designed specifically for use with hPSC-derived HPCs, or MethoCult™ H4435 Enriched medium. HPCs generated with the STEMdiff™ Hematopoietic Kit may be further differentiated using the STEMdiff™ Microglia Differentiation Kit or STEMdiff™ Monocyte Kit. HPCs and downstream cells in the erythroid lineage may be obtained directly using the STEMdiff™ Erythroid Kit, and HPC and immune cell types in the lymphoid lineages may be obtained using the STEMdiff™ NK and T Cell Kits.
A B
80 1x106 ™ 8x105 Why Use the STEMdiff 60
6x105 Cells Hematopoietic Kit? + Cells/Well
40 + 4x105
CD45 CONSISTENT. Reduce variability with a serum- and + 20 CD45 5 + 2x10 feeder-free formulation. 0 0 % CD34 H1 H9 # CD34 H1 H9 EASY-TO-USE. Produce HPCs in suspension for easy WLS-1C WLS-1C STiPS-F016 STiPS-B004 STiPS-F016 STiPS-B004 STiPS-M001 STiPS-M001 harvest with a simple monolayer protocol. ES iPS ES iPS RAPID. Generate HPCs in 12 days. Figure 46. Efficient and Robust Generation of CD34+CD4+ HPCs HIGH YIELD. Generate 4 - 18 million CD34+CD45+ HPCs Human ES and iPS cells were cultured for 12 days in single wells of 12-well with one kit. plates using the STEMdiff™ Hematopoietic Kit. At the end of the culture period, cells in suspension were harvested, stained, and analyzed by flow cytometry for FLEXIBLE. Generate HPCs across multiple human ES and the expression of hematopoietic cell surface markers CD34 and CD45. iPS cell lines. (A,B) Percentages and total numbers of CD34+CD45+ cells in cultures of human ES or iPS cells are shown for 6 cell lines. Data shown as mean ± SEM; n ≥ 3.
Schematic for Hematopoietic Differentiation Day 0 Day 3 Day 7 Day 12 Harvest hematopoietic Plate aggregates Half-medium Half-medium Half-medium Half-medium cells from at low density change change change change supernatant
Day
-1 0 2 3 5 7 10 12
Culture TeSR™ Medium A Medium B Medium Medium*
*mTeSR™1, mTeSR™ Plus, or TeSR™-E8™
Figure 47. Schematic for Hematopoietic Differentiation
One day before the differentiation protocol, hPSC colonies are harvested and seeded as small aggregates (100 - 200 μm in diameter) at 10 - 20 aggregates/cm2 in a TeSR™ maintenance medium (mTeSR™ Plus, mTeSR™1, or TeSR™-E8™). After one day, TeSR™ medium is replaced with Medium A to begin inducing the cells towards a mesoderm- like state (day 0). On day 2, a half-medium change is performed with fresh Medium A. On day 3, the medium is changed to Medium B with half-medium changes on days 5, 7, and 10 to promote further hematopoietic differentiation. Typically, by day 12, large numbers of HPCs can be harvested from the culture supernatant.
Learn more at www.stemcell.com/STEMdiffHeme
28 STEMCELL Technologies Inc. STEMdiff™ Monocyte Kit STEMdiff™ Erythroid Kit
Feeder-free and serum-free conditions provided by this kit ensure Differentiate hPSCs to erythroid progenitor cells (erythroblasts) a robust differentiation of hPSC-derived monocytes. Further expressing Glycophorin A and CD71. hPSCs are induced toward differentiation to dendritic cells or macrophages can be achieved erythroid-biased hematopoietic progenitor cells, and then further using ImmunoCult™ Dendritic Cell Culture Kit or ImmunoCult™-SF differentiated to erythroid progenitor cells (Day 10 - 24). Cells Macrophage Medium, respectively. generated using the STEMdiff™ Erythroid Kit can be further matured into normoblasts and reticulocytes once moved to For differentiation to microglia, please see page 41. appropriate culture conditions for maturation.
Why Use the STEMdiff™ Why Use the STEMdiff™ Monocyte Kit? Erythroid Kit?
HIGH YIELD. Generate up to 7 million CD14+ monocytes SIMPLE. Differentiate to erythroblasts in two stages, per plate in just 14 - 23 days. without feeders or serum.
RELIABLE. Eliminate variation introduced by serum and HIGH YIELD. Expand hematopoietic progenitor cells by feeder cells by using serum- and feeder-free conditions. > 200-fold during erythroid differentiations, with > 70% yield for CD71+GlyA+ erythroblasts. EASY-TO-USE. Produce monocytes in a simple monolayer culture for easier harvest of suspended cells. COMPATIBLE. Human iPSC/ESC-derived erythroblasts can be matured further using StemSpan™ SFEM II medium with appropriate supplements.
ield of D
er late A Mono te Mono te D
Hemoglobin Expression
S S (Relative to Housekeeping Genes) STi S M STi S M ell ine B C Figure 48. STEMdiff™ Monocyte Kit Enables Robust and Efficient Generation of CD14+ Monocytes
hPSCs were differentiated using the STEMdiff™ Monocyte Kit and harvested every 2 - 3 days between days 17 and 23. The average frequency of viable CD14+ monocytes at the peak harvest was 61 - 78% and the average yield of + 6 CD14 monocytes produced per 6-well plate was between 1.6 x 10 and Figure 49. hES and hiPS Cell-Derived Erythroid Cells Are 7.1 x 106 cells. Hemoglobinized and Display Typical Erythroid Morphology
(A) Erythroid cells generated with the STEMdiff™ Erythroid Kit express a mix of primitive (embryonic) and definitive (fetal, adult) hemoglobin. Shown Learn more at are the results of qPCR analysis for globin gene expression after 24 days of www.stemcell.com/STEMdiff-Monocyte culture. (B) A picture of the cell pellet shows that cells produced in culture are hemoglobinized. (C) Cells display typical basophilic erythroblast morphology after 24 days of culture using the STEMdiff™ Erythroid Kit (40X magnification, May-Grunwald Giemsa stain).
Learn more at www.stemcell.com/erythro-diff
WWW.STEMCELL.COM 29 HUMAN PLURIPOTENT STEM CELLS
STEMdiff™ NK Cell Kit STEMdiff™ T Cell Kit
Feeder-free and serum-free conditions ensure a robust Obtain high yields of CD4+CD8+ double-positive (DP) T cells by differentiation of hPSC-derived NK cells for developing adoptive differentiating from hPSCs in feeder-free and serum-free conditions immunotherapies in cancer patients as well as for research into the with this kit. Additionally, generate CD8+ single-positive (SP) T cells basic biology of these cells. with an optional protocol.
Why Use the STEMdiff™ NK Cell Kit? Why Use the STEMdiff™ T Cell Kit?
HIGH YIELD. Produce approximately 230 CD56+ NK cells HIGH YIELD. Produce approximately 60 CD4+CD8+ DP T per input hPSC-derived CD34+ cell. cells per input hPSC-derived CD34+ cell.
CONSISTENT. Reduce variability by producing uniform CONSISTENT. Reduce variability by producing uniform aggregates for embryoid body (EB) formation with aggregates for embryoid body (EB) formation with AggreWell™. AggreWell™.
SIMPLE. Avoid the extra passaging steps required with SIMPLE. Avoid the extra passaging steps required with stromal cell-based cultures. stromal cell-based cultures.
D D A B A B ield of D D D T ell
er n t D er n t D ield of D ell D T αβ
ell
D
ell D ell ell D D D T
D D Yield of CD4 C ield of D D D T ell D er n t D per Input CD34 S S S S STi S STi S STi S M STi S M STi S STi S M STi S M STi S ell
ell ell ine +
CD8 DP T Cells + +
DP T Cells + CD8 + + Cell
Figure 50. hPSCs Differentiate into CD56 NK Cells After 40 Days ell
of Culture D D D T %CD4 hPSCs were cultured using the STEMdiff™ NK Cell Kit for a total of 40 days.
Cells were harvested and analyzed for expression of CD56 and CD16 by flow
cytometry. (A) Representative flow cytometry plot is shown for ES (H1)-derived S S STi S STi S cells. (B) After 40 days of culture, the average frequency of viable CD56+ NK STi S M STi S M cells from hPSC-derived CD34+ cells ranged between 79% and 94%. The ell ine average yield of CD56+ cells produced per hPSC-derived CD34+ cell was + + between 108 and 404. Data are shown as mean ± SEM (n = 7 - 18). Figure 51. Generation of CD4 CD8 DP T Cells from Human hPSCs After a Total of 40 Days of Culture with the STEMdiff™ T Cell Kit
CD4+CD8+ DP T cells were differentiated from hPSCs using the STEMdiff™ T Cell Kit. Cells were harvested and analyzed for expression of CD3, CD4, CD8, and TCRαβ by flow cytometry. (A,B) Representative flow cytometry plots are shown for ES (H1)-derived cells. (C) The average frequency of viable CD4+CD8+ DP T cells on day 28 ranged between 23% and 58%, and the average yield of DP T cells produced per input hPSC-derived CD34+ cell was between 12 and 108. Data are shown as mean ± SEM (n = 6 - 17).
Learn more at www.stemcell.com/STEMdiff-NK Learn more at www.stemcell.com/STEMdiff-T
30 STEMCELL Technologies Inc. STEMdiff™ Cardiomyocyte System Standardize Your hPSC-Derived Cardiomyocyte Why Use the STEMdiff™ Workflow Cardiomyocyte Differentiation Kit?
The STEMdiff™ Cardiomyocyte Differentiation Kit consists COMPLETE. Supports the entire hPSC-derived of serum-free basal media and reagents optimized for a cardiomyocyte workflow. standardized, 15-day differentiation protocol. Resulting EASY-TO-USE. Simple monolayer protocol produces cardiomyocytes can be identified by the expression of a key cardiomyocytes in 15 days. marker, cardiac troponin T (cTnT). Contracting hPSC-derived HIGH YIELD. One kit generates over 50 million cardiomyocytes can be seen as early as day 8. cardiomyocytes expressing cTnT. Atrial cardiomyocytes expressing high levels of atrial-specific STANDARDIZED. Robust performance with minimal genes and expressing dominant atrial-like phenotypes, including variability across multiple hPSC lines. faster beat frequency and decreased field potential duration, can be generated with the STEMdiff™ Atrial Cardiomyocyte Differentiation Kit. After differentiation, the resulting cardiomyocytes can be used for additional downstream assays, such as disease modeling, drug discovery, and cardiotoxicity screening. Differentiated cardiomyocytes and atrial cardiomyocytes can be maintained long-term with the STEMdiff™ Cardiomyocyte Maintenance Kit. The STEMdiff™ Cardiomyocyte Dissociation Kit allows for standardized harvesting of hPSC-derived cardiomyocytes that are ready for use in downstream applications, analysis, and cryopreservation. hPSC‑derived cardiomyocytes can be cryopreserved using STEMdiff™ Cardiomyocyte Freezing Medium. Figure 53. hPSC-Derived Cardiomyocytes Exhibit a Robust and STEMdiff™ Cardiomyocyte Support Medium maintains the viability Stable Excitability Profile of cardiomyocytes after thawing or during dissociation, harvesting, Microelectrode array (MEA) voltage recordings of hPSC-derived cardiomyocytes or re-plating. (day 27) generated and maintained with the STEMdiff™ Cardiomyocyte Differentiation and Maintenance Kits showed a characteristic electrical profile and A B stable beat rate. A large depolarization spike followed by a smaller repolarization deflection was observed. 2.5x106 100
6 80 2.0x10 Cells
+ 1.5x106 Cells 60 + Learn more at www.stemcell.com/cardio-diff 6 40 1.0x10 % cTnT Total cTnT Total 5 20 5x10
H9 H9
WLS-1C WLS-1C STiPS-M001 STiPS-M001
Figure 52. Efficient and Robust Generation of cTnT-Positive Cardiomyocytes hPSCs were cultured for 15 days in single wells of 12-well plates using the STEMdiff™ Cardiomyocyte Differentiation Kit. At the end of the culture period, cells were harvested and analyzed by flow cytometry for expression of cell marker cTnT. (A) Percentages and (B) total numbers of cells expressing cTnT in cultures of human ES (H9) or iPS (WLS-1C and STiPS-M001) cells are shown. Data shown as mean ± SEM; n = 3.
WWW.STEMCELL.COM 31 HUMAN PLURIPOTENT STEM CELLS
STEMdiff™ Myogenic System Generate hPSC-Derived Myogenic Progenitors Why Use the STEMdiff™ Myogenic and Myotubes Progenitor Kit?
STEMdiff™ Myogenic Progenitor Supplement Kit consists of serum- OPTIMIZED. Enjoy compatible reagents designed to free supplements intended for use with DMEM/F12 to differentiate support the entire hPSC-derived myogenic progenitor hPSCs to myogenic progenitor cells. The latter, which are workflow. characterized by myogenic cell markers such as CD56 and CD82, RELEVANT. hPSC-derived myotubes display organized can be culture-expanded for 5+ passages using the MyoCult™-SF sarcomeric proteins and contractility. Expansion Supplement Kit (Human) and further differentiated to functional multinucleated MyHC+ myotubes at high efficiency using the MyoCult™ Differentiation Kit (Human). These myotubes can be used in various downstream applications and analyses.
A B A
B
C D
C
Figure 55. hPSC-Derived Myotubes Generated Using the STEMdiff™ Myogenic Progenitor Kit Are Efficiently Differentiated and Functionally Contractile Figure 54. STEMdiff™ Myogenic Progenitor Kit Generates hPSC-derived myogenic progenitors were generated from the M001 cell line Expandable hPSC-Derived Myogenic Progenitors using the STEMdiff™ Myogenic Progenitor Kit and then induced to differentiate into myotubes using MyoCult™ Differentiation Medium (Human). (A) After 8 (A) Representative image of proliferating sub-cultured hPSC-derived myogenic days, myotubes were fixed and stained for MyHC and MyoD. (B) Multiple hPSC progenitors generated using the STEMdiff™ Myogenic Progenitor Kit. (B) cell lines differentiated and induced using said method exhibited high fusion Expansion rates of hPSC-derived myogenic progenitors (hSPC-MP) over 5 indices similar to primary human myoblasts. (Numbers in bars represent the n passages across multiple hPSC lines are comparable to human primary myoblasts. number and dots represent technical replicates.) (C) hPSC-derived myotubes Error bars represent standard error of mean, n = 3. (C) hPSC-derived myogenic were stained for alpha-actinin and displayed organized sarcomeric structures as progenitors harvested at passage 5 expressed human myoblast markers CD56 indicated by the zoomed-in area. (D) Spontaneous field potential recordings of and CD82. hPSC-derived myotubes using a microelectrode assay plate indicated that the derived myotubes are contractile.
Learn more at www.stemcell.com/myo-diff
32 STEMCELL Technologies Inc. STEMdiff™ Kidney Organoid Kit Differentiate Kidney Organoids from hPSCs
Directed differentiation of hPSCs into kidney organoids allows researchers to work with an in vitro model culture system that has direct relevance to the developing human kidney. Kidney organoids form large (~150 - 400 μm), branched structures containing endothelial cells, podocytes, and epithelial cells of the proximal and distal tubules, mimicking nephron-like structure and segmentation. Kidney organoids modeling both health and disease in specific genetic backgrounds can be created by reprogramming patient‑derived cells. These in vitro models can be further manipulated by introducing or correcting mutations through CRISPR-Cas9 gene editing prior to differentiation. This approach has successfully been used to model polycystic kidney disease and podocyte organization during development.16,17 Like other hPSC‑derived organoid systems, kidney organoids resemble the first trimester kidney and display markers of the developing kidney as well as markers of differentiation.18,19 The STEMdiff™ Kidney Organoid Kit enables growth of tubular kidney organoids from ES and iPS cells in 21 days. These organoids are suitable for a wide range of experimental contexts, including developmental and cell biology, disease modeling, drug screening and nephrotoxicity assessment, and cell therapy research.
A Why Use the STEMdiff™ Kidney Organoid Kit?
RELEVANT. Produces human kidney organoids that model the developing nephron and associated endothelium.
B C D STRAIGHTFORWARD. Minimizes culture manipulations with a two-stage culture system and easy-to-follow protocol.
RELIABLE. Provides low experimental variability through an optimized formulation and rigorous quality controls. Figure 56. Kidney Organoids Display Distinct Domains of the Developing Nephron HIGH THROUGHPUT. Reproducibly generates organoids in 96- and 384-well formats. hPSC-derived kidney organoids incorporate cells and organization mimicking the structure and segmentation of the developing nephron. (A, B) Branched, tubular organoids displaySchematic markers of proximal fortubules Differentiation(LTL, green), distal tubules (ECAD, from hPSCs to Human Kidney white), and podocytes (PODXL, red), while DAPI (blue) shows the nuclei of all cells, including (C)Organoids endothelial cells (CD31, white)with and (D)the mesenchyme STEMdiff (VIM, white; Kidney Organoid Kit Meis 1/2/3, red). Scale bar = 200 µm.
Seed human ECM Initiate Late primitive Tubular kidney ES/iPS cells sandwich differentiation streak organoids
Day
-3 -2 -1 0 1.5 18
Culture Medium mTeSR™1 STEMdiff™ Kidney Basal Medium
Stage Human ES/iPS Cells Cavitated PSC Stage 1 Stage 2 Spheroids
Figure 57. Schematic for Differentiation from hPSCs to Human Kidney Organoids with the STEMdiff™ Kidney Organoid Kit
(A) hPSC cultures progress through a simple three-stage process to generate kidney organoids. hPSCs are plated and overlaid with Corning® Matrigel® to form cavitated spheroids. These are induced towards the late primitive streak and intermediate mesoderm, forming tubular kidney organoids by day 18 of differentiation.
Learn more at www.stemcell.com/STEMdiffKidney
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Ectoderm Differentiation Pathways
Flexible Products for Differentiation OCT4 • BrainPhys™ Neuronal Medium TRA-1-60 SSEA-3 • BrainPhys™ Without Phenol Red Human Pluripotent • NeuroCult™ SM1 Neuronal Supplement Stem Cells (hPSCs) • NeuroCult™ SM1 Without Vitamin A • NeuroCult™ SM1 Without Antioxidants • N2 Supplement-A • N2 Supplement-B Ectoderm • Cytokines • Small Molecules
Neural Epithelium Neural Crest Neuroectoderm
PAX6 PAX6 MAP2 MAP2 NKX2.1 SOX10 PAX6 TBR1 TBR1 LHX6 CD271 SOX1 CTIP2 CTIP2 SST PAX7 NESTIN SATB2 SATB2 GAD Dorsal Forebrain Ventral Forebrain Cerebral Organoid Neural Crest Cells Neural Progenitor Cells Organoid Organoid
STEMdiff™ STEMdiff™ SMADi Neural STEMdiff™ STEMdiff™ Neural Crest Induction Kit STEMdiff™ Cerebral Dorsal Forebrain Ventral Forebrain Differentiation Kit ™ Organoid Kit Organoid Organoid STEMdiff Neural Differentiation Kit Differentiation Kit Progenitor Medium
GFAP * S100B Other Neurons Astrocytes
MAP2 VGLUT1 STEMdiff™ Astrocyte TH FOXA2 GAD67 Differentiation Kit Midbrain Forebrain Spinal Motor Striatal Medium * * Dopaminergic Glutamatergic and STEMdiff™ Astrocyte Neurons Spiny Neurons* Neurons* * Maturation Kit GABAergic Neurons
STEMdiff™ STEMdiff™ Midbrain Neuron Forebrain Neuron Differentiation Kit Differentiation Kit STEMdiff™ STEMdiff™ Midbrain Neuron Forebrain Neuron Maturation Kit Maturation Kit
*Flexible products for differentiation can be used for these cell types.
Other Products for Differentiation
• STEMdiff™ Microglia Differentiation Kit
34 STEMCELL Technologies Inc. STEMdiff™ Neural System Differentiate hPSCs to Neural Progenitor Cells, Why Use the STEMdiff™ Neurons, and Glia Neural System?
The STEMdiff™ SMADi Neural Induction Kit combines DEVELOPMENTALLY RELEVANT. Follow the in vivo STEMdiff™ Neural Induction Medium with STEMdiff™ SMADi developmental program with a small molecule-based Neural Induction Supplement, which directs differentiation by system, with no introduction of foreign genetic material. blocking TGF-b and BMP-dependent SMAD signaling, resulting in VERSATILE. Configure your NPC protocol with or efficient neural induction of even hard-to-differentiate cell lines. without dual SMADi inhibition, and use embryoid body or Neural progenitor cells (NPCs) can be generated using the monolayer protocols for workflow flexibility. STEMdiff™ SMADi Neural Induction Kit with either an embryoid SCALABLE. Save time and effort by expanding NPCs body (EB) protocol or monolayer culture protocol. STEMdiff™ several fold without loss of differentiation potential, and Neural Rosette Selection Reagent allows rapid and efficient cryopreserve NPCs for additional flexibility. isolation of neural rosettes to enrich for CNS-type NPCs. COMPATIBLE. Seamlessly transition from TeSR™ hPSC NPCs generated using the STEMdiff™ SMADi Neural Induction maintenance media to STEMdiff™ neural induction. Kit can be efficiently expanded and cryopreserved in serum‑free STEMdiff™ Neural Progenitor Medium and STEMdiff™ Neural Progenitor Freezing Medium, respectively. STEMdiff™ Forebrain NPCs cultured in STEMdiff™ Neural Progenitor Medium display typical NPC morphology (Figure 58D) and can be consistently Differentiation and Maturation Kits expanded three- to five-fold upon each passage to generate a A mixed population of excitatory and inhibitory forebrain-type large number of cells. NPCs generated using the STEMdiff™ (FOXG1+) neurons can be generated using the serum-free STEMdiff™ SMADi Neural Induction Kit can be differentiated to functional Forebrain Neuron Differentiation Kit and STEMdiff™ Forebrain neuronal subtypes using the lineage-specific STEMdiff™ Neuron Maturation Kit. The basal medium for the maturation kit Differentiation and Maturation Kits. is BrainPhys™, a neuronal medium designed to support electrical activity and neuronal maturation for functional neurons. A B A B
500 µm 100 µm 50 µm 50 µm
C D C D
100 µm 50 µm 50 µm DAPI PAX6 SOX1 Nestin Figure 59. Downstream Differentiation of Neural Progenitor Cells Figure 58. Neural Induction Using the STEMdiff™ SMADi to Neurons Is Possible Using the STEMdiff™ Differentiation and Neural Induction Kit and STEMdiff™ Neural Progenitor Medium Maturation Kits Generates Neural Rosettes and Enriches for CNS-type Neural (A) NPCs generated from STiPS-R038 hPSCs in mTeSR™1 using the STEMdiff™ Progenitor Cells SMADi Neural Induction Kit EB protocol were differentiated and matured to Starting hPSCs were maintained in mTeSR™1 and differentiated using an cortical neurons using STEMdiff™ Forebrain Neuron Differentiation Kit for 7 EB protocol. (A) Morphologically distinct neural rosettes (arrowheads) are days and STEMdiff™ Forebrain Neuron Maturation Kit for 14 days. The resulting clearly visible two days after replating EBs. (B,C) NPCs express CNS-type NPC cultures contain a highly pure population of (B) class III β-tubulin-positive markers PAX6 (B,C; green), SOX1 (B,C; red) and Nestin (C; purple). Nuclei are neurons (green) with less than 10% GFAP-positive astrocytes (not shown). (C) counterstained with DAPI. (D) NPCs maintained in STEMdiff™ Neural Progenitor The generated neurons are also positive for FOXG1 expression (red), indicating a Medium (C) display typical NPC morphology (shown at day 6 of passage 1). forebrain-type identity. (D) Nuclei are labeled with Hoechst (blue).
Learn more at Learn more at www.stemcell.com/STEMdiff-NIM-SMADi www.stemcell.com/STEMdiff-Neuron
WWW.STEMCELL.COM 35 HUMAN PLURIPOTENT STEM CELLS
STEMdiff™ Midbrain STEMdiff™ Astrocyte Differentiation and Maturation Kits Differentiation and Maturation Kits
Dopaminergic neurons can be generated using the serum-free A highly pure population of astrocytes can be generated using the STEMdiff™ Midbrain Neuron Differentiation Kit and STEMdiff™ STEMdiff™ Astrocyte Differentiation Kit and STEMdiff™ Astrocyte Midbrain Neuron Maturation Kit. The midbrain-patterned cell Maturation Kit. Matured astrocytes are functional, as assayed by population produced contains FOXA2-, LMX1A-positive neuronal calcium imaging (data not shown) and can be used for co-culture precursors, yielding neurons that can be maintained long‑term in applications. culture (Figure 60).
A A
B C B C
D E D E
Figure 60. Midbrain-Type Neurons Express Tyrosine Hydroxylase Figure 61. Cortical-Type Astrocytes Are Generated After Culture and Dopamine Transporters (DAT) After Differentiation and in STEMdiff™ Astrocyte Differentiation and Maturation Kits
Maturation in STEMdiff™ Midbrain Neuron Kits (A) NPCs generated from hPSCs in TeSR™-E8™ using the STEMdiff™ SMADi (A) NPCs generated from H9 hPSCs in mTeSR™1 using the STEMdiff™ SMADi Neural Induction Kit embryoid body (EB) protocol were differentiated and Neural Induction Kit monolayer protocol were differentiated and matured to matured to cortical-type astrocytes after culturing with the STEMdiff™ Astrocyte midbrain-type neurons using the STEMdiff™ Midbrain Neuron Differentiation Differentiation Kit for 3 weeks and STEMdiff™ Astrocyte Maturation Kit for 3 Kit for 12 days and STEMdiff™ Midbrain Neuron Maturation Kit for 14 days. weeks. (B) Nuclei are labeled with DAPI (gray). The resulting cultures contain a The resulting cultures contain a population of (B) class III β-tubulin-positive highly pure population of astrocytes, which are (C) more than 60% GFAP- neurons (red), which (C) express DAT in blue, and (E) more than 15% tyrosine positive (green) and (D) more than 70% S100B-positive (magenta), with (E) hydroxylase-positive cells (green). (D) Nuclei are labeled with DAPI (white). fewer than 15% neurons (DCX-positive cells, cyan).
Learn more at Learn more at www.stemcell.com/STEMdiff-Dopa www.stemcell.com/STEMdiff-Astro
36 STEMCELL Technologies Inc. STEMdiff™ Cerebral Organoid Kit
Cerebral organoids are three-dimensional in vitro cultures that recapitulate the developmental processes and organization of the developing human brain. The STEMdiff™ Cerebral Organoid Kit is designed to generate unpatterned, multi-layered neural organoids from human ES and iPS cells. For extended periods of organoid culture, the kit components required for organoid maturation are available separately as the STEMdiff™ Cerebral Organoid Maturation Kit.
A B Why Use the STEMdiff™ Cerebral Organoid Kit?
UNPATTERNED. Allow differentiation to occur spontaneously to generate multiple brain regions within the same organoid.
C D FLEXIBLE. Culture under matrix droplet embedding or liquid matrix conditions.
OPTIMIZED. Enjoy increased efficiency of organoid formation with a formulation based on a popular published protocol.20
COMPATIBLE. Use as a platform for generating new or E F modified organoid models.
Learn more at www.stemcell.com/COKit
Figure 62. Cerebral Organoids Contain Multiple Layered Regions That Recapitulate the Cortical Lamination Process Observed During In Vivo Human Brain Development
(A) A representative phase-contrast image of a whole cerebral organoid at day 40 generated using the STEMdiff™ Cerebral Organoid Kit. Cerebral organoids at this stage are made up of phase-dark structures that may be surrounded by regions of thinner, more translucent structures that display layering (arrowheads). (B) Immunohistological analysis on cryosections of cerebral organoids reveals cortical regions within the organoid labeled by the apical progenitor marker PAX6 (red) and neuronal marker b-tubulin III (green). (C-F) Inset of boxed region from (B). (C) PAX6+ apical progenitors (red, enclosed by dotted line) are localized to a ventricular zone-like region. b-tubulin III+ neurons (green) are adjacent to the ventricular zone. (D) CTIP2, a marker of the developing cortical plate, co-localizes with b-tubulin III+ neurons in a cortical plate-like region. Organization of the layers recapitulatesSchematic early corticogenesis for observed the during STEMdiff human brain development. Cerebral (E) Proliferating Organoid progenitor cells labeled Kit by Ki-67 (green) localize along the ventricle, nuclei are counterstained with DAPI (blue). (F) An additional population of Ki-67+ cells is found in an outer subventricular zone-like region (arrowheads). Scale bars = (A) 1 mm, (B) 500 µm, and (C-F) 200 µm.
Embryoid body Resuspend Matrigel® embed Switch media Assay formation EBs EBs organoids
Day
0 5 7 10 40+ hPSC Change medium Change medium maintenance every 2 days every 3 - 4 days
Culture TeSR™ EB Formation Induction Medium Expansion Medium Maturation Medium Medium Medium* Medium
*mTeSR™1, mTeSR™ Plus, or TeSR™-E8™
Figure 63. Schematic for Generating Unpatterned Neural Organoids Using the STEMdiff™ Cerebral Organoid Kit
The protocol for generating human cerebral organoids using the STEMdiff™ Cerebral Organoid Kit involves EB formation followed by neural induction. After embedding in extracellular matrix, the neuroepithelia proliferate and expand. Organoids are then matured and can be maintained for extended periods over 40 days with the STEMdiff™ Cerebral Organoid Maturation Kit. Based on the protocol published by MA Lancaster and JA Knoblich.20
WWW.STEMCELL.COM 37 HUMAN PLURIPOTENT STEM CELLS
STEMdiff™ Dorsal and Ventral Forebrain Organoid Kits Why Use the STEMdiff™ Dorsal and Ventral Forebrain Organoid Kits? Robustly generate three-dimensional, patterned brain organoid cultures from human pluripotent stem cells without matrix PATTERNED. Direct differentiation to brain regions of embedding. The STEMdiff™ Dorsal and Ventral Forebrain interest with small molecule patterning factors, based on Organoid Differentiation Kits are serum-free cell culture media a popular published protocol.21 A Reliable,A Reliable,Efficient, Efficient, andA Reliable, Feeder-Free andthat Efficient, workFeeder-Free with Method AggreWell™-generated and Feeder-FreetoMethod Generate embryoid to bodies Generate MethodBrain-Region-Specific toBrain-Region-Specific GenerateREPRODUCIBLE. TakeBrain-Region-Specific Dorsal advantage andof reproducibleDorsal Ventral and Ventral Dorsal and Ventral (EBs) to differentiate brain-region-specific organoids that are structural morphology between lines and individual ForebrainForebrain Organoids Organoids ForebrainFrom Human From Organoidsrepresentative Pluripotent Human of the developingFrom Pluripotent Stem humanHuman forebrain. Cells StemPluripotent to Model Cells to EarlyStem Modelorganoids HumanCells Earlyto detect to Brainsubtle ModelHuman disease Development phenotypes.Early Brain Human Development Brain Development eon e eon da on e o e da Eloi eon on e o Mer ier e eanne Eloi da Mer ier an on e o Mar eanne ill Eloi an ErinMer ier Mar Kno ill eanne llen Erin an Kno Ea e Mar llen Terr ill E Ea e Erin T o a Kno Terr and llen E S aron T o a Ea e o i and Terr S aron E T o a o i and S aron o i The STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit SCALABLE. Use with AggreWell™800 to generate over STEM E Te nologie an o er STEM E anada Te nologie Terr o an o er a orator anada STEM E an er Terr gen Te nologie o a orator an o er an o er an er anada gen anada an o er Terr o a orator anada an er gen an o er anada generates tissue of the early developing dorsal pallium,PAX6 whileMAP2 PAX6 MAP2 500B PorganoidsAX6 MAP2 perPAX6 kitMAB PforP2AX6 MAscreeningP2 C CorTIP2 moreSATB2 TB R1detailedB PAX6 MAC P 2CTIP2 SATB2 TBR1 C CTIP2 SATB2 TBR1 INTRODUCTION INTRODUCTION INTRODUCTION the STEMdiff™ Ventral Forebrain Organoid Differentiation Kit longitudinal study. generates tissue of the early developing ventral subpallium.
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Ste Ste r Ste r r r development. These brain organoid models have enabled scientists to study early fetal human brain developmentSeed ell in S en ion in vitro lt re for theSeed ell in S en iona l lt re Seed ell in S en ion lt re s t r t r t r O ggre ell™ ggre ell™ ggre ell™ O O O
first time. For many of these methods, however, variability in the quality and consistency of organoids generated within and e n e n e n Do r V V V among experiments has hampered their widespread adoption as a research tool. Here eedingwe present work ll edi demonstrating eeding ll edi that the ll edi ll edi eeding ll edi ll edi recently developed STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit and STEMdiff™ Ventral ange dail Forebrain ange e er Organoid da ange dail ange e er da ange dail ange100µm e er D da ay 25 Day 100 100 µm 250 µm 250 µm 100 µm D100250 µ50m µm D150 100 µmD20050 µm 250 µm 100 µm 100 µm 50 µm 10020 100µ µmm µm 20 µm 100 µm 20 µm Differentiation Kit can be used to robustly generate homogeneous brain-region-specific lt re Medi tissue representing lt re Medi the dorsal lt re Medi TeS ™ or ation E an ion TeS ™ Medi or ationDifferentiationE an ion Medi Medi TeS ™ Maintenan eDifferentiation or ationDa yMedi 25Medi E an ion Medi Maintenan e Medi Differentiation Medi Day 50 Maintenan e Medi Day 100 Day 150 Day 200 neocortex and ventral telencephalon, respectively. The forebrain organoids can be characterizedTeS ™ E ™ forMedi electrical activityTeS ™ E ™ usingMedi TeS ™ E ™ Medi Day 25 Day 25 Day 50 Day 25Day 50 Day 75 Day 50 Day 75 Day 75 TeS ™ l TeS ™ l TeS ™ l multielectrode array (MEA) and further used in co-culture to form assemblies or “assembloids” with other 2D or 3D systems. D NKX2.1 DAPI E SST NXK2.1 FIGURE 4. DorsalF SandST NKX2 VentralFIGURE.1 Forebrain 4. Dorsal Organoidsand Ventral Express ForebrainFIGURE Appropriate 4.Organoids Dorsal andMarkers Express Ventral by Appropriate ImmunostainingForebrain OrganoidsMarkers by Express Immunostaining Appropriate Markers by Immunostaining Together, these kits enable the further study of the complexity of brain development To and generate disorders entral fore rain in organoid vitro. To e generate E an ion entral Medi fore rain entral organoid S le ent e E an ion To Medi generate entralFigure entral S le ent fore rain 64. organoid Dorsal e E an ion Forebrain Medi entral S le ent Organoids Exhibit(A) DayCortical 25 dorsal Layering, forebrain(A) organoids Dayand 25 Both dorsal display forebrainDorsal multiple organoidsand cortical-like (A)Ventral Day display regions25 Organoidsdorsal multiple marked forebrain cortical-like by Express radializedorganoids regions Markers PAX6+display marked cellsmultiple byCharacteristic surrounded radialized cortical-like PAX6+ by MAP2regions of cells neurons. marked surrounded by radialized by MAP2 PAX6+ neurons. cells surrounded by MAP2 neurons. FIGURE 1. Workflow forFIGURE STEMdiff™ 1. Workflow Dorsal Forebrainfor STEMdiff™ OrganoidFIGURE Dorsal 1. Kit Workflow Forebrain and STEMdiff™ for Organoid STEMdiff™Their Ventral Kit Respective and Dorsal Forebrain STEMdiff™ Forebrain Organoid Brain Ventral Organoid Regions Kit Forebrain Kit and Organoid STEMdiff™ Kit (B)Ventral Day 50Forebrain dorsal forebrain Organoid(B) Dayorganoids Kit 50 dorsal continue forebrain to display organoids(B) multiple Day continue 50 cortical-like dorsal to display forebrain regions multiple organoids marked cortical-like bycontinue PAX6 regions andto display MAP2 marked multiple. (C) by Dorsal PAX6 cortical-like forebrain and MAP2 regions . (C) markedDorsal forebrainby PAX6 and MAP2. (C) Dorsal forebrain METHODS hPSCs maintained in mTeSR™1hPSCs maintained (6 cell lines: in 3mTeSR™1 ES, 3 iPS), (6 hPSCsTeSR™-E8™ cell lines: maintained 3 ES,(3 cell 3 iniPS), lines: mTeSR™1 TeSR™-E8™ 2 ES, 1(6 iPS), cell (3 orlines: cell mTeSR™ lines:3 ES, 23 Plus ES,iPS), 1(2 TeSR™-E8™iPS), cell lines:or mTeSR™ 1 ES,(3 cell 1 Plus iPS) lines: (2 cell 2 ES, lines:organoids 1 iPS), 1 ES, or 1mTeSR™cultured iPS) for Plus 100organoids (2 - cell200 lines:days cultured 1show ES, forincreasing1 iPS) 100 - 200 separation daysorganoids show of increasing deep-layercultured for separation neurons 100 - 200 (CTIP2 ofdays deep-layer ,show TBR1 increasing) fromneurons upper-layer (separationCTIP2, TBR1neurons of deep-layer) from (SATB2 upper-layer neurons). neurons (CTIP2 ,( SATB2TBR1) from). upper-layer neurons (SATB2). were dissociated and seededwere atdissociated a density ofand 3,000,000 seeded atcells/well a weredensity dissociatedin ofFormation 3,000,000 and Medium cells/well seededa i n + 10(A) atin µMFormation aDay density rho-kinase 25 dorsalof Medium 3,000,000 inhibitor forebrain + 10 cells/well(ROCKi) µM organoidsrho-kinase inin AgFormationgreWell™800 inhibitor display Medium (ROCKi) multiple + 10 in cortical-likeAg(D)µMgreWell™800 rho-kinaseVentral forebrain regions inhibitor organoids marked (ROCKi)(D) Ventral by inat Agradializedday forebraingreWell™800 25 exhibit organoidsPAX6 a high+ cells atlevel (D)surroundedday ofVentral 25 expression exhibit forebrain by a MAP2highof NKX2.1. organoids level neurons. of (E) expression at and day (B) (F) 25Day Somatostatin ofexhibit 50 NKX2.1. dorsal a high (E) forebrain(SST level and)-positive of(F) expression Somatostatin GABAergic of NKX2.1. (SST )-positive (E) and GABAergic (F) Somatostatin (SST)-positive GABAergic
0 6 25 43 ds Da plates. Cultures were fedplates. daily withCultures Formation were fedMedium daily withoutwithplates. Formation ROCKi. Cultures Medium After were 6 days, withoutfedeb r daily aggregates ROCKi. with Formation After were 6 transferreddays, Medium aggregates without to a 6-well ROCKi.were plate transferred After in either 6 days, to a aggregates6-wellinterneurons plate werein either aretransferred not observedinterneurons to a 6-well at day plate are50 but notin either canobserved be seen at daybyinterneurons day 50 but75. can are be not seen observed by day at75. day 50 but can be seen by day 75.
o r Dorsal Forebrain Organoids Exhibit Cortical Layering and
F organoids continue to display multiple cortical-like regions marked by PAX6 and MAP2. (C) Dorsal forebrain organoids cultured for 100 - 200 days show increasing separation Dorsal Expansion MediumDorsal or Ventral Expansion Expansion Medium Medium. or Ventral BothDorsal Expansion dorsal Expansion and Medium. ventral Medium organoidsBothano i or dorsalVentral were and Expansion fed ventral every organoidsMedium. 2 - 3 days Both were until dorsal fed day every 25, and at 2ventral which - 3 days organoids until day were 25, at fed which every 2 - 3 days until day 25, at which g B B C B C C Ste a l Seed ell in S en ion lt re r Week 1 WWeekeek 4 1 Week 4 Week 1 Week 4 t r ggre ell™ point organoids were fedpoint with organoids Differentiation were fedMedium with Differentiationuntilpoint day organoids 43. FromMedium were day untilfed43O withonward,dayof deep-layer Differentiation43. organoidsFrom dayneurons Mediumwere43 onward, (CTIP2,fed until every organoids dayTBR1) 2 43.- 3from Fromdays were upper-layer withday fed 43every onward, neurons2 - 3 organoidsdays (SATB2). with were (D) fedVentral every forebrain 2 - 3 days organoids with at day 25 exhibit a high level of expression of NKX2.1. (E) Somatostatin e n
Maintenance Medium. MaintenanceOrganoids were Medium. harvested Organoids for MaintenanceRNA were or harvestedfixed Medium. in 4% for paraformaldehydeV RNAOrganoids or fixed were in harvested4%forBoth subsequentparaformaldehyde for RNADorsal cryosectioning or fixedfor subsequent in 4%and paraformaldehyde cryosectioning Ventral andfor subsequent Organoids cryosectioning andExpress Markers a i n a i n a i n a i n a i n a i n eeding ll edi ll edi (SST)-positive GABAergic interneurons are can be seen by daya i n 75. a i n a i n d d d d d d immunostaining. immunostaining. immunostaining. d d d
ange dail ange e er da eb r eb r eb r eb r eb r eb r 250 µm 100 µm eb r 100 µm eb r 20 µm eb r 50 µm o r o r o r ano i ano i ano i o r o r o r o r o r o r F F F ano i ano i ano i ano i ano i ano i g g g F F F
Characteristic of TheirF RespectiveF Brain RegionsF g g g g g g lt re Medi r r r r r r r r r a l a l a l a l a l a l O O O a l a l a l s s s O O O O O O s s s TeS ™ or ation E an ion Medi Differentiation Medi Maintenan e Medi s s s TeS ™ E ™ Medi RESULTS RESULTS RESULTS Day 25 Day 50 Day 75 Do r Do r Do r Do r Do r Do r TeS ™ l Do r Transfer EBs Do r to Do r FIGURE 4. Dorsal and Ventral Forebrain Organoids Express Appropriate Markers by Immunostaining Seed cells in suspension To generate entral fore rain organoid e E an ion Medi entral S le ent Da B Da Da B C Da Da Da C(A)D DayDa B25Da dorsal Da forebrain DE organoidsDa Da C displayDa multipleE cortical-likeDa D regionsDa marked byE radializedDa PAX6+ cells surrounded by MAP2 neurons. AggreWell™800 culture plate a i n a i n a i n
FIGURE 1. Workflow for STEMdiff™ Dorsal Forebrain Organoid Kit and STEMdiff™ Ventral Forebrain Organoid Kit a i n a i n a i n (B) Day 50 dorsal forebrain organoids continue to display multiple cortical-likea i n regions marked by a i n PAX6 and MAP2. (C) Dorsal forebraina i n d d d d d d eb r eb r eb r d d d eb r eb r eb r hPSCs maintained in mTeSR™1 (6 cell lines: 3 ES, 3 iPS), TeSR™-E8™ (3 cell lines: 2 ES, 1 iPS), or mTeSR™ Plus (2 cell lines: 1 ES, 1 iPS) organoids cultured for 100 - 200 days show increasing separation of deep-layereb r neurons (CTIP2, TBR1eb r ) from upper-layer neurons (SATB2eb r ). o r o r o r o r o r o r o r o r o r F F F ano i ano i ano i F F F F F F were dissociated and seeded at a density of 3,000,000 cells/well in Formation Medium + 10 µM rho-kinase inhibitor (ROCKi) in AggreWell™800 ano i ano i ano i (D) ano i NKX2.1. (E) (F) ano i SST ano i Ventral forebrain organoidsDay at day 25 exhibit a high level of expression of and Somatostatin ( )-positive GABAergic g g g g g g g g g a l a l a l r r r a l a l a l r r r a l a l a l r r r t r t r t r O O O t r t r t r plates. Cultures were fed daily with Formation Medium without ROCKi. After 6 days, aggregates were transferred to a 6-well plate in either O O O t r t r t r interneurons are not observed at day 50 but can be seen by day 75. O O O e n e n e n e n e n e n e n e n e n
Dorsal Expansion Medium or Ventral Expansion Medium. Both dorsal and ventral organoids were fed every 2 - 3 days until day 25, at which 0 1 6 25 43V V V V V V B V C WeekV 1 V point organoids were fed with Differentiation Medium until day 43. From day 43 onward, organoids were fed every 2 - 3 days with Week 4
Dor al ore rain Dor al ore rain Dor al ore rain Partial-medium Full-medium change Full-medium change Full-medium change Maintenance Medium. Organoids were harvested for RNA or fixed in 4% paraformaldehyde for subsequent cryosectioning and a i n a i n
a i n change daily every 2 days every 2 days every 2 - 3 days
D d D D E E E d immunostaining. d eb r eb r eb r o r ano i o r o r F ano i ano i g F F g g r r r a l a l O a l s O O s s Forebrain RESULTS Culture TeSR™ Seeding Forebrain Organoid Forebrain Organoid Forebrain Organoid Do r Do r Do r Organoid Medium Medium* Medium Expansion Medium Differentiation Medium Maintenance Medium Formation Medium Da B Da C Da D Da E Da entral ore rain entral ore rain entral ore rain a i n a i n a i n d d eb r d eb r
F F G F G eb r H G H H o r o r o r F ano i F F ano i ano i
*mTeSR™1, mTeSR™ Plus, or TeSR™-E8™ g g g a l FIGURE 5. Dorsalr ForebrainFIGURE Organoids 5. Dorsal But Forebrain Not Ventral Organoids FIGUREForebrain But 5. Dorsal OrganoidsNot Ventral Forebrain Display Forebrain Organoids Early Organoids Network But Not DisplayBursting Ventral Early Activity Forebrain Network Organoids Bursting DisplayActivity Early Network Bursting Activity a l r a l r t r O t r O t r O Day 50 dorsal and ventralDay 50 forebrain dorsal andorganoids ventral wereforebrainDay plated 50 organoids dorsalon an and Axionwere ventral electrodeplated forebrain on arrayan organoids Axion(CytoView electrode were MEA plated array96) coatedon(CytoView an withAxion MEA electrode 96) coated array with(CytoView MEA 96) coated with e n e n e n V V V Figure 65. Schematic for the STEMdiff™ Dorsal0.1% and polyethyleneimine Ventral Forebrain0.1% in borate polyethyleneimine Organoidbuffer and 20Differentiationin borateµg/mL0.1% bufferCellAdhere™ polyethyleneimine and Kits 20 Laminin-521 µg/mL in CellAdhere™borate and buffermeasured Laminin-521 and once20 µg/mL perand weekCellAdhere™measured for 4 onceweeks. Laminin-521 per week andfor 4measured weeks. once per week for 4 weeks.
Dor al ore rain (A) Representative bright(A) field Representative image of dorsal bright and field ventral image(A) forebrainRepresentative of dorsal organoids and brightventral on field forebrainelectrode image organoids ofarray. dorsal Representative andon electrode ventral forebrainspike array. activity Representative organoids heat on electrodespike activity array. heat Representative spike activity heat map is shown. Scale barmap = 1 ismm. shown. (B) Representative Scale bar = 1 wavemm.map (B) form Representative is of shown. electrode Scale marked wave bar =form in 1 (A).mm. of electrode(C) (B) Raster Representative marked plot of spike in (A).wave activity (C) form Raster shows of electrode plot increasing of spike marked activity in (A). shows (C) increasingRaster plot of spike activity shows increasing D Human ES or iPS cell-derived dorsal forebrain or ventral organoids can be generatedE in 43 days. Embryoid bodies can be created in 6 days with AggreWell™800 plates. The network bursting for dorsalnetwork forebrain bursting organoids for dorsal at weekforebrain network4, which organoids burstingis not atobserved forweek dorsal 4, inwhich forebrainthe ventralis not organoids observedforebrain at organoid.in week the ventral4, which(D) forebrainMean is not firing observedorganoid. rate in(D) the Mean ventral firing forebrain rate organoid. (D) Mean firing rate EBs are then cultured in suspension, allowing growth and(average subsequent ± SEM; patterning 3 - 6 organoids(average to the ±per SEM;dorsal timepoint) 3 forebrain.- 6 organoids shows Forincreasing (averageper patterning timepoint) mean± SEM; to showsfiring ventral 3 - 6rate increasing organoids forebrain,for dorsal meanper forebrainthe timepoint) firing protocol rateorganoids shows for differs dorsal increasingbut only forebrainnot byventral meana organoids forebrain firing rate but for not dorsal ventral forebrain forebrain organoids but not ventral forebrain supplement added to Forebrain Organoid Expansion Medium.organoids For over the 4 long-termweeks oforganoids measurements. maintenance over 4 weeks (E) and Synchrony offurther measurements. maturationindexorganoids (average (E) over ofSynchrony ± dorsal4 SEM; weeks 3and index -of 6 measurements. organoidsforebrain (average perorganoids, ± SEM; timepoint) (E) Synchrony3 - 6 seeorganoids exhibits the index Product increasing per (average timepoint) synchrony ± SEM; exhibits 3 - 6 increasing organoids synchronyper timepoint) exhibits increasing synchrony of firing for dorsal forebrainof firing organoids for dorsal compared forebrain to ventralorganoidsof forebrain firing compared for organoids. dorsal to ventralforebrain forebrain organoids organoids. compared to ventral forebrain organoids. Information Sheet. Adapted from protocols by Sergiu Pa�ca21. FIGURE 2. STEMdiff™ DorsalFIGURE Forebrain 2. STEMdiff™ Organoid Dorsal Kit Forebrain andFIGURE STEMdiff™ Organoid2. STEMdiff™ Ventral Kit and Dorsal Forebrain STEMdiff™ Forebrain Organoid Ventral Organoid Kit Forebrain Support Kit and theOrganoid STEMdiff™ Generation Kit VentralSupport of Forebrain the Generation Organoid of Kit Support the Generation of B Day 0 DBay 5 (post fuDsiaon)y 0 CDay 5 (post fuBsion) Day 0 C Day 5 (post fusion) C
entral ore rain Homogeneous OrganoidsHomogeneous Organoids Homogeneous Organoids Ventral Ventral Ventral Ventral Ventral Ventral organoid organoid organoid Forebrain ForebraAinss embloid AssembFlooridebrain AssembloIBidA1 MAP2 IBA1 MAP2 IBA1 MAP2 F G (A) Representative morphologyH (A) Representative of neural aggregates morphology formed of(A) neural in Representative AggreWell™800 aggregates morphologyformed exhibit in uniformAggreWell™800 of neural size aggregates and exhibitshape formedatuniform day 6 in size(top: AggreWell™800 and aggregates shape at in dayexhibit 6 (top: uniform aggregatesOr gsizeanoid and in shape at day O6rg (top:anoid aggregates in Organoid AggreWell™800, bottom:AggreWell™800, aggregates in suspension).bottom: aggregates RepresentativeAggreWell™800, in suspension). morphology bottom: Representative ofaggregates hESC H9-derived morphology in suspension). organoids of hESC Representative at H9-derived days (B) 25morphologyorganoids (C) 50 at of days hESC (B) H9-derived 25 (C) 50 organoids at days (B) 25 (C) 50 FIGURE 5. Dorsal Forebrain Organoids But Not Ventral Forebrain Organoids Display Early Network Bursting Activity (D) 75 and (E) 100. Top(D) row: 75 dorsal and (E) forebrain 100. Top organoids, row: dorsal (D)bottom forebrain75 and row: (E) ventralorganoids, 100. forebrainTop bottomrow: organoids.dorsal row: forebrainventral Scale forebrain organoids, bar = 1organoids. mm. bottom (F) Both Scalerow: dorsalventral bar = and forebrain1 mm. (F) organoids. Both dorsal Scale and bar = 1 mm. (F) Both dorsal and Day 50 dorsal and ventral forebrain organoids were plated on an Axion electrode array (CytoView MEA 96)500µm coated Dwithorsal 500µm Dorsal 500µm Dorsal ventral forebrain organoidsventral exhibit forebrain homogeneous organoids size exhibit overventral homogeneousmultiple forebrain cell lines size organoids (average over multiple exhibit ±Learn SD, cell usinghomogeneous lines more11 (average cell lines size at ±with overSD, www.stemcell.com/dorsal-forebrain-organoid 3 usingmultiple- 5 organoids 11 cellcell lineslines counted (averagewith 3 - 5 ±organoids SD, using counted 11 cell lines with 3 - 5 organoids counted organoid organoid 50 µmorganoid 50 µm 50 µm 0.1% polyethyleneimine in borate buffer and 20 µg/mL CellAdhere™ Laminin-521 and measured once per week for 4 weeks. per cell line and timepoint).per (G)cell Dorsal line and forebrain timepoint). organoids (G) Dorsalper and cell forebrain(H) line ventral and organoids forebraintimepoint). and organoids (G) (H) Dorsal ventral compared forebrain forebrain to organoids anorganoids American and compared dime(H) ventral for toscale. anforebrain American organoids dime for compared scale. to an American dime for scale. (A) Representative bright field image of dorsal and ventral forebrain organoidsDorsal on electrode array.Dorsal Representative spike activityDor sheatal map is shown. Scale bar = 1 mm. (B) Representative wave form of electrodeFore bmarkedrain in (A). (C) RasterForeb rplotain of spike activity shows increasingForebrain FIGURE 3. Dorsal and VentralFIGURE Forebrain 3. Dorsal Organoidsand Ventral ForebrainFIGURE O3.Organoidsrg aDorsalnoid and Ventral ForebrainOrganoid Organoids Organoid network bursting for dorsal forebrain organoids at week 4, which is not observed in the ventral forebrain organoid. (D) Mean firing rate Express Appropriate MarkersExpress of Appropriate Their Respective Markers ofExpress Their Respective Appropriate Markers of Their Respective 500 µm 500 µm 500 µm (averageTissue ± SEM; Over 3 -Time 6 organoidsTissue per timepoint)Over Time shows increasingTissue mean Over firing Time rate for dorsal forebrain organoids but not ventral forebrain organoids38RNA over from 4 weeks a STEMCELLsingle of measurements. organoidRNA Technologies fromwas (E)harvesteda single Synchrony organoid Inc.at eachindex was (averageRNA harvested from ± SEM; a atsingle 3each - 6 organoids organoid perwas timepoint) harvested exhibits at each increasing synchrony of firing for dorsal forebrain organoids compared to ventral forebrain organoids.hPSC- hPSC- hPSC- respective timepoint andrespective subsequently timepoint assayed and either subsequently respective assayedDer itimepointved either and subsequentlyDerived assayed either De100rive dµm 100 µ100m µm 100 µm 50 µ100m µm 10050 µµm 50 µm FIGURE 2. STEMdiff™ Dorsal Forebrain Organoid Kit and STEMdiff™ Ventral Forebrain Organoid Kit Support the Generation of using RT-qPCR or bulkusing RNA-seq. RT-qPCR (A) Bor RT-qPCR bulk DaRNA-seq.y 0 using (A)DRT-qPCRaM y ic5RT-qPCRr ogli(poast fu sioron) bulk RNA-seq.C Microgli a (A) RT-qPCR Microglia Homogeneous Organoids Ventranalysisal shows upregulationanalysis of PAX6 shows and upregulation TBR1Ven trinal the of PAX6analysis and TBR1shows in upregulation the of PAX6 and TBR1 in the organoid FIGURE 6. Brain-Region-SpecificFIGURE 6. Organoids Brain-Region-Specific Can Be Used FIGUREOrganoids in Co-Culture 6. Brain-Region-Specific Can toBe Form Used Assembloidsin Co-Culture Organoids to Form Can Assembloids Be Used in Co-Culture to Form Assembloids Forebrain Assembloid IBA1 MAP2 (A) Representative morphology of neural aggregates formed in AggreWell™800 exhibit uniform size and shape at day 6 (top: aggregates in Organdorsaloid forebrain organoidsdorsal while forebrain NKX2.1 organoids and LHX6 while dorsalNKX2.1(A) forebrain Schematic and LHX6 organoids depicting while(A)the Schematicformation NKX2.1 of depictingand a dorsal LHX6 the(red) formation and(A) ventral Schematic of a (blue) dorsal forebraindepicting (red) and organoid theventral formation (blue) assembloid offorebrain a dorsal and organoid (red) a dorsal and assembloid forebrainventral (blue) org andanoid forebrain a dorsal and organoid forebrain assembloid organoid and and a dorsal forebrain organoid and AggreWell™800, bottom: aggregates in suspension). Representative morphology of hESC H9-derived organoids at days (B) 25 (C) 50 were upregulated in thewere ventral upregulated forebrain inorganoids. the ventral forebrainweremicroglia upregulated organoids. co-culture. in the (B)ventralmicroglia A day forebrain 30 co-culture.dorsal organoids. forebrain (B) A organoidday 30microglia dorsal and ventralforebrain co-culture. forebrain organoid (B) organoid A andday 30ventral (expressing dorsal forebrain forebrain endogenous organoid organoid (expressing GFP) and ventralwere endogenousallowed forebrain to organoid GFP) were (expressing allowed to endogenous GFP) were allowed to (D) 75 and (E) 100. Top row: dorsal forebrain organoids, bottom row: ventral forebrain organoids. Scale bar = 1 mm. (F) Both dorsal and Both MAP2 and FOXG1Both wereMAP2 not and differentially FOXG1 wereBoth not MAP2differentially and FOXG1 were not differentially 500µm Dorsal fuse together over 5 days.fuse On together day 5, over GFP-expressing 5 days. On daycellsfuse 5, from togetherGFP-expressing the ventralover 5 forebraindays. cells Onfrom organoid day the 5, ventral GFP-expressing had forebrain migrated organoid intocells the from dorsalhad the migrated ventralforebrain forebrain into the organoiddorsal forebrain had migrated into the dorsal forebrain ventral forebrain organoids exhibit homogeneous size over multiple cell lines (average ± SD, using 11 cell lines with 3 - 5 organoids counted expressed between dorsalexpressed and ventralbetween forebrain dorsal andorexpressedga noidventralorganoid forebrainbetween (red and dorsal orangeorganoid and boxes). ventral (red (C) and forebrainA dayorange 20050 µ m boxes).dorsalorganoid forebrain(C) A day(red organoid 200and dorsalorange was forebrain incubatedboxes). (C)organoid with A day250,000 was 200 incubated dorsalmicroglia forebrain withgenerated 250,000 organoid using microglia was incubated generated with using 250,000 microglia generated using per cell line and timepoint). (G) Dorsal forebrain organoids and (H) ventral forebrain organoids compared to an American dime for scale. + + + Dorsaorganoids.l Average ± SEM;organoids. n = 3 -Average 20 organoids ± SEM; per n = 3organoids. - 20STEMdiff organoids Average™ Microgliaper ± SEM; Differentiation STEMdiffn = 3 - 20™ organoids MicrogliaKit. Within Differentiationper one week, IBA1STEMdiff Kit. Withincells™ withMicroglia one stereotypical week, Differentiation IBA1 microglia cells withKit. morphology Withinstereotypical one week, have microglia IBA1been incorporatedmorphologycells with stereotypical haveinto been microgliaincorporated morphology into have been incorporated into Forebtimepointrain using 11 hPSCtimepoint lines; Data using are 11normalized hPSC lines; to Datatimepoint arethe normalized dorsal using forebrain 11 to hPSC organoid. lines;the Data dorsal are forebrain normalized organoid. to the dorsal forebrain organoid. Organoid FIGURE 3. Dorsal andCondition Ventral3 Condition Forebrain OrganoidsCondition 3 Condition Condition 3 Condition Dorsal.d25 Dorsal.d25 18S/TBP andDorsal.d25 compared18S/TBP to an undifferentiated and compared hPSC to an undifferentiated18S/TBP and comparedhPSC to an undifferentiated hPSC B B POU5F1 BPOU5F1 POU5F1 2 Dorsal.d50 C 2 Dorsal.d50 C 2 Dorsal.d50 C Dorsal.d75 Dorsal.d75 Dorsal.d75 Express AppropriateNANOG Markers1 of Their RespectiveNANOG 1 NANOG 1 PSC.d0 PSC.d0 control. (B) HeatPSC.d0 map control.of select (B) genes Heat showsmap ofthat select control. genes (B)shows Heat that map of select500 µmgenes shows that 0 Ventral.d25 0 Ventral.d25 0 Ventral.d25 NEUROG1 NEUROG1 NEUROG1 Summary Summary Summary Tissue Over Time −1 Category −1 Category −1 Category NES CORTEX NES CORTEX dorsal forebrainNES CORTEX organoidsdorsal have forebrain an increasing organoids amount have andorsal increasing forebrain amount organoids have an increasing amount −2 Early Neurogenesis −2 Early Neurogenesis −2 Early Neurogenesis MAP2 MAP2 MAP2 GABAergic GABAergic GABAergic RNA from a single organoid−3 was harvested at−3 each −3 STEMdiff™ Dorsal Forebrain STEMdiff™ Organoid Dorsal Differentiation Forebrain Organoid Kit and STEMdiff™ STEMdiff™ Differentiation Dorsal Ventral Forebrain Kit Forebrainand STEMdiff™ Organoid Organoid Differentiation Ventral Differentiation Forebrain Kit and Organoid Kit STEMdiff™generate Differentiation Ventral Forebrain Kit generate Organoid Differentiation Kit generate Glutamatergic Neuron Glutamatergic Neuron Glutamatergic Neuron SOX2 SOX2 of cortex-SOX2 and glutaminergicof cortex- neuron-specific and glutaminergic genes neuron-specificof cortex- and genes glutaminergic neuron-specific genes MGE MGE hPSC- MGE Pluripotent Pluripotent Pluripotent FOXG1 FOXG1 FOXG1 100 µm 100 µm respective timepoint and Progenitorsubsequently assayed eitherProgenitor Derivfromed day 25 toProgenitor 75. Day 25from ventral day 25forebrain to 75. Dayorganoids 25 ventral fromforebrain day 25organoids tissue to 75. resembling Day 25 ventral the dorsalforebrain tissue forebrain resembling organoids (PAX6,50 the µm dorsalTBR1) forebrainand tissueventral (PAX6, resembling forebrain TBR1) (NKX2.1, the and dorsal ventral LHX6), forebrain forebrain respectively (PAX6, (NKX2.1, TBR1) LHX6), and ventral respectively forebrain (NKX2.1, LHX6), respectively DLX1 DLX1 Microglia DLX1 using RT-qPCR orATP5MC3 bulk RNA-seq. (A) ATP5MC3RT-qPCR exhibit highATP5MC3 expression exhibitof markers high ofexpression the MGE ofand markers exhibit of the high MGE Dorsal expression and forebrain of markersorganoids Dorsal of werethe forebrain MGE observed and organoids to exhibit were network observed Dorsal bursting forebrain to exhibit earlier organoids networkthan ventral were bursting forebrain observed earlier organoids to than exhibit ventral network forebrain bursting organoids earlier than ventral forebrain organoids NKX2−1 NKX2−1 NKX2−1 analysis shows upregulationNKX6−2 of PAX6 and TBR1NKX6−2 in the FIGUREGABAergic 6. Brain-Region-SpecificNKX6−2 neurons. (C)GABAergic OrganoidsVenn diagram neurons. Can comparingBe Used(C) Venn in Co-Culture diagramGABAergic tocomparing Formneurons. Assembloids (C) Venn diagram comparing dorsal forebrain organoidsFOXA2 while NKX2.1 andFOXA2 LHX6 (A) FOXA2 Dorsal and ventral forebrain Dorsal organoids and ventral can forebrain form assembloids organoids Dorsal to can andstudy form ventral interneuron assembloids forebrain migration organoidsto study and interneuron can interactions form assembloids migration with microglia and to interactionsstudy interneuron with microglia migration and interactions with microglia NEUROG2 NEUROG2 Schematicday 25 depictingNEUROG2 dorsal theforebrain formationday and25 of a dorsalventral (red) forebrainforebrain and ventral andday (blue)ventral 25 forebrain dorsalforebrain organoid forebrain assembloid and ventral and a dorsalforebrain forebrain organoid and were upregulated inPAX6 the ventral forebrain organoids.PAX6 microglia co-culture.PAX6 (B) A day 30 dorsal forebrain organoid and ventral forebrain organoid (expressing endogenous GFP) were allowed to TBR1 TBR1 organoidsTBR1 shows a high organoidsdegree of showsoverlap a ofhigh markers degree oforganoids overlapFeatures of shows markers ofa highSTEMdiff™ degree Forebrain Featuresof overlap ofOrganoid of STEMdiff™ markers Kits: Forebrain FeaturesOrganoid of Kits: STEMdiff™ Forebrain Organoid Kits: Both MAP2 and BCL11BFOXG1 were not differentiallyBCL11B fuse together overBCL11B 5 days. On day 5, GFP-expressing cells from the ventral forebrain organoid had migrated into the dorsal forebrain SATB2 SATB2 that wereSATB2 differentially upregulatedthat were differentially in these organoids upregulated that in thesewere differentiallyorganoids upregulated in these organoids RELIABILITY: Optimized RELIABILITY: and matrix-free Optimized formulations and matrix-free for patterning RELIABILITY: formulations of brain-region-specific Optimized for patterning and matrix-free oforganoids brain-region-specific formulations improves for reproducibility patterning organoids of improves brain-region-specific reproducibility organoids improves reproducibility expressed betweenEMX1 dorsal and ventral EMX1forebrain organoidcompared (red andEMX1 orangeto boxes).hPSCs.compared (C) ADorsal dayto 200 forebraindorsalhPSCs. forebrain comparedDorsal organoid forebrain towas incubatedhPSCs. withDorsal 250,000 forebrain microglia generated using SLC32A1 SLC32A1 SLC32A1 + across experiments and across cell lines experiments and cell lines across experiments and cell lines organoids. AverageGAD1 ± SEM; n = 3 - 20 organoidsGAD1 per STEMdifforganoid-specific™ MicrogliaGAD1 Differentiation genesorganoid-specific are Kit. Withinrelated one to genesweek, pallium IBA1are cellsrelatedorganoid-specific with tostereotypical pallium genes microglia are morphologyrelated to havepallium been incorporated into GAD2 GAD2 GAD2 timepoint using 11 hPSCSLC17A7 lines; Data are normalizedSLC17A7 to the dorsaldevelopment forebrainSLC17A7 organoid. of the developmentneocortex whereas of the ventralneocortex development whereas SCALABILITY: ventralof the neocortex High-throughput SCALABILITY:whereas organoid ventral High-throughput generation in AggreWell™800organoid SCALABILITY: generation High-throughput in AggreWell™800 organoid generation in AggreWell™800 SLC17A6 SLC17A6 SLC17A6
Condition 3 Condition Category 10−SC−L 6 7 2 5−SC−L 6−SC−L 7−SC−L 11−SC−L 12−SC−L 21−SC−L Category 10−SC−L 22−SC−L 6 23−SC−L 7 2 5−SC−L 6−SC−L 7−SC−L 11−SC−L 12−SC−L 21−SC−L 22−SC−L 23−SC−L Category 10−SC−L 6 7 2 5−SC−L 6−SC−L 7−SC−L 11−SC−L 12−SC−L 21−SC−L 22−SC−L 23−SC−L Dorsal.d25 18S/TBP and compared to an undifferentiated hPSC B POU5F1 2 forebrain organoid-specificforebrain genes organoid-specific are related to genesforebrain are relatedorganoid-specific to genes are related to Dorsal.d50 C MODULARITY: Ease of use MODULARITY: in generating Ease co-culture of use in models generating MODULARITY: (assembloids) co-culture Ease models of use (assembloids)in generating co-culture models (assembloids) Dorsal.d75 NANOG 1 PSC.d0 control. (B) Heat map of select genes shows that 0 Ventral.d25 subpallium developmentsubpallium of the ventral development telencephalon. of the ventralsubpallium telencephalon. development of the ventral telencephalon. NEUROG1 Summary −1 Category NES CORTEX dorsal forebrain organoids have an increasing amount −2 Early Neurogenesis MAP2 GABAergic −3 STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit and STEMdiff™ Ventral Forebrain Organoid Differentiation Kit generate Glutamatergic Neuron SOX2 of cortex- and glutaminergic neuron-specific genes MGE Pluripotent FOXG1 T EE E T EE E E T E EE E STE M E M T EE STE S M E T S MTE M E T E S M STET S M TE E M E M M T T E T DS E T ST S T S TE M STEM E MTE T DEMT S S T STEM E M T T DET S S T STEM E M Progenitor from day 25 to 75. Day 25 ventral forebrain organoids tissue resembling the dorsal forebrain (PAX6, TBR1) and ventral forebrain (NKX2.1, LHX6), respectively DLX1 ATP5MC3 exhibit high expression of markers of theFOR MGERESEARCH and USE ONLY. NOT INTENDEDFOR RESEARCH Dorsal FOR HUMAN forebrainUSE ONLY. OR NOTANIMAL organoids INTENDED DIAGNOSTIC FOR wereFOR HUMAN OR RESEARCH observed THERAPEUTIC OR ANIMAL USE to ONLY. USES. exhibitDIAGNOSTIC NOT STEMCELL INTENDED network OR TECHNOLOGIES THERAPEUTIC FOR bursting HUMAN USES.INC.’S ORearlier ANIMAL STEMCELLQUALITY than DIAGNOSTIC MANAGEMENTTECHNOLOGIES ventral OR forebrain THERAPEUTIC SYSTEM INC.’S QUALITY IS organoids CERTIFIED USES. MANAGEMENT STEMCELL TO ISO 13485 TECHNOLOGIES SYSTEM MEDICAL IS CERTIFIED DE ICE INC.’S QUALITYSTANDARDS. TO ISO 13485 MANAGEMENT MEDICAL DE ICESYSTEM STANDARDS. IS CERTIFIED TO ISO 13485 MEDICAL DE ICE STANDARDS. NKX2−1 NKX6−2 GABAergic neurons. (C) Venn diagram comparing FOXA2 Dorsal and ventral forebrain organoids can form assembloids to study interneuron migration and interactions with microglia NEUROG2 day 25 dorsal forebrain and ventral forebrain PAX6 TBR1 organoids shows a high degree of overlap of markers Features of STEMdiff™ Forebrain Organoid Kits: BCL11B
SATB2 that were differentially upregulated in these organoids RELIABILITY: Optimized and matrix-free formulations for patterning of brain-region-specific organoids improves reproducibility EMX1 compared to hPSCs. Dorsal forebrain SLC32A1 across experiments and cell lines GAD1 organoid-specific genes are related to pallium GAD2 SLC17A7 development of the neocortex whereas ventral SCALABILITY: High-throughput organoid generation in AggreWell™800 SLC17A6 Category 10−SC−L 6 7 2 5−SC−L 6−SC−L 7−SC−L 11−SC−L 12−SC−L 21−SC−L 22−SC−L 23−SC−L forebrain organoid-specific genes are related to MODULARITY: Ease of use in generating co-culture models (assembloids) subpallium development of the ventral telencephalon.
T EE E E STEM E M TE S T STEM E M T T DET S S T STEM E M FOR RESEARCH USE ONLY. NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES. STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485 MEDICAL DE ICE STANDARDS. BrainPhys™ Neuronal Medium Culture Active Neurons Under Why Use BrainPhys™ Neuronal Physiological Conditions Medium?
Neurons can be generated efficiently from hPSC-derived NPCs PHYSIOLOGICAL. More representative of the brain’s using BrainPhys™ Neuronal Medium and supplements. Using extracellular environment. BrainPhys™ Neuronal Medium as the basal medium for hPSC- ACTIVE. Improved neuronal function and a higher derived NPC differentiation and neuronal maturation will proportion of synaptically active neurons. generate a more neurophysiologically active culture that better STREAMLINED. Perform functional assays without represents the human brain environment.22 replacing media.
Published protocols using a basal medium together with neural VERSATILE. Supports long-term culture of hPSC- and supplements, such as NeuroCult™ SM1 Neuronal Supplement CNS-derived neurons. (based on the published B27 formulation23) and N2 supplement,24 as well as various cytokines and small molecules, are available for the generation of many neuronal subtypes.
BrainPhys™ Neuronal Medium may also be used to culture induced neurons derived through lineage conversion of somatic NeuroFluor™ NeuO cells (i.e. without transitioning through an hPSC intermediate) or through forced Ngn2 expression in hPSCs.22 Selectively Label Live Neurons
NeuroFluor™ NeuO is a membrane-permeable fluorescent probe that selectively labels primary and pluripotent stem A AMPA B AMPA cell-derived neurons in live cultures.25 Labeling with this probe is non-permanent; it can be washed off, providing unlabeled, viable 15 pA cells for downstream applications. 1 s C GABA D GABA
15 pA A B 1 s
Figure 66. hPSC-Derived Neurons Matured in BrainPhys™ Neuronal Medium Show Improved Excitatory and Inhibitory Synaptic Activity by 44 Days
NPCs were generated from H9 cells using STEMdiff™ Neural Induction Medium in an EB-based protocol. Next, NPCs were cultured for 44 days in vitro in (A,C) BrainPhys™ Neuronal Medium, supplemented with 2% NeuroCult™ SM1 Figure 67. NeuroFluor™ NeuO Selectively Labels hPSC-Derived Supplement, 1% N2 Supplement-A, 20 ng/mL GDNF, 20 ng/mL BDNF, 1 mM Neurons db-cAMP and 200 nM ascorbic acid to initiate neuronal differentiation, or (B,D) in DMEM/F12 under the same supplementation conditions. (A,C) Neurons matured in (A) Neuronal precursors generated from hPSC-derived (XCL-1) NPCs were BrainPhys™ Neuronal Medium showed spontaneous excitatory (AMPA‑mediated; cultured in STEMdiff™ Neuron Maturation Medium. After 18 days of culture, A) and inhibitory (GABA-mediated; C) synaptic events as measured by patch hPSC-derived neurons were labeled with NeuroFluor™ NeuO (green). (B) The clamp analysis. The frequency and amplitude of spontaneous synaptic events same culture was later fixed and immunostained for class III b-tubulin (red). is consistently greater in neuronal cultures matured in BrainPhys™ Neuronal Nuclei are counterstained with DAPI. The images show that NeuroFluor™ NeuO Medium, compared to neurons plated and matured in DMEM/F12 (B,D). Traces specifically labels class III b-tubulin-positive neurons. are representative. hPSC-derived neurons have been successfully matured in BrainPhys™ Neuronal Medium for up to 126 days in vitro. Learn more at www.stemcell.com/NeuO-imaging Learn more at www.BrainPhys.com
WWW.STEMCELL.COM 39 HUMAN PLURIPOTENT STEM CELLS
STEMdiff™ Neural Crest Differentiation Kit Why Use the STEMdiff™ Generate Pure Populations of Neural Crest Cells Neural Crest Differentiation Kit? RAPID. Generates neural crest cells in an easy-to-use The STEMdiff™ Neural Crest Differentiation Kit consists of a monolayer protocol in only 6 days. serum-free basal medium and supplement for highly efficient and reproducible differentiation of hPSCs into neural crest cells EFFICIENT. Obtain greater than 70% purity of + + (NCCs). Due to their migratory capabilities and transience during multipotent SOX10 CD271 neural crest cells. development, NCCs remain difficult to isolate from tissues. MULTIPOTENT. Produces cells capable of downstream With the STEMdiff™ Neural Crest Differentiation Kit, multipotent differentiation, including to peripheral neurons, NCCs can be generated from hPSC monolayers in 6 days. Further chondrocytes, or osteoblasts. expansion of this population is possible for up to 3 passages using VERSATILE. Compatible with human ES and iPS cells the STEMdiff™ Neural Crest Differentiation Kit or MesenCult™-ACF maintained in mTeSR™ Plus, mTeSR™1, or TeSR™-E8™. Plus Medium, depending on the desired downstream application. The NCCs produced using this kit are multipotent and can be further differentiated to cell types of both the neural and ecto-mesenchymal lineages. Peripheral neurons expressing peripherin and BRN3A may be generated using established protocols (Figure 69B), with potential A applications for pain and sensory neuron research. Passaging NCCs into MesenCult™-ACF Plus Medium allows for differentiation to the chondrogenic lineage after using the MesenCult™-ACF Chondrogenic Differentiation Kit (Figure 69C), to the osteogenic lineage after using MesenCult™ Osteogenic Differentiation Kit (Figure 69D), and to the adipogenic lineage using MesenCult™ Adipogenic Differentiation Kit. B C D
A B C
Figure 69. NCCs Generated with the STEMdiff™ Neural Crest Differentiation Kit Are Multipotent
NCCs (A) were cultured for 6 days and display typical morphology. (B) Culturing D E NCCs using established protocols generates peripheral neurons (Peripherin, green; BRN3a, red; DAPI, blue). (C) Passaging NCCs into MesenCult™-ACF Plus Medium and then into the MesenCult™-ACF Chondrogenic Differentiation Kit generates a chondrocyte pellet (Alcian Blue, Nuclear Fast Red) with deposition of cartilage around the cells. (D) Passaging NCCs into MesenCult™-ACF Plus Medium and then into the MesenCult™ Osteogenic Differentiation Kit (Human) generates an osteoblast culture with high levels of alizarin red-positive mineral deposition. Scale bar = (A-C) 500 μm, (D) 1 mm.
Figure 68. STEMdiff™ Neural Crest Differentiation Kit Generates a Highly Pure Population of NCCs with Minimal CNS-type Progenitors
After 6 days in culture, neural crest cells (SOX10+, red; CD271+, light blue) Learn more at www.stemcell.com/NCKit outnumber CNS-type progenitors (PAX6+, green). (A) Channel merge of cells fixed 2 days after being passaged on day 6. Individual immunofluorescence channels show (B) DAPI, (C) PAX6, (D) SOX10, and (E) CD271. The NCCs also express expected neural crest markers, such as FOXD3 and HNK1 (CD57) (data not shown). Scale bar = 100 μm.
40 STEMCELL Technologies Inc. STEMdiff™ Microglia Culture System Differentiate to Microglia from hPSCs Why Use the STEMdiff™ Microglia Differentiation and Maturation Kits? The STEMdiff™ Microglia Differentiation and Maturation Kits consist of a serum-free basal medium and supplements for highly EFFICIENT. Differentiate greater than 80% of efficient and reproducible generation of microglia from hPSCs via a source HPCs into microglia, with few contaminating hematopoietic progenitor cell (HPC) intermediate. macrophages or monocytes. These kits are optimized for use on HPCs generated with the OPTIMIZED. Upstream-compatible with HPCs generated STEMdiff™ Hematopoietic Kit, taking 28 days to generate functional using STEMdiff™ Hematopoietic Kit. microglia. FUNCTIONAL. Produce microglia capable of phagocytosis Microglia produced using the STEMdiff™ Microglia Culture System and activation. are versatile tools for studying human neurological development, CONVENIENT. Easy-to-use culture system can replace neuroimmune responses, and disease, in particular for modeling workflows based on various published differentiation neuroinflammation and neurodegeneration. Cells can also be applied protocols. in both 2D and 3D co-culture with other neuronal cell types.
Figure 70. Microglia Generated with STEMdiff™ Microglia Culture System Show Expected Morphology and Markers
Microglia (STiPS-M001 cell line) cultured for 24 days in STEMdiff™ Microglia Differentiation Medium followed by 4 days in STEMdiff™ Microglia Maturation Medium express IBA1 (magenta; DAPI, blue). Based on the protocol from the laboratory of Mathew Blurton-Jones,26 the resulting cells are a highly pure population of microglia (at least 80% CD45/CD11b-positive, and at least 50% TREM2-positive cells) with no more than 20% morphologically distinct monocytes or macrophages. The microglia also express other expected markers, such as TMEM119, and APOE (data not shown). PC2: 16% Variance
PC1: 32% Variance
Figure 71. Microglia Generated with STEMdiff™ Microglia Culture System Are Typical PSC-Derived Microglial Cells
RNA-seq datasets of hPSC-derived and primary microglia and other immune cell types were extracted from 4 different publications (Protocols A-D). Principal component analysis (PCA) was performed on these data along with RNA-seq data from microglia generated with the STEMdiff™ Microglia Culture System. The hPSC-derived microglia from STEMdiff™ Microglia Culture System plot most closely to those from Protocols A and B.
Learn more at www.stemcell.com/microglia WWW.STEMCELL.COM 41 HUMAN PLURIPOTENT STEM CELLS
Endoderm Differentiation Pathways
Flexible Products for Differentiation OCT4 TRA-1-60 SSEA-3 • STEMdiff™ APEL™2 Human Pluripotent • STEMdiff™ APEL™2-LI Stem Cells (hPSCs) • TeSR™-E6 • TeSR™-E5 • hPSC-Derived Endoderm qPCR Array SOX17 FOXA2 CXCR4 • Cytokines C-KIT • Small Molecules Definitive Endoderm
STEMdiff™ Definitive Endoderm Kit
CDX2 GATA4 Foregut Mid-/Hindgut
PDX1 PTF1α HNF1B CDX2 Anterior Foregut Posterior Foregut
SOX2 SOX9 NKX2.1 NKX2.1 PDX1 Lung CPM PTF1α Lung Pancreatic NKX6.1 Epithelium VIM Extrahepatic Progenitors Progenitors Hepatoblasts and Mesenchyme Cholangiocytes
™ ™ ™ STEMdiff STEMdiff STEMdiff CDX2 Branching Lung Lung Pancreatic KLF5 Organoid Kit Progenitor Kit Progenitors Intestinal SOX9 Epithelium
STEMdiff™ Intestinal Organoid Kit Acinar Cells
Distal Airway Proximal Airway Ductal Cells LGR5 Cells Cells OLFM4 Intestinal Stem Cells
VIL1 Endocrine Cells SLC2A2 Enterocytes LYSO MMP7 Paneth Cells
MUC2 CHGA KRT20 NTS Enteroendocrine Goblet Cells Cells
42 STEMCELL Technologies Inc. STEMdiff™ Definitive Endoderm Kit Quickly and Easily Differentiate Definitive Endoderm
The STEMdiff™ Definitive Endoderm Kit is a serum-free, animal component-free system that enables differentiation of hPSCs to multipotent definitive endoderm cells using a short and simple protocol. This product is available in formulations optimized for use with hPSCs cultured in mTeSR™ Plus, mTeSR™1, or TeSR™-E8™. Definitive endoderm cells generated with this kit can be further differentiated to multiple downstream endodermal cell types, including hepatic27 and pancreatic28 progenitor cells for drug development, toxicity testing, research for development of cell-based therapies, or studying developmental pathways.
TM ™ TM TM Why Use the STEMdiff Definitive Endoderm Kit? F 2ANIMAL COMPONENT-FREE. Serum-free erge and animal component-free formulation.
OPTIMIZED. Compatible with hPSCs cultured in mTeSR™ Plus, mTeSR™1, or TeSR™-E8™.
ROBUST. Reproducible differentiation of multiple human ES and iPS cell lines. Figure 72. Definitive Endoderm Differentiation Is Efficient MULTIPOTENT. Generate functional endoderm capable Across Multiple Human ES and iPS Cell Lines, Regardless of hPSC of downstream differentiation to multiple lineages. Maintenance Medium
Quantitative analysis of definitive endoderm formation in multiple human ES (H1 and H9) and iPS (WLS-4D1 and STiPS-M001) cell lines, as measured by co-expression of CXCR4 and SOX17. Cells maintained in mTeSR™1 medium were differentiated using STEMdiff™ Definitive Endoderm Kit, and cells A B maintained in TeSR™-E8™ were differentiated using STEMdiff™ Definitive Endoderm Kit (TeSR™-E8™-Optimized). Data are expressed as the mean percentage of cells expressing both markers. Error bars indicate SEM; n = 44 to 18 per cell line.
Learn more at www.stemcell.com/STEMdiff-DE 100 µm
Representative images of SOX17 and FigureFOXA2 73. hPSCs immunoreactivity Differentiated with STEMdiff™ in Definitive hES (H9) and hiPS (4D1) cells Endoderm Kit Are Highly Enriched for Expression of Key following 4 days of differentiation to definitiveDefinitive endoderm Endoderm Markers using STEMdiff™ Definitive Endoderm. Merged (A) Representative density plot showing CXCR4 and SOX17 expression images show extensive co-localization ofin mTeSR™1-culturedthese two H1 markers. ES cells, following 5Scale days of differentiation bar, 100 µm. (B) Representative image of FOXA2 (green) and SOX17 (red) in WLS-4D1 iPS cells following 4 days of differentiation. Yellow indicates cells co-expressing FOXA2 and SOX17. hPSC-Derived Endoderm qPCR Array
The hPSC-Derived Endoderm qPCR Array provides a validated 90‑gene assay to characterize definitive endodermal progenitor cells and their differentiated progeny, including pancreatic, hepatic, and intestinal cells. Housekeeping controls and a synthetic DNA positive control are included. Data analysis is streamlined with our flexible online app www.stemcell.com/qPCRanalysis( ).
Learn more at www.stemcell.com/DE-array
WWW.STEMCELL.COM 43 HUMAN PLURIPOTENT STEM CELLS
STEMdiff™ Intestinal Organoid Kit Differentiate Human ES and iPS Cell Lines to Intestinal Organoids
hPSC-derived organoids provide direct relevance to human tissues while retaining the genotype and phenotype of donor cells. The STEMdiff™ Intestinal Organoid Kit enables the culture of intestinal organoids from embryonic stem (ES) or induced pluripotent stem (iPS) cells within 30 days. These organoids incorporate the key cell types and features of the developing intestinal epithelium, including the incorporation of some mesenchymal components. Intestinal organoids can be expanded and maintained in culture through passaging, or cryopreserved for future experiments.
Why Use the STEMdiff™ Intestinal Organoid Kit?
RELEVANT. Enables generation of small intestinal organoid cultures that model the developing intestinal epithelium and associated mesenchyme.
ROBUST. Supports efficient differentiation of human ES and iPS cell lines to intestinal organoids.
CONVENIENT. Intestinal organoids can be maintained 100 µm long-term through passaging or cryopreserved for experimental flexibility. Figure 74. hPSC-Derived Intestinal Organoids Incorporate Features of the Intestinal Epithelium and Mesenchyme SERUM-FREE. Optimized formulation for low experimental variability. Organoids grown using STEMdiff™ Intestinal Organoid Kit display markers of the intestinal epithelium (EPCAM, CDX2, MUC2). Organoids also exhibit markers for intestinal mesenchyme and intestinal progenitor cells.
Schematic for Differentiating from hPSCs to Human Intestinal Organoids with the STEMdiff Intestinal Organoid Kit 500 µm 500 µm 500 µm 500 µm Day 0 Day 3 Day 7 P3 (Day 7)
Seed human Initiate Definitive Mid-/Hindgut Long-term ES/iPS cells differentiation endoderm organoid culture
Day
-2 -1 0 3 9 Change medium every 3-4 days Stage Human ES/iPS Cells Stage 1 Stage 2 Stage 3
Figure 75. Schematic for Differentiating from hPSCs to Human Intestinal Organoids with the STEMdiff™ Intestinal Organoid Kit
hPSC cultures progress through a three-stage differentiation process to generate human intestinal organoids. By day 3 of the protocol, cultures exhibit characteristics typical of definitive endoderm and mid-/hindgut differentiation is initiated. During mid-/hindgut differentiation (days 5 - 7), cells form mid-/hindgut spheroids that are released from the cell monolayer into the culture medium. These spheroids are collected, embedded in extracellular matrix, and cultured in STEMdiff™ Intestinal Organoid Growth Medium to mature into intestinal organoids. Days in parentheses indicate days post-embedding in a given passage.
Learn more at www.stemcell.com/STEMdiff-HIO
44 STEMCELL Technologies Inc. STEMdiff™ Pancreatic Progenitor Kit Produce Pancreatic Progenitor Cells from hPSCs
The STEMdiff™ Pancreatic Progenitor Kit is a serum-free medium that supports efficient and reproducible generation of pancreatic progenitor cells from hPSCs. The kit directs efficient differentiation from multiple hPSC lines through definitive endoderm, primitive gut tube, and posterior foregut endoderm before transitioning to pancreatic progenitor cells. The differentiated cells are characterized by the expression of key transcription factors, including PDX-1, NKX6.1, and NEUROD1, and by the upregulation of insulin and glucagon (Figures 76,77). The resulting pancreatic progenitor cells can be further differentiated to both exocrine and endocrine cell fates, making them useful research tools for studying diabetes and b-cell maturation, disease modeling, and studying pancreatic cancer.
H1 H9 104 104 A 72.6% 73.2% 103 103 Why Use the STEMdiff™ Pancreatic 102 102 Progenitor Kit?
1 1
PDX-1-PE 10 10 PDX-1-PE SERUM-FREE. Optimized formulation for low 100 100 100 101 102 103 104 100 101 102 103 104 experimental variability. NKX6.1-APC NKX6.1-APC ROBUST. Reproducible differentiation of multiple WLS-4D1 WLS-1C 104 104 70.0% 78.9% human ES and iPS cell lines.
3 3 10 10 EFFICIENT. Greater than 65% PDX-1+/NK X6.1+ cells 102 102 in differentiated cultures.
PDX-1-PE 1 1 10 PDX-1-PE 10 FUNCTIONAL. Produces pancreatic progenitor cells 100 100 b 100 101 102 103 104 100 101 102 103 104 capable of differentiating toward insulin-producing -cells NKX6.1-APC NKX6.1-APC or other endocrine and exocrine pancreatic cell fates.
B 100 PDX-1 80 NKX6.1 1010 PDX-1/ 60 NKX6.1 108 Undifferentiated WLS-4D1 hPSCs Definitive Endoderm 6 Pancreatic Progenitor 40 10 104 % of Total Cells Total % of 20 102 Undifferentiated Cells Undifferentiated 0 to Change Relative Fold 100 H1 H9 WLS-4D1 WLS-1C PDX-1 NKX6.1 NEUROD1 SOX9 NGN3 INS GCG Figure 76. STEMdiff™ Pancreatic Progenitor Kit Efficiently Generates PDX-1, NKX6.1-Positive Progenitors Across Multiple Figure 77. Gene Expression Profile Indicates Transition to hPSC Lines Pancreatic Progenitor Cell
PDX-1 and NKX6.1 expression measured in pancreatic progenitor cells Gene expression profile of key transcription factors or hormones (INS: insulin, derived from four different hPSC lines (H1, H9, WLS-4D1, and WLS-1C). GCG: glucagon) expressed in pancreatic progenitor cells (mean ± SEM; (A) Representative flow cytometry plots for PDX-1 and NKX6.1 expression at n = 3 - 7 experiments on WLS-4D1 cells). Expression was first normalized to 18S the end of Stage 4. (B) Cumulative quantitative data for PDX-1 and NKX6.1 ribosomal RNA and then to the expression level found in undifferentiated cells. co‑expression at the end of Stage 4 of differentiation (mean ± SD; n = 3 - 5 Gene expression is shown for WLS-4D1 cells at the end of Stage 1 (Definitive per cell line). The average efficiency of differentiation ranges from 66.5% to Endoderm) and at the end of Stage 4 (Pancreatic Progenitor). Expression pattern 30 74.5% depending on the cell line. The efficiency of conversion from definitive is consistent with published data. N.D.: Not Determined. endoderm to pancreatic progenitor ranges from 77.3% to 96.3%. In addition, nearly all NKX6.1+ cells co-express PDX-1 as observed in the developing human pancreas.29
Learn more at www.stemcell.com/STEMdiff-Pancreatic
WWW.STEMCELL.COM 45 HUMAN PLURIPOTENT STEM CELLS
STEMdiff™ Lung Progenitor Kit Generate hPSC-Derived Lung Progenitor Cells Why Use the STEMdiff™ Lung Progenitor Kit? The STEMdiff™ Lung Progenitor Kit is a serum-free culture medium system for efficient and reproducible generation of lung REPRODUCIBLE. Maximize experimental reproducibility progenitor cells from human ES and iPS cells. Differentiated cells with an optimized, serum-free formulation. will express NKX2.1, a key marker of lung progenitor cells. The RELEVANT. Obtain lung progenitor cells capable of resulting cells can be further matured toward proximal or distal downstream differentiation to proximal and distal airway airway cells, using published protocols, for the study of lung cells. diseases and lung development. CONVENIENT. Follow an easy-to-use, 15-day protocol. Schematic for Generating Lung Progenitor Cells from Human ES/iPS Cells Using STEMdiff™ Lung Progenitor Kit
Seed human Initiate ES/iPS cells differentiation
Day
0 1 2 4 5 7 15
mTeSR™1 Medium Culture Medium Medium Medium Medium or mTeSR™ LP-1 Medium DE-1 DE-2 LP-1 LP-2 Plus + Y-27632
Stage Human Stage 1: Stage 2: Stage 3: ES/iPS Cells Definitive Endoderm Anterior Foregut Endoderm Lung Progenitors
Figure 78. Schematic for Generating Lung Progenitor Cells from Human ES/iPS Cells Using STEMdiff™ Lung Progenitor Kit
hPSC cultures progress through a simple three-stage process to generate lung progenitor cells. hPSC clumps are first seeded in mTeSR™1. On Day 1, differentiation is initiated with Medium DE-1 (STEMdiff™ Endoderm Basal Medium containing Supplement MR and Supplement CJ). Subsequently on day 2 and 3, the medium is changed to Medium DE-2 (STEMdiff™ Endoderm Basal Medium containing Supplement CJ) for definitive endoderm patterning. On Day 4, to initiate anterior foregut endoderm patterning, the endoderm monolayer is passaged in Medium LP-1 (STEMdiff™ Lung Basal Medium, Lung Supplement (10X), and Supplement 1) and Y-27632. Finally, at Day 7, the cells are differentiated into the lung progenitor stage with Medium LP-2 (STEMdiff™ Lung Basal Medium, Lung Supplement (10X), and Supplement 2).
Learn more at www.stemcell.com/STEMdiff-Lung-Progenitor
46 STEMCELL Technologies Inc. STEMdiff™ Branching Lung Organoid Kit Generate hPSC-Derived Branching Lung Organoids
The STEMdiff™ Branching Lung Organoid Kit supports the efficient and reproducible generation of branching lung organoids from human embryonic stem (ES) and induced pluripotent stem Why Use the STEMdiff™ Branching (iPS) cells through four stages of differentiation: 1) definitive Lung Organoid Kit? endoderm, 2) anterior foregut endoderm, 3) lung bud organoids, and 4) branching lung organoids. This serum-free medium RELEVANT. Generate organoids that recapitulate in vivo system generates organoids that develop proximal and distal-like airway branching morphogenesis and proximodistal branching airway epithelial structures expressing EPCAM, NKX2.1, specification. SOX2, SOX9, MUC1, and P63. When matured for extended periods CONVENIENT. Get experimental flexibility with the ability (> 28 days) of organoid culture using the STEMdiff™ Branching to cryopreserve at intermediate stages. Lung Organoid Maturation Kit, there is an increase in expression levels of markers for more mature lung cells, such as SFTPC, SFTPB, and ABCA3.
A B D F H
C E G I
Figure 79. Branching Lung Organoids Cultured in STEMdiff™ Branching Lung Organoid Kit Feature Key Protein Markers and Exhibit Branching Morphogenesis
(A) Branching lung organoids express lung progenitor marker NKX2.1 throughout their branching structures and (B, C) demonstrate the presence of alveolar type II-like cells with Pro-surfactant protein B and C expressions. (D, E) These organoids undergo proximodistal differentiation demonstrated by the differential expression of SOX2 and SOX9. (F) MUC1 can be found luminally expressed while the (G) organoids are surrounded by VIM-expressing mesenchyme. (H, I) Branching lung organoids generated with the STEMdiff™ Branching Lung Organoid Kit also express proteins associated with SARS-CoV-2 entry, ACE2, and TMPRSS2. Protein expression was visualized by immunohistochemistry and confocal microscopy of branching lung organoids on day 63.
A B Figure 80. Branching Lung Organoids Cultured in STEMdiff™ Branching Lung Organoid Maturation Kit for Extended Periods Express More Mature Lung Markers
(A) The branching tips of branching lung organoids derived from peripheral blood endothelial progenitor cell-derived iPS cells (iPSC-1), peripheral blood mononuclear cell-derived iPS cells (iPSC-2), or embryonic stem cells (H1 and H9) continue to grow and branch up to day 101. (B) The expression levels of more mature distal lung markers ABCA3, SFTPC, and SFTPB increase overtime. Morphology and gene expression of branching lung organoids cultured up to day 105 were assessed by RT-qPCR. RQ-values were normalized to TBP and compared to commercially available lung RNA.
Learn more at www.stemcell.com/STEMdiff-Branching-Lung-Organoid
WWW.STEMCELL.COM 47 HUMAN PLURIPOTENT STEM CELLS
Flexible User-Directed Differentiation STEMdiff™ APEL™2 and APEL™2-LI Why Use STEMdiff™ APEL™2? STEMdiff™ APEL™2 Medium is a fully defined, serum-free, and animal component-free medium for differentiation of human ANIMAL COMPONENT-FREE. Defined, versatile, growth embryonic stem (ES) and induced pluripotent stem (iPS) cells. It is factor-free formulation. 31 based on the APEL formulation published by Ng et al. and lacks NO LINEAGE BIAS. Unsupplemented medium supports undefined components, such as protein-free hybridoma medium. cell survival without lineage bias. This medium can be used in adherent or embryoid body (EB)- based protocols, such as with AggreWell™ plates. Appropriate FLEXIBLE DIFFERENTIATION. Can be used to direct induction factors must be added before use. differentiation to a variety of cell lineages. STEMdiff™ APEL™2-LI Medium does not contain growth factors VERSATILE. Can be used in adherent- or EB-based or cytokines, and appropriate induction factors must be added protocols. before use. The low insulin content of this medium makes it particularly useful for differentiation to lineages in which insulin is a known inhibitor, such as cardiomyocytes.32
A B TeSR™-E5 and TeSR™-E6 Media 647 ® TeSR™-E5 and TeSR™-E6 are defined, serum-, and xeno‑free media that are based on the formulation of TeSR™-E8™, but do not contain transforming growth factor b (TGFb) or basic
VEGFR2 Alexa Fluor fibroblast growth factor (bFGF). Additionally, TeSR™-E5 does not contain insulin. These formulations may be used as basal media 50 µm CD105 PE for differentiation of human ES and iPS cells, or other applications 104 where removal of the above cytokines and insulin C D 6.10% 5.30% is desirable. 103
102 Count CD45 APC Learn more at www.STEMdiff.com/#custom 101
069.5% 101 102 103 19 1%104 100 FL1-H:: cTnT FITC CD34 PE
Figure 81. STEMdiff™ APEL™ and STEMdiff™ APEL™-LI Media Can Be Used for Customized Differentiation to Various Mesodermal Cell Lineages
(A) Endothelial differentiation of STiPS-F001 human iPS cells using STEMdiff™ APEL™ medium*, based on methods by Tan et al.33 (B) Immunocytochemistry image of CD31 (green; nuclei shown in blue) in endothelial cells differentiated from H1 cells using STEMdiff™ APEL™ medium. Image courtesy of the Cao Tong lab, University of Singapore. (C) Cardiomyocyte differentiation of WLS-4D1 iPS cells based on methods by Yang et al.,34 using 6 ng/mL Activin A and 10 ng/mL BMP4, with the following changes: (1) STEMdiff™ APEL™-LI medium* was used as the basal medium; (2) EBs were formed in AggreWell™400 at 1,000 cells/EB; (3) 100 ng/mL Wnt3a was added on days 1 - 4. On day 15, 77.3% (± 1.4%, n = 3) of the cells expressed cardic troponin T (cTnT; solid line). Filled histogram represents unlabeled negative control. (D) Hematopoietic differentiation of H9 cells, based on methods by Ng et al.31 and Chadwick et al.35 with the following changes: (1) STEMdiff™ APEL™ medium was used as the basal medium; (2) prior to differentiation, cells were maintained in mTeSR™1 on Matrigel®; (3) differentiation was performed in adherent cell culture on a Matrigel®-coated surface, instead of using an EB‑based method. *STEMdiff™ APEL™ and STEMdiff™ APEL™-LI have been updated to STEMdiff™ APEL™2 and STEMdiff™ APEL™2-LI, respectively, and the formulations now do not contain the undefined component, protein-free hybridoma medium.
48 STEMCELL Technologies Inc. Accessory Products Small Molecules Cytokines
Small molecules are increasingly being used as critical tools Cytokines are commonly used tools in lineage-specific to understand stem cell biology. Whether used to affect differentiation protocols, as well as for self-renewal of reprogramming, self-renewal, or differentiation, the right small hPSCs. For a complete listing of cytokines available, including molecule can transform a research project. STEMCELL Technologies animal‑component free (ACF) versions, please visit offers small molecules that are being widely used in high impact www.stemcell.com/cytokines. research to target the key pathways in stem cell biology. For a complete listing and more details on the small molecules Most Popular Cytokines available, and to see how they are being used in high-impact studies, visit www.stemcell.com/smallmolecules. CATALOG # PRODUCT Most Popular Small Molecules Non-ACF ACF *
1 PATHWAY/ Activin A 78001 78132 MOLECULE APPLICATIONS CATALOG # TARGET B18R Protein 78075 - RHO/ROCK Y-27632 pathway inhibitor Maintenance 72302 bFGF 78003 78134 Inhibits ROCK BMP-2 78004 78135 WNT pathway Reprogramming, CHIR99021 activator Maintenance, 72052 BMP-4 78211 - Inhibits GSK3 Differentiation DKK-1 78208 - WNT pathway Maintenance, 1 IWP-2 inhibitor 72122 EGF 78006 78136 Differentiation Inhibits Porcupine EGFR 78171 - BMP pathway inhibitor Flt3/Flk-2 Ligand 78009 78137 LDN193189 Differentiation 72147 Inhibits ALK2, LIF 78055 78149 ALK3, ALK6 TGF-β inhibitor Reprogramming, Noggin 78060 - SB431542 Inhibits ALK5, Differentiation, 72232 SCF 78062 78155 ALK4, ALK7 Maintenance β 1 RHO/ROCK TGF- 1 78067 - Reprogramming, Thiazovivin pathway inhibitor 72252 Maintenance VEGF-165 78073 78159 Inhibits ROCK MEK/ERK VEGF-121 78127 78158 Reprogramming, PD0325901 pathway inhibitor 72182 Maintenance PDGF-DD 78222 - Inhibits MEK Hedgehog pathway IL-18 BPa 78209 - activator Purmorphamine Differentiation 72202 TPO 78210 - Activates Smoothened BMPR-1A Fc 78212 - Notch pathway Differentiation, R-Spondin-1 78213 - DAPT inhibitor 72082 Maintenance Inhibits g-secretase RANKL 78214 78215 Prostanoid pathway activator *All ACF cytokines are human recombinant proteins produced in E. coli and are Prostaglandin Activates Differentiation, 72192 guaranteed free of animal or human components. E2 prostaglandin Maintenance 1International Units (IU) data available. Visit www.stemcell.com/IU-data. receptors EP1, EP2, EP3 and EP4 TGF-β inhibitor Reprogramming, A 83-01 Inhibits ALK5, 72022 Maintenance ALK4, ALK7
Inhibits SU5402 VEGFR2, FGFR1, Maintenance 73912 and PDGFRβ
WWW.STEMCELL.COM 49 HUMAN PLURIPOTENT STEM CELLS
AggreWell™ Plates Reproducible Production of Uniform Embryoid Bodies
Many hPSC differentiation protocols begin with the formation of three-dimensional aggregates of cells called embryoid bodies (EBs). Conventional EB formation methods36 result in EBs that are heterogeneous in size and shape (Figure 82A), leading to inefficient and uncontrolled differentiation.37 AggreWell™ plates provide an easy and standardized approach to the production of EBs. Each well contains microwells of defined size, making it easy to produce large numbers of highly uniform EBs (Figure 82B) and to ensure reproducibility of differentiation experiments.38
A B
Why Use AggreWell™?
EASY-TO-USE. Simple EB generation.
Figure 82. AggreWell™ Plates Are Used to Generate Uniform EBs REPRODUCIBLE. Produce large numbers of uniformly sized EBs. (A) Human EBs formed using conventional methods are heterogeneous in size and shape. (B) Human EBs formed using AggreWell™ plates are uniform in size CONTROL OF EB SIZE. 50 to 20,000 cells per EB. and consistently spherical in shape. Shown are EBs generated with 2000 cells using AggreWell™400. CONSISTENCY. Reduces variability in differentiation protocols.
HIGH YIELD. A B Up to 5900 EBs per well.
Learn more at www.stemcell.com/AggreWell
Figure 83. The Size of EBs Can Be Controlled in AggreWell™
Starting from a single cell suspension, hPSCs form EBs after 24 hours in AggreWell™. The size of the EB can be easily modified by adjusting the seeding density. Shown are EBs in AggreWell™400 (A) 250 cells per microwell and (B) 1000 cells per microwell.
AggreWell™ is available in 2 sizes of microwells: 400 µm (AggreWell™400) or 800 µm (AggreWell™800).
NUMBER OF PRODUCT MICROWELL SIZE CELL RANGE PLATE FORMAT CATALOG # EMBRYOID BODIES
50 - 3000 cells 24-well plate ~ 1200 per well 34411/34415 AggreWell™400 400 μm per EB 6-well plate ~ 5900 per well 34421/34425
3000 - 20,000 24-well plate ~ 300 per well 34811/34815 AggreWell™800 800 μm cells per EB 6-well plate ~1500 per well 34821/34825
Anti-Adherence Rinsing Solution (Catalog #07010) is required for optimal performance.
50 STEMCELL Technologies Inc. Antibodies GloCell™ Fixable Viability Dyes For Human Pluripotent Stem Cells and For Live/Dead Cell Staining Differentiated Cells GloCell™ Fixable Viability Dyes are fluorescent amine-labeling dyes Be confident in your experimental results, save valuable research for live/dead staining of mammalian cells, allowing clear exclusion time, and ensure experimental reproducibility by choosing antibodies of dead cells from flow cytometry data. These dyes are resistant to from STEMCELL Technologies. STEMCELL’s high-quality primary and washing and fixation and are compatible with intracellular antibody secondary antibodies are verified to work with our pluripotent stem staining protocols. Stained cells can also be cryopreserved without cell reagents in specific applications, ensuring that your downstream loss of fluorescence intensity. cell analysis, including phenotyping and purity assessments, works consistently. A Unfixed B Fixed
Dead ell
Dead ell Popular Antibodies for hPSC Research l l l l e e
f f i e ell o o
r TARGET ANTIGEN CLONE ISOTYPE CATALOG # r e e i e ell OCT4 (OCT3) 3A2A20 Mouse IgG2b 60093
OCT4 (OCT3) 40 Mouse IgG1 60059 SSEA-1 (CD15) MC-480 Mouse IgM 60060 lo ell™ i a le ia ilit D e iolet
SSEA-3 MC-631 Rat IgM 60061 Figure 84. Preservation of Fluorescence Signals in Unfixed and Fixed Cells Stained with GloCell™ Fixable Viability Dye Violet 450 SSEA-4 MC-813-70 Mouse IgG3 60062 A mixture of live and dead (heat-shocked at 95°C for 30 minutes) WLS-1C human SSEA-5 8e11 Mouse IgG1 60063 iPS cells were stained with GloCell™ Fixable Viability Dye Violet 450 (Catalog #75009) with or without fixation in 4% paraformaldehyde. After staining, (A) TRA-1-60 TRA-1-60R Mouse IgM 60064 unfixed and (B) fixed cells were immediately analyzed by flow cytometry.
TRA-1-81 TRA-1-81 Mouse IgM 60065
TRA-2-49 TRA-2-49/6E Mouse IgG1 60066 PRODUCT SIZE CATALOG #
TRA-2-54 TRA-2-54/2J Mouse IgG1 60067 GloCell™ Fixable Viability Dye 100 tests 75006.1 Red 710 5 x 100 tests 75006
For a complete listing of antibodies and conjugates available, visit GloCell™ Fixable Viability Dye 100 tests 75007.1 www.stemcell.com/antibodies. Red 780 5 x 100 tests 75007
GloCell™ Fixable Viability Dye 100 tests 75008.1 UV 450 5 x 100 tests 75008
GloCell™ Fixable Viability Dye 100 tests 75009.1 Violet 450 5 x 100 tests 75009
GloCell™ Fixable Viability Dye 100 tests 75010.1 Violet 510 5 x 100 tests 75010
GloCell™ Fixable Viability Dye 100 tests 75011.1 Violet 540 5 x 100 tests 75011
Learn more at www.stemcell.com/GloCell.
WWW.STEMCELL.COM 51 HUMAN PLURIPOTENT STEM CELLS
Annexin V Dyes
For Detection of Early-Stage Cell Apoptosis
Annexin V is a characteristic cell death marker that can be used to specifically detect early apoptotic mammalian cells. The Annexin V Apoptosis Detection Kit can be used for the combined detection of early-stage cell apoptosis using Annexin V and late-stage cell apoptosis or necrosis using both Annexin V and 7-Aminoactinomycin D (7-AAD).
PRODUCT SIZE CATALOG #
APC, 25 tests 100-0328 APC, 100 tests 100-0329
PE, 25 tests 100-0330 Annexin V PE, 100 tests 100-0331
FITC, 25 tests 100-0332 FITC, 100 tests 100-0333
Annexin V Binding Buffer 50 mL 100-0334
FITC, 1 Kit 100-0338
Annexin V Apoptosis PE, 1 Kit 100-0337 Detection Kit with 7-AAD
APC, 1 Kit 100-0339
52 STEMCELL Technologies Inc. Product Listing
Reprogramming Products for Naïve Induction and Maintenance
PRODUCT SIZE CATALOG # PRODUCT SIZE CATALOG #
RSeT™ Feeder-Free ReproRNA™-OKSGM Kit1 1 Kit 05930 500 mL Kit 05975 Medium Kit
ReproRNA™-OKSGM 12 μg 05931 NaïveCult™ Induction Kit 1 Kit 05580
NaïveCult™ Expansion ReproTeSR™ 500 mL 05920 1 Kit 05590 Medium
Erythroid Progenitor For reprogramming hPSC Naïve State qPCR 05924 96-well Plate 07521 Reprogramming Kit 10 mL of blood Array
CD34+ Progenitor For reprogramming 05925 Reprogramming Kit 80 mL of blood Matrices
TeSR™-E7™ 500 mL 05914 PRODUCT SIZE CATALOG #
1. Kit includes the ReproRNA™-OKSGM vector, ReproRNA™ Transfection 2 mL 07180 Reagent, ReproRNA™ Transfection Supplement, and Recombinant B18R Protein. Vitronectin XF™ 10 X 2 mL 100-0763
100 μg 77003 Maintenance hPSC Maintenance Media CellAdhere™ Laminin-521 10 x 100 μg 77004
PRODUCT SIZE CATALOG # 500 μg 200-0117
mTeSR™ Plus 500 mL Kit, cGMP 100-0276 Dissociation Reagents TeSR™-AOF 500 mL Kit, cGMP 100-0401 PRODUCT SIZE CATALOG #
500 mL Kit, cGMP 85850 100 mL 05872 mTeSR™1 ReLeSR™ 1 L Kit, cGMP 85857 500 mL, cGMP 100-0484
Gentle Cell Dissociation TeSR™-E8™ 500 mL Kit 05990 100 mL, cGMP 100-0485 Reagent
100 mL 07920 Products for Scale-Up ACCUTASE™ 500 mL 07922 PRODUCT SIZE CATALOG #
mTeSR™3D1 1 Kit 03950 10 mg 07446 Dispase, ACF
TeSR™-E8™3D1 1 Kit 03990 50 mg 100-0396
Dispase (1 U/mL) 100 mL 07923 1. Kits include Seed Medium (basal and 5X supplement) and Feed Medium. Components not available for individual sale. Dispase (5 U/mL) 100 mL 07913
WWW.STEMCELL.COM 53 HUMAN PLURIPOTENT STEM CELLS
Genome Editing Cell Quality Characterization
PRODUCT SIZE CATALOG # PRODUCT SIZE CATALOG #
10 mL 05888 hPSC Genetic Analysis Kit 1 Kit 07550 CloneR™ STEMdiff™ Trilineage 5 x 10 mL 05889 1 Kit 05230 Differentiation Kit hPSC Trilineage Purified Cas9 Proteins 384-well Plate 07515 Differentiation qPCR Array
100 μg 76002 ArciTect™ Cas9 Nuclease 300 μg 76004 Cryopreservation ArciTect™ Cas9-eGFP 100 μg 76006 Nuclease PRODUCT SIZE CATALOG # Guide RNA (gRNA) 50 mL 05855 mFreSR™ ArciTect™ sgRNA 4 nmol 200-0013 10 x 5 mL Tubes 05854
2 nmol 76010 FreSR™-S 50 mL 05859
ArciTect™ crRNA 10 nmol 76011 100 mL, cGMP 07930
20 nmol 76012 5 x 16 mL Vials, 07931 cGMP 5 nmol Kit 76016 1000 mL Bag, 07940 cGMP CryoStor® CS10 ArciTect™ tracrRNA Kit 10 nmol Kit 76017 100 mL Bag, cGMP 07955
20 nmol Kit 76018 5 x 10 mL Vials, 07959 cGMP T7 Endonuclease I Assay Reagents 16 x 10 mL Vials, 07952 cGMP 25 Reactions 76021 ThawSTAR® CFT2 1 Each 100-0650 ArciTect™ T7 Automated Thawing System Endonuclease I Kit 50 Reactions 76022 ThawSTAR® CFT2 1 Each 100-0642 Transporter ArciTect™ High-Fidelity 1 Kit 76026 ThawSTAR® CFT2 DNA Polymerase Kit 20 Vials 100-0643 Confirmation Vials Positive Control Kits ThawSTAR® CFT2 IOPQ Kit 1 Kit 100-0730 ArciTect™ Human CRISPR 1 Kit 100-0470 Optimization Kit, APC ArciTect™ Human CRISPR 1 Kit 100-0472 Optimization Kit, FITC ArciTect™ Human CRISPR 1 Kit 100-0471 Optimization Kit, PE ArciTect™ Human HPRT 1 Kit 76013 Positive Control Kit
54 STEMCELL Technologies Inc. Cell Quality Characterization Differentiation Mesoderm Differentiation Products Ectoderm Differentiation Products
PRODUCT SIZE CATALOG # PRODUCT SIZE CATALOG #
100 mL 05220 STEMdiff™ Mesoderm Neural Differentiation Induction Medium 500 mL 05221 STEMdiff™ SMADi Neural 1 Kit 08581 Induction Kit STEMdiff™ Mesenchymal 1 Kit 05240 STEMdiff™ Neural Induction Progenitor Kit 250 mL 05835 Medium Endothelial Differentiation STEMdiff™ Neural Rosette 100 mL 05832 STEMdiff™ Endothelial Selection Reagent 1 Kit 08005 Differentiation Kit STEMdiff™ Neural Progenitor 1 Kit 05833 Cryopreservation STEMdiff™ Endothelial Medium 1 Kit 08007 Expansion Medium Kit STEMdiff™ Forebrain Neuron 1 Kit 08600 Hematopoietic and Immune Differentiation Differentiation Kit STEMdiff™ Forebrain Neuron 1 Kit 08605 STEMdiff™ Hematopoietic Kit1 1 Kit 05310 Maturation Kit STEMdiff™ Midbrain Neuron 1 Kit 100-0038 STEMdiff™ Monocyte Kit 1 Kit 05320 Differentiation Kit STEMdiff™ Microglia STEMdiff™ Midbrain Neuron 1 Kit 100-0019 1 Kit 100-0041 Differentiation Kit Maturation Kit STEMdiff™ Microglia STEMdiff™ Astrocyte 1 Kit 100-0020 1 Kit 100-0013 Maturation Kit Differentiation Kit STEMdiff™ Erythroid Kit 1 Kit 100-0074 STEMdiff™ Astrocyte 1 Kit 100-0016 Maturation Kit 100 mL 09605 STEMdiff™ Neural Progenitor 100 mL 05838 StemSpan™ SFEM II Freezing Medium 500 mL 09655 STEMdiff™ Cerebral Organoid 1 Kit 08570 STEMdiff™ NK Cell Kit 1 Kit 100-0170 Kit STEMdiff™ Cerebral Organoid 1 Kit 08571 STEMdiff™ T Cell Kit 1 Kit 100-0194 Maturation Kit STEMdiff™ Dorsal Forebrain Cardiomyocyte Differentiation 1 Kit 08620 Organoid Differentiation Kit STEMdiff™ Cardiomyocyte STEMdiff™ Ventral Forebrain 1 Kit 05010 1 Kit 08630 Differentiation Kit Organoid Differentiation Kit STEMdiff™ Cardiomyocyte 1 Kit 05020 STEMdiff™ Neural Organoid Maintenance Kit 1 Kit 100-0120 Maintenance Kit STEMdiff™ Cardiomyocyte 1 Kit 05027 Support Medium Customizable Neural Differentiation and Characterization STEMdiff™ Cardiomyocyte 1 Kit 05025 Dissociation Kit BrainPhys™ Neuronal Medium 500 mL 05790 STEMdiff™ Atrial Cardiomyocyte 1 Kit 100-0215 Differentiation Kit BrainPhys™ Without Phenol Red 500 mL 05791 STEMdiff™ Cardiomyocyte 50 mL 05030 Freezing Medium BrainPhys™ Imaging Optimized 500 mL 05796
Myogenic Differentiation BrainPhys™ Neuronal Medium 1 Kit 05792 and SM1 Kit STEMdiff™ Myogenic Progenitor 1 Kit 100-0151 Supplement Kit BrainPhys™ Neuronal Medium 1 Kit 05793 MyoCult™ Differentiation Kit N2-A & SM1 Kit 1 Kit 05965 (Human) BrainPhys™ hPSC Neuron Kit 1 Kit 05795 MyoCult™-SF Expansion 1 Kit 05980 Supplement Kit (Human) NeuroFluor™ NeuO 0.1 mL 01801 Kidney Differentiation
STEMdiff™ Kidney Organoid Kit 1 Kit 05160
1. Kit includes basal medium and supplements A and B. WWW.STEMCELL.COM 55 HUMAN PLURIPOTENT STEM CELLS
Ectoderm Differentiation Products (continued) Endoderm Differentiation Products
PRODUCT SIZE CATALOG # PRODUCT SIZE CATALOG # STEMdiff™ Definitive Endoderm 1 Kit 05110 Neural Crest Differentiation Kit STEMdiff™ Neural Crest STEMdiff™ Definitive Endoderm 1 Kit 08610 1 Kit 05115 Differentiation Kit Kit (TeSR™-E8™-Optimized) MesenCult™-ACF Plus Medium hPSC-Derived Endoderm qPCR 1 Kit 05445 96-well Plate 07531 Kit Array MesenCult™-ACF Chondrogenic Differentiation 1 Kit 05455 Intestinal Differentiation Kit MesenCult™ Osteogenic STEMdiff™ Intestinal Organoid 1 Kit 05465 1 Kit 05140 Differentiation Kit (Human) Kit MesenCult™ Adipogenic STEMdiff™ Intestinal Organoid 1 Kit 05412 1 Kit 05145 Differentiation Kit (Human) Growth Medium Neuroimmune Cell Differentiation Pancreatic Differentiation
STEMdiff™ Hematopoietic Kit 1 Kit 05310 STEMdiff™ Pancreatic 1 Kit 05120 Progenitor Kit STEMdiff™ Microglia 1 Kit 100-0019 Differentiation Kit Lung Differentiation STEMdiff™ Microglia 1 Kit 100-0020 Maturation Kit STEMdiff™ Lung Progenitor Kit 1 Kit 100-0230
STEMdiff™ Branching Lung 1 Kit 100-0195 Organoid Kit STEMdiff™ Branching Lung 1 Kit 100-0528 Organoid Maturation Kit
56 STEMCELL Technologies Inc. Products for Flexible User-Directed Differentiation
PRODUCT SIZE CATALOG #
STEMdiff™ APEL™2 Medium 100 mL 05270
STEMdiff™ APEL™2-LI Medium 100 mL 05271
TeSR™-E5 1 Kit 05916
TeSR™-E6 1 Kit 05946
WWW.STEMCELL.COM 57 HUMAN PLURIPOTENT STEM CELLS
References
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DOC#29063 VERSION 13.0.0 JUNE 2021