HUMAN PLURIPOTENT STEM CELLS Products for Your Research Scientists Helping Scientists™ | WWW.STEMCELL.COM HUMAN PLURIPOTENT STEM CELLS 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 WWW.STEMCELL.COM 3 HUMAN PLURIPOTENT STEM CELLS 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. 1 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 WWW.STEMCELL.COM 5 HUMAN PLURIPOTENT STEM CELLS 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