Robust Media to Culture Intestinal, Hepatic, Pancreatic, Kidney, Airway, and Cerebral Organoids

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2 STEMCELL Technologies Inc. TABLE OF CONTENTS

4 Introduction

5 Intestinal Organoids 6 IntestiCult™ Growth Medium (Mouse) 7 IntestiCult™ Organoid Growth Medium (Human) 8 IntestiCult™ Organoid Differentiation Medium (Human) 11 STEMdiff™ Intestinal Organoid Kit

13 Hepatic Organoids 13 HepatiCult™ Organoid Growth Medium (Mouse)

16 Pancreatic Organoids 16 PancreaCult™ Organoid Growth Medium (Mouse)

19 Kidney Organoids 19 STEMdiff™ Kidney Organoid Kit

22 Airway Organoids 22 PneumaCult™ Airway Organoid Kit

24 Cerebral Organoids 24 STEMdiff™ Kit

26 Product Information & References

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Introduction

One of the most impactful advances in stem research of Removal of confounding variables inherent in animal models while the past decade has been the development of organoid culture providing increased complexity compared with homogeneous techniques. Organoids are three-dimensional cell cultures that cell cultures gives organoid cultures specific experimental recapitulate some of the key cell types and structural features advantages. By combining a high level of physiological relevance of the represented . These “mini-organs” enable scientists with amenability to manipulation in vitro, organoids have the to investigate complex research questions in vitro by providing potential to complement or replace in vitro experiments using highly relevant model systems. Since landmark publications primary cells or immortalized cell lines. There is also a movement describing intestinal1 and cerebral organoids,2 organoid culture to use organoids to replace animal experimentation in many techniques have been developed for multiple species and tissues to circumstances. Additionally, organoids demonstrate high genetic derive organoids from either adult stem cells (ASCs) or pluripotent stability in culture, retaining the genotype and phenotype of the stem cells (PSCs). source tissue. Organoids therefore provide faithful models of disease and have been used to model disease mechanisms and While each culture system is distinct, organoids generally contain progression, as well as to predict patient-specific response to drug multiple different organ-specific cell types capable of recapitulating treatment. some functions of that organ. During organoid culture, stem cells (PSCs or ASCs) are provided with appropriate signaling factors Continued development of organoid methodologies is proving that mimic the in vivo environment. Exposure to these conditions transformative to the research landscape. In many labs, directs stem cells to divide and differentiate into the cell types of organoids have complemented model systems already in place, the represented organ. In general, organoids display architecture while in others they have enabled investigations previously not similar to what is observed in vivo, such as polarization of the possible. Development and standardization of organoid culture apical and basolateral surfaces (epithelial organoids), appropriate techniques will enable even greater accessibility of organoids. cellular stratification (cerebral organoids), or re-creation of complex domains such as the intestinal crypt-villus structure observed in mouse intestinal organoids. In epithelial organoid systems, the presence of actively dividing adult stem cells enable the cultures to be expanded and maintained in long-term culture through serial passaging. Organoid cultures have seen quick and widespread adoption for a variety of applications, from basic research to translational and industrial uses. The roots of organoid technology lie in , and organoids are now feeding back valuable information about the development of the tissues that they model, especially in the case of PSC-derived organoids. As organoids recapitulate many of the signaling and cell-cell interactions observed in vivo, they are also invaluable models for studying , including investigation into tissue regeneration mechanisms, maintenance of the stem cell niche, and interactions with bacteria, viruses, and cells from other tissues.

4 STEMCELL Technologies Inc. Intestinal Organoids

Intestinal organoids incorporate key intestinal cell types including long‑term maintenance of ASC-derived intestinal organoids enterocytes, goblet cells, enteroendocrine cells, transit amplifying from mouse and human cells, respectively. STEMdiff™ Intestinal cells, and intestinal stem cells, recapitulating the cellular Organoid Kit can be used to direct human PSCs through a three- complement of the intestinal . The presence of an actively stage differentiation process to intestinal organoids that can dividing stem cell population enables expansion and maintenance of be maintained and matured in STEMdiff™ Intestinal Organoid intestinal organoids in long-term culture. Growth Medium. Intestinal organoids grown using these media are convenient, flexible, and relevant intestinal tissue models that can IntestiCult™ Organoid Growth Medium (Mouse) and IntestiCult™ increase experimental impact and reduce laboratory animal use. Organoid Growth Medium (Human) support establishment and

Table 1. Comparison of Intestinal Organoid Culture Systems

INTESTINAL ORGANOID TYPE MOUSE (ASC-DERIVED) HUMAN (ASC-DERIVED) HUMAN (PSC-DERIVED)

Representative Image

200 µm 200 µm 500 µm

Organoid Cellular Composition Organoids simultaneously After 1 - 3 passages organoids Organoids simultaneously incorporate an active intestinal are composed primarily of cells incorporate an active intestinal stem cell compartment and in a progenitor/stem-like state; stem cell compartment, differentiated cell types in an organoids can be differentiated differentiated intestinal epithelium that recapitulates the to mature intestinal cells in an epithelial cell types and in vivo intestine. appropriate medium. associated mesenchymal cells. Organoids are phenotypically fetal.

Starting Material • Mouse intestinal or • Human intestinal or • Human induced pluripotent colonic crypts colonic crypts stem cells (iPSC) or embryonic • Mouse Intestinal Organoids • Established ASC-derived stem cell (ESC) lines (Catalog #70931) intestinal organoids • Established PSC-derived intestinal organoids

Organoid Maintenance Organoids can be maintained Organoids can be maintained Organoids can be maintained through long-term passaging through long-term passaging through long-term passaging or cryopreserved. or cryopreserved. or cryopreserved.

Genetic Considerations • Established common healthy • Donor-specific genetic • Donor-specific genetic and disease model strains background background • Tools for in vivo genetic • Targeted in vitro gene editing • Targeted in vitro gene editing manipulation possible possible

Compatible Culture Media IntestiCult™ Organoid IntestiCult™ Organoid STEMdiff™ Intestinal Growth Medium (Mouse) Growth Medium (Human) Organoid Kit (Catalog #05140) (Catalog #06005) (Catalog #06010) STEMdiff™ Intestinal IntestiCult™ Organoid Organoid Growth Medium Differentiation Medium (Catalog #05145) (Human) (Catalog #100- 0214); for differentiation in 3D organoids and as monolayers in submerged or air-liquid interface cultures.

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IntestiCult™ Organoid Why Use IntestiCult™ Organoid Growth Medium (Mouse) Growth Medium (Mouse)? Medium for RELEVANT. Enables generation of intestinal organoid cultures that recapitulate the identity and organization of Establishment and Maintenance the adult intestinal epithelium.

of Mouse Intestinal Organoids ROBUST. Supports efficient establishment of organoids from mouse intestinal crypts in less than one week. IntestiCult™ Organoid Growth Medium (Mouse) is a complete, serum‑free organoid growth medium that enables researchers to SIMPLE. Convenient format and easy-to-follow protocol. easily and reproducibly generate experiment-ready organoids in SERUM-FREE. Optimized formulation for low 5 - 7 days. experimental variability.

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Mouse intestinal organoids were cultured in IntestiCult™ Organoid Growth 88000000

Medium (Mouse) and imaged on day 5 of passage 0. s 77000000 d i

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Figure 2. IntestiCult™ Organoid Growth Medium (Mouse) Supports Efficient Organoid Establishment and Expansion

(A) Established organoids can be passaged efficiently over an indefinite number of passages. (B) Starting from a single well containing 100 organoids and passaging at a 1 in 4 split ratio, organoid count increases an average of 4.2-fold per passage.

6 STEMCELL Technologies Inc. IntestiCult™ Organoid Why Use IntestiCult™ Organoid Growth Medium (Human) Growth Medium (Human)? Cell Culture Medium for ROBUST. Reliable and efficient expansion of intestinal stem cells across donor samples. Establishment and Maintenance RELEVANT. Enables generation of intestinal organoid of Human Intestinal Organoids cultures that can be used to model the adult intestinal epithelium. IntestiCult™ Organoid Growth Medium (Human) is a complete cell culture medium for efficient establishment and long-term COMPLETE. Two-component medium system does not maintenance of human intestinal epithelial organoids derived from require additional growth factors to support organoid primary intestinal or colonic crypts or previously frozen colonic growth. organoids. CONSISTENT. Quality-controlled medium generates consistent results between experiments and between laboratories.

A B DAPI EPCAM

Ki67 MERGE

200 µm

Figure 3. Human Colonic Organoids Grown in IntestiCult™ Organoid Growth Medium (Human)

(A) Colonic organoids grown in IntestiCult™ Organoid Growth Medium (Human) and imaged on day 7 of passage 0. (B) Immunofluorescence of intestinal organoids showing DAPI (blue), EPCAM (red), Ki67 (green), and the merged image.

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104 Control p0 p1 p2 p3 p4 p5 p6 p7 p8 p9 p10 Passage Number

Figure 4. Intestinal Organoids Can be Maintained in Long-Term Figure 5. Forskolin-Induced Swelling of Intestinal Organoids

Culture Through Passaging Organoids cultured in IntestiCult™ Organoid Growth Medium (Human) were Organoids cultured in IntestiCult™ Organoid Growth Medium (Human) show treated with (A) 5 µM Forskolin or (B) DMSO. Organoid area was measured at efficient growth throughout passaging. Cultures were split with an average split 0 minutes and 120 minutes. Forskolin-treated organoids increased in size by ratio of 1 in 6 at each passage. Error bars represent standard error. 33.5 ± 3.8% in size compared to a 7.5 ± 0.8% increase for control organoids (n=3). Representative images are shown.

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IntestiCult™ Organoid Why Use IntestiCult™ Organoid Differentiation Medium Differentiation Medium? (Human) RELEVANT. Enables generation of intestinal organoid cultures with physiological proportions of differentiated Complete Culture Medium for the and stem cell populations. Differentiation of Intestinal Organoids. FLEXIBLE. Allows easy access to the apical surface by transitioning intestinal organoids into monolayer and ALI IntestiCult™ Organoid Differentiation Medium (Human; ODM) is a cultures. complete culture medium that supports the further differentiation REPRODUCIBLE. Generates consistent results across of intestinal organoids in three dimensions (3D), or in 2D as passages and replicates. submerged monolayers or air-liquid interface (ALI) cultures. These cultures can be initiated from intestinal organoids derived from human intestinal crypts, or passaged organoids that have been cultured with IntestiCult™ Organoid Growth Medium (Human; OGM; Catalog #06010).

A B Figure 6. Differentiated Human Intestinal Organoids Display a Budded Morphology

(A) Organoids grown in IntestiCult™ OGM are primarily cystic. (B) When switched to IntestiCult™ ODM, organoids develop a thickened epithelium with a pronounced, budded morphology indicative of a more differentiated state. Organoids were imaged on day 5 of expansion or differentiation respectively.

200 µm 200 µm

A B Figure 7. Intestinal Organoids Contain a Higher Proportion of Mature Cell Types Following Differentiation in IntestiCult™ ODM

(A, B) Organoids grown in IntestiCult™ OGM are enriched for Ki-67+ proliferative cells (A), while containing few differentiated cell types such as goblet cells (MUC2, MUC2 CHGA A), enterocytes (KRT20, B), and enteroendocrine cells (CHGA, B). (C, D) When Ki-67 KRT20 + E-cadherin E-cadherin switched to IntestiCult™ ODM, organoids contain a small number of Ki-67 DAPI 100 µm DAPI 100 µm proliferative cells (C, arrows), with more physiological proportions of goblet cells (MUC2, C), enterocytes (KRT20, D), and chromogranin A- (CHGA-)positive enteroendocrine cells (D, arrow). C D

MUC2 CHGA Ki-67 KRT20 E-cadherin E-cadherin DAPI 100 µm DAPI 100 µm

8 STEMCELL Technologies Inc. A B C 1.5 Lgr5 1.5 Axin2 600 KRT20

1.0 1.0 400 RQ RQ RQ

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0.0 0.0 0 r r r d e er d er d er y y ye y ye noi a noi a a noi a a olay ol ol nol ga n ga n Orga on MUC2 Or CHG-A onol Or onol M Mo Mo Mo I IM IM D 40000 GM E 30000 GM M IntestiCult™GM OGMM Organoid O DM AL O D AL O D AL 30000 O ™ O ™ O ™ t ™ M t ™ M lt ™ DM 20000l t D l t D 20000 lt O l O l O ™ IntestiCult™ ODM™ Monolayer tiCu ™ tiCu t tiCu t s 10000 tiCu lt s tiCu l s tiCu l e s e 10000 s e s 120 e nt e nt e Int tiCu I 6000 nt tiCu I IntestiCult™nt ODMtiCu ALI Monolayer Int s I s I s 100 e e e 80 nt 4000 Int Int

RQ I RQ 60 2000 40 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 d r r er d er y ye y ye noi a a noi a a ol ol F ga n G ga n Or onol Or onol Mo Mo IM IM GM M GM M O D AL O D AL ™ O M MUC2™ O t ™ t ™ M l lt D l t D O E-cadherin l O ™ tiCu t tiCu ™ s tiCu l Ds API tiCu lt e s e s nt e e I nt tiCu Int tiCu I s Int s e e Int Int

MUC2 E-cadherin DAPI 30 µm 30 µm

Figure 8. Differentiation of Intestinal Epithelium at the Air-Liquid Interface (ALI) Using IntestiCult™ ODM

(A – E) Growing organoid-derived monolayers as ALI cultures drives further differentiation of intestinal epithelial cultures as seen by changes in gene expression measured by RT-qPCR. Relative quantification (RQ) for each marker is shown relative to actB and TBP housekeeping genes and normalized with respect to undifferentiated organoids grown in IntestiCult OGM (Human). Progenitor markers (A) Lgr5 and (B) Axin2 are significantly reduced in both submerged monolayers and ALI cultures, while markers of enterocytes (KRT20, C), goblet cells (MUC2, D), and enteroendocrine cells (CHGA, E) are significantly increased. Further reduction in Axin2 is seen in ALI monolayers with an increase in expression of KRT20, MUC2, and CHGA. (F, G) Comparing cross-sections of organoid monolayers grown in IntestiCult™ ODM as (F) submerged culture or (G) at the ALI shows further differentiation of the intestinal epithelium with an increased proportion of goblet cells and extracellular mucus (MUC2, green).

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A 2500 * *

0 220 IntestiCult™OGM DMSO OGM DMSO

2000 oT 200 IntestiCult™OGM Forskolin OGM Forskolin et 2 180 IntestiCult™ ODM DMSO m 1500 ODM DMSO

elativ 160 xc IntestiCult™ODM Forskolin ODM Forskolin 1000

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500 120 wellin 100 0 %S I 80 0 MIN 20 MIN 40 MIN 60 MIN co-2 AL Ca DM O Incubation Time t™ DM Monolayer l O tiCu ™ s lt e B IntestiCult™ ODM C Caco-2 Int tiCu s e Int IBMX/Forskolin CFTR inhibitor-172 40 IBMX/Forskolin CFTRinh172

100 30 2 2 Figure 9. Differentiated Organoid-Derived Monolayers and ALI 20 A/cm Cultures Display More Physiological Trans-Epithelial Electrical A/cm 50 10 µ µ

Resistance (TEER) than Caco-2 Cells 0

Differentiated organoid-derived monolayers grown as a submerged monolayer 0 -10 Time (IntestiCult™ ODM Monolayer), or at the ALI (IntestiCult™ ODM ALI), show higher Time TEER values as compared to Caco-2 cultures.Organoid-derived monolayers grown at the ALI show a loosening of tight junctions due to further differentiation of the D E IBMX/ForskolinIBMX/Forskolin CFTRCFTR Inhibitor-172 inhibitor-172 brush border, and thus lower TEER values are observed. * p < 0.0001 100 p < 0.001 100 p < 0.001 2) 2) 80 80

60 60 A/cm A/cm

40 40 C( C( IS IS 20 20

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M M OD OD -2 Cells -2 Cells lt™ u lt™ C aco u aco i C iC C

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Figure 10. Differentiated Intestinal Organoids Provide a Suitable Model for Studying CFTR Response In Vitro

(A) Organoids differentiated further in IntestiCult™ ODM show a comparable degree of swelling when treated with forskolin as compared to organoids grown in IntestiCult™ OGM, demonstrating suitability for use in forskolin-induced swelling assays. (B – E) Ussing chamber analysis of submerged (B) organoid-derived monolayers and (C) Caco-2 cultures demonstrate increased sensitivity of organoid-derived monolayers to CFTR activation and inhibition by IBMX/Forskolin and CFTR Inhibitor-172 respectively. (D, E) Analysis of CFTR modulation by IBMX/ Forskolin and CFTR Inhibitor-172 show significantly greater (D) activation and (E) inhibition of CFTR activity in organoid-derived monolayers as compared to Caco-2 cultures (p < 0.001 for both).

10 STEMCELL Technologies Inc. STEMdiff™ Intestinal Why Use STEMdiff™ Intestinal Organoid Kit Organoid Kit? Cell Culture Medium Kit for RELEVANT. Enables generation of small intestinal organoid cultures that model the developing intestinal epithelium Differentiation of PSC-Derived and associated . Human Intestinal Organoids ROBUST. Supports efficient differentiation of human ES and iPS cell lines to intestinal organoids. The STEMdiff™ Intestinal Organoid Kit supports efficient establishment of PSC-derived small intestinal organoid cultures CONVENIENT. Intestinal organoids can be maintained from embryonic stem (ES) cells or induced pluripotent stem (iPS) long‑term through passaging or cryopreserved for cells within 30 days. This serum-free medium kit is based on the experimental flexibility. 3 formulation published by Spence et al. and has been optimized SERUM-FREE. Optimized formulation for low experimental to increase efficiency and reproducibility of organoid formation variability. and expansion.

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Seed Initiate Definitive Mid-/ Small Intestinal Human PSCs Differentiation Hindgut Organoids

Day -2 -1 0 3 5 7 9 Long-Term Organoid Culture

Human Stage STAGE 1 STAGE 2 STAGE 3 PSCs

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P0 (Day 0) P0 (Day 8) P1 (Day 7) P3 (Day 7)

Figure 11. Generation of Human Intestinal Organoid Cultures Using the STEMdiff™ Intestinal Organoid Kit

(A) Human PSC 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 - 9), cells form mid-/hindgut spheroids that are released from the cell monolayer into the culture medium. These spheroids are collected and embedded in . (B) Embedded mid-/hindgut spheroids cultured in STEMdiff™ Intestinal Organoid Growth Medium mature into intestinal organoids (days in parentheses indicate days post-embedding in a given passage). Once established, intestinal organoids can be maintained and expanded in culture by passaging every 7 - 10 days. After multiple passages, the organoids generally exhibit less sinking within the matrix dome and a lower proportion of mesenchymal cells.

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A B C

12001200 6000 H9 120 10001000 5000 H7 100100 WLS-1C + s

+ 800800 4000 STiPS-M001 8080

OX17 600600 3000 6060 / SOX17 pheroid /S + +

#S 400400 2000 4040 Cell Yield (x1000) Cell Yield OXA2

2020 # Spheroids per Well 200200 1000 %F %FOXA2 00 00 0 C ) ) C 09) 07) O1 -1 WA WA LS-1 MO WA09 WA07 LS 9 ( 7 ( W W S-MOO1 H H WLS-1C 9 ( 7 ( WLS-1C TiPS- H H P Spheroids Passage 1 Passage 2 Passage 3 H7 (WA07) S H7 (WA07) Ti H9 (WA09) STiPS-M001 H9 (WA09) SSTiPS-M001 Human Intestinal Organoids

Figure 12. STEMdiff™ Intestinal Organoid Kit Supports Robust Differentiation and Expansion Across ESC and iPSC Lines

STEMdiff™ Intestinal Organoid Kit enables high-efficiency generation of intestinal organoids from both ES cells (H9, H7) and iPS cells (WLS-1C, STiPS-M001). (A) Organoids initiated from a variety of cell lines show efficient induction of definitive endoderm, measured by co-expression of FOXA2 and SOX17 on day 3 of differentiation. (B) Both ES and iPS cell‑derived cultures demonstrate efficient spheroid formation upon mid-/hindgut induction. The total number of spheroids obtained per well in a given differentiation is shown. (C) Organoids cultured from either ES or iPS cells can be expanded and maintained over multiple passages. Shown is the total cell yield per passage. Organoids were passaged every 7 - 10 days with a split ratio between 1 in 2 and 1 in 4. Data points represent the mean of 3 biological replicates. Error bars throughout represent standard deviation of the mean.

Figure 13. Characteristics of Intestinal Organoids Cultured Using A B STEMdiff™ Intestinal Organoid Growth Medium

Intestinal organoids were stained and examined for marker expression by immunocytochemistry. (A,B) Intestinal organoids express markers of intestinal progenitor cells including CDX2 and the intestinal crypt marker SOX9. The organoids incorporate a DAPI polarized epithelium, visualized by the localization of EPCAM to the exterior (basolateral) CDX2 DAPI MUC2 CHGA surface of the organoids (A), and express markers typical of mature cell types including EPCAM 100 µm SOX9 100 µm MUC2 (goblet cells, A) and CHGA (enteroendocrine cells, B). (C,D) Observation of desmin (C) and vimentin (D) in intestinal organoids demonstrates incorporation of mesenchymal C D cells in the organoid cultures, while KRT20 (C) and Ki67 (D) are markers of differentiated intestinal cells and actively dividing progenitor cells, respectively. Images are digital cross- sections of whole-mount imaging of intact organoids.

DAPI DAPI KRT20 Ki67 Desmin Vimentin EPCAM 100 µm EPCAM 100 µm

12 STEMCELL Technologies Inc. Hepatic Organoids

The development of hepatic organoid culture techniques has provided the hepatic research field with a convenient method for sustained maintenance of cells in vitro.4,5 Hepatic progenitor organoids are cultured by isolating and expanding liver stem and progenitor cells, which are postulated to reside in hepatic ducts. Culture of hepatic progenitor cells as organoids produces spherical organoids consisting primarily of progenitor cells that can be further differentiated to either hepatocytes or cholangiocytes.6

HepatiCult™ Organoid Why Use HepatiCult™ Organoid Growth Medium (Mouse) Growth Medium (Mouse)? Cell Culture Medium for Establishment CONVENIENT. In vitro system for generating organoids within a week. and Maintenance of Mouse Hepatic STEP-BY-STEP PROTOCOL. No injury models, hand- Organoids picking of ducts, or cell sorting required.

HepatiCult™ Organoid Growth Medium (Mouse) is a serum-free SIMPLE, TWO-COMPONENT FORMAT. Serum-free medium that allows for rapid generation of hepatic progenitor medium formulation. organoids from mouse liver tissue. Organoid culture may be initiated FLEXIBLE PROTOCOL. Organoids be can grown from duct from hepatic ducts, duct fragments, single cells, or cryopreserved fragments or single cells and cultured in matrix domes or ® ® organoids and cultured in Corning domes or as a dilute suspension. Matrigel® suspension.

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Figure 14. Mouse Hepatic Progenitor Organoids Can Be Initiated from a Variety of Starting Materials

HepatiCult™ Organoid Growth Medium (Mouse) enables the initiation of hepatic progenitor organoids from (A) duct fragments, (B) single cells or (C) cryopreserved organoids. All organoids were grown in Matrigel® domes and imaged on day 7 of primary culture or the first passage post thaw (cryopreserved organoids).

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Figure 15. Hepatic Organoids Can Be Grown in Matrigel® Domes or in a Dilute Matrigel® Suspension

Hepatic progenitor organoids cultured from freshly isolated mouse hepatic duct fragments in HepatiCult™ Organoid Growth Medium (Mouse) can be plated in (A) Matrigel® domes or (B) a dilute Matrigel® suspension. Organoids grown in either culture condition are ready for passage within 4-7 days.

Dome-Expanded Suspension-Expanded 10251025 Hepatic Organoids Hepatic Organoids ield Proliferation Mki67 ly Axin2 201020 Cd24a 10 cel Cd44 Prom1 15 15 Sox9 total 10 10 Wnt Pathway Lgr5 Epcam Epithelial Tjp1 10101010 Acox2 Cumulative Total Cell Yield Cumulative Total Foxa2 Hnf1α cumulative 105 105 Hnf4α p0 p1 p2 p3 p4 p5 p6 p7 p8 p9 p10

Hepatic Pparg p0 p1 p2 p3 p4 p5 p6 p7 p8 p9 p10 Progenitor Tbx3 Passage Number Ttr Hnf1β Figure 17. Expansion of Organoids Grown in HepatiCult™ Ductal Krt7 Progenitor Organoid Growth Medium (Mouse) Krt19 Afp Organoids cultured with HepatiCult™ Organoid Growth Medium (Mouse) show Ldlr -2 Abcc4 efficient growth over multiple passages.Cultures were split with an average split )

2 Alb ratio of 1:25 at each passage. Asgr1 Cyp3a11 Hepatic 8 Fah Glul Ppara Serpina1a

Gene Expression(rlog Gene Ggt1 16 Notch2 Onecut1 Ductal Sstr2 Pou5f1 Pluripotency Sox2

Figure 16. Analysis of Organoid Gene Expression

Analysis of marker expression by RNA-seq shows organoids grown in HepatiCult™ Organoid Growth Medium (Mouse) in either Matrigel® domes or in a dilute Matrigel® suspension express markers associated with hepatic stem and progenitor cells. The organoids also express low levels of genes associated with mature hepatic cell types, including cholangiocytes and hepatocytes. Columns represent biological replicates at passages ranging from passage 1 - 40. 14 STEMCELL Technologies Inc. A B A Hnf4α B Alb nα l . RQ RQ

EM M EM M EM M EM M ME SSESI ME SSESI 100 µm 10 µm

C Sox9 D Axin2 So Axin2 C . . . . 0.02x 0.04x . . RQ RQ RQ . .

DAPI 100 µm . . EM M EM M EM M EM M ME SSESI ME SSESI

E Krt19 F Hnf1β nβ

. . .

MRP4 100 µm MERGE 100 µm RQ RQ . D EM M EM M EM M EM M ME SSESI ME SSESI

Figure 19. Differentiation of Hepatic Progenitor Organoids

Hepatic progenitor organoids grown in HepatiCult™ Organoid Growth Medium DAPI 100 µm (Mouse) (EM) can be differentiated to resemble more mature cell types when switched to a differentiation medium (DM). After switching to published differentiation medium, hepatic organoids show the upregulation of mature hepatic markers including (A) Hnf4α and (B) Alb, downregulation of hepatic stem cell and progenitor markers (C) Sox9 and (D) Axin2 and upregulation of ductal markers (E) Krt19 and (F) Hnf1β. Relative quantification (RQ) for each marker is reported relative to 18S and TBP housekeeping genes and normalized with respect to hepatic progenitor organoids grown in HepatiCult™ Organoid Growth Ki67 100 µm MERGE 100 µm Medium (Mouse) and cultured in Matrigel® domes.

Figure 18. Organoids Grown in HepatiCult™ Organoid Growth Medium (Mouse) Display Some Characteristics Typical of the Mature Hepatic Epithelium

(A) Hepatic progenitor organoids exhibit the polygonal morphology typical of the hepatic epithelium. (B) Hepatic progenitor organoids show binucleation (arrows), a common feature of mature hepatocytes. (C) Immunocytochemistry analysis shows localization of MRP4 (green), a membrane-bound, unidirectional efflux transporter, along the exterior of the organoids and DAPI (red) localized to the cellular nuclei. This indicates cellular polarization of the organoids with the basolateral surface of the epithelium distal from the lumen. (D) Hepatic organoids contain an actively dividing progenitor population, shown by the expression of Ki67 (red). Cell nuclei are stained with DAPI (blue).

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Pancreatic Organoids

Pancreatic organoids are cultured through isolation and expansion of pancreatic stem and progenitor cells from pancreatic ducts.7 When cultured with the appropriate growth factors and extracellular matrix, pancreatic progenitor cells form organoids resembling the exocrine component of the pancreas. In addition, isolation of primary tissue from mouse primary tumors and metastases allows for the growth of tumor- derived organoids, providing a model for pancreatic carcinomas and pancreatic ductal adenocarcinoma progression. These organoids retain key characteristics of the parental tumor, including genotype and phenotype, providing a convenient model system for in vitro experimentation.

PancreaCult™ Organoid Why Use PancreaCult™ Organoid Growth Medium (Mouse) Growth Medium (Mouse)? Cell Culture Medium for Establishment CONVENIENT. In vitro system for generating organoids within a week. and Maintenance of Mouse Pancreatic STEP-BY-STEP PROTOCOL. No injury models, hand- Exocrine Organoids picking of ducts, or cell sorting required.

PancreaCult™ Organoid Growth Medium (Mouse) enables the SIMPLE, TWO-COMPONENT FORMAT. Serum-free growth of pancreatic exocrine organoids from pancreatic ducts, medium formulation. duct fragments, single cells, or organoid fragments. Pancreatic FLEXIBLE PROTOCOL. Organoids can be grown from duct organoids can be maintained through extended passaging or fragments or single cells and cultured in matrix domes or cryopreservation, providing a readily available source of cells for suspension. future experiments.

A B C

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Figure 20. Mouse Pancreatic Organoids Initiated from a Variety of Starting Materials

PancreaCult™ Organoid Growth Medium (Mouse) enables the initiation of pancreatic exocrine organoids from (A) duct fragments, (B) single cells, and (C) cryopreserved organoid fragments. All organoids were grown in Matrigel® domes. Organoids were imaged on day 4 or day 5 of primary culture (duct fragments and single cells, respectively) or day 3 of the first passage post-thaw (cryopreserved organoids).

16 STEMCELL Technologies Inc. A

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Day 0 Day 4 Day 6

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Day 4 Day 5 Day 6

Figure 21. Pancreatic Organoids Grown in Matrigel® Domes or as a Dilute Matrigel® Suspension

Organoids cultured using PancreaCult™ Organoid Growth Medium (Mouse) from freshly isolated pancreatic tissue fragments and plated in (A) Matrigel® domes or (B) as a dilute Matrigel® suspension. Organoids grown in either culture condition are typically ready for passage within 3 - 6 days.

Figure 22. Pancreatic Exocrine Organoids Display Markers of DAPI KRT19 Pancreatic Progenitor and Ductal Cells

Pancreatic exocrine organoids grown in PancreaCult™ Organoid Growth Medium (Mouse) and stained for nuclei (DAPI, blue), ductal marker KRT19 (green) and pancreatic progenitor marker PDX1 (red). Organoids were imaged during passage 12 on day 5.

Note: The folded appearance of epithelium is a function of cryosectioning and not representative of the shape of proliferating organoids.

PDX1 MERGE

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A Axin2 B Krt19 A Axin2 Krt19 8080 10001000 ancreas ancreas 800800 6060 6p 6p 600600

RQ 4040 RQ C57/Bl C57/Bl 400400 to to 2020 200200

00 00 Relative Relative e e ts e e ts ag ag as ag ag as 1000 µm pass ass robl pass ass robl ib ib e p FFibroblasts e p FFibroblasts arly at arly at EEarly passage L Late passage EEarly passage L Late passage

C Muc1 D Pdx1 B 55 1515 ancreas 44 ancreas 6p 6p 1010 33 RQ RQ C57/Bl 22 C57/Bl 55 to to 11

00 00 Relative Relative e e ts e e ts ag ag as ag ag as pass ass robl pass ass robl ib ib e p FFibroblasts e p FFibroblasts arly at arly at 1000 µm EEarly passage L Late passage EEarly passage L Late passage

Figure 23. Pancreatic Exocrine Organoids Retain Pancreatic Figure 25. Pancreatic Exocrine Organoids Provide a Model for Marker Expression During Passaging Pancreatic Carcinomas

Pancreatic organoids express stem cell markers and those typical of the pancreatic PancreaCult™ Organoid Growth Medium (Mouse) supports the growth of exocrine system, including (A) Axin2, (B) Krt19, (C) Muc1, and (D) Pdx1. Relative organoids from pancreatic carcinomas. Pancreatic ducts were isolated from quantification (RQ) of each marker is reported relative to the 18S and TBP KPC mice (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre) and cultured in PancreaCult™ housekeeping genes and normalized to C57/Bl6 pancreatic tissue. Marker Organoid Growth Medium (Mouse). Organoids were imaged on (A) day 4 of expression was assayed during early passages (passage 1-5) and late passages primary culture and (B) day 4 after the first passage. An activated KRAS genotype (passage 6-10). was retained in organoids during culture. Data used with permission from Dr. David Tuveson.

10 201020 d Yiel 10 151015 Cell

10 10

Total 10 10

10 5105 Cumulative Total Cell Yield Cumulative Total Cumulative

10 0100 p0 p1p1 p2 p3p3 p4 p5p5 p6p6 p7p7 p8p8 p9 p1p100pp1111 PassagePassage NumberNumber

Figure 24. Expansion of Organoids Grown in PancreaCult™ Organoid Growth Medium (Mouse)

Organoids cultured with PancreaCult™ Organoid Growth Medium (Mouse) show efficient growth over multiple passages. Cultures were split with an average split ratio of 1:16 at each passage.

18 STEMCELL Technologies Inc. Kidney Organoids

The kidney plays an important physiological role in the body, such that nephrotoxicity is a critical consideration during drug screening and development. Despite this, the limited availability of relevant in vitro models has presented challenges for studying kidney health and function. Kidney organoids are addressing this challenge by providing efficient in vitro culture systems that mimic the structure and cellular architecture of the nephron and associated mesenchyme and endothelium.8 These kidney organoids are composed of convoluted tubular structures with typical nephron-like segmentation marked by the expression of podocyte (podocalyxin [PODXL]), proximal (lotus tetragonolobus lectin [LTL]), and distal tubule (E-cadherin [ECAD]) markers.

STEMdiff™ Kidney Why Use the STEMdiff™ Kidney Organoid Kit Organoid Kit? Serum-Free Medium Kit for the Culture RELEVANT. Enables generation of human kidney organoids that model the developing nephron and of Kidney Organoids from PSCs associated endothelium and mesenchyme.

STEMdiff™ Kidney Organoid Kit is a complete, serum-free SIMPLE. Minimizes culture manipulations with a cell culture medium system that supports highly efficient and two-stage culture system and easy-to-follow protocol. reproducible generation of human pluripotent stem cell RELIABLE. Provides low experimental variability through (PSC)-derived kidney organoids in a simple two-stage differentiation an optimized formulation and rigorous quality controls. protocol. It has an optimized, quality controlled formulation to HIGH THROUGHPUT. Generates organoids in 96- and increase reproducibility and efficiency across multiple pluripotent 384-well formats. cell lines. This kit has been optimized for the differentiation of PSCs previously cultured in mTeSR™1 (Catalog #85850).

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Figure 26. Differentiation of PSCs to Human Kidney Organoids with the STEMdiff™ Kidney Organoid Kit

Human pluripotent stem cell cultures progres through a simple two-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, posterior intermediate , and metanephric mesenchyme and form tubular kidney organoids by day 18 of differentiation.

WWW.STEMCELL.COM 19 ORGANOIDS

A B Figure 27. Representative Images of Kidney Organoids

The STEMdiff™ Kidney Organoid Kit facilitates the of PSCs to form kidney organoids that model the developing nephron. Pictured are human kidney organoids grown using the STEMdiff™ Kidney Organoid Kit differentiated from (A) iPS (WLS-1C) or (B) ES (H9) cells and imaged on day 12 and day 18 of differentiation, respectively.

200 µm 200 µm

nieenie OCT4_POU5F1

BRACHYYURY

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ooces PODXL

oil Tles SLC3

isl Tles ECAD

Mesenche VIM

Enoheli CD31 Day 0 Day 1.5 Day 4 Day 6 Day 8 Day 10 Day 12 Day 14 Day 17 Day 20

Figure 28. Kidney Organoids Grown Using the STEMdiff™ Kidney Organoid Kit Show the Expected Changes in Gene Expression During Differentiation

As organoids progress through the stages of differentiation to more mature renal cell types, gene expression shifts from markers of pluripotency (day 0) to the mesoderm (day 1.5 - 4) and to the intermediate mesoderm/metanephric mesenchyme by day 4 - 12. Markers of the podocytes, proximal and distal tubules, mesenchyme and endothelium are observable starting by day 14. Marker levels were assessed in four independent experiments by RT-qPCR and normalized to expression levels of undifferentiated cells.

20 STEMCELL Technologies Inc. 60 STEMdiffTM Kidney r STEMdiff™ Kidney e Organoid Kit

a 50 p 60 Organoid Kit TM r e s STEMdiff Kidney r

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40 e HomemadeHome-Made Medium Medium Organoid Kit h

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m 30 r r g r o c e O

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Figure 29. Efficient Differentiation of Human Pluripotent Stem Cells into Self-Organizing Kidney Organoids

The STEMdiff™ Kidney Organoid Kit enables high-efficiency generation of kidney organoids from both ES (A, H1; B, H9) and iPS (C, WLS-1C; D, STiPS-M001) cell lines. (E) Kidney organoids were grown using the STEMdiff™ Kidney Organoid Kit or using homemade medium, and the number of organoids per well of a 96-well plate was quantified on Day 18. All four tested cell lines were capable of differentiating into self-organizing kidney organoids that form convoluted tubular structures with high efficiency (mean ± SD).

A

Podocytes (PODXL)

Proximal Tubules (LTL)

Distal Tubules (ECAD) PODXL LTL PODXL ECAD Loop-of-Henle LTL DAPI 200 µm ECAD DAPI 200 μm Collecting Duct

B C D

CD31 PODXL MEIS1/2/3LTL LTL CD31 LTLWT1 ECAD LTL LTL PODXL VIMDAPI DAPI DAPI DAPI 200 µm 200 µm 200 µm

Figure 30. Kidney Organoids Form Convoluted Tubular Structures with Typical Nephron-Like Segmentation

(A) During differentiation, kidney organoids form convoluted tubular structures that resemble the structure and segmentation of the developing nephron. These organoids express markers of the (B) renal epithelium including podocalyxin (PODXL), lotus tetragonolobus lectin (LTL), and E-cadherin (ECAD), as well as markers of the (C) PODXL endothelium (platelet endothelial molecule, CD31), and (D) mesenchyme (vimentin, VIM; Meis homeobox family,MEIS1 /MEIS1/2/3).2/3 LTL LTL ECAD VIM DAPI ECAD DAPI DAPI WWW.STEMCELL.COM 21

ORGANOIDS

Airway Organoids

Three-dimensional human airway organoids can be generated by differentiation of human airway epithelial cells. Fully differentiated human airway organoids represent a versatile and physiologically relevant organotypic model system for studying the airway epithelium, consisting of basal cells, ciliated cells, club cells, and goblet cells. Airway organoid cultures can be used for in vitro study of the cell biology of the respiratory epithelium, and to understand the underlying mechanisms of chronic respiratory diseases such as (CF), chronic obstructive pulmonary disease (COPD), asthma, idiopathic pulmonary fibrosis (IPF), lung , and infectious diseases. Furthermore, airway organoids can also be used for high-throughput screening of efficacy and toxicity of compounds.

PneumaCult™ Airway Why Use PneumaCult™ Airway Organoid Kit Organoid Kit? Cell Culture Medium Kit for Generation PHYSIOLOGICAL. Three-dimensional in vitro system recapitulates the in vivo human airway. of Human Airway Organoids OPTIMIZED. Complete system for expansion and PneumaCult™ Airway Organoid Kit is a novel, serum-free medium differentiation of human airway epithelial cells, compatible kit that supports the efficient generation of fully differentiated and with PneumaCult™-Ex Plus. functional airway organoids from both healthy and disease samples. RELIABLE. Rigorous raw material screening and extensive Airway organoid cultures provide an alternative method to air-liquid quality control testing ensure reproducibility and minimal interface (ALI)-based cultures for in vitro human airway modeling. lot-to-lot variability. Since this culture system does not require the use of cell culture USER-FRIENDLY. Convenient format and easy-to-use inserts, it is amenable to high-throughput drug screening and can protocol. be used in large-scale screening for CF transmembrane conductance regulator (CFTR) modulators. PneumaCult™ Airway Organoid Kit provides the necessary components to prepare PneumaCult™ Airway Organoid Seeding Medium, that allows for initiation of 3D organoid culture, and PneumaCult™ Airway Organoid Differentiation Medium, to further obtain morphologically representative and fully differentiated human airway organoids.

2D Culture Expansion Airway Organoid Culture

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ole cell Mei

PneumaCult ™-Ex Plus Medium PneumaCult ™ Airway Organoid PneumaCult ™ Airway Organoid Cl cell Seeding Medium Differentiation Medium sl cell

Figure 31. Schematic of Human Airway Organoid Culture Workflow

In the early two-dimensional expansion phase of the human airway organoid culture procedure, human airway epithelial cells (HAECs) are expanded using PneumaCult™-Ex Plus Medium. The HAECs are then embedded into a Matrigel® dome and expanded for 4 - 7 days using PneumaCult™ Airway Organoid Seeding Medium. Following the expansion, the HAECs are differentiated using PneumaCult™ Airway Organoid Differentiation Medium for an additional 21+ days.

22 STEMCELL Technologies Inc. Expansion in PneumaCult™ Airway Differentiation in PneumaCult™ Airway Organoid Seeding Medium Organoid Differentiation Medium

A B C

200 µm 200 µm 25 µm

Figure 32. Fully Differentiated Human Airway Organoids Generated Using PneumaCult™ Airway Organoid Kit

(A) Bright-field image of airway organoids growing in PneumaCult™ Airway Organoid Seeding Medium at day 7 exhibit basal cell spheroid morphology.(B) Bright-field image of airway organoids differentiated in PneumaCult™ Airway Organoid Differentiation Medium at day 21 exhibit hollow lumens. (C) Airway organoid stained for ZO-1 (junction protein marker, red), MUC5AC (goblet cell marker, purple), AC-Tubulin (ciliated cell marker, green), and DAPI (nuclei, blue).

A B DMSO Control Amiloride + Forskolin + Genistein 0 hours 6 hours

C D

Degree of Swelling (%) Degree of Swelling (%) 200 µm 200 µm

Healthy Donor CF Donor

Figure 33. Forskolin-Induced Swelling of Airway Organoids

(A) Forskolin-treated organoids derived from healthy donors increased in size compared to the DMSO control, indicating functional CFTR protein expression. (B) Forskolin- induced swelling is lost in organoids derived from CF donors, but re-established in VX-809-treated airway organoids. Error bars represent ± 95% confidence interval for the mean (n=3). Bright-field images of airway organoids taken during the Forskolin swelling assay at (C) 0 hours and (D) 6 hours show organoid swelling after treatment.

Ciliated Cell Percentage

A B Donor 1 Donor 2

Figure 34. Fully Differentiated Airway Organoids Retain Morphological Characteristics at Different Passages

The ciliated cell percentage in organoids grown from (A) healthy and (B) CF donors using PneumaCult™ Airway Organoid Kit increased from P3 to P5. The total and ciliated cells were counted using a hemocytometer. Error bars represent ± 95% confidence interval for the mean (n=3). Ciliated Cell Percentage (%) Ciliated Cell Percentage (%)

Healthy Cystic Fibrosis

WWW.STEMCELL.COM 23 ORGANOIDS

Cerebral Organoids

Differentiation of cerebral organoids from PSCs generates three-dimensional in vitro cultures that recapitulate the developmental processes and organization of the developing human . These “mini-” provide unique, physiologically relevant in vitro model systems for the study of human neurological development and disease. Cerebral organoids have important applications in studying human brain development and neurological disorders such as autism, schizophrenia, or brain defects caused by Zika virus infection.

STEMdiff™ Cerebral Why Use STEMdiff™ Cerebral Organoid Kit Organoid Kit? Cell Culture Medium Kit for PHYSIOLOGICAL. Three-dimensional in vitro system recapitulates the developmental processes and Establishment and Maturation of organization of the developing brain.

Human Cerebral Organoids INNOVATIVE. Serum-free human pluripotent stem cell‑based model enables the study of development and Based on the formulation published by Lancaster and Knoblich,8 the disease processes. STEMdiff™ Cerebral Organoid Kit is a serum-free culture system that is designed to generate cerebral organoids from human ES OPTIMIZED. Formulation is optimized for increased or iPS cells. Large, optically dense organoids that display multiple efficiency of organoid formation. cortical-like regions when cryosectioned and immunostained RELIABLE. Rigorous raw material screening and extensive are observed in 40 days, providing relevant models for the early quality control testing ensure reproducibility and minimal developing human cortex. lot-to-lot variability.

SIMPLE. Convenient format and easy-to-use protocol.

Embryoid Body Formation Induction Organoid Expansion Organoid Maturation

300 µm 300 µm 300 µm 1 mm

Embryoid Body Resuspend Matrigel® Switch Assay Formation EBs Media Organoids PSC Change medium Embed EBs Change medium maintenance every 2 days every 3 - 4 days

Day 0 5 7 10 40+

mTeSRTM1 EB Formation Medium Induction Medium Expansion Medium Maturation Medium

Figure 35. Schematic for the STEMdiff™ Cerebral Organoid Kit

Human pluripotent stem cells (either embryonic or induced pluripotent stem cells) maintained in mTeSR1™ were dissociated into single-cell suspensions using Gentle Cell Dissociation Reagent (GCDR) and seeded at a density of 9,000 cells/well in a U-Bottom 96-well Ultra-Low Attachment Plate (Corning®) in embryoid body (EB) Formation Medium + 10 µM Rho‑Kinase Inhibitor (ROCKi). EBs were fed every 2 days with EB Formation Medium without ROCKi. After 5 days, EBs were transferred to Induction Medium in a 24-well Ultra‑Low Attachment Plate (Corning®). EBs were cultured for an additional 2 days and were then embedded in liquid Matrigel® (Growth Factor Reduced, Corning®). They were then transferred to a non-tissue culture treated 6-well plate (12 -16 organoids/well). Embedded organoids were maintained in Expansion Medium for 3 days. On Day 10, organoids were switched to Maturation Medium and cultured on an orbital shaker set at 57-95 RPM (Infors HT). Organoids were fed every 3 – 4 days with Maturation Medium. On Day 40, organoids were processed for analysis by RT-qPCR or immunostaining followed by cryosectioning.

24 STEMCELL Technologies Inc. H9 H7 5.0 5.0 4.5 4.5 A B 4.0 4.0 3.5 3.5 3.0 3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0

Log (Fold Change (ΔΔCt)) 0.5

Log (Fold Change (ΔΔCt)) 0.5 0.0 0.0 100 µm 500 µm TBR1 CTIP2 MAP2 TUJ1 TBR2 PAX6 TBR1 CTIP2 MAP2 TUJ1 TBR2 PAX6

WLS-1C STiPS-F019 5.0 5.0 4.5 4.5 C D 4.0 4.0 3.5 3.5 3.0 3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0

Log (Fold Change (ΔΔCt)) 0.5

Log (Fold Change (ΔΔCt)) 0.5 0.0 0.0 200 µm 200 µm TBR1 CTIP2 MAP2 TUJ1 TBR2 PAX6 TBR1 CTIP2 MAP2 TUJ1 TBR2 PAX6

Figure 36. Day 40 Cerebral Organoids Contain Progenitor and E F Populations That Organize into Distinct Layers

RT-qPCR analysis showed upregulation of both neural progenitor and mature neuron transcripts as Log(Fold Change (∆∆Ct)) (Average ± SEM n = 2 per cell line, ≥ 3 organoids per analysis). Data were normalized to 18S/TBP and compared to undifferentiated PSC control. 200 µm 200 µm

Figure 38. Characterization of Cerebral Organoids Generated Using the STEMdiff™ Cerebral Organoid Kit

0 (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 -25 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

PC2: 9% variance -50 apical progenitor marker PAX6 (red) and neuronal marker class III β-tubulin (TUJ- 1) (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. Class

-50 -25 0 25 III β-tubulin+ (green) are adjacent to the ventricular zone. (D) CTIP2, a marker of the developing cortical plate, co-localizes with class III β-tubulin+ PC1: 80% variance neurons in a cortical plate-like region. Organization of the layers recapitulates Condition 50 early corticogenesis observed during human brain development. (E) Proliferating Day 40 Organoid PSC Primary Embryonic Brain progenitor cells labeled by Ki-67 (green) localize along the ventricle, nuclei are counterstained with DAPI (blue). (F) An additional population of Ki-67+ cells is Source found in an outer subventricular zone-like region (arrowheads). Luo 20162 STEMCELL

Figure 37. Organoids Generated with STEMdiff™ Cerebral Organoid Kit Are Similar to Organoids Produced in Literature

Principal component analysis of PSC and cerebral organoid transcriptomes. Cerebral organoids generated using the STEMdiff™ Cerebral Organoid Kit (filled blue circles) clustered together, and clustered with previously published cerebral organoids (open blue circles). The first principal component accounted for the majority of variance observed (PC1; 80%) and distinguished the cerebral organoid samples from the PSCs (green circles). The second principal component accounted for only 9% of the variation, and highlighted the modest difference in gene expression between cultured organoids and primary embryonic fetal brain samples (19 post-conceptional weeks, brown circles).

WWW.STEMCELL.COM 25 ORGANOIDS

Product Information WEBINAR PRODUCT SIZE CATALOG # From Stem Cells to Organoids: Modeling the Gastrointestinal Tract IntestiCult™ Organoid 100 mL 06005 Growth Medium (Mouse) www.stemcell.com/huch_webinar

200 Mouse Intestinal Organoids 70931 Organoids WALLCHART Building 3D Human Brain Organoids IntestiCult™ Organoid 100 mL 06010 www.stemcell.com/BrainOrganoidPoster Growth Med ± ium (Human)

IntestiCult™ Organoid 100 mL 100-0214 TECH RESOURCE CENTER Differentiation Medium Learn More About Organoids and Their Applications STEMdiff™ Intestinal 1 Kit 05140 Organoid Kit www.stemcell.com/discover_organoids

STEMdiff™ Intestinal 1 Kit 05145 Organoid Growth Medium References HepatiCult™ Organoid 100 mL 06030 Growth Medium (Mouse) 1. Sato T et al. (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459(7244): 262–5. Mouse Hepatic Organoids 2 Culture Wells 70932 2. Lancaster MA et al. (2013) Cerebral organoids model human brain development and microcephaly. Nature 501(7467): 373–9. PancreaCult™ Organoid 100 mL 06040 Growth Medium (Mouse) 3. Spence JR et al. (2011) Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 200 Mouse Pancreatic Organoids 70933 470(7332): 105–9. Organoids 4. Huch M et al. (2013) In vitro expansion of single Lgr5+ liver stem STEMdiff™ Kidney Organoid cells induced by Wnt-driven regeneration. Nature 494(7436): 1 Kit 05160 Kit 247–50. 5. Huch M et al. (2015). Long-Term Culture of Genome-Stable PneumaCult™ Airway 1 Kit 05060 Organoid Kit Bipotent Stem Cells from Adult Human Liver. Cell 160(1-2): 299–312. STEMdiff™ Cerebral 1 Kit 08570 6. Broutier L et al. (2016) Culture and establishment of self-renewing Organoid Kit human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat Protoc 11(9): 1724–43. STEMdiff™ Cerebral 1 Kit 08571 Organoid Maturation Kit 7. Huch M et al. (2013) Unlimited in vitro expansion of adult bi- potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J 32(20): 2708–21 mTeSR™1 500 mL 85850 8. Lancaster MA & Knoblich JA. (2014) Generation of cerebral mTeSR™ Plus 1 Kit 05825 organoids from human pluripotent stem cells. Nat Protoc 9(10): 2329–40. 9. Freedman B, et al. (2015). Modelling kidney disease with CRISPR- mutant kidney organoids derived from human pluripotent spheroids. Nature Communications. 6(8715): 1–13.

26 STEMCELL Technologies Inc. Copyright © 2020 by STEMCELL Technologies Inc. All rights reserved including graphics and images. STEMCELL Technologies & Design, STEMCELL Shield Design, Scientists Helping Scientists, Intesticult, Hepaticult, Pancreacult, PneumaCult, and STEMdiff are trademarks of STEMCELL Technologies Canada Inc. mTeSR is a trademark of WARF. Corning and Matrigel are registered trademarks of Corning Incorporated. 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.

PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED. FOR ADDITIONAL INFORMATION ON QUALITY AT STEMCELL, REFER TO WWW.STEMCELL.COM/COMPLIANCE.

WWW.STEMCELL.COM 27 ORGANOIDS Robust Media to Culture Intestinal, Hepatic, Pancreatic, Kidney, Airway, and Cerebral Organoids

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DOC#27132 VERSION 2.0.1 FEB 2021