Highly efficient differentiation of human pluripotent stem cells to multipotent definitive endoderm using the serum-free and animal component-free STEMdiff ™ Definitive Endoderm Culture System Michael J. Riedel 1 , Stephanie Lam 1 , Terry E. Thomas 1 , Allen C. Eaves 1,2 , and Sharon A. Louis 1 1 STEMCELL Technologies Inc., Vancouver, BC, Canada 2 Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada FIGURE 2: Differentiation is accompanied by loss of pluripotency markers and gain of definitive endoderm markers H9 WLS- 4D1 SOX17 FOXA2 Merged Introduction The formation of definitive endoderm (DE) from human embryonic stem (hES) and human induced pluripotent stem (hiPS) cells is a required intermediate step in the development of more specialized cells of endoderm organs. Recent studies show that hES and hiPS cells can be differentiated towards cell types of the liver, pancreas, lung, and intestine. Critical in these processes is the initial development of a highly pure population of DE. Current state-of-the-art protocols for DE differentiation often require fetal bovine serum to allow for high efficiency differentiation across multiple hPSC lines. However, inclusion of animal-derived serum may contribute to inconsistent performance and create a barrier to clinical translation. Furthermore, the use of patient-specific hiPS cells for disease modeling, drug screening, and cell-based therapies will require a protocol for efficient DE differentiation across multiple hiPS cell lines. To meet the need in the field for improved and standardized reagents and protocols for DE differentiation, we developed STEMdiff ™ Definitive Endoderm, a fully defined serum-free and animal component-free medium that promotes highly efficient DE differentiation across multiple hPSC lines. Cells derived using STEMdiff ™ Definitive Endoderm express high levels of DE-specific markers including CXCR4, SOX17, and FOXA2 and show reduced expression of pluripotent markers OCT4 and SSEA-3. Reduced expression of SOX2 following differentiation to DE suggests a lack of biasing towards anterior foregut endoderm lineages. Subsequent directed differentiation towards both anterior and posterior foregut endoderm using published protocols indicates that cells derived using STEMdiff ™ Definitive Endoderm retain multipotency and can therefore be reliably used in studies aimed at generating cells of pulmonary, hepatic, or pancreatic lineages. Product Format and Protocol STEMdiff ™ Definitive Endoderm is a fully defined, animal component-free medium formulation that allows for reproducible differentiation of hES and hiPS cells to definitive endoderm. Differentiation is highly efficient and reproducible across multiple hES and hiPS cell lines, yielding cells that express multiple markers of definitive endoderm including CXCR4, SOX17, and FOXA2. These cells maintain their ability to be further directed towards pulmonary, pancreatic, and hepatic lineages. Efficiency of differentiation to the endoderm lineage using STEMdiff ™ Definitive Endoderm is equally efficient with human pluripotent stem cells maintained in mTeSR ™ 1, TeSR ™ 2, or TeSR ™ -E8 ™ using Matrigel ™ or human recombinant vitronectin as a matrix. STEMdiff ™ Definitive Endoderm can reliably be used as a starting point for studies aimed at the formation of endoderm cell lineages from hES and hiPS cells. Results FIGURE 1: Efficient differentiation to definitive endoderm in multiple hES and hiPS cell lines Representative dot plots showing CXCR4 and SOX17 co-expression in hES cells (H1 and H9) and hiPS cells (WLS-4D1 and A13700) following 5 days of differentiation using STEMdiff ™ Definitive Endoderm. Isotype controls were used to set quadrant gates. Quantitative analysis of CXCR4/SOX17 co-expression in multiple differentiated hES and hiPS cell lines previously cultured in A) mTeSR ™ 1, B) TeSR ™ 2, or C) TeSR ™ -E8 ™ . For high efficiency DE differentiation of TeSR ™ -E8 ™ cultures, protocol optimization was required (C; *P < 0.05 vs. mTeSR ™ 1). D) Definitive Endoderm differentiation of human pluripotent stem cells maintained in TeSR ™ 2 is equally efficient when using human recombinant vitronectin as a matrix as compared to Matrigel ™ , thus providing a completely defined, animal protein-free culture system for definitive endoderm formation. Data are expressed as mean ±SEM; n values are indicated by the number within each bar. A) Quantitative analysis of undifferentiated (Ai, H9; Bi, WLS-4D1) and Day 5 differentiated (Aii, H9; Bii, WLS-4D1) human pluripotent stem cells indicates a down-regulation of the pluripotency and anterior foregut endoderm marker SOX2 with a concomitant up-regulation in the definitive endoderm markers SOX17 and FOXA2. C) Quantitative analysis of marker expression at daily intervals during differentiation of H9 cells to definitive endoderm reveals up-regulation of DE markers (CXCR4/SOX17) by Day 3 and a loss of pluripotency markers (OCT-4/SSEA-3) by Day 4. Transient expression of PDGFR-α on Day 3 suggests a transition through a mesendoderm state. D) Verification of SOX17/FOXA2 expression by immunocytochemistry. Scale bars = 100 μm. FIGURE 3: Definitive endoderm formation is efficient when using human pluripotent stem cells cultured with multiple maintenance media and matrices 0 20 40 60 80 100 H1 H9 A13700 WLS-4D1 WLS-1C WLS-1D % CXCR4 + /SOX17 + Cells 0 20 40 60 80 100 H1 H9 A13700 WLS-4D1 0 20 40 60 80 100 Matrigel ™ Vitronectin mTeSR ™ TeSR ™ -E8 ™ TeSR ™ -E8 ™ (modified protocol) 0 20 40 60 80 100 % CXCR4 + /SOX17 + Cells % CXCR4 + /SOX17 + Cells % CXCR4 + /SOX17 + Cells A B C D C 10 3 10 4 10 5 10 6 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 10 3 10 4 10 5 10 6 2.9 x 10 5 4.9 x 10 6 9.5 x 10 6 98.29% 10 2 10 3 10 4 10 5 99.55% 0.27% 0.12% 0.06% SOX2 PE FOXA2 PE 9.7 x 10 4 4.8 x 10 6 9.4 x 10 6 5.58% 10 2 10 3 10 4 10 5 6.52% 0.86% 2.16% 90.46% FSC FSC SOX17 PerCP SOX17 PerCP A % of Cells Exhibiting Marker Expression Protocol Day 0 20 40 60 80 100 1 2 3 4 OCT4 SSEA-3 CXCR4 SOX17 PDGFR-α CXCR4/SOX17 5 * SOX2 PE FOXA2 PE 12 18 6 17 1 1 4 11 1 6 8 7 4 2 2 Day 0: High quality clump cultures of hPSCs are dissociated to single cells and plated at high density (2.1 x 10 5 cells/cm 2 ) onto Matrigel ™ (BD Biosciences) or vitronectin in mTeSR ™ 1 or TeSR ™ 2 with Y-27632. Day 1: Remove medium and wash cells once with DMEM/F12, then add STEMdiff ™ Definitive Endoderm Medium 1 (1 in 100 dilution of Supplement A and Supplement B in Basal Medium). Days 2-4: Replace medium with STEMdiff ™ Definitive Endoderm Medium 2 (1 in 100 dilution of Supplement B in Basal Medium). Day 5: Cells are ready to be assayed for definitive endoderm formation by flow cytometry or immunocytochemistry and carried forward to more specific lineages. Ai Aii H9 WLS-4D1 10 3 10 4 10 5 10 6 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 10 3 10 4 10 5 10 6 3.1 x 10 5 4.9 x 10 6 9.5 x 10 6 98.95% 10 2 10 3 10 4 10 5 97.02% 1.09% 1.68% 0.21% SOX2 PE FOXA2 PE 9.96% 10 2 10 3 10 4 10 5 9.6 x 10 4 4.7 x 10 6 9.3 x 10 6 1.52% 84.01% 10.73% 3.74% SOX17 PerCP SOX17 PerCP FSC FSC SOX2 PE FOXA2 PE da d a n a C , C B B r, e v u o co n a V Vancouver, BC, Canada , Allen C s 1 , Terry E. Thomas 1 , Stephanie Lam 1 , Michael J. Riedel M an , 1,2 . Eaves er Te 2 T 2 a d a n a C C C, BC B r, er ve uv ou co nc an Va V ., c n I s e gi og lo o no hn ch ec Te T LL EL CE C M EM TE ST S 1 Ter 2 T 2 STEMCELL Technologies Inc., Vancouver, BC, Canada S 1 o b a L x ox Fo F y rry Fox Labo 1 nd Sharon A. Louis V y, cy n en ge Ag A r e nc n a C C BC B , ry or to at ratory, BC Cancer Agency, V tent stem cells to multipotent defin nitive st ys y Sy S S e e re ur u u t lt ul u Cu C C m m m rm er e de d d o o do d nd n En E E e e ve v iv ti t i n n fin fi efi e De D D D ™ ™ Definitive Endoderm Culture Syst ™ ™ Definitive Endoderm Culture Syst m m m em e te t tem tem endoderm usi ing the ing the Highly efficient differentiation Highly efficient differentiation n of hu o o po p mp m m om o o c c l al a ma m m m i n n an a a d d nd n an a a e e e e r fr -f - m m m um u ru r e e s s serum-free and animal compo s serum-free and animal compo s f - t nt n en e ne n nent-f uman pluripot f ™ f f if di d Md M M M E E T T ST S e e e e r fr f ™ free STEMdiff free STEMdiff fin fi efi e de d d t nt n en e te t o o po p ip ti t l u u mu m m m o o o t t s ls ll el e ce c c m m m em e te t st s t t n n en e te e e ve v iv ti t i n n si us u u m m m rm er e de d d o o do d nd n en e e e e he h th t g g n n in n o o io ti at a ia ti t nt n en e re r e e f ff f i d d t nt n en e ie i c fic fi fi f ef e e y y ly hl h gh g ig Hi H H u h h f of o o n n ot o o p p ip ri ur u u l p p n n an a ma m m um u FIGURE 4: Genome-wide expression profile indicates that STEMdiff ™ Definitive Endoderm directs differentiation of human pluripotent stem cells specifically to the endoderm lineage A) Expression pattern of key pluripotency (Pluri), endoderm (Endo), mesoderm (Meso), and ectoderm (Ecto) lineage markers and cell adhesion (Adh) genes of H9 hES cells are shown. Data were acquired using the Illumina HumanHT-12 v4 BeadChip and are expressed as relative fold change in expression compared to undifferentiated H9 cells (n = 3 - 4 for each group). Human pluripotent stem cells were either formed into embryoid bodies and differentiated spontaneously for 5 days in the presence of 10% serum or directed to definitive endoderm using the STEMdiff ™ Definitive Endoderm Kit. B) A heat map representation of the data depicted in A. Highly expressed genes are shown in red, genes with minimal expression are shown in green. STEMdiff ™ DE Embryoid Body (spontaneous) -500 0 500 1000 1500 2000 4000 6000 POU5F1 SOX2 NANOG CDH1 CXCR4 SOX17 FOXA2 FOXA1 EOMES CER1 GSC MIXL1 GATA4 GATA6 KIT KNF1A HNF1B HNF4A AFP CD34 CDH2 ENG MESP1 PCDH12 PDGFRA PECAM1 SALL4 SOX7 T KDR PAX6 SOX1 OTX2 SOX10 TFAP2A ITGA6 ITGA5 ITGB1 SDC1 10 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7 4.46% 93.45% 0.73% 1.36% 4.26% 0.38% 94.31% 1.05% 5.95% 1.38% 85.28% 7.40% 5.81% 2.57% 89.39% 2.24% SOX17 APC SOX17 APC SOX17 APC SOX17 APC CXCR4 PE CXCR4 PE CXCR4 PE CXCR4 PE H1 H9 4D1 A13700 Following differentiation to definitive endoderm using the STEMdiff ™ Definitive Endoderm Kit, cells were further differentiated using published protocols to pancreatic progenitors (Rezania et al., Diabetes 2012), pulmonary progenitors (Wong et al., Nature Biotechnology, 2012), or hepatocyte progenitors (Hay et al., Stem Cells, 2008). A) Representative flow cytometry analysis of PDX-1 and NKX6.1 co-expression (circled) following differentiation of H9-derived definitive endoderm to pancreatic progenitors. B) Representative flow cytometry analysis of SOX2 expression following differentiation of WLS-4D1-derived definitive endoderm to pulmonary progenitors. Isotype control is shown in grey and was used to set the gate. C) Representative image depicting human serum albumin (HSA) immunoreactivity of hepatocyte progenitors following differentiation of H9-derived definitive endoderm. Scale bars = 100 μm. FIGURE 5: STEMdiff ™ Definitive Endoderm yields DE that is capable of downstream differentiation to multiple lineages B 0 44.0 25 50 75 100 10 0 10 1 10 2 10 3 SOX2 PE Count 66.30 10 2 NKX6.1 APC A 10 3 10 4 10 5 10 6 10 2 10 3 10 4 10 5 10 6 PDX-1 PE C B D Bi Bii A B Undifferentiated STEMdiff ™ DE Embryoid Body POU5F1 SOX2 NANOG CDH1 CXCR4 SOX17 FOXA2 FOXA1 EOMES CER1 GSC MIXL1 GATA4 GATA6 KIT HNF1A HNF1B HNF4A AFP CD34 CDH2 ENG MESP1 PCDH12 PDGFRA PECAM1 SALL4 SOX7 T KDR PAX6 SOX1 OTX2 SOX10 TFAP2A ITGA6 ITGA5 ITGB1 SDC1 Undifferentiated Day 5 Differentiated HSA DAPI Summary Pluri Endo Meso Ecto Adh Pluri Endo Meso Ecto Adh Fold Change in Expression vs. Undifferentiated Cells
Transcript
Highly efficient differentiation of human pluripotent stem cells to multipotent definitive endoderm using the serum-free and animal component-free STEMdiff™ Definitive Endoderm Culture SystemMichael J. Riedel1, Stephanie Lam1, Terry E. Thomas1, Allen C. Eaves1,2, and Sharon A. Louis1
1 STEMCELL Technologies Inc., Vancouver, BC, Canada 2 Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada
FIGURE 2: Differentiation is accompanied by loss of pluripotency markers and gain ofdefinitive endoderm markers
H9
WLS-4D1
SOX17 FOXA2 Merged
IntroductionThe formation of definitive endoderm (DE) from human embryonic stem (hES) and human induced pluripotent stem (hiPS) cells is a required intermediate step in the development of more specialized cells of endoderm organs. Recent studies show that hES and hiPS cells can be differentiated towards cell types of the liver, pancreas, lung, and intestine. Critical in these processes is the initial development of a highly pure population of DE. Current state-of-the-art protocols for DE differentiation often require fetal bovine serum to allow for high efficiency differentiation across multiple hPSC lines. However, inclusion of animal-derived serum may contribute to inconsistent performance and create a barrier to clinical translation. Furthermore, the use of patient-specific hiPS cells for disease modeling, drug screening, and cell-based therapies will require a protocol for efficient DE differentiation across multiple hiPS cell lines. To meet the need in the field for improved and standardized reagents and protocols for DE differentiation, we developed STEMdiff™ Definitive Endoderm, a fully defined serum-free and animal component-free medium that promotes highly efficient DE differentiation across multiple hPSC lines. Cells derived using STEMdiff™ Definitive Endoderm express high levels of DE-specific markers including CXCR4, SOX17, and FOXA2 and show reduced expression of pluripotent markers OCT4 and SSEA-3. Reduced expression of SOX2 following differentiation to DE suggests a lack of biasing towards anterior foregut endoderm lineages. Subsequent directed differentiation towards both anterior and posterior foregut endoderm using published protocols indicates that cells derived using STEMdiff™ Definitive Endoderm retain multipotency and can therefore be reliably used in studies aimed at generating cells of pulmonary, hepatic, or pancreatic lineages.
Product Format and Protocol
STEMdiff™ Definitive Endoderm is a fully defined, animal component-free medium formulation that allows for reproducible differentiation of hES and hiPS cells to definitive endoderm.
Differentiation is highly efficient and reproducible across multiple hES and hiPS cell lines, yielding cells that express multiple markers of definitive endoderm including CXCR4, SOX17, and FOXA2. These cells maintain their ability to be further directed towards pulmonary, pancreatic, and hepatic lineages.
Efficiency of differentiation to the endoderm lineage using STEMdiff™ Definitive Endoderm is equally efficient with human pluripotent stem cells maintained in mTeSR™1, TeSR™2, or TeSR™-E8™ using Matrigel™ or human recombinant vitronectin as a matrix.
STEMdiff™ Definitive Endoderm can reliably be used as a starting point for studies aimed at the formation of endoderm cell lineages from hES and hiPS cells.
ResultsFIGURE 1: Efficient differentiation to definitive endoderm in multiple hES and hiPS cell lines
Representative dot plots showing CXCR4 and SOX17 co-expression in hES cells (H1 and H9) and hiPS cells (WLS-4D1 and A13700) following 5 days of differentiation using STEMdiff™ Definitive Endoderm. Isotype controls were used to set quadrant gates.
Quantitative analysis of CXCR4/SOX17 co-expression in multiple differentiated hES and hiPS cell lines previously cultured in A) mTeSR™1, B) TeSR™2, or C) TeSR™-E8™. For high efficiency DE differentiation of TeSR™-E8™ cultures, protocol optimization was required (C; *P < 0.05 vs. mTeSR™1). D) Definitive Endoderm differentiation of human pluripotent stem cells maintained in TeSR™2 is equally efficient when using human recombinant vitronectin as a matrix as compared to Matrigel™, thus providing a completely defined, animal protein-free culture system for definitive endoderm formation. Data are expressed as mean ±SEM; n values are indicated by the number within each bar.
A) Quantitative analysis of undifferentiated (Ai, H9; Bi, WLS-4D1) and Day 5 differentiated (Aii, H9; Bii, WLS-4D1) human pluripotent stem cells indicates a down-regulation of the pluripotency and anterior foregut endoderm marker SOX2 with a concomitant up-regulation in the definitive endoderm markers SOX17 and FOXA2. C) Quantitative analysis of marker expression at daily intervals during differentiation of H9 cells to definitive endoderm reveals up-regulation of DE markers (CXCR4/SOX17) by Day 3 and a loss of pluripotency markers (OCT-4/SSEA-3) by Day 4. Transient expression of PDGFR-α on Day 3 suggests a transition through a mesendoderm state. D) Verification of SOX17/FOXA2 expression by immunocytochemistry. Scale bars = 100 µm.
FIGURE 3: Definitive endoderm formation is efficient when using human pluripotent stemcells cultured with multiple maintenance media and matrices
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H1 H9
A1370
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99.55% 0.27%
0.12% 0.06%
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102 103 104 105
97.02% 1.09%
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PE
FOXA
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WLS-4D1
9.7 x 104 4.8 x 106 9.4 x 106
5.58%
102 103 104 105
6.52% 0.86%
2.16% 90.46%
9.96%
102 103 104 1059.6 x 104 4.7 x 106 9.3 x 106
1.52% 84.01%
10.73%3.74%
FSC
FSC
SOX17 PerCP
SOX17 PerCP
SOX17 PerCP
SOX17 PerCP
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FOXA
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12 18 6 17 1 1 4 11 1 6 8 7 4 2 2
Day 0: High quality clump cultures of hPSCs are dissociated to single cells and plated at high density (2.1 x 105 cells/cm2) onto Matrigel™ (BD Biosciences) or vitronectin in mTeSR™1 or TeSR™2 with Y-27632.
Day 1: Remove medium and wash cells once with DMEM/F12, then add STEMdiff™ Definitive Endoderm Medium 1 (1 in 100 dilution of Supplement A and Supplement B in Basal Medium).
Days 2-4: Replace medium with STEMdiff™ Definitive Endoderm Medium 2 (1 in 100 dilution of Supplement B in Basal Medium).
Day 5: Cells are ready to be assayed for definitive endoderm formation by flow cytometry or immunocytochemistry and carried forward to more specific lineages.
Ai
Aii
H9 WLS-4D1
103
104
105
106
102
103
104
105
102
103
104
105
103
104
105
106
3.1 x 105 4.9 x 106 9.5 x 106
98.95%
102 103 104 105
97.02% 1.09%
1.68% 0.21%
SOX2
PE
FOXA
2 PE
9.96%
102 103 104 1059.6 x 104 4.7 x 106 9.3 x 106
1.52% 84.01%
10.73%3.74%
SOX17 PerCP
SOX17 PerCP
FSC
FSC
SOX2
PE
FOXA
2 PE
Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada
, Allen C. Eaves, Terry E. Thomas1, Terry E. Thomas1, Terry E. Thomas, Stephanie Lam1, Stephanie LamMichael J. RiedelMichael J. Riedel , Allen C. Eaves, Allen C. Eaves, Allen C. Eaves, Allen C. Eaves, Allen C. Eaves, Allen C. Eaves, Allen C. Eaves, Terry E. Thomas, Terry E. Thomas, Terry E. Thomas, Terry E. Thomas, Terry E. Thomas, Terry E. Thomas, Terry E. Thomas, Terry E. Thomas, Terry E. Thomas, Terry E. Thomas, Terry E. Thomas, Terry E. Thomas, Terry E. Thomas, Terry E. Thomas, Terry E. Thomas, Terry E. Thomas, Stephanie Lam, Stephanie Lam, Stephanie Lam, Stephanie Lam, Stephanie Lam, Stephanie Lam, Stephanie Lam, Stephanie Lam, Stephanie Lam, Stephanie Lam, Stephanie Lam, Stephanie Lam, Stephanie Lam, Stephanie Lam , and Sharon A. Louis, and Sharon A. Louis1,2, Allen C. Eaves , and Sharon A. Louis, and Sharon A. Louis, and Sharon A. Louis, Allen C. Eaves, Allen C. Eaves, Allen C. Eaves, Allen C. Eaves, Allen C. Eaves, Allen C. Eaves
Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada2 Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada2 STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada 1 Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada2 Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada2 STEMCELL Technologies Inc., Vancouver, BC, Canada STEMCELL Technologies Inc., Vancouver, BC, Canada 1 Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada
1, and Sharon A. Louis, and Sharon A. Louis, and Sharon A. Louis, and Sharon A. Louis, and Sharon A. Louis, and Sharon A. Louis, and Sharon A. Louis, and Sharon A. Louis, and Sharon A. Louis, and Sharon A. Louis, and Sharon A. Louis, and Sharon A. Louis, and Sharon A. Louis, and Sharon A. Louis, and Sharon A. Louis, and Sharon A. Louis
Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, CanadaTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada
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Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System Definitive Endoderm Culture System™ Definitive Endoderm Culture System™ Definitive Endoderm Culture System™ Definitive Endoderm Culture System™ Definitive Endoderm Culture System Definitive Endoderm Culture SystemHighly efficient differentiation of human pluripotent stem cells to multipotent definitive endoderm using the
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FIGURE 4: Genome-wide expression profile indicates that STEMdiff™ Definitive Endodermdirects differentiation of human pluripotent stem cells specifically to the endoderm lineage
A) Expression pattern of key pluripotency (Pluri), endoderm (Endo), mesoderm (Meso), and ectoderm (Ecto) lineage markers and cell adhesion (Adh) genes of H9 hES cells are shown. Data were acquired using the Illumina HumanHT-12 v4 BeadChip and are expressed as relative fold change in expression compared to undifferentiated H9 cells (n = 3 - 4 for each group). Human pluripotent stem cells were either formed into embryoid bodies and differentiated spontaneously for 5 days in the presence of 10% serum or directed to definitive endoderm using the STEMdiff™ Definitive Endoderm Kit. B) A heat map representation of the data depicted in A. Highly expressed genes are shown in red, genes with minimal expression are shown in green.
Following differentiation to definitive endoderm using the STEMdiff™ Definitive Endoderm Kit, cells were further differentiated using published protocols to pancreatic progenitors (Rezania et al., Diabetes 2012), pulmonary progenitors (Wong et al., Nature Biotechnology, 2012), or hepatocyte progenitors (Hay et al., Stem Cells, 2008). A) Representative flow cytometry analysis of PDX-1 and NKX6.1 co-expression (circled) following differentiation of H9-derived definitive endoderm to pancreatic progenitors. B) Representative flow cytometry analysis of SOX2 expression following differentiation of WLS-4D1-derived definitive endoderm to pulmonary progenitors. Isotype control is shown in grey and was used to set the gate. C) Representative image depicting human serum albumin (HSA) immunoreactivity of hepatocyte progenitors following differentiation of H9-derived definitive endoderm. Scale bars = 100 µm.
FIGURE 5: STEMdiff™ Definitive Endoderm yields DE that is capable of downstreamdifferentiation to multiple lineages
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