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  • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

• the present invention relates to a method to differentiate pluripotent stem cells to a primitive streak cell population, in a stepwise manner for further maturation todefinitive endoderm.
• DE cells from pluripotent embryonic stem (ES) cells has been reported for both mouse and human in, e.g., WO2005/1 16073, WO2005/063971 , and US 2006/0148081 .
• the generation of pancreatic endoderm (PE) cells from DE cells is necessary for the generation of insulin-producing beta cells for the treatment of diabetes.
• CHIR is a glycogen synthase kinase 3 beta (Gsk3b) inhibitor anda known compo- nent of a defined tissue culture medium to maintain mouse embryonic stem cells in the pluri- potent state (Ying et al Nature 453, 519-523) .
• Gsk3b has multiple targets but is mainly known to regulate degradation and/or nuclear transfer of beta-catenin.
• the role of stabilized beta-catenin in PS formation from human embryonic stem cells (hESC) has been described using other glycogen synthase kinase 3 beta (Gsk3b) inhibitors such as BIO and
• the present invention relates to the utilization of CHIR withou- tAA, at adefined concentration range to treat pluripotent stem cells prior to AA-mediated definitive endoderm induction compared to the conventional induction protocol for obtaining DE (D'Amour protocol, as described in Kroon et al. 2008).
• This sequential exposure, first to CHIR and then to AA, is essentialand reflects successive PS formation (induced by CHIR) followed by more efficient and rapid DE formation by AA.
• the present invention relates to the discrete and successive control of induction of first PS then followed by DE leading to an overall more efficient, with earlier peak of SOX17 expression and robust DE protocol.
• Ctrl No Chir treatment D1. Cells are left in RPMI during priming period, followed by AA D2 and AA + serum replacement (B27) from D3 onwards.
• Fig. 1 shows the CHIR DEprotocol nomenclature and overview. The protocol has been confirmed in 7 different pluripotent stem cell lines: SA121 , SA181 , SA461 , SA167 (human ESCs) and chlPS2, chlPS3, chlPS4 (human iPSCs).
• Fig. 2 shows transcriptionalexpression of PS markers Brachyury (T), MIXL1 , EOMES and Goosecoid (GSC) after 24h of CHIR treatment (D1 ) in hES SA121 (Fig. 2A) and chlPS4 (Fig. 2B), compared to non-treated cells. T protein levels were also confirmed with ICC (Fig. 2C). T fold inductions after 24h CHIR treatment have spanned between 500-100000 compared to undifferentiated cells (n > 60). Bars show Log10 values relative to undifferentiated cells DO.
• Fig. 3 shows geneexpression of DE markers (SOX17, CXCR4, FOXA2.CER) one day after replacing CHIR with ActivinA (AA) compared to with cells that were not pretreated with CHIR. Expression is shown both in hESC SA121 (Fig. 3A) and iPSC chlPS4 (Fig. 3B). Bars show Log10 values relative to undifferentiated cells DO.
• Fig. 4 showshESC SA121 (4A-B) and iPSC chlPS4 (4C) cells treated with either CHIR (3 ⁇ ), BIO (0,5uM), Wnt3a (200ng/ml) or AA alone (1 OOng/ml) for 24h (D1 ) prior to 2d AA treatment (D3). Additionally, D'Amour (protocol as described in Kroon et al 2008) was analyzed.. Expression of Brachyury and SOX17 was analyzed. hES cells treated with AA or BIO for the first 24h did not survive until day 3.
• Fig. 5 shows gene expression levels of DE markers SOX17, FOXA2 and CXCR4 (Fig. 5A-C) and OCT4 (Fig. 5E) after CHIR induction compared to D'Amour and quantified ICC protein levels of SOX17/OCT4 in chlPS4 cells(Fig. 5D, F). Corresponding % of cells positively stained for OCT4 and SOX17 (D1 -3) are also indicated in Table 2.chlPS/SA121 DE cells, using either of the two protocols, were further differentiated towards pancreatic endoderm (PE) using a previously published PE differentiation protocol (Ameri et al 2010). Cells were analyzed after seven days of PE induction using ICC for PDX1 protein levels.
• PE pancreatic endoderm
• Fig. 6 shows geneexpression of SOX17 D3 in hES SA121 (Fig. 6A) or chlPS4 (Fig. 6C) treated with either only RPMI, AA and Wnt3a or CHIR D1 followed by treatment with either AA or a combination of AA and Wnt3a D2 and AA D3 (Fig 6A).
• Fig. 7 shows thatCHIR was titrated at concentrations between 1 -7uM and analysed after 24h (D1 ) and after 2d of following AA treatment (D3). CHIR 0,5-1 uM and CHIR 7uM did not survive D3. Brachyury expression was analysed after 24h treatment in SA121 (Fig. 7A) and chlPS cells (Fig. 7C). SOX17 expression was analysed D3 in SA121 (Fig. 7B) and chlPS cells (Fig. 7D)).
• Fig. 8 shows gene expression of SOX17 D3 after CHIR pre-treatment or after the D'Amour protocol in the mTeSR system (Fig. 8).
• Fig. 9 shows gene expression levels of markers Brachyury and SOX17 at DO, D2 and D3 when cultured on MEFfeeders.
• the present invention also relates to a method for differentiation of stem cells into definitive endoderm comprising the steps of:
• first priming step of incubating stem cells in a medium comprising at least 2 ⁇ CHIR, wherein activin A is not present during said priming step;
• the present invention relates to a faster and more pronounced peak of SOX17 expression and a more robust method of differentiating human pluripotent stem cells to obtain definitive endoderm.
• the present invention relates to a method for differentiation of stem cells into definitive endoderm comprising the steps of incubating pluripotent stem cells in a medium comprising at least 2 ⁇ CHIR and subsequently incubating these cells in a medium comprising Activin A (AA).
• AA Activin A
• the present inventors have found that using CHIR alone for 24 h prior to further addition of AA, induced a significant and dose dependent upregulation of PS markers such as Brachyury, MixH , Eomes and Goosecoid (GSC) during these 24h.
• PS markers such as Brachyury, MixH , Eomes and Goosecoid (GSC) during these 24h.
• the present inventors have surprisingly found that in the subsequent incubation with AA to induce DE, DE induction as measured by SOX17 expression, peaked at an earlier time pointand with higher fold change when compared to cultures without CHIR pre-incubation or when compared to the conventional D'Amour protocol (Novocell, Nature Biotec 2006, 2008).
• the present inventors surprisingly found that the method of the present invention was more efficient and showed a faster induction of Sox17 mRNA expression than other known proto- cols for DE Induction.
• the CHIR-mediated effect has been reproduced both in other culture systems (mTeSR media and conventional ES media on mouse embryonic feeder (MEF) cells) and across different cell lines (SA121 , SA181 , SA461 , SA167, chlPS2, chlPS3, chlPS4), showing that the effect is not dependent on the cell line or the DEF media culture system.
• Human em- bryonic stem cells may be derived from single blastomeres without the destruction of the embryo (Klimanskaya et al. 2006; Chung et al. 2008; Geens et al. 2009).
• Cell lines chlPS2, chlPS3, chlPS4 are iPS cell lines.
• CHIR CHIR promotes high cell densities after AA is added. Dose dependency of CHIR in PS induction will allow future optimization of possible pre-patterning influences when initiating AA treatment. The subsequent order of added factors decreases the amount of signaling pathways that areactivated in parallel. Minimizing the amount of factors that may influence the efficiency should promote robustness and reproducibility.
• the present inventors have surprisingly found that the CHIR-priming step is unique for DE induction and was superior when compared to BIO or Wnt3a when it comes to efficiency peak of SOX17 expression and robustness.
• hESCs Human embryonic stem cells
• iPSCs human induced pluripotent stem cells
• a method for differentiation of stem cells into definitive endoderm comprising the steps of: c. first priming step of incubating stem cells in a medium comprising at least 2 ⁇ CHIR, wherein activin A is not present during said priming step; and
• stem cells are embryonic stem cellsor induced pluripotent stem cells.
• stem cells are embryonic stem cells.
• stem cells are human embryonic stem cells.
• the concentration of CHIR is at least about 2,5 ⁇ , or at least 3 ⁇ , such as in arange of about 2-20uM, such as in a range of about 3,1 -15 ⁇ , or such as in a range of about 3,5-7 ⁇ or such as in arange ofabout 3,5-6 ⁇ , or such as in arange ofabout 3,5-5 ⁇ .
• a method according to embodiment 7, wherein the concentration of CHIR is about 5 ⁇ A method according to any one of the preceding embodiments, wherein said incubation with CHIR is at least 12 hours.
• a method according to embodiment 37, wherein said incubation with CHIR is 48 hours.
• endoderm cells are hepatic endoderm cells, pancreatic endoderm cells, intestinal endoderm cells and/or lung endoderm cells.
• Endodermal cells obtainable by the methods of embodiments 1 -64.
• Endodermal cells according to embodiment 66, wherein said cells are hepatic endoderm cells.
• Endodermal cells according to embodiment 66, wherein said cells are pancreatic endo- derm cells.
• Use of CHIR in a concentration of CHIR in the culture medium is at least 2 ⁇ CHIR, such as in the range of 3,1 -15 ⁇ , or such as 3,1 -7 ⁇ , or such as 3,5-15 uM, or such as 72.
• Use of CHIR in a concentration of at least 3,1 uM in the culture medium to induce primitive streak cells from embryonic stem cells.
• CHIR CHIR in a concentration in the range of 3,5-7uM to induce definitive endoderm cells from embryonic stem cells.
• 75 Use of CHIR in a concentration of 3,5 uM to induce definitive endoderm cells from embryonic stem cells.
• CHIR Use of CHIR in a concentration of 4,5 uM to induce definitive endoderm cells from embryonic stem cells.
• CHIR Use of CHIR in a concentration of 5 uM to induce definitive endoderm cells from embryonic stem cells.
• a method for differentiation of stem cells into definitive endoderm comprising the steps of: a. first priming step of incubating stem cells in a medium comprising at least 2 ⁇ CHIR, wherein activin A is not present during said priming step; and
• a method for differentiation of stem cells into definitive endoderm comprising the steps of: a. first priming step of at least 12 hours, of incubating stem cells in a medium comprising at least 2 ⁇ CHIR, wherein activin A is not present during said priming step; and
• a method for differentiation of stem cells into definitive endoderm comprising the steps of: b. first priming step of at least 24 hours, of incubating stem cells in a medium comprising at least 2 ⁇ CHIR, wherein activin A is not present during said priming step; and
• a method for differentiation of stem cells into definitive endoderm comprising the steps of: a. first priming step of incubating stem cells in a medium comprising at least 3,1 ⁇ CHIR, wherein activin A is not present during said priming step; and b. second subsequent step of incubating stem cells in a medium comprising at least 25ng/ml activin A.
• a method for differentiation of stem cells into definitive endoderm comprising the steps of: a. first priming step of incubating stem cells in a medium comprising at least 3,5 ⁇ CHIR, wherein activin A is not present during said priming step; and
• stem cells are embryonic stem cells or induced pluripotent stem cells.
• stem cells are human embryonic stem cells.
• stem cells are induced pluripotent stem cells.
• 3,1 ⁇ such as in the range of about 3,1 -15 ⁇ , or such as about 3,5-7 ⁇ or such as about 3,5-6 ⁇ , or such as about 3,5-5 ⁇ .
• endoderm cells are hepatic endoderm cells, pancreatic endoderm cells, intestinal endoderm cells and/or lung endoderm cells.
• Endodermal cells obtainable by the methods of embodiments 81 -122.
• Endodermal cells according to embodiment 124, wherein said cells are hepatic endoderm cells.
• Endodermal cells according to embodiment 124, wherein said cells are pancreatic endoderm cells.
• CHIR Use of CHIR in a specific concentration to induce primitive streak cells from stem cells.
• CHIR CHIR in a concentration in the range of 3,5-7 uM to induce definitive endoderm cells from embryonic stem cells.
• the pancreatic endocrine cells obtainable by the method according to the invention are insulin producing cells, optionally together with cells differentiated to- wards glucagon, somatostatin, pancreatic polypeptide, and/or ghrelin producing cells.
• insulin producing cells refers to cells that produce and store or secrete detectable amounts of insulin.
• Insulin producing cells can be individual cells or collections of cells.
• the cell population comprising pancreatic cells is obtained from a somatic cell population.
• the somatic cell population has been in- prised to de-differentiate in to an embryonic-like stem (ES, e.g., a pluripotent) cell.
• ES embryonic-like stem
• IPS induced pluripotent stem cells
• the cell population comprising pancreatic cells is obtained from embryonic stem (ES, e.g., pluripotent) cells.
• ES embryonic stem
• the cell population comprising pancreatic cells is pluripotent cells such as ES like-cells.
• the cell population comprising pancreatic cells is embryonic differentiated stem (ES or pluripotent) cells. Differentiation takes place in embryoid bodies and/or in monolayer cell cultures or a combination thereof.
• ES embryonic differentiated stem
• the cell population is a population of stem cells.
• the cell population is a population of stem cells differentiated to the pancreatic endo- crine lineage.
• Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts.
• Stem cells are classified by their developmental potential as: (1 ) totipotent, meaning able to give rise to all embryonic and extraembryonic cell types; (2) pluripotent, meaning able to give rise to all embryonic cell types; (3) multi-potent, meaning able to give rise to a subset of cell lineages, but all within a particular tissue, organ, or physiological system (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self-renewal), blood cell restricted oligopotent progenitors and all cell types and elements (e.g., platelets) that are normal components of the blood); (4) oligopotent, meaning able to give rise to a more re- stricted subset of cell lineages than multi-potent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage (e.g., spermatogenic stem cells).
• HSC hematopoietic stem cells
• a protocol for obtaining pancreatic cells from stem cells is exemplified by, but not limited to, the protocols described in D'Amour, K. A. et al. (2006), Nat Biotechnol 24, 1392- 401 ; Jiang, J. et al. (2007), Stem Cells 25, 1940-53; and Kroon, E. et al. (2008), Nat Biotech- nol 26, 443 - 452.
• a protocol for obtaining pancreatic cells from somatic cells or somatic cells induced to de-differentiate into pluripotent cells such as ES like-cells is exemplified by, but not limited to, the protocols described in Aoi, T. et al. (2008), Science 321 (no. 5889), 699 - 702;
• differentiate refers to a process where cells progress from an undifferentiated state to a differentiated state, from an immature state to a less immature state or from an immature state to a mature state.
• characteristics markers like Pdx1 , Nkx6.1 , and Ptfl a.
• Mature or differentiated pancreatic cells do not proliferate and do secrete high levels of pancreatic endocrine hormones or digestive enzymes. E.g., fully differentiated beta cells secrete insulin at high levels in response to glucose.
• differentiation factor refers to a compound added to pancreatic cells to enhance their differentiation to mature endocrine cells also containing insulin producing beta cells.
• exemplary differentiation factors include hepatocyte growth factor, keratinocyte growth factor, exendin-4, basic fibroblast growth factor, insulin-like growth fac- tor-1 , nerve growth factor, epidermal growth factor platelet-derived growth factor, and gluca- gon-like peptide 1.
• differentiation of the cells comprises culturing the cells in a medium comprising one or more differentiation factors.
• human pluripotent stem cells refers to cells that may be derived from any source and that are capable, under appropriate conditions, of producing human progeny of different cell types that are derivatives of all of the 3 germinal layers (endoderm, mesoderm, and ectoderm). hPS cells may have the ability to form a teratoma in 8-12 week old SCID mice and/or the ability to form identifiable cells of all three germ layers in tissue culture. Included in the definition of human pluripotent stem cells are embryonic cells of various types including human blastocyst derived stem (hBS) cells in 30 literature often denoted as human embryonic stem (hES) cells, (see, e.g., Thomson et al.
• hBS human blastocyst derived stem
• hES human embryonic stem
• hPS cells suitable for use may be obtained from
• suitable hPS cells may be obtained from established cell lines and/or human induced pluripotent stem (hiPS) cells.
• hiPS cells refers to human induced pluripotent stem cells.
• blastocyst-derived stem cell is denoted BS cell, and the human form is termed "hBS cells".
• BS cell the human form
• hBS cells the cells are often referred to as embryonic stem cells, and more specifically human embryonic stem cells (hESC).
• the pluripotent stem cells used in the present invention can thus be embryonic stem cells prepared from blastocysts, as described in e.g. WO 03/055992 and WO 2007/042225, or be
• any human pluripotent stem cell can be used in the present invention, including differentiated adult cells which are reprogrammed to pluripotent cells by e.g. the treating adult cells with certain transcription factors, such as OCT4, SOX2, NANOG, and LIN28 as disclosed in Yu, et al., 2007, Takahashi et al. 2007 and Yu et al 2009.
• feeder cells are intended to mean supporting cell types used alone or in combination.
• the cell type may further be of human or other species origin.
• the tissue from which the feeder cells may be derived include embryonic, fetal, neonatal, juvenile or adult tissue, and it further includes tissue derived from skin, including foreskin, umbilical chord, muscle, lung, epithelium, placenta, fallopian tube, glandula, stroma or breast.
• the feeder cells may be derived from cell types pertaining to the group consisting of human fibroblasts, fibrocytes, myocytes, keratinocytes, endothelial cells and epithelial cells.
• Examples of specific cell types that may be used for deriving feeder cells include
• embryonic fibroblasts extraembryonic endodermal cells, extraembryonic mesoderm cells, fetal fibroblasts and/or fibrocytes, fetal muscle cells, fetal skin cells, fetal lung cells, fetal endothelial cells, fetal epithelial cells, umbilical chord mesenchymal cells, placental fibroblasts and/or fibrocytes, placental endothelial cells,
• MEF cells is intended to mean mouse embryonic fibroblasts.
• CHIR glycogen synthase kinase 3 beta
• a method for differentiation of stem cells into definitive endoderm comprising the steps of:
• stem cells are embryonic stem cells.
• a method according to any of embodiments 136-141 wherein said incubation with activin A is in the range of 12 hours to 5 days, such as at least 12 hours, such as at least 24 hours or at least 48 hours, or such as 3-4 days.
• concentration of CHIR in the culture medium is at least 2 ⁇ CHIR, such as in the range of 2-7 ⁇ , such as 3 ⁇ or such as 4 ⁇ .
• CHIR Use of CHIR in a concentration in the range of 2-7 uM to induce definitive endoderm cells from embryonic stem cells.
• CHIR Use of CHIR in a concentration in the range of 3-7 uM to induce definitive endoderm cells from embryonic stem cells.
• bFGF basic fibroblast growth factor (FGF2)
• Gsk3b glycogen synthase kinase 3 beta
• hBSC human blastocyst-derived stem cells
• hESC human embryonic stem cells
• hIPSC human induced pluripotent cells
• hPSC human pluripotent stem cells
• RNA ribonucleic acid
• SOX17 SRY (sex determining region Y)-box 17 T: Brachyury
• Example 1 In vitro culture of human ES/iPS cells
• the DE protocol was confirmed in two different feeder free (DEF, mTeSR) culture systems and one feeder dependent (MEF-ES) culture system.
• hES cells SA121 and chlPS4 were grown on human fi- bronectin (Sigma) in DEF culture media (Cellartis) with 30 ng/ml bFGF (Invitrogen) and 10 ng/ml Noggin (Peprotech) in 6-96 well plates.
• Cells were single cell passaged with Rock inhibitor Y-27632 (Calbiochem) and seeded at a density of 40000 cells/cm2 for experiments. Experiments were initiated 4d after passage.
• hES cells were cluster passaged from feeders (MEFs) to Matrigel (BD Biosciences) in mTeSRI media (Cell Signaling Techologies) and passaged further with Dispase (BD Biosciences) according to the culture system protocol. DE was initiated once clusters started to touch eachother MEF-ES
• hES cells (SA121 ) were passaged on gelatine-coated plates pre-seeded with MEFs in hES media (KO-DMEM, PEST, Glutamax, NEAA, 2-mercaptoethanol, KO serum replacement, 10ng/ml bFGF). DE was initiated at 80% confluency.
• CHIR DE cell culture protocol a protocol that has been confirmed in 7 different cell lines: SA121 , SA181 , SA461 , SA167 (hESC) and chlPS2, chlPS3, chlPS4 (hiPSC). Table 1. CHIR DE cell culture protocol
• Day 1 Cells were washed carefully in prewarmed (37°C) RPMI before starting the differentiation. Day1 medium was premixed (CHIR 3 ⁇ in prewarmed RPMI + PEST) and gently added to cell culture. Volumes were adjusted according to plate formats.
• Day 2 Cells were washed carefully in prewarmed (37°C) RPMI before media change.
• Day2 medium was premixed (100ng/ml Activin A in prewarmed RPMI + 0,1 %PEST) and gently added to cell culture.
• Day 3 Day3&4 mediums were premixed (100ng/ml Activin A and 2% B27 in prewarmed RPMI + 0,1 %PEST) and gently added to cell culture.
• RNA samples were collected after 24h pre-treatment (D1 ), after 1 day of AA treatment (D2) and after 1 -2d of AA+B27 treatment (D3-4). Total RNA was extracted with the Rneasy Plus Mini kit (Qiagen) and quantitative real-time PCR was performed using the StepOnePLus system (Applied Biosystems).
• Undifferentiated SA121 hES and chlPS4 iPS cells in DEF media (DO) were cultured either in RPMI without CHIR (Ctrl D1 ) or treated for 24h with CHIR in RPMI (CHIR D1 ). The cells were then washed to ensure absence of CHIR and then both conditions were treated with 1 d AA (Ctrl and CHIR d2). After 1 d CHIR treatment, PS markers such as Brachyury (T),
• MIXL1 .EOMES and GSC were highly upregulated compared to non pre-treated cells (Fig. 2A-C). T fold inductions after 24h CHIR treatment spanned between 500-100000 compared to undifferentiated cells (dO). T protein levels were also confirmed with ICC (Fig. 2C). 1 d after AA addition (D2), markers that together are indicative of DE formation such as SOX17, CXCR4, FOXA2 and CER were also highly upregulated compared to non-pre- treated cells despite addition of AA to both conditions (Fig. 3A,B), Sox17-fold inductions after CHIR pretreatment spanned between 10000-3500000 compared to undifferentiated cells (DO, n>60).
• the efficiency levels for both T and SOX17 were highly robust (n > 60) and confirmed with IHC and SOX17 quantifications (Fig. 2C and Table 2.).
• the CHIR pre-treatment step resulted in a remarkably rapid induction of SOX17 after AA treatment.
• SOX17 expression peaked already after one day of AA treatment when cells were subjected to CHIR for 24h before AA induction.
• Cells in AA-containing media that were not pre-treated with CHIR (ctrl) did not show same speed or efficiency in Sox17 induction.
• CHIR has a direct capacity to induce PS markers after 24h treatment (D1 ) and resulted in a remarkably higher ability for AA to induce DE when added (D2). Brachyury was greatly repressedonce AA was added indicating that the cells were differentiating in a highly stepwise order.
• Table 2 shows D1 -3 of DE induction according to D'Amour (protocol as described in Kroon et al 2008) and cells treated with 3uM CHIR (CHIR D1 ) prior to addition of ActivinA (CHIR D2- 3).
• hES SA121 and ChlPS4 cells were treated with either CHIR (3 ⁇ ), BIO (0,5uM), Wnt3a
• Undifferentiated ChlPS4 cells were eitherpre-treated for 24h with CHIR (3uM) or directly exposed to AA (100ng/ml) and Wnta3a (25ng/ml) according to Kroon et al 2008. The cells were then washed and AA was added for 1 -4d. See Tablel for detailed setup.
• CHIR treatment was superior in upregulating both T (mRNA D1 ) and SOX17 after CHIR pre-treatment (mRNA, ICC D3) compared to D'Amour (Fig. 5 and Table 2).
• mRNA D1 mRNA D1
• SOX17 SOX17 after CHIR pre-treatment
• Fig. 5 and Table 2 D'Amour
• chlPS4 cells differentiated to DE were further differentiated towards pancreatic endoderm (PE) by using a previously published PE differentiation proto- col (Ameri et al 2010). Seven days after the induction of PE differentiation the cells were fixed and analyzed by ICC using a PDX1 specific antibody. The results showed very modest PDX1 -staining in the D'Amour-treated cells whereas the staining was evident in the CHIR pre-treated cells (data not shown). In parallel, mRNA was collected from the cells and the PDX1 expression was analyzed by real-time PCR.
• Cells were pre-treated 1 d with either RPMI (ctrl), CHIR 3uM, CHIR 3uM+AA 100ng/ml or 100ng/ml AA + 25ng/ml Wnt3a (D'Amour)prior to addition of AA +/- Wnt3a (25ng/ml) d2 and AA d3 (Table 3).
• B27 was added in Ctrl and CHIR d3 and FBS to D'Amour d2-3.
• CHIR and exogenous AA may have counteracting effects that reduce overall effi- ciency and/or target different cell populations differently, resulting in higher levels of heterogeneity within the cell culture and increased inter-experimental variability. This suggests that PS induction/transition before adding AA to the media is an important and novel step to enhance protocol efficiency and robustness.
• Chir was titrated at concentrations between 1 -7uM and analyzed after 24h Chir treatment (D1 ) and after 2d of following AA treatment (D3). CHIR 0,5-1 uM and CHIR 7uM did not sur- make d3.
• FGF2 specifies hESC-derived definitive endoderm into foregut/midgut cell lineages in a concentration-independent manner.

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