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• W3040382034 abstract "•Method development for addressing multifactorial problems in directed differentiation•Generation of endodermal populations without the use of TGF-β agonism•Small-molecule-based pancreatic differentiation protocol The derivation of endoderm and descendant organs, such as pancreas, liver, and intestine, impacts disease modeling and regenerative medicine. Use of TGF-β signaling agonism is a common method for induction of definitive endoderm from pluripotency. By using a data-driven, High-Dimensional Design of Experiments (HD-DoE)-based methodology to address multifactorial problems in directed differentiation, we found instead that optimal conditions demanded BMP antagonism and retinoid input leading to induction of dorsal foregut endoderm (DFE). We demonstrate that pancreatic identity can be rapidly, and robustly, induced from DFE and that such cells are of dorsal pancreatic identity. The DFE population was highly competent to differentiate into both stomach organoids and pancreatic tissue types and able to generate fetal-type β cells through two subsequent differentiation steps using only small molecules. This alternative, rapid, and low-cost basis for generating pancreatic insulin-producing cells may have impact for the development of cell-based therapies for diabetes. The derivation of endoderm and descendant organs, such as pancreas, liver, and intestine, impacts disease modeling and regenerative medicine. Use of TGF-β signaling agonism is a common method for induction of definitive endoderm from pluripotency. By using a data-driven, High-Dimensional Design of Experiments (HD-DoE)-based methodology to address multifactorial problems in directed differentiation, we found instead that optimal conditions demanded BMP antagonism and retinoid input leading to induction of dorsal foregut endoderm (DFE). We demonstrate that pancreatic identity can be rapidly, and robustly, induced from DFE and that such cells are of dorsal pancreatic identity. The DFE population was highly competent to differentiate into both stomach organoids and pancreatic tissue types and able to generate fetal-type β cells through two subsequent differentiation steps using only small molecules. This alternative, rapid, and low-cost basis for generating pancreatic insulin-producing cells may have impact for the development of cell-based therapies for diabetes. Endoderm is the germ layer that creates the majority of cells within most of the internal organ systems, such as lung, stomach, pancreas, liver, and gut. The ability to robustly generate endodermal descendant tissues will impact the studies and therapy modalities of multiple human diseases. Until present, almost all efforts on inducing endoderm from pluripotent cells have relied on using a TGF-β pathway agonist, most commonly Activin A (AA), to push pluripotent cells through an in vitro gastrulation event (D'amour et al., 2005D'amour K.A. Agulnick A.D. Eliazer S. Kelly O.G. Kroon E. Baetge E.E. Efficient differentiation of human embryonic stem cells to definitive endoderm.Nat. Biotechnol. 2005; 23: 1534-1541Crossref PubMed Scopus (1273) Google Scholar; Gadue et al., 2006Gadue P. Huber T.L. Paddison P.J. Keller G.M. Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells.Proc. Natl. Acad. Sci. U S A. 2006; 103: 16806-16811Crossref PubMed Scopus (425) Google Scholar). This results in an endodermal population that can be successfully used for generating multiple descendant fates including intestinal (Spence et al., 2011Spence J.R. Mayhew C.N. Rankin S.A. Kuhar M.F. Vallance J.E. Tolle K. Hoskins E.E. Kalinichenko V.V. Wells S.I. Zorn A.M. et al.Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.Nature. 2011; 470: 105-109Crossref PubMed Scopus (1109) Google Scholar), pancreatic (Kroon et al., 2008Kroon E. Martinson L.A. Kadoya K. Bang A.G. Kelly O.G. Eliazer S. Young H. Richardson M. Smart N.G. Cunningham J. et al.Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo.Nat. Biotechnol. 2008; 26: 443-452Crossref PubMed Scopus (1339) Google Scholar; Rezania et al., 2014Rezania A. Bruin J.E. Arora P. Rubin A. Batushansky I. Asadi A. O'dwyer S. Quiskamp N. Mojibian M. Albrecht T. et al.Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.Nat. Biotechnol. 2014; 32: 1121-1133Crossref PubMed Scopus (835) Google Scholar; Pagliuca et al., 2014Pagliuca F.W. Millman J.R. Gurtler M. Segel M. van Dervort A. Ryu J.H. Peterson Q.P. Greiner D. Melton D.A. Generation of functional human pancreatic beta cells in vitro.Cell. 2014; 159: 428-439Abstract Full Text Full Text PDF PubMed Scopus (1158) Google Scholar), and liver (Sampaziotis et al., 2015Sampaziotis F. de Brito M.C. Madrigal P. Bertero A. Saeb-Parsy K. Soares F.A.C. Schrumpf E. Melum E. Karlsen T.H. Bradley J.A. et al.Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation.Nat. Biotechnol. 2015; 33: 845-852Crossref PubMed Scopus (215) Google Scholar). Generation of more anterior endodermal fates, such as lung, has been achieved by providing patterning inputs at a subsequent stage (Green et al., 2011Green M.D. Chen A. Nostro M.C. D'souza S.L. Schaniel C. lemischka I.R. Gouon-Evans V. Keller G. Snoeck H.W. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells.Nat. Biotechnol. 2011; 29: 267-272Crossref PubMed Scopus (253) Google Scholar). However, recent studies argue that initial patterning of definitive endoderm may occur during its generation (Matsuno et al., 2016Matsuno K. Mae S.I. Okada C. Nakamura M. Watanabe A. Toyoda T. Uchida E. Osafune K. Redefining definitive endoderm subtypes by robust induction of human induced pluripotent stem cells.Differentiation. 2016; 92: 281-290Crossref PubMed Scopus (16) Google Scholar; Loh et al., 2014Loh K.M. Ang L.T. Zhang J. Kumar V. Ang J. Auyeong J.Q. Lee K.L. Choo S.H. Lim C.Y. Nichane M. et al.Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations.Cell Stem Cell. 2014; 14: 237-252Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The pancreas is of particular interest for cell-based therapy in diabetes, which is characterized by defects in, or loss of, insulin-producing cells. The pancreas is formed from two spatially distinct primordia arising on the dorsal and ventral sides of the primitive gut tube, which subsequently fuse. Although both pancreatic buds are capable of generating all lineages of the adult pancreas (Matsuura et al., 2009Matsuura K. Katsumoto K. Fukuda K. Kume K. Kume S. Conserved origin of the ventral pancreas in chicken.Mech. Dev. 2009; 126: 817-827Crossref PubMed Scopus (17) Google Scholar), distinct transcriptional programs control the initial induction of the pancreatic domains on opposing sides of the gut tube. In mice, the ventral pancreatic bud forms first at approximately embryonic day 8.5 (E8.5) from a region of endoderm possessing bipotential competence for pancreas and liver (Angelo et al., 2012Angelo J.R. Guerrero-Zayas M.I. Tremblay K.D. A fate map of the murine pancreas buds reveals a multipotent ventral foregut organ progenitor.PLoS One. 2012; 7: e40707Crossref PubMed Scopus (29) Google Scholar; Deutsch et al., 2001Deutsch G. Jung J. Zheng M. Lora J. Zaret K.S. A bipotential precursor population for pancreas and liver within the embryonic endoderm.Development. 2001; 128: 871-881Crossref PubMed Google Scholar; Tremblay and Zaret, 2005Tremblay K.D. Zaret K.S. Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues.Dev. Biol. 2005; 280: 87-99Crossref PubMed Scopus (217) Google Scholar). This early ventral endoderm field consists of a progenitor population that co-expresses Pdx1/Sox17 transiently, which by E9.5 splits to form the ventral pancreas and the extra-hepatobiliary system, respectively (Spence et al., 2009Spence J.R. Lange A.W. Lin S.C. Kaestner K.H. Lowy A.M. Kim I. Whitsett J.A. Wells J.M. Sox17 regulates organ lineage segregation of ventral foregut progenitor cells.Dev. Cell. 2009; 17: 62-74Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Specification of the ventral pancreas relies on HHex expression. Gene ablation models have demonstrated complete ventral agenesis without affecting dorsal pancreatic bud formation (Bort et al., 2004Bort R. Martinez-Barbera J.P. Beddington R.S. Zaret K.S. Hex homeobox gene-dependent tissue positioning is required for organogenesis of the ventral pancreas.Development. 2004; 131: 797-806Crossref PubMed Scopus (203) Google Scholar). In contrast, the dorsal pancreatic bud in mice emerges at approximately embryonic day 9.0 and forms from an outgrowth caudal to the antral stomach region. Studies in mice have also identified factors involved in dorsal pancreatic specification with no effect on ventral organogenesis. Mnx1 (Hlxb9) knockout models have shown dorsal agenesis occurs without a ventral phenotype (Li et al., 1999Li H. Arber S. Jessell T.M. Edlund H. Selective agenesis of the dorsal pancreas in mice lacking homeobox gene Hlxb9.Nat. Genet. 1999; 23: 67-70Crossref PubMed Scopus (23) Google Scholar). Mnx1 expression is observed in the ventral field but only following Pdx1 expression, whereas in the dorsal field, Mnx1 precedes Pdx1 expression. Raldh2 knockout models resulted in a dorsal-specific agenesis attributed to the loss of Pdx1 and Prox1 expression in the dorsal bud (Martin et al., 2005Martin M. Gallego-Llamas J. Ribes V. Kedinger M. Niederreither K. Chambon P. Dolle P. Gradwohl G. Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice.Dev. Biol. 2005; 284: 399-411Crossref PubMed Scopus (182) Google Scholar; Molotkov et al., 2005Molotkov A. Molotkova N. Duester G. Retinoic acid generated by Raldh2 in mesoderm is required for mouse dorsal endodermal pancreas development.Dev. Dyn. 2005; 232: 950-957Crossref PubMed Scopus (154) Google Scholar). Furthermore, studies in chick have shown that the initial budding of the dorsal pancreas is dependent on the selective inhibition of SHH within the dorsal midgut (Hebrok et al., 1998Hebrok M. Kim S.K. Melton D.A. Notochord repression of endodermal Sonic hedgehog Permits pancreas development.Genes Dev. 1998; 12: 1705-1713Crossref PubMed Scopus (475) Google Scholar). Although it is unclear if the murine system is conserved between species, a recent study using laser capture followed by deep sequencing analysis described some fundamental differences between the ventral and dorsal pancreas during human development (Jennings et al., 2017Jennings R.E. Berry A.A. Gerrard D.T. Wearne S.J. Strutt J. Withey S. Chhatriwala M. Piper Hanley K. Vallier L. Bobola N. Hanley N.A. Laser capture and deep sequencing reveals the transcriptomic programmes regulating the onset of pancreas and liver differentiation in human embryos.Stem Cell Rep. 2017; 9: 1387-1394Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Despite differential pathway utilization and distinct cell intrinsic factors the dorsal and ventral pancreatic programs have much in common. HNF1β (Tcf2) is required for pancreas specification in both pancreatic buds and is critical through pancreatic development. Tcf2 knockout mice fail to generate a ventral pancreas and have a greatly reduced dorsal bud incapable of differentiating or proliferating (Haumaitre et al., 2005Haumaitre C. Barbacci E. Jenny M. Ott M.O. Gradwohl G. Cereghini S. Lack of TCF2/vHNF1 in mice leads to pancreas agenesis.Proc. Natl. Acad. Sci. U S A. 2005; 102: 1490-1495Crossref PubMed Scopus (194) Google Scholar). HNF1β is expressed in the pre-pancreatic foregut and functions at the apex of a sequential transcriptional cascade resulting in the activation of Hnf6 (Oc1) followed by Pdx1 (Poll et al., 2006Poll A.V. Pierreux C.E. Lokmane L. Haumaitre C. Achouri Y. Jacquemin P. Rousseau G.G. Cereghini S. Lemaigre F.P. A vHNF1/TCF2-HNF6 cascade regulates the transcription factor network that controls generation of pancreatic precursor cells.Diabetes. 2006; 55: 61-69Crossref PubMed Scopus (77) Google Scholar). Conditional inactivation of HNF1β results in a loss of Glis3 and Ngn3 expression and results in a pancreas characterized with cystic ducts and a loss of the pro-endocrine field (de Vas et al., 2015de Vas M.G. Kopp J.L. Heliot C. Sander M. Cereghini S. Haumaitre C. Hnf1b controls pancreas morphogenesis and the generation of Ngn3+ endocrine progenitors.Development. 2015; 142: 871-882Crossref PubMed Scopus (69) Google Scholar). In human development, the importance of HNF1β is highlighted by the occurrence of “maturity-onset diabetes of the young type 5” (MODY5) syndrome, a condition attributed to mutations in the HNF1β gene. Although a heterozygous mutation in HNF1β does not display a phenotype in mouse studies, in humans heterozygous mutation of HNF1β have been shown to be associated with MODY5 or complete pancreatic agenesis suggesting a more important role for HNF1β in human pancreatic development than in mouse (Body-Bechou et al., 2014Body-Bechou D. Loget P. D'herve D. le Fiblec B. Grebille A.G. le Guern H. Labarthe C. Redpath M. Cabaret-Dufour A.S. Sylvie O. et al.TCF2/HNF-1beta mutations: 3 cases of fetal severe pancreatic agenesis or hypoplasia and multicystic renal dysplasia.Prenat Diagn. 2014; 34: 90-93Crossref PubMed Scopus (20) Google Scholar). Most current laboratory efforts at directing the differentiation of pluripotent stem cells rely on emulating developmental signaling event(s) leading to the generation of desired cell type. This is generally accomplished by assaying a single pro-differentiation factor at time. The limitation of this methodology is that it relies on studying each pro-differentiation factor individually, hindering the detection of synergistic or systemic influences. A way to address this problem is using a systems biology approach (Kitano, 2002Kitano H. Computational systems biology.Nature. 2002; 420: 206-210Crossref PubMed Scopus (1517) Google Scholar; Carinhas et al., 2012Carinhas N. Oliveira R. Alves P.M. Carrondo M.J. Teixeira A.P. Systems biotechnology of animal cells: the road to prediction.Trends Biotechnol. 2012; 30: 377-385Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) capable of assaying multiple factors simultaneously in a manner capable of elucidating individual and synergistic effects. Because experimental design size increases exponentially as additional factors are incorporated, this greatly limits traditional methods from approaching a systems biology level of interrogation. To overcome this limitation, we have developed a novel approach focusing on key aspects of a manufacturing process (key concepts are defined within Box 1). Using Design-of-Experiments (DoE) mathematics (Chakrabarty et al., 2013Chakrabarty A. Buzzard G.T. Rundell A.E. Model-based design of experiments for cellular processes.Wiley Interdiscip. Rev. Syst. Biol. Med. 2013; 5: 181-203Crossref PubMed Scopus (22) Google Scholar), we are able to greatly increase the dimensionality of our differentiation experiments by relying on a compression of the design space (Gerin et al., 2014Gerin S. Mathy G. Franck F. Modeling the dependence of respiration and photosynthesis upon light, acetate, carbon dioxide, nitrate and ammonium in Chlamydomonas reinhardtii using design of experiments and multiple regression.BMC Syst. Biol. 2014; 8: 96Crossref PubMed Scopus (13) Google Scholar; Mendes et al., 2016Mendes L.F. Tam W.L. Chai Y.C. Geris L. Luyten F.P. Roberts S.J. Combinatorial Analysis of Growth Factors Reveals the Contribution of Bone Morphogenetic Proteins to Chondrogenic Differentiation of Human Periosteal Cells.Tissue Eng. Part C Methods. 2016; 22: 473-486Crossref PubMed Scopus (25) Google Scholar). This systematic approach minimizes the number of experimental runs needed to interrogate multiple parameters simultaneously within a single experimental design (Rathore et al., 2014Rathore A.S. Mittal S. Pathak M. Arora A. Guidance for performing multivariate data analysis of bioprocessing data: pitfalls and recommendations.Biotechnol. Prog. 2014; 30: 967-973Crossref PubMed Scopus (22) Google Scholar). Combined with a deep set of lineage-informative transcript level measurements a better understanding of the cell culture behavior is obtained within the design space (Mercier et al., 2013Mercier S.M. Diepenbroek B. Dalm M.C. Wijffels R.H. Streefland M. Multivariate data analysis as a PAT tool for early bioprocess development data.J. Biotechnol. 2013; 167: 262-270Crossref PubMed Scopus (42) Google Scholar). Such an approach is integral to a Quality-by-Design (QbD) process (Juran, 1993Juran J. Made in the U. S. A.: a renaissance in quality.Harv. Business Rev. 1993; 71: 42-47PubMed Google Scholar; McConnell et al., 2010McConnell J. Nunnally B.K. McGarvey B. The forgotten origins of quality by design.J. Validation Technol. 2010; : 30-34Google Scholar; Swain et al., 2018Swain S. Padhy R. Jena B.R. Babu S.M. Quality by design: concept to applications.Curr. Drug Discov. Technol. 2018; 16: 240-250Crossref Scopus (12) Google Scholar). It provides process understanding, allowing for consistent product manufacturing (Kumar et al., 2014Kumar V. Bhalla A. Rathore A.S. Design of experiments applications in bioprocessing: concepts and approach.Biotechnol. Prog. 2014; 30: 86-99Crossref PubMed Scopus (80) Google Scholar; Lipsitz et al., 2016Lipsitz Y.Y. Timmins N.E. Zandstra P.W. Quality cell therapy manufacturing by design.Nat. Biotechnol. 2016; 34: 393-400Crossref PubMed Scopus (136) Google Scholar). In the present study, we use DoE-based optimization to produce a pancreatic directed differentiation protocol defining the critical process parameters relevant to the differentiation process and future manufacturing. We monitor gene expression throughout the differentiation process as a critical material attribute that defines the cellular phenotype. We then identify which pathway control elements are the critical process parameters that must be controlled to ensure proper differentiation. Contrary to most methods of endoderm induction, we demonstrate that effective and regionalized patterned endoderm can be robustly differentiated directly from pluripotency without the use of TGF-β agonism. Exploring for optimal endodermal fate conversion conditions, we used HNF1β expression as an initial waypoint for pancreas and other endodermal derivatives. This led to a novel and highly robust protocol for inducing specialized human endoderm representative of the dorsal foregut region of the gut tube. We demonstrate that this population can be effectively converted into dorsal pancreatic progenitors that subsequently are able to adopt endocrine fates, including the generation of fetal-like beta cells. By inspecting the critical process parameters we created a three-stage protocol that converts PSCs into fetal-like beta cells using a series of small molecules. We combined a high-dimensional application of Design of Experiments (HD-DoE) methodology with deep response set measurements to generate predictive models for pluripotent culture forward differentiation. This method allowed interrogation of pathway interactions, providing a more comprehensive understanding of the biological inputs impacting endodermal differentiation. By challenging pluripotent cultures with a perturbation matrix composed of multiple morphogen pathway agonists and antagonists simultaneously, and measuring multiple fate determining genes, we extracted a systems-level effector/response dynamic. The application of DoE substantially compressed the number of experimental runs compared with a full factorial design. Yet it remained possible to statistically determine any first-order pathway/pathway interactions. It also revealed the system behavior covered by the tested dimensions within the concentration ranges used (i.e., “known space”). The morphogens (effectors) used in initial experimental designs were AA, BMP4, FGF2, WNT3a, SHH, and RA. We also included respective small-molecule antagonists for each pathway except RA. These factors were chosen because previous publications within the stem-cell field have shown these inputs to elicit forward differentiation from pluripotency into various descendant fates. We generated D-optimal DoE designs to test all of these effectors in a single experiment. Designs specified the combinations of effectors to be included in each well of a 96-well plate. The designs were constrained to prevent unproductive combinations of agonist/antagonist of the same pathway being used together in the individual reactions. Robotic assembly of experimental media (i.e., the Perturbation Matrix) eliminated human errors while ensuring accuracy. Response measurements were custom-chosen early lineage-determining genes. Following experiment execution, a multivariable regression model was generated for each response gene as a function of effector contribution. All response data were mathematically fitted to maximize predictive power (Q2 maximization). As a result, we obtained an in silico representation of the behavior of each response gene as related to each effector input. These models allowed us to predict conditions that would achieve desirable induction, or suppression, of any of the genes monitored. We refer to these interrogations as in silico predictive analysis (ISPA), noting that such predictions were calculated on the basis of statistical models resting on the entire set of the DoE design. For ISPA, various tools were needed to achieve specific desirable outcomes and identify critical process parameters. Coefficient Plots may be generated for each individual response gene. These plots display the coefficient for each effector term in the regression model for the respective gene. coefficient plots are scaled and centered and thus also provide graphical representations of model term significance. We used coefficient plots to inspect individual effectors' contributions to the activation of individual genes. The “optimizer” function used the regression models to identify which media compositions contributed to a desired differentiation event. The optimizer provides the relative Factor Contribution (FC) for each effector. FC is proportional to how important the individual effector is to the differentiation event. FC thereby helps identify criticality of a process parameter. For practical proposes, we considered FC < 10 as low relevance, 10–20 as relevant, and >20 as highly relevant. Particularly valuable, we used the “Dynamic Profiling” to visualize expression behavior for multiple genes simultaneously at any given input condition (set point). We initially set out to predict conditions needed to define a definitive endoderm population as suggested by the literature. This was accomplished by defining an anterior primitive streak (APS) population by modeling for the maximal expression of MESP1, EOMES, and BRACHYURY/T while minimizing EVX1 (posterior primitive streak marker) (Loh et al., 2014Loh K.M. Ang L.T. Zhang J. Kumar V. Ang J. Auyeong J.Q. Lee K.L. Choo S.H. Lim C.Y. Nichane M. et al.Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations.Cell Stem Cell. 2014; 14: 237-252Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Through ISPA, the conditions predicted to generate this differentiation event consisted of low tolerance to Wnt inhibition (FC = 18.65) and high levels of AA (FC = 18.85) (Figures 1A and 1B ). These conditions agree with current protocols for generating definitive endoderm (DE) (D'amour et al., 2005D'amour K.A. Agulnick A.D. Eliazer S. Kelly O.G. Kroon E. Baetge E.E. Efficient differentiation of human embryonic stem cells to definitive endoderm.Nat. Biotechnol. 2005; 23: 1534-1541Crossref PubMed Scopus (1273) Google Scholar). Indeed, when using this condition to differentiate pluripotent cultures, a FOXA2+/SOX17 + population was obtained within a 3-day period (Figure 1C). Through ISPA, a number of other genes were predicted to be highly expressed under these conditions including COL6A1, HHEX, MESP2, SOX17 (Figure 1D and data not shown). These genes are all known to be elevated in definitive endoderm. However, through ISPA, we noted that not all known early endoderm-expressed genes were uniquely maximized through the APS conditions. Inspecting the known space from the aforementioned experiment, a quite different solution set could be obtained focusing on HNF1β (TCF2) expression, also known to be expressed in definitive endoderm. Maximizing for HNF1β expression also led to expression of accompanying genes such as FOXA2, HNF4A, MNX1, CXCR4, MTF1, all known markers of endoderm, whereas expression of HHEX and SOX17 remained low (ISPA results not shown). We went on to characterize these distinct states and the requirements for their induction. Using ISPA, we inspected the fundamental logics governing endodermal gene induction. For the APS-derived DE, it was clear that many early endoderm genes were under the direct control of TGF-β signaling, displaying strong and positive coefficient terms from AA in their complex regulatory models. These genes included, but were not limited to, SOX17, CXCR4, LEFTY1, MIXL1, and HHEX (Figure 1D). This was not the case for multiple other known endoderm markers. A sub-group of endoderm genes did not respond to AA stimulation but were directly dependent on RA signaling and to a lesser extent required the inhibition of the BMP pathway. These genes included FOXA2, EPCAM, ONECUT1, CDX2, and MNX1 (Figure 1D) and were predicted to be directly controlled through the synergistic effects of retinoic acid and BMP inhibition with high factor contributions including FOXA2 (FC for RA = 24.8, BMPi = 25.3), HNF1β (FC for RA = 30.1, BMPi = 31.7), and MNX1 (FC for RA = 22.7, BMPi = 30.5) (Figure 1B and data not shown). Of note, AA was predicted to have no contribution to activating these genes; rather, inhibition of the TGF-β pathway was predicted to benefit the expression of these genes. Factor contributions for Alk5i were 7.59 for HNF1β, 8.88 for FOXA2, and 18.98 for MNX1 (Figure 1B and data not shown). To assess the robustness of the methodology, the same perturbation matrix design was used on a second pluripotent cell line, H9 (female) (Figure S1). Both the BMPi (Factor Contribution of 11.0) and the provision of RA (Factor Contribution 29.9) were again shown to be critical factors for the activation of HNF1β. Although the factor contribution for BMPi decreased, there was a corresponding increased importance in the absence of AA (Factor Contribution 20.1 that AA is not included) (Figures S1A’–S1B′). Altogether, this suggests that retinoic acid input when provided in the absence of TGF-β signaling is a critical process parameter for induction of HNF1β. Comparative analysis using dynamic profiling at the HNF1β set point revealed that effectors controlling HNF1β, MNX1 (both retinoic acid and TGF-β inhibition responsive), and HHEX (AA responsive) were very similar between these cell lines, which differ in sex and prior culturing conditions (the male H1 in Essential 8 and the female H9 in mTesR media) (Figures S1C and 1C′). The predicted conditions for HNF1β optimization (HNF1βOpt) were tested on differentiating pluripotent cells for validation. Based on protein expression, HNF1β, MNX1, and FOXA2 could all be activated as expected. The target gene for optimization, HNF1β, was present in 97.9 ± 0.7% of the cells (Figures S2A and 2C). The two paths to endoderm activation were fundamentally distinct and rested on conflicting input logic suggesting that the pathways were mutually exclusive. We tested this by creating hybrid protocols assaying the effects of RA and BMPi in the presence of AA (Figure 2A). Inclusion of RA into the APS-based DE-generating protocols (D'amour et al., 2005D'amour K.A. Agulnick A.D. Eliazer S. Kelly O.G. Kroon E. Baetge E.E. Efficient differentiation of human embryonic stem cells to definitive endoderm.Nat. Biotechnol. 2005; 23: 1534-1541Crossref PubMed Scopus (1273) Google Scholar) proved to only moderately increase gene expression for retinoic acid-responsive genes; only CDX2 (data not shown) and OSR1 (Figure 2B) were significantly up-regulated in this manner. Conversely, known TGF-β-responsive genes were shown to be significantly down-regulated when retinoic acid was included in APS-type DE-generating reactions including HHEX, SOX17, and GSC (Figure 2B and data not shown). This demonstrates that presence of either of the key protocol drivers (AA versus RA) will suppress the other. Furthermore, also as predicted by ISPA, the genes up-regulated in the presence of retinoic acid and LDN3189 were activated more efficiently when AA was excluded from these reactions. Importantly, the two key protocol inputs for the HNF1βOPT conditions, RA and LDN3189, sufficed to initiate differentiation comparable with the full-input HNF1βOpt conditions (Figure 2B) with 97.1 ± 1.8% of the cells within the culture expressing HNF1β (Figures S2A and S2C). To gain a better understanding of the differing nature of the endodermal populations, we subjected cultures for RNA sequencing. Common endodermal genes were expressed at similar levels in both populations; these included CXCR4, FOXA2, EPCAM, GATA4, and GATA6 (Figure 2C). However, significant differences were observed for genes associated with patterning revealing that HNF1βOpt induced endoderm was enriched in genes characteristic of known dorsal (MNX1 and PAX6) and foregut endoderm (HOXA1, HOXA3, HNF4A, and HNF1β) (Figure 2C). In contr" @default.
• W3040382034 created "2020-07-10" @default.
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• W3040382034 title "High-Dimensional Design-Of-Experiments Extracts Small-Molecule-Only Induction Conditions for Dorsal Pancreatic Endoderm from Pluripotency" @default.
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