Lay Teng Ang

All Publications

• Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells. Developmental cell Fowler, J. L., Zheng, S. L., Nguyen, A., Chen, A., Xiong, X., Chai, T., Chen, J. Y., Karigane, D., Banuelos, A. M., Niizuma, K., Kayamori, K., Nishimura, T., Cromer, M. K., Gonzalez-Perez, D., Mason, C., Liu, D. D., Yilmaz, L., Miquerol, L., Porteus, M. H., Luca, V. C., Majeti, R., Nakauchi, H., Red-Horse, K., Weissman, I. L., Ang, L. T., Loh, K. M. 2024

  Abstract

  The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.

  View details for DOI 10.1016/j.devcel.2024.03.003

  View details for PubMedID 38569552

• Monolayer platform to generate and purify primordial germ-like cells in vitro provides insights into human germline specification. Nature communications Vijayakumar, S., Sala, R., Kang, G., Chen, A., Pablo, M. A., Adebayo, A. I., Cipriano, A., Fowler, J. L., Gomes, D. L., Ang, L. T., Loh, K. M., Sebastiano, V. 2023; 14 (1): 5690

  Abstract

  Generating primordial germ cell-like cells (PGCLCs) from human pluripotent stem cells (hPSCs) advances studies of human reproduction and development of infertility treatments, but often entails complex 3D aggregates. Here we develop a simplified, monolayer method to differentiate hPSCs into PGCs within 3.5 days. We use our simplified differentiation platform and single-cell RNA-sequencing to achieve further insights into PGCLC specification. Transient WNT activation for 12h followed by WNT inhibition specified PGCLCs; by contrast, sustained WNT induced primitive streak. Thus, somaticcells (primitive streak) and PGCLCs are related-yet distinct-lineages segregated by temporally-dynamic signaling. Pluripotency factors including NANOG are continuously expressed during the transition from pluripotency to posterior epiblast to PGCs, thus bridging pluripotent and germline states. Finally, hPSC-derived PGCLCs can be easily purified by virtue of their CXCR4+PDGFRA-GARP- surface-marker profile and single-cell RNA-sequencing reveals that they harbor transcriptional similarities with fetal PGCs.

  View details for DOI 10.1038/s41467-023-41302-w

  View details for PubMedID 37709760

• Building human artery and vein endothelial cells from pluripotent stem cells, and enduring mysteries surrounding arteriovenous development. Seminars in cell & developmental biology Loh, K. M., Ang, L. T. 2023

  Abstract

  Owing to their manifold roles in health and disease, there have been intense efforts to synthetically generate blood vessels in vitro from human pluripotent stem cells (hPSCs). However, there are multiple types of blood vessel, including arteries and veins, which are molecularly and functionally different. How can we specifically generate either arterial or venous endothelial cells (ECs) from hPSCs in vitro? Here, we summarize how arterial or venous ECs arise during embryonic development. VEGF and NOTCH arbitrate the bifurcation of arterial vs. venous ECs in vivo. While manipulating these two signaling pathways biases hPSC differentiation towards arterial and venous identities, efficiently generating these two subtypes of ECs has remained challenging until recently. Numerous questions remain to be fully addressed. What is the complete identity, timing and combination of extracellular signals that specify arterial vs. venous identities? How do these extracellular signals intersect with fluid flow to modulate arteriovenous fate? What is a unified definition for endothelial progenitors or angioblasts, and when do arterial vs. venous potentials segregate? How can we regulate hPSC-derived arterial and venous ECs in vitro, and generate organ-specific ECs? In turn, answers to these questions could avail the production of arterial and venous ECs from hPSCs, accelerating vascular research, tissue engineering, and regenerative medicine.

  View details for DOI 10.1016/j.semcdb.2023.06.004

  View details for PubMedID 37393122

• Variation in CFHR3 determines susceptibility to meningococcal disease by controlling factor H concentrations AMERICAN JOURNAL OF HUMAN GENETICS Kumar, V., Pouw, R. B., Autio, M., Sagmeister, M. G., Phua, Z., Borghini, L., Wright, V. J., Hoggart, C., Pan, B., Tan, A., Binder, A., Brouwer, M. C., Pinnock, E., De Groot, R., Hazelzet, J., Emonts, M., van der Flier, M., Reiter, K., Nothen, M. M., Hoffmann, P., Schlapbach, L. J., Bellos, E., Anderson, S., Secka, F., Martinon-Torres, F., Salas, A., Fink, C., Carrol, E. D., Pollard, A. J., Coin, L. J., Zenz, W., Wouters, D., Ang, L., Hibberd, M. L., Levin, M., Kuijpers, T. W., Davila, S., EUCLIDS Consortium 2022; 109 (9): 1680-1691

  Abstract

  Neisseria meningitidis protects itself from complement-mediated killing by binding complement factor H (FH). Previous studies associated susceptibility to meningococcal disease (MD) with variation in CFH, but the causal variants and underlying mechanism remained unknown. Here we attempted to define the association more accurately by sequencing the CFH-CFHR locus and imputing missing genotypes in previously obtained GWAS datasets of MD-affected individuals of European ancestry and matched controls. We identified a CFHR3 SNP that provides protection from MD (rs75703017, p value = 1.1 × 10-16) by decreasing the concentration of FH in the blood (p value = 1.4 × 10-11). We subsequently used dual-luciferase studies and CRISPR gene editing to establish that deletion of rs75703017 increased FH expression in hepatocyte by preventing promotor inhibition. Our data suggest that reduced concentrations of FH in the blood confer protection from MD; with reduced access to FH, N. meningitidis is less able to shield itself from complement-mediated killing.

  View details for DOI 10.1016/j.ajhg.2022.08.001

  View details for Web of Science ID 000877624100010

  View details for PubMedID 36007525

  View details for PubMedCentralID PMC9502058

• Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses. Cell Ang, L. T., Nguyen, A. T., Liu, K. J., Chen, A., Xiong, X., Curtis, M., Martin, R. M., Raftry, B. C., Ng, C. Y., Vogel, U., Lander, A., Lesch, B. J., Fowler, J. L., Holman, A. R., Chai, T., Vijayakumar, S., Suchy, F. P., Nishimura, T., Bhadury, J., Porteus, M. H., Nakauchi, H., Cheung, C., George, S. C., Red-Horse, K., Prescott, J. B., Loh, K. M. 2022

  Abstract

  Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.

  View details for DOI 10.1016/j.cell.2022.05.024

  View details for PubMedID 35738284

• Dach1 Extends Artery Networks and Protects Against Cardiac Injury. Circulation research Raftrey, B., Williams, I. M., Rios Coronado, P. E., Fan, X., Chang, A. H., Zhao, M., Roth, R. K., Trimm, E., Racelis, R., D'Amato, G., Phansalkar, R., Nguyen, A., Chai, T., Gonzalez, K. M., Zhang, Y., Ang, L. T., Loh, K., Bernstein, D., Red-Horse, K. 2021

  Abstract

  Rationale: Coronary artery disease (CAD) is the leading cause of death worldwide, but there are currently no methods to stimulate artery growth or regeneration in diseased hearts. Studying how arteries are built during development could illuminate strategies for re-building these vessels during ischemic heart disease. We previously found that Dach1 deletion in mouse embryos resulted in small coronary arteries. However, it was not known whether Dach1 gain-of-function would be sufficient to increase arterial vessels and whether this could benefit injury responses. Objective: We investigated how Dach1 overexpression in endothelial cells affected transcription and artery differentiation, and how it influenced recovery from myocardial infarction (MI). Methods and Results: Dach1 was genetically overexpressed in coronary endothelial cells (ECs) in either developing or adult hearts using ApjCreER. This increased the length and number of arterial end branches expanded arteries during development, in both the heart and retina, by inducing capillary ECs to differentiate and contribute to growing arteries. Single-cell RNA sequencing (scRNAseq) of ECs undergoing Dach1-induced arterial specification indicated that it potentiated normal artery differentiation, rather than functioning as a master regulator of artery cell fate. ScRNAseq also showed that normal arterial differentiation is accompanied by repression of lipid metabolism genes, which were also repressed by Dach1. In adults, Dach1 overexpression did not cause a statistically significant change artery structure prior to injury, but increased the number of perfused arteries in the injury zone post-MI. Conclusions: Our data demonstrate that increasing Dach1 is a novel method for driving artery specification and extending arterial branches, which could be explored as a means of mitigating the effects of CAD.

  View details for DOI 10.1161/CIRCRESAHA.120.318271

  View details for PubMedID 34383559

• Controversies surrounding the origin of hepatocytes in adult livers and the in vitro generation or propagation of hepatocytes. Cellular and molecular gastroenterology and hepatology Qian Pek, N. M., Liu, K. J., Nichane, M. n., Ang, L. T. 2020

  Abstract

  Epithelial cells in the liver (known as hepatocytes) are high-performance engines of myriad metabolic functions and versatile responders to liver injury. As hepatocytes metabolize amino acids, alcohol, drugs and other substrates, they produce and are exposed to a milieu of toxins and harmful byproducts that can damage themselves. In the healthy liver, hepatocytes generally divide slowly. However, after liver injury, hepatocytes can ramp up proliferation to regenerate the liver. Yet, upon extensive injury, regeneration falters and liver failure ensues. It is therefore critical to understand the mechanisms underlying liver regeneration, and in particular, which liver cells are mobilized during liver maintenance and repair. Controversies continue to surround the very existence of hepatic stem cells and, if they exist, their spatial location, multipotency, degree of contribution to regeneration, ploidy, and susceptibility to tumorigenesis. This review discuss these controversies. Finally, we highlight how insights into hepatocyte regeneration and biology in vivo can inform in vitro studies to propagate primary hepatocytes with signals in liver regeneration and to generate hepatocytes de novo from pluripotent stem cells.

  View details for DOI 10.1016/j.jcmgh.2020.09.016

  View details for PubMedID 32992051

• Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells. Journal of visualized experiments : JoVE Loh, K. M., Palaria, A., Ang, L. T. 2019

  Abstract

  The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure-which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults-is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

  View details for DOI 10.3791/58975

  View details for PubMedID 31259908

• A critical look: Challenges in differentiating human pluripotent stem cells into desired cell types and organoids. Wiley interdisciplinary reviews. Developmental biology Fowler, J. L., Ang, L. T., Loh, K. M. 2019: e368

  Abstract

  Too many choices can be problematic. This is certainly the case for human pluripotent stem cells (hPSCs): they harbor the potential to differentiate into hundreds of cell types; yet it is highly challenging to exclusively differentiate hPSCs into a single desired cell type. This review focuses on unresolved and fundamental questions regarding hPSC differentiation and critiquing the identity and purity of the resultant cell populations. These are timely issues in view of the fact that hPSC-derived cell populations have or are being transplanted into patients in over 30 ongoing clinical trials. While many in vitro differentiation protocols purport to "mimic development," the exact number and identity of intermediate steps that a pluripotent cell takes to differentiate into a given cell type in vivo remains largely unknown. Consequently, most differentiation efforts inevitably generate a heterogeneous cellular population, as revealed by single-cell RNA-sequencing and other analyses. The presence of unwanted cell types in differentiated hPSC populations does not portend well for transplantation therapies. This provides an impetus to precisely control differentiation to desired ends-for instance, by logically blocking the formation of unwanted cell types or by overexpressing lineage-specifying transcription factors-or by harnessing technologies to selectively purify desired cell types. Conversely, approaches to differentiate three-dimensional "organoids" from hPSCs intentionally generate heterogeneous cell populations. While this is intended to mimic the rich cellular diversity of developing tissues, whether all such organoids are spatially organized in a manner akin to native organs (and thus, whether they fully qualify as organoids) remains to be fully resolved. This article is categorized under: Adult Stem Cells > Tissue Renewal > Regeneration: Stem Cell Differentiation and Reversion Gene Expression > Transcriptional Hierarchies: Cellular Differentiation Early Embryonic Development: Gastrulation and Neurulation.

  View details for DOI 10.1002/wdev.368

  View details for PubMedID 31746148

• A Roadmap for Human Liver Differentiation from Pluripotent Stem Cells CELL REPORTS Ang, L., Tan, A., Autio, M. I., Goh, S., Choo, S., Lee, K., Tan, J., Pan, B., Lee, J., Lum, J., Lim, C., Yeo, I., Wong, C., Liu, M., Oh, J., Chia, C., Loh, C., Chen, A., Chen, Q., Weissman, I. L., Loh, K. M., Lim, B. 2018; 22 (8): 2190–2205

  Abstract

  How are closely related lineages, including liver, pancreas, and intestines, diversified from a common endodermal origin? Here, we apply principles learned from developmental biology to rapidly reconstitute liver progenitors from human pluripotent stem cells (hPSCs). Mapping the formation of multiple endodermal lineages revealed how alternate endodermal fates (e.g., pancreas and intestines) are restricted during liver commitment. Human liver fate was encoded by combinations of inductive and repressive extracellular signals at different doses. However, these signaling combinations were temporally re-interpreted: cellular competence to respond to retinoid, WNT, TGF-β, and other signals sharply changed within 24 hr. Consequently, temporally dynamic manipulation of extracellular signals was imperative to suppress the production of unwanted cell fates across six consecutive developmental junctures. This efficiently generated 94.1% ± 7.35% TBX3+HNF4A+ human liver bud progenitors and 81.5% ± 3.2% FAH+ hepatocyte-like cells by days 6 and 18 of hPSC differentiation, respectively; the latter improved short-term survival in the Fah-/-Rag2-/-Il2rg-/- mouse model of liver failure.

  View details for PubMedID 29466743

• Isolation and 3D expansion of multipotent Sox9+ mouse lung progenitors. Nature methods Nichane, M., Javed, A., Sivakamasundari, V., Ganesan, M., Ang, L. T., Kraus, P., Lufkin, T., Loh, K. M., Lim, B. 2017; 14 (12): 1205-1212

  Abstract

  Multiple adult tissues are maintained by stem cells of restricted developmental potential which can only form a subset of lineages within the tissue. For instance, the two adult lung epithelial compartments (airways and alveoli) are separately maintained by distinct lineage-restricted stem cells. A challenge has been to obtain multipotent stem cells and/or progenitors that can generate all epithelial cell types of a given tissue. Here we show that mouse Sox9+ multipotent embryonic lung progenitors can be isolated and expanded long term in 3D culture. Cultured Sox9+ progenitors transcriptionally resemble their in vivo counterparts and generate both airway and alveolar cell types in vitro. Sox9+ progenitors that were transplanted into injured adult mouse lungs differentiated into all major airway and alveolar lineages in vivo in a region-appropriate fashion. We propose that a single expandable embryonic lung progenitor population with broader developmental competence may eventually be used as an alternative for region-restricted adult tissue stem cells in regenerative medicine.

  View details for DOI 10.1038/nmeth.4498

  View details for PubMedID 29106405

• Evaluating the regenerative potential and functionality of human liver cells in mice DIFFERENTIATION Tan, A., Loh, K. M., Ang, L. 2017; 98: 25–34

  Abstract

  Liver diseases afflict millions of patients worldwide. Currently, the only long-term treatment for liver failure is the transplantation of a new liver. However, intravenously transplanting a suspension of human hepatocytes might be a less-invasive approach to partially reconstitute lost liver functions in human patients as evinced by promising outcomes in clinical trials. The purpose of this essay is to emphasize outstanding questions that continue to surround hepatocyte transplantation. While adult primary human hepatocytes are the gold standard for transplantation, hepatocytes are heterogeneous. Whether all hepatocytes engraft equally and what specifically defines an "engraftable" hepatocyte capable of long-term liver reconstitution remains unclear. To this end, mouse models of liver injury enable the evaluation of human hepatocytes and their behavior upon transplantation into a complex injured liver environment. While mouse models may not be fully representative of the injured human liver and human hepatocytes tend to engraft mice less efficiently than mouse hepatocytes, valuable lessons have nonetheless been learned from transplanting human hepatocytes into mouse models. With an eye to the future, it will be crucial to eventually detail the optimal biological source (whether in vivo- or in vitro-derived) and presumptive heterogeneity of human hepatocytes and to understand the mechanisms through which they engraft and regenerate liver tissue in vivo.

  View details for PubMedID 29078082

• An atlas of transcriptional, chromatin accessibility, and surface marker changes in human mesoderm development SCIENTIFIC DATA Koh, P. W., Sinha, R., Barkal, A. A., Morganti, R. M., Chen, A., Weissman, I. L., Ang, L. T., Kundaje, A., Loh, K. M. 2016; 3

  Abstract

  Mesoderm is the developmental precursor to myriad human tissues including bone, heart, and skeletal muscle. Unravelling the molecular events through which these lineages become diversified from one another is integral to developmental biology and understanding changes in cellular fate. To this end, we developed an in vitro system to differentiate human pluripotent stem cells through primitive streak intermediates into paraxial mesoderm and its derivatives (somites, sclerotome, dermomyotome) and separately, into lateral mesoderm and its derivatives (cardiac mesoderm). Whole-population and single-cell analyses of these purified populations of human mesoderm lineages through RNA-seq, ATAC-seq, and high-throughput surface marker screens illustrated how transcriptional changes co-occur with changes in open chromatin and surface marker landscapes throughout human mesoderm development. This molecular atlas will facilitate study of human mesoderm development (which cannot be interrogated in vivo due to restrictions on human embryo studies) and provides a broad resource for the study of gene regulation in development at the single-cell level, knowledge that might one day be exploited for regenerative medicine.

  View details for DOI 10.1038/sdata.2016.109

  View details for PubMedID 27996962

• Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types CELL Loh, K. M., Chen, A., Koh, P. W., Deng, T. Z., Sinha, R., Tsai, J. M., Barkal, A. A., Shen, K. Y., Jain, R., Morganti, R. M., Shyh-Chang, N., Fernhoff, N. B., George, B. M., Wernig, G., Salomon, R. E., Chen, Z., Vogel, H., Epstein, J. A., Kundaje, A., Talbot, W. S., Beachy, P. A., Ang, L. T., Weissman, I. L. 2016; 166 (2): 451-467

  Abstract

  Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence, comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end, here, we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%-99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively, this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. VIDEO ABSTRACT.

  View details for DOI 10.1016/j.cell.2016.06.011

  View details for PubMedID 27419872

• Ex uno plures: molecular designs for embryonic pluripotency. Physiological reviews Loh, K. M., Lim, B., Ang, L. T. 2015; 95 (1): 245-295

  Abstract

  Pluripotent cells in embryos are situated near the apex of the hierarchy of developmental potential. They are capable of generating all cell types of the mammalian body proper. Therefore, they are the exemplar of stem cells. In vivo, pluripotent cells exist transiently and become expended within a few days of their establishment. Yet, when explanted into artificial culture conditions, they can be indefinitely propagated in vitro as pluripotent stem cell lines. A host of transcription factors and regulatory genes are now known to underpin the pluripotent state. Nonetheless, how pluripotent cells are equipped with their vast multilineage differentiation potential remains elusive. Consensus holds that pluripotency transcription factors prevent differentiation by inhibiting the expression of differentiation genes. However, this does not explain the developmental potential of pluripotent cells. We have presented another emergent perspective, namely, that pluripotency factors function as lineage specifiers that enable pluripotent cells to differentiate into specific lineages, therefore endowing pluripotent cells with their multilineage potential. Here we provide a comprehensive overview of the developmental biology, transcription factors, and extrinsic signaling associated with pluripotent cells, and their accompanying subtypes, in vitro heterogeneity and chromatin states. Although much has been learned since the appreciation of mammalian pluripotency in the 1950s and the derivation of embryonic stem cell lines in 1981, we will specifically emphasize what currently remains unclear. However, the view that pluripotency factors capacitate differentiation, recently corroborated by experimental evidence, might perhaps address the long-standing question of how pluripotent cells are endowed with their multilineage differentiation potential.

  View details for DOI 10.1152/physrev.00001.2014

  View details for PubMedID 25540144

• Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations. Cell stem cell Loh, 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., Tan, J., Noghabi, M. S., Azzola, L., Ng, E. S., Durruthy-Durruthy, J., Sebastiano, V., Poellinger, L., Elefanty, A. G., Stanley, E. G., Chen, Q., Prabhakar, S., Weissman, I. L., Lim, B. 2014; 14 (2): 237-252

  Abstract

  Human pluripotent stem cell (hPSC) differentiation typically yields heterogeneous populations. Knowledge of signals controlling embryonic lineage bifurcations could efficiently yield desired cell types through exclusion of alternate fates. Therefore, we revisited signals driving induction and anterior-posterior patterning of definitive endoderm to generate a coherent roadmap for endoderm differentiation. With striking temporal dynamics, BMP and Wnt initially specified anterior primitive streak (progenitor to endoderm), yet, 24 hr later, suppressed endoderm and induced mesoderm. At lineage bifurcations, cross-repressive signals separated mutually exclusive fates; TGF-β and BMP/MAPK respectively induced pancreas versus liver from endoderm by suppressing the alternate lineage. We systematically blockaded alternate fates throughout multiple consecutive bifurcations, thereby efficiently differentiating multiple hPSC lines exclusively into endoderm and its derivatives. Comprehensive transcriptional and chromatin mapping of highly pure endodermal populations revealed that endodermal enhancers existed in a surprising diversity of "pre-enhancer" states before activation, reflecting the establishment of a permissive chromatin landscape as a prelude to differentiation.

  View details for DOI 10.1016/j.stem.2013.12.007

  View details for PubMedID 24412311

• Pluripotency factors regulate definitive endoderm specification through eomesodermin GENES & DEVELOPMENT Teo, A., Arnold, S. J., Trotter, M. B., Brown, S., Ang, L., Chng, Z., Robertson, E. J., Dunn, N., Vallier, L. 2011; 25 (3): 238-250

  Abstract

  Understanding the molecular mechanisms controlling early cell fate decisions in mammals is a major objective toward the development of robust methods for the differentiation of human pluripotent stem cells into clinically relevant cell types. Here, we used human embryonic stem cells and mouse epiblast stem cells to study specification of definitive endoderm in vitro. Using a combination of whole-genome expression and chromatin immunoprecipitation (ChIP) deep sequencing (ChIP-seq) analyses, we established an hierarchy of transcription factors regulating endoderm specification. Importantly, the pluripotency factors NANOG, OCT4, and SOX2 have an essential function in this network by actively directing differentiation. Indeed, these transcription factors control the expression of EOMESODERMIN (EOMES), which marks the onset of endoderm specification. In turn, EOMES interacts with SMAD2/3 to initiate the transcriptional network governing endoderm formation. Together, these results provide for the first time a comprehensive molecular model connecting the transition from pluripotency to endoderm specification during mammalian development.

  View details for DOI 10.1101/gad.607311

  View details for Web of Science ID 000286880600006

  View details for PubMedID 21245162

  View details for PubMedCentralID PMC3034899