Contributions of Mammalian Chimeras to Pluripotent Stem Cell Research.

Chimeras are widely acknowledged as the gold standard for assessing stem cell pluripotency, based on their capacity to test donor cell lineage potential in the context of an organized, normally developing tissue. Experimental chimeras provide key insights into mammalian developmental mechanisms and offer a resource for interrogating the fate potential of various pluripotent stem cell states. We highlight the applications and current limitations presented by intra- and inter-species chimeras and consider their future contribution to the stem cell field. Despite the technical and ethical demands of experimental chimeras, including human-interspecies chimeras, they are a provocative resource for achieving regenerative medicine goals.British Heart Foundation Centre of Regenerative Medicine, Wellcome Trust, Medical Research Council Cambridge Stem Cell Institute, Cambridge NIHR Biomedical Research CentreThis is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.stem.2016.07.01

Human-Mouse Chimerism Validates Human Stem Cell Pluripotency.

Pluripotent stem cells are defined by their capacity to differentiate into all three tissue layers that comprise the body. Chimera formation, generated by stem cell transplantation to the embryo, is a stringent assessment of stem cell pluripotency. However, the ability of human pluripotent stem cells (hPSCs) to form embryonic chimeras remains in question. Here we show using a stage-matching approach that human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) have the capacity to participate in normal mouse development when transplanted into gastrula-stage embryos, providing in vivo functional validation of hPSC pluripotency. hiPSCs and hESCs form interspecies chimeras with high efficiency, colonize the embryo in a manner predicted from classical developmental fate mapping, and differentiate into each of the three primary tissue layers. This faithful recapitulation of tissue-specific fate post-transplantation underscores the functional potential of hPSCs and provides evidence that human-mouse interspecies developmental competency can occur.This work was supported by National Institutes of Health grant No. 1R21ID012228 (R.A.P.); Medical Research Council/British Heart Foundation grant No. G1000847 (R.A.P.); British Heart Foundation Ph.D. studentship (V.L.M.); British Heart Foundation Centre of Regenerative Medicine (Oxford grant RM/13/3/3015); core support from the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute; and the Cambridge NIHR Biomedical Research Centre.This is the final version of the article. It first appeared from Cell Press via http://dx.doi.org/10.1016/j.stem.2015.11.01

Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells.

The epicardium has emerged as a multipotent cardiovascular progenitor source with therapeutic potential for coronary smooth muscle cell, cardiac fibroblast (CF) and cardiomyocyte regeneration, owing to its fundamental role in heart development and its potential ability to initiate myocardial repair in injured adult tissues. Here, we describe a chemically defined method for generating epicardium and epicardium-derived smooth muscle cells (EPI-SMCs) and CFs from human pluripotent stem cells (HPSCs) through an intermediate lateral plate mesoderm (LM) stage. HPSCs were initially differentiated to LM in the presence of FGF2 and high levels of BMP4. The LM was robustly differentiated to an epicardial lineage by activation of WNT, BMP and retinoic acid signalling pathways. HPSC-derived epicardium displayed enhanced expression of epithelial- and epicardium-specific markers, exhibited morphological features comparable with human foetal epicardial explants and engrafted in the subepicardial space in vivo. The in vitro-derived epicardial cells underwent an epithelial-to-mesenchymal transition when treated with PDGF-BB and TGFβ1, resulting in vascular SMCs that displayed contractile ability in response to vasoconstrictors. Furthermore, the EPI-SMCs displayed low density lipoprotein uptake and effective lowering of lipoprotein levels upon treatment with statins, similar to primary human coronary artery SMCs. Cumulatively, these findings suggest that HPSC-derived epicardium and EPI-SMCs could serve as important tools for studying human cardiogenesis, and as a platform for vascular disease modelling and drug screening.This work was supported by the British Heart Foundation (BHF) [NH/11/1/28922], by the UK Medical Research Council and BHF [G1000847 to S.S. and R.A.P.), and by Cambridge Hospitals National Institute for Health Research Biomedical Research Centre funding (S.S. and R.A.P.). D.I. is on a University of Cambridge Commonwealth Scholarship. S.S. and F.S. are supported by the BHF [FS/13/29/ 30024]. L.G. is supported by the BHF [RM/l3/3/30159]. W.G.B. and V.L.M. are supported by BHF PhD studentships [FS/11/77/29327 and FS/10/48/28674].This is the final published version. It first appeared at http://dev.biologists.org/content/early/2015/03/25/dev.119271.abstract

NANOG and CDX2 pattern distinct subtypes of human mesoderm during exit from pluripotency

SummaryMesoderm is induced at the primitive streak (PS) and patterns subsequently into mesodermal subtypes and organ precursors. It is unclear whether mesoderm induction generates a multipotent PS progenitor or several distinct ones with restricted subtype potentials. We induced mesoderm in human pluripotent stem cells with ACTIVIN and BMP or with GSK3-β inhibition. Both approaches induced BRACHYURY+ mesoderm of distinct PS-like identities, which had differing patterning potential. ACTIVIN and BMP-induced mesoderm patterned into cardiac but not somitic subtypes. Conversely, PS precursors induced by GSK3-β inhibition did not generate lateral plate and cardiac mesoderm and favored instead somitic differentiation. The mechanism of these cell fate decisions involved mutual repression of NANOG and CDX2. Although NANOG was required for cardiac specification but blocked somitic subtypes, CDX2 was required for somitic mesoderm but blocked cardiac differentiation. In sum, rather than forming a common PS progenitor, separate induction mechanisms distinguish human mesoderm subtypes

Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells

This is the final published version. It first appeared at http://dev.biologists.org/content/early/2015/03/25/dev.119271.abstract.The epicardium has emerged as a multipotent cardiovascular progenitor source with therapeutic potential for coronary smooth muscle cell, cardiac fibroblast (CF) and cardiomyocyte regeneration, owing to its fundamental role in heart development and its potential ability to initiate myocardial repair in injured adult tissues. Here, we describe a chemically defined method for generating epicardium and epicardium-derived smooth muscle cells (EPI-SMCs) and CFs from human pluripotent stem cells (HPSCs) through an intermediate lateral plate mesoderm (LM) stage. HPSCs were initially differentiated to LM in the presence of FGF2 and high levels of BMP4. The LM was robustly differentiated to an epicardial lineage by activation of WNT, BMP and retinoic acid signalling pathways. HPSC-derived epicardium displayed enhanced expression of epithelial- and epicardium-specific markers, exhibited morphological features comparable with human foetal epicardial explants and engrafted in the subepicardial space in vivo. The in vitro-derived epicardial cells underwent an epithelial-to-mesenchymal transition when treated with PDGF-BB and TGF?1, resulting in vascular SMCs that displayed contractile ability in response to vasoconstrictors. Furthermore, the EPI-SMCs displayed low density lipoprotein uptake and effective lowering of lipoprotein levels upon treatment with statins, similar to primary human coronary artery SMCs. Cumulatively, these findings suggest that HPSC-derived epicardium and EPI-SMCs could serve as important tools for studying human cardiogenesis, and as a platform for vascular disease modelling and drug screening.This work was supported by the British Heart Foundation (BHF) [NH/11/1/28922], by\ud the UK Medical Research Council and BHF [G1000847 to S.S. and R.A.P.), and by\ud Cambridge Hospitals National Institute for Health Research Biomedical Research\ud Centre funding (S.S. and R.A.P.). D.I. is on a University of Cambridge\ud Commonwealth Scholarship. S.S. and F.S. are supported by the BHF [FS/13/29/\ud 30024]. L.G. is supported by the BHF [RM/l3/3/30159]. W.G.B. and V.L.M. are\ud supported by BHF PhD studentships [FS/11/77/29327 and FS/10/48/28674]

Brachyury and SMAD signalling collaboratively orchestrate distinct mesoderm and endoderm gene regulatory networks in differentiating human embryonic stem cells

The transcription factor brachyury (T, BRA) is one of the first markers of gastrulation and lineage specification in vertebrates. Despite its wide use and importance in stem cell and developmental biology, its functional genomic targets in human cells are largely unknown. Here, we use differentiating human embryonic stem cells to study the role of BRA in activin A-induced endoderm and BMP4-induced mesoderm progenitors. We show that BRA has distinct genome-wide binding landscapes in these two cell populations, and that BRA interacts and collaborates with SMAD1 or SMAD2/3 signalling to regulate the expression of its target genes in a cell-specific manner. Importantly, by manipulating the levels of BRA in cells exposed to different signalling environments, we demonstrate that BRA is essential for mesoderm but not for endoderm formation. Together, our data illuminate the function of BRA in the context of human embryonic development and show that the regulatory role of BRA is context dependent. Our study reinforces the importance of analysing the functions of a transcription factor in different cellular and signalling environments

Retinoic acid signaling modulation guides in vitro specification of human heart field-specific progenitor pools.

Acknowledgements: We would like to acknowledge Birgit Campbell, Christina Scherb, and Marco Crovella for their technical assistance, Gabrielle Lederer (Cytogenetic Department, TUM) for karyotyping, Dr. Rupert Öllinger (TUM, Germany) for sequencing, Dr. David Elliott for sharing the ES03 and ES03-NKX2.5eGFP cell lines, Drs. Ed Stanley and Andrew Elefanty (MCRI, Australia) for advice in construct design and gene targeting, and Dr. Sasha Mendjan for advice and discussion. This work was supported by the European Research Council (ERC) (grant 788381 to A.Mo. and grant 261053 to K-.L.L.), the Else-Kroener-Fresenius Stiftung (EKFS, to A.G.), the German Research Foundation (grant GO3220/1-1 to A.G.; Transregio Research Unit 152 to A.Mo. and K-.L.L.; Transregio Research Unit 267 to A.Mo., K-.L.L., and P.G.), the German Centre for Cardiovascular Research (DZHK) (grant FKZ 81Z0600601 to A.Mo. and K-.L.L.; grant 81X3600607 to J.K.), the Fondazione Umberto Veronesi (to G.S.).Funder: German Centre for Cardiovascular ReserachFunder: Else Kröner-Fresenius-Stiftung (Else Kroner-Fresenius Foundation); doi: https://doi.org/10.13039/501100003042Cardiogenesis relies on the precise spatiotemporal coordination of multiple progenitor populations. Understanding the specification and differentiation of these distinct progenitor pools during human embryonic development is crucial for advancing our knowledge of congenital cardiac malformations and designing new regenerative therapies. By combining genetic labelling, single-cell transcriptomics, and ex vivo human-mouse embryonic chimeras we uncovered that modulation of retinoic acid signaling instructs human pluripotent stem cells to form heart field-specific progenitors with distinct fate potentials. In addition to the classical first and second heart fields, we observed the appearance of juxta-cardiac field progenitors giving rise to both myocardial and epicardial cells. Applying these findings to stem-cell based disease modelling we identified specific transcriptional dysregulation in first and second heart field progenitors derived from stem cells of patients with hypoplastic left heart syndrome. This highlights the suitability of our in vitro differentiation platform for studying human cardiac development and disease

Retinoic acid signaling modulation guides in vitro specification of human heart field-specific progenitor pools.

Cardiogenesis relies on the precise spatiotemporal coordination of multiple progenitor populations. Understanding the specification and differentiation of these distinct progenitor pools during human embryonic development is crucial for advancing our knowledge of congenital cardiac malformations and designing new regenerative therapies. By combining genetic labelling, single-cell transcriptomics, and ex vivo human-mouse embryonic chimeras we uncovered that modulation of retinoic acid signaling instructs human pluripotent stem cells to form heart field-specific progenitors with distinct fate potentials. In addition to the classical first and second heart fields, we observed the appearance of juxta-cardiac field progenitors giving rise to both myocardial and epicardial cells. Applying these findings to stem-cell based disease modelling we identified specific transcriptional dysregulation in first and second heart field progenitors derived from stem cells of patients with hypoplastic left heart syndrome. This highlights the suitability of our in vitro differentiation platform for studying human cardiac development and disease