Roger Pedersen

All Publications

• Allele-specific RNA-seq expression profiling of imprinted genes in mouse isogenic pluripotent states. Epigenetics & chromatin Dirks, R. A., van Mierlo, G., Kerstens, H. H., Bernardo, A. S., Kobolák, J., Bock, I., Maruotti, J., Pedersen, R. A., Dinnyés, A., Huynen, M. A., Jouneau, A., Marks, H. 2019; 12 (1): 14

  Abstract

  Genomic imprinting, resulting in parent-of-origin specific gene expression, plays a critical role in mammalian development. Here, we apply allele-specific RNA-seq on isogenic B6D2F1 mice to assay imprinted genes in tissues from early embryonic tissues between E3.5 and E7.25 and in pluripotent cell lines to evaluate maintenance of imprinted gene expression. For the cell lines, we include embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) derived from fertilized embryos and from embryos obtained after nuclear transfer (NT) or parthenogenetic activation (PGA).As homozygous genomic regions of PGA-derived cells are not compatible with allele-specific RNA-seq, we developed an RNA-seq-based genotyping strategy allowing identification of informative heterozygous regions. Global analysis shows that proper imprinted gene expression as observed in embryonic tissues is largely lost in the ESC lines included in this study, which mainly consisted of female ESCs. Differentiation of ESC lines to embryoid bodies or NPCs does not restore monoallelic expression of imprinted genes, neither did reprogramming of the serum-cultured ESCs to the pluripotent ground state by the use of 2 kinase inhibitors. Fertilized EpiSC and EpiSC-NT lines largely maintain imprinted gene expression, as did EpiSC-PGA lines that show known paternally expressed genes being silent and known maternally expressed genes consistently showing doubled expression. Notably, two EpiSC-NT lines show aberrant silencing of Rian and Meg3, two critically imprinted genes in mouse iPSCs. With respect to female EpiSC, most of the lines displayed completely skewed X inactivation suggesting a (near) clonal origin.Altogether, our analysis provides a comprehensive overview of imprinted gene expression in pluripotency and provides a benchmark to allow identification of cell lines that faithfully maintain imprinted gene expression and therefore retain full developmental potential.

  View details for DOI 10.1186/s13072-019-0259-8

  View details for PubMedID 30767785

  View details for PubMedCentralID PMC6376749

• A novel piperidine identified by stem cell-based screening attenuates pulmonary arterial hypertension by regulating BMP2 and PTGS2 levels. The European respiratory journal Xing, Y., Zhao, S., Wei, Q., Gong, S., Zhao, X., Zhou, F., Ai-Lamki, R., Ortmann, D., Du, M., Pedersen, R., Shang, G., Si, S., Morrell, N. W., Yang, J. 2018; 51 (4)

  Abstract

  Genetic defects in bone morphogenetic protein type II receptor (BMPRII) signalling and inflammation contribute to the pathogenesis of pulmonary arterial hypertension (PAH). The receptor is activated by bone morphogenetic protein (BMP) ligands, which also enhance BMPR2 transcription. A small-molecule BMP upregulator with selectivity on vascular endothelium would be a desirable therapeutic intervention for PAH.We assayed compounds identified in the screening of BMP2 upregulators for their ability to increase the expression of inhibitor of DNA binding 1 (Id1), using a dual reporter driven specifically in human embryonic stem cell-derived endothelial cells. These assays identified a novel piperidine, BMP upregulator 1 (BUR1), that increased endothelial Id1 expression with a half-maximal effective concentration of 0.098??mol·L-1 Microarray analyses and immunoblotting showed that BUR1 induced BMP2 and prostaglandin-endoperoxide synthase 2 (PTGS2) expression. BUR1 effectively rescued deficient angiogenesis in autologous BMPR2+/R899X endothelial cells generated by CRISPR/Cas9 and patient cells.BUR1 prevented and reversed PAH in monocrotaline rats, and restored BMPRII downstream signalling and modulated the arachidonic acid pathway in the pulmonary arterial endothelium in the Sugen 5416/hypoxia PAH mouse model.In conclusion, using stem cell technology we have provided a novel small-molecule compound which regulates BMP2 and PTGS2 levels that might be useful for the treatment of PAH.

  View details for DOI 10.1183/13993003.02229-2017

  View details for PubMedID 29449428

• Mammalian embryo comparison identifies novel pluripotency genes associated with the naïve or primed state. Biology open Bernardo, A. S., Jouneau, A., Marks, H., Kensche, P., Kobolak, J., Freude, K., Hall, V., Feher, A., Polgar, Z., Sartori, C., Bock, I., Louet, C., Faial, T., Kerstens, H. H., Bouissou, C., Parsonage, G., Mashayekhi, K., Smith, J. C., Lazzari, G., Hyttel, P., Stunnenberg, H. G., Huynen, M., Pedersen, R. A., Dinnyes, A. 2018; 7 (8)

  Abstract

  During early mammalian development, transient pools of pluripotent cells emerge that can be immortalised upon stem cell derivation. The pluripotent state, 'naïve' or 'primed', depends on the embryonic stage and derivation conditions used. Here we analyse the temporal gene expression patterns of mouse, cattle and porcine embryos at stages that harbour different types of pluripotent cells. We document conserved and divergent traits in gene expression, and identify predictor genes shared across the species that are associated with pluripotent states in vivo and in vitro Amongst these are the pluripotency-linked genes Klf4 and Lin28b The novel genes discovered include naïve- (Spic, Scpep1 and Gjb5) and primed-associated (Sema6a and Jakmip2) genes as well as naïve to primed transition genes (Dusp6 and Trip6). Both Gjb5 and Dusp6 play a role in pluripotency since their knockdown results in differentiation and downregulation of key pluripotency genes. Our interspecies comparison revealed new insights of pluripotency, pluripotent stem cell identity and a new molecular criterion for distinguishing between pluripotent states in various species, including human.

  View details for DOI 10.1242/bio.033282

  View details for PubMedID 30026265

• Inducible and Deterministic Forward Programming of Human Pluripotent Stem Cells into Neurons, Skeletal Myocytes, and Oligodendrocytes STEM CELL REPORTS Pawlowski, M., Ortmann, D., Bertero, A., Tavares, J. M., Pedersen, R. A., Vallier, L., Kotter, M. R. 2017; 8 (4): 803-812

  Abstract

  The isolation or in vitro derivation of many human cell types remains challenging and inefficient. Direct conversion of human pluripotent stem cells (hPSCs) by forced expression of transcription factors provides a potential alternative. However, deficient inducible gene expression in hPSCs has compromised efficiencies of forward programming approaches. We have systematically optimized inducible gene expression in hPSCs using a dual genomic safe harbor gene-targeting strategy. This approach provides a powerful platform for the generation of human cell types by forward programming. We report robust and deterministic reprogramming of hPSCs into neurons and functional skeletal myocytes. Finally, we present a forward programming strategy for rapid and highly efficient generation of human oligodendrocytes.

  View details for DOI 10.1016/j.stemcr.2017.02.016

  View details for Web of Science ID 000401129100001

  View details for PubMedID 28344001

  View details for PubMedCentralID PMC5390118

• Roles of H3K27me2 and H3K27me3 Examined during Fate Specification of Embryonic Stem Cells CELL REPORTS Juan, A. H., Wang, S., Ko, K. D., Zare, H., Tsai, P., Feng, X., Vivanco, K. O., Ascoli, A. M., Gutierrez-Cruz, G., Krebs, J., Sidoli, S., Knight, A. L., Pedersen, R. A., Garcia, B. A., Casellas, R., Zou, J., Sartorelli, V. 2016; 17 (5): 1369-1382

  Abstract

  The polycomb repressive complex 2 (PRC2) methylates lysine 27 of histone H3 (H3K27) through its catalytic subunit Ezh2. PRC2-mediated di- and tri-methylation (H3K27me2/H3K27me3) have been interchangeably associated with gene repression. However, it remains unclear whether these two degrees of H3K27 methylation have different functions. In this study, we have generated isogenic mouse embryonic stem cells (ESCs) with a modified H3K27me2/H3K27me3 ratio. Our findings document dynamic developmental control in the genomic distribution of H3K27me2 and H3K27me3 at regulatory regions in ESCs. They also reveal that modifying the ratio of H3K27me2 and H3K27me3 is sufficient for the acquisition and repression of defined cell lineage transcriptional programs and phenotypes and influences induction of the ESC ground state.

  View details for DOI 10.1016/j.celrep.2016.09.087

  View details for Web of Science ID 000386527100015

  View details for PubMedID 27783950

  View details for PubMedCentralID PMC5123747

• Contributions of Mammalian Chimeras to Pluripotent Stem Cell Research CELL STEM CELL Mascetti, V. L., Pedersen, R. A. 2016; 19 (2): 163-175

  Abstract

  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.

  View details for DOI 10.1016/j.stem.2016.07.018

  View details for Web of Science ID 000381622000011

  View details for PubMedID 27494674

  View details for PubMedCentralID PMC5366358

• Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming NATURE COMMUNICATIONS Moreau, T., Evans, A. L., Vasquez, L., Tijssen, M. R., Yan, Y., Trotter, M. W., Howard, D., Colzani, M., Arumugam, M., Wu, W. H., Dalby, A., Lampela, R., Bouet, G., Hobbs, C. M., Pask, D. C., Payne, H., Ponomaryov, T., Brill, A., Soranzo, N., Ouwehand, W. H., Pedersen, R. A., Ghevaert, C. 2016; 7

  Abstract

  The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.

  View details for DOI 10.1038/ncomms11208

  View details for Web of Science ID 000373826000001

  View details for PubMedID 27052461

  View details for PubMedCentralID PMC4829662

• Human-Mouse Chimerism Validates Human Stem Cell Pluripotency CELL STEM CELL Mascetti, V. L., Pedersen, R. A. 2016; 18 (1): 67-72

  Abstract

  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.

  View details for DOI 10.1016/j.stem.2015.11.017

  View details for Web of Science ID 000372321300012

  View details for PubMedID 26712580

  View details for PubMedCentralID PMC4712187

• Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus. Methods in molecular biology (Clifton, N.J.) Soares, F. A., Pedersen, R. A., Vallier, L. 2016; 1357: 23-31

  Abstract

  This protocol describes the efficient isolation of peripheral blood mononuclear cells from circulating blood via density gradient centrifugation and subsequent generation of integration-free human induced pluripotent stem cells. Peripheral blood mononuclear cells are cultured for 9 days to allow expansion of the erythroblast population. The erythroblasts are then used to derive human induced pluripotent stem cells using Sendai viral vectors, each expressing one of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc.

  View details for DOI 10.1007/7651_2015_202

  View details for PubMedID 25687300

• Activin/Nodal Signaling Supports Retinal Progenitor Specification in a Narrow Time Window during Pluripotent Stem Cell Neuralization STEM CELL REPORTS Bertacchi, M., Lupo, G., Pandolfini, L., Casarosa, S., D'Onofrio, M., Pedersen, R. A., Harris, W. A., Cremisi, F. 2015; 5 (4): 532-545

  Abstract

  Retinal progenitors are initially found in the anterior neural plate region known as the eye field, whereas neighboring areas undertake telencephalic or hypothalamic development. Eye field cells become specified by switching on a network of eye field transcription factors, but the extracellular cues activating this network remain unclear. In this study, we used chemically defined media to induce in vitro differentiation of mouse embryonic stem cells (ESCs) toward eye field fates. Inhibition of Wnt/?-catenin signaling was sufficient to drive ESCs to telencephalic, but not retinal, fates. Instead, retinal progenitors could be generated from competent differentiating mouse ESCs by activation of Activin/Nodal signaling within a narrow temporal window corresponding to the emergence of primitive anterior neural progenitors. Activin also promoted eye field gene expression in differentiating human ESCs. Our results reveal insights into the mechanisms of eye field specification and open new avenues toward the generation of retinal progenitors for translational medicine.

  View details for DOI 10.1016/j.stemcr.2015.08.011

  View details for Web of Science ID 000364990900008

  View details for PubMedID 26388287

  View details for PubMedCentralID PMC4624997

• Brachyury and SMAD signalling collaboratively orchestrate distinct mesoderm and endoderm gene regulatory networks in differentiating human embryonic stem cells DEVELOPMENT Faial, T., Bernardo, A. S., Mendjan, S., Diamanti, E., Ortmann, D., Gentsch, G. E., Mascetti, V. L., Trotter, M. W., Smith, J. C., Pedersen, R. A. 2015; 142 (12): 2121-?

  Abstract

  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.

  View details for DOI 10.1242/dev.117838

  View details for Web of Science ID 000357122600006

  View details for PubMedID 26015544

  View details for PubMedCentralID PMC4483767

• Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells DEVELOPMENT Iyer, D., Gambardella, L., Bernard, W. G., Serrano, F., Mascetti, V. L., Pedersen, R. A., Talasila, A., Sinha, S. 2015; 142 (8): 1528-1541

  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.

  View details for DOI 10.1242/dev.119271

  View details for Web of Science ID 000353590900015

  View details for PubMedID 25813541

  View details for PubMedCentralID PMC4392600

• Activin/Nodal signaling and NANOG orchestrate human embryonic stem cell fate decisions by controlling the H3K4me3 chromatin mark GENES & DEVELOPMENT Bertero, A., Madrigal, P., Galli, A., Hubner, N. C., Moreno, I., Burks, D., Brown, S., Pedersen, R. A., Gaffney, D., Mendjan, S., Pauklin, S., Vallier, L. 2015; 29 (7): 702-717

  Abstract

  Stem cells can self-renew and differentiate into multiple cell types. These characteristics are maintained by the combination of specific signaling pathways and transcription factors that cooperate to establish a unique epigenetic state. Despite the broad interest of these mechanisms, the precise molecular controls by which extracellular signals organize epigenetic marks to confer multipotency remain to be uncovered. Here, we use human embryonic stem cells (hESCs) to show that the Activin-SMAD2/3 signaling pathway cooperates with the core pluripotency factor NANOG to recruit the DPY30-COMPASS histone modifiers onto key developmental genes. Functional studies demonstrate the importance of these interactions for correct histone 3 Lys4 trimethylation and also self-renewal and differentiation. Finally, genetic studies in mice show that Dpy30 is also necessary to maintain pluripotency in the pregastrulation embryo, thereby confirming the existence of similar regulations in vivo during early embryonic development. Our results reveal the mechanisms by which extracellular factors coordinate chromatin status and cell fate decisions in hESCs.

  View details for DOI 10.1101/gad.255984.114

  View details for Web of Science ID 000352161600004

  View details for PubMedID 25805847

  View details for PubMedCentralID PMC4387713

• An Important Role of Endothelial Hairy-Related Transcription Factors in Mouse Vascular Development GENESIS Morioka, T., Sakabe, M., Ioka, T., Iguchi, T., Mizuta, K., Hattammaru, M., Sakai, C., Itoh, M., Sato, G. E., Hashimoto, A., Fujita, M., Okumura, K., Araki, M., Xin, M., Pedersen, R. A., Utset, M. F., Kimura, H., Nakagawa, O. 2014; 52 (11): 897-906

  Abstract

  The Hairy-related transcription factor family of Notch- and ALK1-downstream transcriptional repressors, called Hrt/Hey/Hesr/Chf/Herp/Gridlock, has complementary and indispensable functions for vascular development. While mouse embryos null for either Hrt1/Hey1 or Hrt2/Hey2 did not show early vascular phenotypes, Hrt1/Hey1; Hrt2/Hey2 double null mice (H1(ko) /H2(ko) ) showed embryonic lethality with severe impairment of vascular morphogenesis. It remained unclear, however, whether Hrt/Hey functions are required in endothelial cells or vascular smooth muscle cells. In this study, we demonstrate that mice with endothelial-specific deletion of Hrt2/Hey2 combined with global Hrt1/Hey1 deletion (H1(ko) /H2(eko) ) show abnormal vascular morphogenesis and embryonic lethality. Their defects were characterized by the failure of vascular network formation in the yolk sac, abnormalities of embryonic vascular structures and impaired smooth muscle cell recruitment, and were virtually identical to the H1(ko) /H2(ko) phenotypes. Among signaling molecules implicated in vascular development, Robo4 expression was significantly increased and activation of Src family kinases was suppressed in endothelial cells of H1(ko) /H2(eko) embryos. The present study indicates an important role of Hrt1/Hey1 and Hrt2/Hey2 in endothelial cells during early vascular development, and further suggests involvement of Robo4 and Src family kinases in the mechanisms of embryonic vascular defects caused by the Hrt/Hey deficiency.

  View details for DOI 10.1002/dvg.22825

  View details for Web of Science ID 000345572700002

  View details for PubMedID 25264302

• NANOG and CDX2 Pattern Distinct Subtypes of Human Mesoderm during Exit from Pluripotency CELL STEM CELL Mendjan, S., Mascetti, V. L., Ortmann, D., Ortiz, M., Karjosukarso, D. W., Ng, Y., Moreau, T., Pedersen, R. A. 2014; 15 (3): 310-325

  Abstract

  Mesoderm 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-? 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-? 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.

  View details for DOI 10.1016/j.stem.2014.06.006

  View details for Web of Science ID 000341441600011

  View details for PubMedID 25042702

• Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be? REPRODUCTION Roberts, R. M., Loh, K. M., Amita, M., Bernardo, A. S., Adachi, K., Alexenko, A. P., Schust, D. J., Schulz, L. C., Telugu, B. P., Ezashi, T., Pedersen, R. A. 2014; 147 (5): D1-D12

  Abstract

  It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.

  View details for DOI 10.1530/REP-14-0080

  View details for Web of Science ID 000336898100001

• Directed differentiation of embryonic origin-specific vascular smooth muscle subtypes from human pluripotent stem cells NATURE PROTOCOLS Cheung, C., Bernardo, A. S., Pedersen, R. A., Sinha, S. 2014; 9 (4): 929-938

  Abstract

  Vascular smooth muscle cells (SMCs) arise from diverse developmental origins. Regional distribution of vascular diseases may, in part, be attributed to this inherent heterogeneity in SMC lineage. Therefore, systems for generating human SMC subtypes of distinct embryonic origins would represent useful platforms for studying the influence of SMC lineage on the spatial specificity of vascular disease. Here we describe how human pluripotent stem cells can be differentiated into distinct populations of SMC subtypes under chemically defined conditions. The initial stage (days 0-5 or 0-7) begins with the induction of three intermediate lineages: neuroectoderm, lateral plate mesoderm and paraxial mesoderm. Subsequently, these precursor lineages are differentiated into contractile SMCs (days 5-19+). At key stages, the emergence of lineage-specific markers confirms recapitulation of embryonic developmental pathways and generation of functionally distinct SMC subtypes. The ability to derive an unlimited supply of human SMCs will accelerate applications in regenerative medicine and disease modeling.

  View details for DOI 10.1038/nprot.2014.059

  View details for Web of Science ID 000333752100014

  View details for PubMedID 24675733

• Investigating the feasibility of scale up and automation of human induced pluripotent stem cells cultured in aggregates in feeder free conditions JOURNAL OF BIOTECHNOLOGY Soares, F. A., Chandra, A., Thomas, R. J., Pedersen, R. A., Vallier, L., Williams, D. J. 2014; 173: 53-58

  Abstract

  The transfer of a laboratory process into a manufacturing facility is one of the most critical steps required for the large scale production of cell-based therapy products. This study describes the first published protocol for scalable automated expansion of human induced pluripotent stem cell lines growing in aggregates in feeder-free and chemically defined medium. Cells were successfully transferred between different sites representative of research and manufacturing settings; and passaged manually and using the CompacT SelecT automation platform. Modified protocols were developed for the automated system and the management of cells aggregates (clumps) was identified as the critical step. Cellular morphology, pluripotency gene expression and differentiation into the three germ layers have been used compare the outcomes of manual and automated processes.

  View details for DOI 10.1016/j.jbiotec.2013.12.009

  View details for Web of Science ID 000331207300009

  View details for PubMedID 24440272

  View details for PubMedCentralID PMC3969287

• Naivete of the human pluripotent stem cell NATURE BIOTECHNOLOGY Mascetti, V. L., Pedersen, R. A. 2014; 32 (1): 68-70

  View details for DOI 10.1038/nbt.2789

  View details for Web of Science ID 000329513000019

  View details for PubMedID 24406934

• Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after Injury CELL STEM CELL Fordham, R. P., Yui, S., Hannan, N. R., Soendergaard, C., Madgwick, A., Schweiger, P. J., Nielsen, O. H., Vallier, L., Pedersen, R. A., Nakamura, T., Watanabe, M., Jensen, K. B. 2013; 13 (6): 734-744

  Abstract

  Regeneration and homeostasis in the adult intestinal epithelium is driven by proliferative resident stem cells, whose functional properties during organismal development are largely unknown. Here, we show that human and mouse fetal intestine contains proliferative, immature progenitors, which can be expanded in vitro as Fetal Enterospheres (FEnS). A highly similar progenitor population can be established during intestinal differentiation of human induced pluripotent stem cells. Established cultures of mouse fetal intestinal progenitors express lower levels of Lgr5 than mature progenitors and propagate in the presence of the Wnt antagonist Dkk1, and new cultures can be induced to form mature intestinal organoids by exposure to Wnt3a. Following transplantation in a colonic injury model, FEnS contribute to regeneration of colonic epithelium by forming epithelial crypt-like structures expressing region-specific differentiation markers. This work provides insight into mechanisms underlying development of the mammalian intestine and points to future opportunities for patient-specific regeneration of the digestive tract.

  View details for DOI 10.1016/j.stem.2013.09.015

  View details for Web of Science ID 000329571700015

  View details for PubMedID 24139758

  View details for PubMedCentralID PMC3858813

• Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/beta-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures OPEN BIOLOGY Lupo, G., Novorol, C., Smith, J. R., Vallier, L., Miranda, E., Alexander, M., Biagioni, S., Pedersen, R. A., Harris, W. A. 2013; 3

  Abstract

  Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/?-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/?-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification.

  View details for DOI 10.1098/rsob.120167

  View details for Web of Science ID 000318269600001

  View details for PubMedID 23576785

  View details for PubMedCentralID PMC3718331

• Conversion from mouse embryonic to extra-embryonic endoderm stem cells reveals distinct differentiation capacities of pluripotent stem cell states DEVELOPMENT Cho, L. T., Wamaitha, S. E., Tsai, I. J., Artus, J., Sherwood, R. I., Pedersen, R. A., Hadjantonakis, A., Niakan, K. K. 2012; 139 (16): 2866-2877

  Abstract

  The inner cell mass of the mouse pre-implantation blastocyst comprises epiblast progenitor and primitive endoderm cells of which cognate embryonic (mESCs) or extra-embryonic (XEN) stem cell lines can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of their in vivo tissue of origin. Recently, we demonstrated that XEN-like cells arise within mESC cultures. This raises the possibility that mESCs can generate self-renewing XEN cells without the requirement for gene manipulation. We have developed a novel approach to convert mESCs to XEN cells (cXEN) using growth factors. We confirm that the downregulation of the pluripotency transcription factor Nanog and the expression of primitive endoderm-associated genes Gata6, Gata4, Sox17 and Pdgfra are necessary for cXEN cell derivation. This approach highlights an important function for Fgf4 in cXEN cell derivation. Paracrine FGF signalling compensates for the loss of endogenous Fgf4, which is necessary to exit mESC self-renewal, but not for XEN cell maintenance. Our cXEN protocol also reveals that distinct pluripotent stem cells respond uniquely to differentiation promoting signals. cXEN cells can be derived from mESCs cultured with Erk and Gsk3 inhibitors (2i), and LIF, similar to conventional mESCs. However, we find that epiblast stem cells (EpiSCs) derived from the post-implantation embryo are refractory to cXEN cell establishment, consistent with the hypothesis that EpiSCs represent a pluripotent state distinct from mESCs. In all, these findings suggest that the potential of mESCs includes the capacity to give rise to both extra-embryonic and embryonic lineages.

  View details for DOI 10.1242/dev.078519

  View details for Web of Science ID 000306706300004

  View details for PubMedID 22791892

  View details for PubMedCentralID PMC3403099

• Genomic Targets of Brachyury (T) in Differentiating Mouse Embryonic Stem Cells PLOS ONE Evans, A. L., Faial, T., Gilchrist, M. J., Down, T., Vallier, L., Pedersen, R. A., Wardle, F. C., Smith, J. C. 2012; 7 (3)

  Abstract

  The T-box transcription factor Brachyury (T) is essential for formation of the posterior mesoderm and the notochord in vertebrate embryos. Work in the frog and the zebrafish has identified some direct genomic targets of Brachyury, but little is known about Brachyury targets in the mouse.Here we use chromatin immunoprecipitation and mouse promoter microarrays to identify targets of Brachyury in embryoid bodies formed from differentiating mouse ES cells. The targets we identify are enriched for sequence-specific DNA binding proteins and include components of signal transduction pathways that direct cell fate in the primitive streak and tailbud of the early embryo. Expression of some of these targets, such as Axin2, Fgf8 and Wnt3a, is down regulated in Brachyury mutant embryos and we demonstrate that they are also Brachyury targets in the human. Surprisingly, we do not observe enrichment of the canonical T-domain DNA binding sequence 5'-TCACACCT-3' in the vicinity of most Brachyury target genes. Rather, we have identified an (AC)(n) repeat sequence, which is conserved in the rat but not in human, zebrafish or Xenopus. We do not understand the significance of this sequence, but speculate that it enhances transcription factor binding in the regulatory regions of Brachyury target genes in rodents.Our work identifies the genomic targets of a key regulator of mesoderm formation in the early mouse embryo, thereby providing insights into the Brachyury-driven genetic regulatory network and allowing us to compare the function of Brachyury in different species.

  View details for DOI 10.1371/journal.pone.0033346

  View details for Web of Science ID 000305339100043

  View details for PubMedID 22479388

  View details for PubMedCentralID PMC3316570

• Human pre-implantation embryo development DEVELOPMENT Niakan, K. K., Han, J., Pedersen, R. A., Simon, C., Pera, R. A. 2012; 139 (5): 829-841

  Abstract

  Understanding human pre-implantation development has important implications for assisted reproductive technology (ART) and for human embryonic stem cell (hESC)-based therapies. Owing to limited resources, the cellular and molecular mechanisms governing this early stage of human development are poorly understood. Nonetheless, recent advances in non-invasive imaging techniques and molecular and genomic technologies have helped to increase our understanding of this fascinating stage of human development. Here, we summarize what is currently known about human pre-implantation embryo development and highlight how further studies of human pre-implantation embryos can be used to improve ART and to fully harness the potential of hESCs for therapeutic goals.

  View details for DOI 10.1242/dev.060426

  View details for Web of Science ID 000300402400001

  View details for PubMedID 22318624

  View details for PubMedCentralID PMC3274351

• Generation of human vascular smooth muscle subtypes provides insight into embryological origin-dependent disease susceptibility NATURE BIOTECHNOLOGY Cheung, C., Bernardo, A. S., Trotter, M. W., Pedersen, R. A., Sinha, S. 2012; 30 (2): 165-173

  Abstract

  Heterogeneity of embryological origins is a hallmark of vascular smooth muscle cells (SMCs) and may influence the development of vascular disease. Differentiation of human pluripotent stem cells (hPSCs) into developmental origin-specific SMC subtypes remains elusive. Here we describe a chemically defined protocol in which hPSCs were initially induced to form neuroectoderm, lateral plate mesoderm or paraxial mesoderm. These intermediate populations were further differentiated toward SMCs (>80% MYH11(+) and ACTA2(+)), which displayed contractile ability in response to vasoconstrictors and invested perivascular regions in vivo. Derived SMC subtypes recapitulated the unique proliferative and secretory responses to cytokines previously documented in studies using aortic SMCs of distinct origins. Notably, this system predicted increased extracellular matrix degradation by SMCs derived from lateral plate mesoderm, which was confirmed using rat aortic SMCs from corresponding origins. This differentiation approach will have broad applications in modeling origin-dependent disease susceptibility and in developing bioengineered vascular grafts for regenerative medicine.

  View details for DOI 10.1038/nbt.2107

  View details for Web of Science ID 000300269100019

  View details for PubMedID 22252507

  View details for PubMedCentralID PMC3272383

• Status of Genomic Imprinting in Epigenetically Distinct Pluripotent Stem Cells STEM CELLS Sun, B., Ito, M., Mendjan, S., Ito, Y., Brons, I. G., Murrell, A., Vallier, L., Ferguson-Smith, A. C., Pedersen, R. A. 2012; 30 (2): 161-168

  Abstract

  Mouse epiblast stem cells (EpiSCs) derived from postimplantation embryos are developmentally and functionally different from embryonic stem cells (ESCs) generated from blastocysts. EpiSCs require Activin A and FGF2 signaling for self-renewal, similar to human ESCs (hESCs), while mouse ESCs require LIF and BMP4. Unlike ESCs, EpiSCs have undergone X-inactivation, similar to the tendency of hESCs. The shared self-renewal and X-inactivation properties of EpiSCs and hESCs suggest that they have an epigenetic state distinct from ESCs. This hypothesis predicts that EpiSCs would have monoallelic expression of most imprinted genes, like that observed in hESCs. Here, we confirm this prediction. By contrast, we find that mouse induced pluripotent stem cells (iPSCs) tend to lose imprinting similar to mouse ESCs. These findings reveal that iPSCs have an epigenetic status associated with their pluripotent state rather than their developmental origin. Our results also reinforce the view that hESCs and EpiSCs are in vitro counterparts, sharing an epigenetic status distinct from ESCs and iPSCs.

  View details for DOI 10.1002/stem.793

  View details for Web of Science ID 000299209200008

  View details for PubMedID 22109880

• BRACHYURY and CDX2 Mediate BMP-Induced Differentiation of Human and Mouse Pluripotent Stem Cells into Embryonic and Extraembryonic Lineages CELL STEM CELL Bernardo, A. S., Faial, T., Gardner, L., Niakan, K. K., Ortmann, D., Senner, C. E., Callery, E. M., Trotter, M. W., Hemberger, M., Smith, J. C., Bardwell, L., Moffett, A., Pedersen, R. A. 2011; 9 (2): 144-155

  Abstract

  BMP is thought to induce hESC differentiation toward multiple lineages including mesoderm and trophoblast. The BMP-induced trophoblast phenotype is a long-standing paradox in stem cell biology. Here we readdressed BMP function in hESCs and mouse epiblast-derived cells. We found that BMP4 cooperates with FGF2 (via ERK) to induce mesoderm and to inhibit endoderm differentiation. These conditions induced cells with high levels of BRACHYURY (BRA) that coexpressed CDX2. BRA was necessary for and preceded CDX2 expression; both genes were essential for expression not only of mesodermal genes but also of trophoblast-associated genes. Maximal expression of the latter was seen in the absence of FGF but these cells coexpressed mesodermal genes and moreover they differed in cell surface and epigenetic properties from placental trophoblast. We conclude that BMP induces human and mouse pluripotent stem cells primarily to form mesoderm, rather than trophoblast, acting through BRA and CDX2.

  View details for DOI 10.1016/j.stem.2011.06.015

  View details for Web of Science ID 000293990900011

  View details for PubMedID 21816365

  View details for PubMedCentralID PMC3567433

• Signaling Pathways Controlling Pluripotency and Early Cell Fate Decisions of Human Induced Pluripotent Stem Cells STEM CELLS Vallier, L., Touboul, T., Brown, S., Cho, C., Bilican, B., Alexander, M., Cedervall, J., Chandran, S., Ahrlund-Richter, L., Weber, A., Pedersen, R. A. 2009; 27 (11): 2655-2666

  Abstract

  Human pluripotent stem cells from embryonic origins and those generated from reprogrammed somatic cells share many characteristics, including indefinite proliferation and a sustained capacity to differentiate into a wide variety of cell types. However, it remains to be demonstrated whether both cell types rely on similar mechanisms to maintain their pluripotent status and to control their differentiation. Any differences in such mechanisms would suggest that reprogramming of fibroblasts to generate induced pluripotent stem cells (iPSCs) results in novel states of pluripotency. In that event, current methods for expanding and differentiating human embryonic stem cells (ESCs) might not be directly applicable to human iPSCs. However, we show here that human iPSCs rely on activin/nodal signaling to control Nanog expression and thereby maintain pluripotency, thus revealing their mechanistic similarity to human ESCs. We also show that growth factors necessary and sufficient for achieving specification of human ESCs into extraembryonic tissues, neuroectoderm, and mesendoderm also drive differentiation of human iPSCs into the same tissues. Importantly, these experiments were performed in fully chemically defined medium devoid of factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Together these data reveal that human iPSCs rely on mechanisms similar to human ESCs to maintain their pluripotency and to control their differentiation, showing that these pluripotent cell types are functionally equivalent.

  View details for DOI 10.1002/stem.199

  View details for Web of Science ID 000271830700003

  View details for PubMedID 19688839

• Early Cell Fate Decisions of Human Embryonic Stem Cells and Mouse Epiblast Stem Cells Are Controlled by the Same Signalling Pathways PLOS ONE Vallier, L., Touboul, T., Chng, Z., Brimpari, M., Hannan, N., Millan, E., Smithers, L. E., Trotter, M., Rugg-Gunn, P., Weber, A., Pedersen, R. A. 2009; 4 (6)

  Abstract

  Human embryonic stem cells have unique value for regenerative medicine, as they are capable of differentiating into a broad variety of cell types. Therefore, defining the signalling pathways that control early cell fate decisions of pluripotent stem cells represents a major task. Moreover, modelling the early steps of embryonic development in vitro may provide the best approach to produce cell types with native properties. Here, we analysed the function of key developmental growth factors such as Activin, FGF and BMP in the control of early cell fate decisions of human pluripotent stem cells. This analysis resulted in the development and validation of chemically defined culture conditions for achieving specification of human embryonic stem cells into neuroectoderm, mesendoderm and into extra-embryonic tissues. Importantly, these defined culture conditions are devoid of factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Importantly, the growth factor roles defined using these culture conditions similarly drove differentiation of mouse epiblast stem cells derived from post implantation embryos, thereby reinforcing the hypothesis that epiblast stem cells share a common embryonic identity with human pluripotent stem cells. Therefore the defined growth factor conditions described here represent an essential step toward the production of mature cell types from pluripotent stem cells in conditions fully compatible with clinical use ant also provide a general approach for modelling the early steps of mammalian embryonic development.

  View details for DOI 10.1371/journal.pone.0006082

  View details for Web of Science ID 000267515700004

  View details for PubMedID 19564924

  View details for PubMedCentralID PMC2700259

• Activin/Nodal signalling maintains pluripotency by controlling Nanog expression DEVELOPMENT Vallier, L., Mendjan, S., Brown, S., Chng, Z., Teo, A., Smithers, L. E., Trotter, M. W., Cho, C. H., Martinez, A., Rugg-Gunn, P., Brons, G., Pedersen, R. A. 2009; 136 (8): 1339-1349

  Abstract

  The pluripotent status of embryonic stem cells (ESCs) confers upon them the capacity to differentiate into the three primary germ layers, ectoderm, mesoderm and endoderm, from which all the cells of the adult body are derived. An understanding of the mechanisms controlling pluripotency is thus essential for driving the differentiation of human pluripotent cells into cell types useful for clinical applications. The Activin/Nodal signalling pathway is necessary to maintain pluripotency in human ESCs and in mouse epiblast stem cells (EpiSCs), but the molecular mechanisms by which it achieves this effect remain obscure. Here, we demonstrate that Activin/Nodal signalling controls expression of the key pluripotency factor Nanog in human ESCs and in mouse EpiSCs. Nanog in turn prevents neuroectoderm differentiation induced by FGF signalling and limits the transcriptional activity of the Smad2/3 cascade, blocking progression along the endoderm lineage. This negative-feedback loop imposes stasis in neuroectoderm and mesendoderm differentiation, thereby maintaining the pluripotent status of human ESCs and mouse EpiSCs.

  View details for DOI 10.1242/dev.033951

  View details for Web of Science ID 000264402400012

  View details for PubMedID 19279133

  View details for PubMedCentralID PMC2687465

• Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration STEM CELLS Smith, J. R., Maguire, S., Davis, L. A., Alexander, M., Yang, F., Chandran, S., Ffrench-Constant, C., Pedersen, R. A. 2008; 26 (2): 496-504

  Abstract

  Silencing and variegated transgene expression are poorly understood problems that can interfere with gene function studies in human embryonic stem cells (hESCs). We show that transgene expression (enhanced green fluorescent protein [EGFP]) from random integration sites in hESCs is affected by variegation and silencing, with only half of hESCs expressing the transgene, which is gradually lost after withdrawal of selection and differentiation. We tested the hypothesis that a transgene integrated into the adeno-associated virus type 2 (AAV2) target region on chromosome 19, known as the AAVS1 locus, would maintain transgene expression in hESCs. When we used AAV2 technology to target the AAVS1 locus, 4.16% of hESC clones achieved AAVS1-targeted integration. Targeted clones expressed Oct-4, stage-specific embryonic antigen-3 (SSEA3), and Tra-1-60 and differentiated into all three primary germ layers. EGFP expression from the AAVS1 locus showed significantly reduced variegated expression when in selection, with 90% +/- 4% of cells expressing EGFP compared with 57% +/- 32% for randomly integrated controls, and reduced tendency to undergo silencing, with 86% +/- 7% hESCs expressing EGFP 25 days after withdrawal of selection compared with 39% +/- 31% for randomly integrated clones. In addition, quantitative polymerase chain reaction analysis of hESCs also indicated significantly higher levels of EGFP mRNA in AAVS1-targeted clones as compared with randomly integrated clones. Transgene expression from the AAVS1 locus was shown to be stable during hESC differentiation, with more than 90% of cells expressing EGFP after 15 days of differentiation, as compared with approximately 30% for randomly integrated clones. These results demonstrate the utility of transgene integration at the AAVS1 locus in hESCs and its potential clinical application.

  View details for DOI 10.1634/stemcells.2007-0039

  View details for Web of Science ID 000253372600023

  View details for PubMedID 18024421

• Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm DEVELOPMENTAL BIOLOGY Smith, J. R., Vallier, L., Lupo, G., Alexander, M., Harris, W. A., Pedersen, R. A. 2008; 313 (1): 107-117

  Abstract

  Nodal, a member of the TGF-beta family of signaling molecules, has been implicated in pluripotency in human embryonic stem cells (hESCs) [Vallier, L., Reynolds, D., Pedersen, R.A., 2004a. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev. Biol. 275, 403-421], a finding that seems paradoxical given Nodal's central role in mesoderm/endoderm specification during gastrulation. In this study, we sought to clarify the role of Nodal signaling during hESC differentiation by constitutive overexpression of the endogenous Nodal inhibitors Lefty2 (Lefty) and truncated Cerberus (Cerb-S) and by pharmacological interference using the Nodal receptor antagonist SB431542. Compared to wildtype (WT) controls, embryoid bodies (EBs) derived from either Lefty or Cerb-S overexpressing hESCs showed increased expression of neuroectoderm markers Sox1, Sox3, and Nestin. Conversely, they were negative for a definitive endoderm marker (Sox17) and did not generate beating cardiomyocyte structures in conditions that allowed mesendoderm differentiation from WT hESCs. EBs derived from either Lefty or Cerb-S expressing hESCs also contained a greater abundance of neural rosette structures as compared to controls. Differentiating EBs derived from Lefty expressing hESCs generated a dense network of beta-tubulin III positive neurites, and when Lefty expressing hESCs were grown as a monolayer and allowed to differentiate, they generated significantly higher numbers of beta-tubulin positive neurons as compared to wildtype hESCs. SB431542 treatments reproduced the neuralising effects of Lefty overexpression in hESCs. These results show that inhibition of Nodal signaling promotes neuronal specification, indicating a role for this pathway in controlling early neural development of pluripotent cells.

  View details for DOI 10.1016/j.ydbio.2007.10.003

  View details for Web of Science ID 000252300500010

  View details for PubMedID 18022151

• Recombination signatures distinguish embryonic stem cells derived by parthenogenesis and somatic cell nuclear transfer CELL STEM CELL Kim, K., Ng, K., Rugg-Gunn, P. J., Shieh, J., Kirak, O., Jaenisch, R., Wakayama, T., Moore, M. A., Pedersen, R. A., Daley, G. Q. 2007; 1 (3): 346-352

  Abstract

  Parthenogenesis and somatic cell nuclear transfer (SCNT) are two methods for deriving embryonic stem (ES) cells that are genetically matched to the oocyte donor or somatic cell donor, respectively. Using genome-wide single nucleotide polymorphism (SNP) analysis, we demonstrate distinct signatures of genetic recombination that distinguish parthenogenetic ES cells from those generated by SCNT. We applied SNP analysis to the human ES cell line SCNT-hES-1, previously claimed to have been derived by SCNT, and present evidence that it represents a human parthenogenetic ES cell line. Genome-wide SNP analysis represents a means to validate the genetic provenance of an ES cell line.

  View details for DOI 10.1016/j.stem.2007.07.001

  View details for Web of Science ID 000251055200016

  View details for PubMedID 18371368

• Derivation of pluripotent epiblast stem cells from mammalian embryos NATURE Brons, I. G., Smithers, L. E., Trotter, M. W., Rugg-Gunn, P., Sun, B., de Sousa Lopes, S. M., Howlett, S. K., Clarkson, A., Ahrlund-Richter, L., Pedersen, R. A., Vallier, L. 2007; 448 (7150): 191-U7

  Abstract

  Although the first mouse embryonic stem (ES) cell lines were derived 25 years ago using feeder-layer-based blastocyst cultures, subsequent efforts to extend the approach to other mammals, including both laboratory and domestic species, have been relatively unsuccessful. The most notable exceptions were the derivation of non-human primate ES cell lines followed shortly thereafter by their derivation of human ES cells. Despite the apparent common origin and the similar pluripotency of mouse and human embryonic stem cells, recent studies have revealed that they use different signalling pathways to maintain their pluripotent status. Mouse ES cells depend on leukaemia inhibitory factor and bone morphogenetic protein, whereas their human counterparts rely on activin (INHBA)/nodal (NODAL) and fibroblast growth factor (FGF). Here we show that pluripotent stem cells can be derived from the late epiblast layer of post-implantation mouse and rat embryos using chemically defined, activin-containing culture medium that is sufficient for long-term maintenance of human embryonic stem cells. Our results demonstrate that activin/Nodal signalling has an evolutionarily conserved role in the derivation and the maintenance of pluripotency in these novel stem cells. Epiblast stem cells provide a valuable experimental system for determining whether distinctions between mouse and human embryonic stem cells reflect species differences or diverse temporal origins.

  View details for DOI 10.1038/nature05950

  View details for Web of Science ID 000247934500042

  View details for PubMedID 17597762

• HOXB4 overexpression promotes hematopoietic development by human embryonic stem cells STEM CELLS Bowles, K. M., Vallier, L., Smith, J. R., Alexander, M. R., Pedersen, R. A. 2006; 24 (5): 1359-1369

  Abstract

  Human embryonic stem cells (hESCs) are a potential source of hematopoietic cells for therapeutic transplantation and can provide a model for human hematopoiesis. Culture of hESCs on murine stromal layers or in stromal-free conditions as embryoid bodies results in low levels of hematopoietic cells. Here we demonstrate that overexpression of the transcription factor HOXB4 considerably augments hematopoietic development of hESCs. Stable HOXB4-expressing hESC clones were generated by lipofection and could be maintained in the undifferentiated state for prolonged passages. Moreover, differentiation of hESCs as embryoid bodies in serum-containing medium without the use of additional cytokines led to sequential expansion of first erythroid and then myeloid and monocytic progenitors from day 10 of culture. These cells retained the capacity to develop into formed blood elements during in vitro culture. Consistent with the development of committed hematopoietic cells, we observed the expression of transcription factors known to be critical for hematopoietic development. We thus demonstrate successful use of enforced gene expression to promote the differentiation of hESCs into a terminally differentiated tissue, thereby revealing an important role for HOXB4 in supporting their in vitro development along the hematopoietic pathway.

  View details for DOI 10.1634/stemcells.2005-0210

  View details for Web of Science ID 000240639200025

  View details for PubMedID 16410392

• Banking on human embryonic stem cells: estimating the number of donor cell lines needed for HLA matching LANCET Taylor, C. J., Bolton, E. M., Pocock, S., Sharples, L. D., Pedersen, R. A., Bradley, J. A. 2005; 366 (9502): 2019-2025

  Abstract

  Human embryonic stem (hES) cells are a promising source for transplantation to replace diseased or damaged tissue, but their differentiated progeny express human leucocyte antigens (HLAs) that will probably cause graft rejection. The creation of a bank of HLA-typed hES cells, from which a best match could be selected, would help reduce the likelihood of graft rejection. We investigated how many hES cell lines would be needed to make matching possible in most cases.The number of hES cell lines needed to achieve varying degrees of HLA match was estimated by use of, as a surrogate for hES-cell donor embryos, blood group and HLA types on a series of 10,000 consecutive UK cadaveric organ donors. The degree of blood group compatibility and HLA matching for a recipient population consisting of 6577 patients registered on the UK kidney transplant waiting list was determined, assuming all donor hES cell lines could provide a transplant for an unlimited number of recipients.A bank of 150 consecutive donors provided a full match at HLA-A, HLA-B, and HLA-DR for a minority of recipients (<20%); a beneficial match (defined as one HLA-A or one HLA-B mismatch only) or better for 37.9% (range 27.9-47.5); and an HLA-DR match or better for 84.9% (77.5-90.0). Extending the number of donors beyond 150 conferred only a very gradual incremental benefit with respect to HLA matching. A panel of only ten donors homozygous for common HLA types selected from 10,000 donors provided a complete HLA-A, HLA-B and HLA-DR match for 37.7% of recipients, and a beneficial match for 67.4%.Approximately 150 consecutive blood group compatible donors, 100 consecutive blood group O donors, or ten highly selected homozygous donors could provide the maximum practical benefit for HLA matching. The findings from these simulations have practical, political, and ethical implications for the establishment of hES-cell banks.

  View details for Web of Science ID 000233826900021

  View details for PubMedID 16338451

• Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells JOURNAL OF CELL SCIENCE Vallier, L., Alexander, M., Pedersen, R. A. 2005; 118 (19): 4495-4509

  Abstract

  Maintenance of pluripotency is crucial to the mammalian embryo's ability to generate the extra-embryonic and embryonic tissues that are needed for intrauterine survival and foetal development. The recent establishment of embryonic stem cells from human blastocysts (hESCs) provides an opportunity to identify the factors supporting pluripotency at early stages of human development. Using this in vitro model, we have recently shown that Nodal can block neuronal differentiation, suggesting that TGFbeta family members are involved in cell fate decisions of hESCs, including preservation of their pluripotency. Here, we report that Activin/Nodal signalling through Smad2/3 activation is necessary to maintain the pluripotent status of hESCs. Inhibition of Activin/Nodal signalling by follistatin and by overexpression of Lefty or Cerberus-Short, or by the Activin receptor inhibitor SB431542, precipitates hESC differentiation. Nevertheless, neither Nodal nor Activin is sufficient to sustain long-term hESC growth in a chemically defined medium without serum. Recent studies have shown that FGF2 can also maintain long-term expression of pluripotency markers, and we find that inhibition of the FGF signalling pathway by the tyrosine kinase inhibitor SU5402 causes hESC differentiation. However, this effect of FGF on hESC pluripotency depends on Activin/Nodal signalling, because it is blocked by SB431542. Finally, long-term maintenance of in-vitro pluripotency can be achieved with a combination of Activin or Nodal plus FGF2 in the absence of feeder-cell layers, conditioned medium or Serum Replacer. These findings suggest that the Activin/Nodal pathway maintains pluripotency through mechanism(s) in which FGF acts as a competence factor and therefore provide further evidence of distinct mechanisms for preservation of pluripotency in mouse and human ESCs.

  View details for DOI 10.1242/jcs.02553

  View details for Web of Science ID 000233258900018

  View details for PubMedID 16179608

• Epigenetic status of human embryonic stem cells NATURE GENETICS Rugg-Gunn, P. J., Ferguson-Smith, A. C., Pedersen, R. A. 2005; 37 (6): 585-587

  Abstract

  We examined the allele-specific expression of six imprinted genes and the methylation profiles of three imprinting control regions to assess the epigenetic status of human embryonic stem cells. We identified generally monoallelic gene expression and normal methylation patterns. During prolonged passage, one cell line became biallelic with respect to H19, but without loss of the gametic methylation imprint. These data argue for a substantial degree of epigenetic stability in human embryonic stem cells.

  View details for DOI 10.1038/ng1556

  View details for Web of Science ID 000229495300009

  View details for PubMedID 15864307

• Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway DEVELOPMENTAL BIOLOGY Vallier, L., Reynolds, D., Pederson, R. A. 2004; 275 (2): 403-421

  Abstract

  Genetic studies in fish, amphibia, and mice have shown that deficiency of Nodal signaling blocks differentiation into mesoderm and endoderm. Thus, Nodal is considered as a major inducer of mesendoderm during gastrulation. On this basis, Nodal is a candidate for controlling differentiation of pluripotent human embryonic stem cells (hESCs) into tissue lineages with potential clinical value. We have investigated the effect of Nodal, both as a recombinant protein and as a constitutively expressed transgene, on differentiation of hESCs. When control hESCs were grown in chemically defined medium, their expression of markers of pluripotency progressively decreased, while expression of neuroectoderm markers was strongly upregulated, thus revealing a neuroectodermal default mechanism for differentiation in this system. hESCs cultured in recombinant Nodal, by contrast, showed prolonged expression of pluripotency marker genes and reduced induction of neuroectoderm markers. These Nodal effects were accentuated in hESCs expressing a Nodal transgene, with striking morphogenetic consequences. Nodal-expressing hESCs developing as embryoid bodies contained an outer layer of visceral endoderm-like cells surrounding an inner layer of epiblast-like cells, each layer having distinct gene expression patterns. Markers of neuroectoderm were not upregulated during development of Nodal-expressing embryoid bodies, nor was there induction of markers for definitive mesoderm or endoderm differentiation. Moreover, the inner layer expressed markers of pluripotency, characteristic of undifferentiated hESCs and of epiblast in mouse embryos. These results could be accounted for by an inhibitory effect of Nodal-induced visceral endoderm on pluripotent cell differentiation into mesoderm and endoderm, with a concomitant inhibition of neuroectoderm differentiation by Nodal itself. There could also be a direct effect of Nodal in the maintenance of pluripotency. In summary, analysis of the Nodal-expressing phenotype suggests a function for the transforming growth factor-beta (TGF-beta) growth factor superfamily in pluripotency and in early cell fate decisions leading to primary tissue layers during in vitro development of pluripotent human stem cells. The effects of Nodal on early differentiation illustrate how hESCs can augment mouse embryos as a model for analyzing mechanisms of early mammalian development.

  View details for DOI 10.1016/j.ydbio.2004.08.031

  View details for Web of Science ID 000224952500010

  View details for PubMedID 15501227

• Enhancing and diminishing gene function in human embryonic stem cells STEM CELLS Vallier, L., Rugg-Gunn, P. J., Bouhon, I. A., Andersson, F. K., Sadler, A. J., Pedersen, R. A. 2004; 22 (1): 2-11

  Abstract

  It is widely recognized that gain- and loss-of-function approaches are essential for understanding the functions of specific genes, and such approaches would be particularly valuable in studies involving human embryonic stem (hES) cells. We describe a simple and efficient approach using lipofection to transfect hES cells, which enabled us to generate hES cell lines expressing naturally fluorescent green or red proteins without affecting cell pluripotency. We used these cell lines to establish a means of diminishing gene function using small interfering (si)RNAs, which were effective at knocking down gene expression in hES cells. We then demonstrated that stable expression of siRNA could knock down the expression of endogenous genes. Application of these gain- and loss-of-function approaches should have widespread use, not only in revealing the developmental roles of specific human genes, but also for their utility in modulating differentiation.

  View details for Web of Science ID 000187497200001

  View details for PubMedID 14688386