Boris Greber

Direct reprogramming of human neural stem cells by OCT4

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by ectopic expression of four transcription factors (OCT4 (also called POU5F1), SOX2, c-Myc and KLF4). We previously reported that Oct4 alone is sufficient to reprogram directly adult mouse neural stem cells to iPS cells. Here we report the generation of one-factor human iPS cells from human fetal neural stem cells (one-factor (1F) human NiPS cells) by ectopic expression of OCT4 alone. One-factor human NiPS cells resemble human embryonic stem cells in global gene expression profiles, epigenetic status, as well as pluripotency in vitro and in vivo. These findings demonstrate that the transcription factor OCT4 is sufficient to reprogram human neural stem cells to pluripotency. One-factor iPS cell generation will advance the field further towards understanding reprogramming and generating patient-specific pluripotent stem cells.

Analysis of Oct4‐Dependent Transcriptional Networks Regulating Self‐Renewal and Pluripotency in Human Embryonic Stem Cells

The POU domain transcription factor OCT4 is a key regulator of pluripotency in the early mammalian embryo and is highly expressed in the inner cell mass of the blastocyst. Consistent with its essential role in maintaining pluripotency, Oct4 expression is rapidly downregulated during formation of the trophoblast lineage. To enhance our understanding of the molecular basis of this differentiation event in humans, we used a functional genomics approach involving RNA interference‐mediated suppression of OCT4 function in a human ESC line and analysis of the resulting transcriptional profiles to identify OCT4‐dependent genes in human cells. We detected altered expression of >1,000 genes, including targets regulated directly by OCT4 either positively (NANOG, SOX2, REX1, LEFTB, LEFTA/EBAF DPPA4, THY1, and TDGF1) or negatively (CDX2, EOMES, BMP4, TBX18, Brachyury [T], DKK1, HLX1, GATA6, ID2, and DLX5), as well as targets for the OCT4‐associated stem cell regulators SOX2 and NANOG. Our data set includes regulators of ACTIVIN, BMP, fibroblast growth factor, and WNT signaling. These pathways are implicated in regulating human ESC differentiation and therefore further validate the results of our analysis. In addition, we identified a number of differentially expressed genes that are involved in epigenetics, chromatin remodeling, apoptosis, and metabolism that may point to underlying molecular mechanisms that regulate pluripotency and trophoblast differentiation in humans. Significant concordance between this data set and previous comparisons between inner cell mass and trophectoderm in human embryos indicates that the study of human ESC differentiation in vitro represents a useful model of early embryonic differentiation in humans.

Chromatin-Remodeling Components of the BAF Complex Facilitate Reprogramming.

Reprogramming of somatic cells achieved by combination of the four transcription factors Oct4, Sox2, Klf4, and c-Myc has very low efficiency. To increase the reprogramming efficiency and better understand the process, we sought to identify factors that mediate reprogramming with higher efficiency. We established an assay to screen nuclear fractions from extracts of pluripotent mouse cells based on Oct4 reactivation. Using proteomics, we identified components of the ATP-dependent BAF chromatin-remodeling complex, which significantly increases reprogramming efficiency when used together with the four factors. The reprogrammed cells could transmit to the germline and exhibited pluripotency. Reprogramming remained highly efficient when c-Myc was not present but BAF components were overexpressed. BAF complex components mediate this effect by facilitating enhanced Oct4 binding to target promoters during reprogramming. Thus, somatic cell reprogramming using chromatin-remodeling molecules represents an efficient method of generating reprogrammed cells.

Conserved and Divergent Roles of FGF Signaling in Mouse Epiblast Stem Cells and Human Embryonic Stem Cells

Mouse epiblast stem cells (EpiSCs) are cultured with FGF2 and Activin A, like human embryonic stem cells (hESCs), but the action of the associated pathways in EpiSCs has not been well characterized. Here, we show that activation of the Activin pathway promotes self-renewal of EpiSCs via direct activation of Nanog, whereas inhibition of this pathway induces neuroectodermal differentiation, like in hESCs. In contrast, the different roles of FGF signaling appear to be only partially conserved in the mouse. Our data suggest that FGF2 fails to cooperate with SMAD2/3 signaling in actively promoting EpiSC self-renewal through Nanog, in contrast to its role in hESCs. Rather, FGF appears to stabilize the epiblast state by dual inhibition of differentiation to neuroectoderm and of media-induced reversion to a mouse embryonic stem cell-like state. Our data extend the current model of cell fate decisions concerning EpiSCs by clarifying the distinct roles played by FGF signaling.

Induction of Pluripotency in Adult Unipotent Germline Stem Cells

Mouse and human stem cells with features similar to those of embryonic stem cells have been derived from testicular cells. Although pluripotent stem cells have been obtained from defined germline stem cells (GSCs) of mouse neonatal testis, only multipotent stem cells have been obtained so far from defined cells of mouse adult testis. In this study we describe a robust and reproducible protocol for obtaining germline-derived pluripotent stem (gPS) cells from adult unipotent GSCs. Pluripotency of gPS cells was confirmed by in vitro and in vivo differentiation, including germ cell contribution and transmission. As determined by clonal analyses, gPS cells indeed originate from unipotent GSCs. We propose that the conversion process requires a GSC culture microenvironment that depends on the initial number of plated GSCs and the length of culture time.

Fibroblast growth factor 2 modulates transforming growth factor β signaling in mouse embryonic fibroblasts and human escs (hESCs) to support hESC self-renewal

Fibroblast growth factor 2 (FGF2) is known to promote self‐renewal of human embryonic stem cells (hESCs). In addition, it has been shown that transforming growth factor β (TGFβ) signaling is crucial in that the TGFβ/Activin/Nodal branch of the pathway needs to be activated and the bone morphogenic protein (BMP)/GDF branch repressed to prevent differentiation. This holds particularly true for Serum Replacement‐based medium containing BMP‐like activity. We have reinvestigated a widely used protocol for conditioning hESC medium with mouse embryonic fibroblasts (MEFs). We show that FGF2 acts on MEFs to release supportive factors and reduce differentiation‐inducing activity. FGF2 stimulation experiments with supportive and nonsupportive MEFs followed by genome‐wide expression profiling revealed that FGF2 regulates the expression of key members of the TGFβ pathway, with Inhba, Tgfb1, Grem1, and Bmp4 being the most likely candidates orchestrating the above activities. In addition, restimulation experiments in hESCs combined with global expression analysis revealed downstream targets of FGF2 signaling in these cells. Among these were the same factors previously identified in MEFs, thus suggesting that FGF2, at least in part, promotes self‐renewal of hESCs by modulating the expression of TGFβ ligands, which, in turn, act on hESCs in a concerted and autocrine manner.

FGF signalling inhibits neural induction in human embryonic stem cells

Human embryonic stem cells (hESCs) can exit the self‐renewal programme, through the action of signalling molecules, at any given time and differentiate along the three germ layer lineages. We have systematically investigated the specific roles of three signalling pathways, TGFβ/SMAD2, BMP/SMAD1, and FGF/ERK, in promoting the transition of hESCs into the neuroectoderm lineage. In this context, inhibition of SMAD2 and ERK signalling served to cooperatively promote exit from hESC self‐renewal through the rapid downregulation of NANOG and OCT4. In contrast, inhibition of SMAD1 signalling acted to maintain SOX2 expression and prevent non‐neural differentiation via HAND1. Inhibition of FGF/ERK upregulated OTX2 that subsequently induced the neuroectodermal fate determinant PAX6, revealing a novel role for FGF2 in indirectly repressing PAX6 in hESCs. Combined inhibition of the three pathways hence resulted in highly efficient neuroectoderm formation within 4 days, and subsequently, FGF/ERK inhibition promoted rapid differentiation into peripheral neurons. Our study assigns a novel, biphasic role to FGF/ERK signalling in the neural induction of hESCs, which may also have utility for applications requiring the rapid and efficient generation of peripheral neurons.

Direct reprogramming of fibroblasts into epiblast stem cells

Epiblast stem cells (EpiSCs) derived from epiblast tissue of post-implantation embryos are pluripotent and can give rise to all three germ layers in teratoma assays. Introduction of the four transcription factors Oct4, Sox2, Klf4 and c-Myc into somatic cells has been shown to generate induced pluripotent stem cells (iPSCs) that are very similar to embryonic stem cells (ESCs) in a number of characteristics. However, generation of EpiSCs by the direct reprogramming of somatic cells using these transcription factors has not been shown to date. Here, we show that these transcription factors can be used to directly generate induced EpiSCs (iEpiSCs) under EpiSC culture conditions. iEpiSCs resemble EpiSCs in morphology, gene expression pattern, epigenetic status and chimaera-forming capability. This study demonstrates that the culture environment in transcription factor-mediated reprogramming determines the cell fate of the reprogrammed cell. We therefore hypothesize that it will eventually be possible to shape the identity of a directly reprogrammed cell simply by modulating culture conditions.

Human adult germline stem cells in question

Arising from: S. Conrad et al. 456, 344–349 (2008)10.1038/nature07404; Conrad et al. replyConrad et al. have generated human adult germline stem cells (haGSCs) from human testicular tissue, which they claim have similar pluripotent properties to human embryonic stem cells (hESCs). Here we investigate the pluripotency of haGSCs by using global gene-expression analysis based on their gene array data and comparing the expression of pluripotency marker genes in haGSCs and hESCs, and in haGSCs and human fibroblast samples derived from different laboratories, including our own. We find that haGSCs and fibroblasts have a similar gene-expression profile, but that haGSCs and hESCs do not. The pluripotency of Conrad and colleagues’ haGSCs is therefore called into question.

Recessive cardiac phenotypes in induced pluripotent stem cell models of Jervell and Lange-Nielsen syndrome: disease mechanisms and pharmacological rescue.

Significance There are few laboratory models that recapitulate human cardiac disease. Here, we created human cell models for Jervell and Lange-Nielsen syndrome (JLNS) in vitro, based on human induced pluripotent stem cells (hiPSCs). JLNS is one of the most severe disorders of heart rhythm and can cause sudden death in young patients. JLNS is inherited recessively and is caused by homozygous mutations in the slow component of the delayed rectifier potassium current, IKs. Cardiomyocytes (CMs) from two independent sets of patient-derived and engineered hiPSCs showed electrophysiological defects that reflect the severity of the condition in patients. Our work allowed better understanding of the mechanisms of recessive inheritance. Furthermore, JLNS-CMs showed increased sensitivity to proarrhythmic drugs, which could be rescued pharmacologically, demonstrating the potential of hiPSC-CMs in drug testing. Jervell and Lange-Nielsen syndrome (JLNS) is one of the most severe life-threatening cardiac arrhythmias. Patients display delayed cardiac repolarization, associated high risk of sudden death due to ventricular tachycardia, and congenital bilateral deafness. In contrast to the autosomal dominant forms of long QT syndrome, JLNS is a recessive trait, resulting from homozygous (or compound heterozygous) mutations in KCNQ1 or KCNE1. These genes encode the α and β subunits, respectively, of the ion channel conducting the slow component of the delayed rectifier K+ current, IKs. We used complementary approaches, reprogramming patient cells and genetic engineering, to generate human induced pluripotent stem cell (hiPSC) models of JLNS, covering splice site (c.478-2A>T) and missense (c.1781G>A) mutations, the two major classes of JLNS-causing defects in KCNQ1. Electrophysiological comparison of hiPSC-derived cardiomyocytes (CMs) from homozygous JLNS, heterozygous, and wild-type lines recapitulated the typical and severe features of JLNS, including pronounced action and field potential prolongation and severe reduction or absence of IKs. We show that this phenotype had distinct underlying molecular mechanisms in the two sets of cell lines: the previously unidentified c.478-2A>T mutation was amorphic and gave rise to a strictly recessive phenotype in JLNS-CMs, whereas the missense c.1781G>A lesion caused a gene dosage-dependent channel reduction at the cell membrane. Moreover, adrenergic stimulation caused action potential prolongation specifically in JLNS-CMs. Furthermore, sensitivity to proarrhythmic drugs was strongly enhanced in JLNS-CMs but could be pharmacologically corrected. Our data provide mechanistic insight into distinct classes of JLNS-causing mutations and demonstrate the potential of hiPSC-CMs in drug evaluation.