Roger A. Pedersen

Derivation of pluripotent epiblast stem cells from mammalian embryos.

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.

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells

Maintenance of pluripotency is crucial to the mammalian embryos 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 TGFβ 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.

Mutations of the homeobox genes Dlx-1 and Dlx-2 disrupt the striatal subventricular zone and differentiation of late born striatal neurons.

The striatum has a central role in many neurobiological processes, yet little is known about the molecular control of its development. Inroads to this subject have been made, due to the discovery of transcription factors, such as the Dlx genes, whose expression patterns suggest that they have a role in striatal development. We report that mice lacking both Dlx-1 and Dlx-2 have a time-dependent block in striatal differentiation. In these mutants, early born neurons migrate into a striatum-like region, which is enriched for markers of the striosome (patch) compartment. However, later born neurons accumulate within the proliferative zone. Several lines of evidence suggest that mutations in Dlx-1 and Dlx-2 produce abnormalities in the development of the striatal subventricular zone and in the differentiation of striatal matrix neurons.

Banking on human embryonic stem cells: estimating the number of donor cell lines needed for HLA matching

BACKGROUND 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. METHODS 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. FINDINGS 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%. INTERPRETATION 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.

Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development

Generation of hepatocytes from human embryonic stem cells (hESCs) could represent an advantageous source of cells for cell therapy approaches as an alternative to orthotopic liver transplantation. However, the generation of differentiated hepatocytes from hESCs remains a major challenge, especially using a method compatible with clinical applications. We report a novel approach to differentiate hESCs into functional hepatic cells using fully defined culture conditions, which recapitulate essential stages of liver development. hESCs were first differentiated into a homogenous population of endoderm cells using a combination of activin, fibroblast growth factor 2, and bone morphogenetic protein 4 together with phosphoinositide 3‐kinase inhibition. The endoderm cells were then induced to differentiate further into hepatic progenitors using fibroblast growth factor 10, retinoic acid, and an inhibitor of activin/nodal receptor. After further maturation, these cells expressed markers of mature hepatocytes, including asialoglycoprotein receptor, tyrosine aminotransferase, α1‐antitrypsin, Cyp7A1, and hepatic transcription factors such as hepatocyte nuclear factors 4α and 6. Furthermore, the cells generated under these conditions exhibited hepatic functions in vitro, including glycogen storage, cytochrome activity, and low‐density lipoprotein uptake. After transduction with a green fluorescent protein–expressing lentivector and transplantation into immunodeficient uPA transgenic mice, differentiated cells engrafted into the liver, grew, and expressed human albumin and α1‐antitrypsin as well as green fluorescent protein for at least 8 weeks. In addition, we showed that hepatic cells could be generated from human‐induced pluripotent cells derived from reprogrammed fibroblasts, demonstrating the efficacy of this approach with pluripotent stem cells of diverse origins. Conclusion: We have developed a robust and efficient method to differentiate pluripotent stem cells into hepatic cells, which exhibit characteristics of human hepatocytes. Our approach should facilitate the development of clinical grade hepatocytes for transplantation and for research on drug discovery. (HEPATOLOGY 2010.)

Integrin α8β1 Is Critically Important for Epithelial–Mesenchymal Interactions during Kidney Morphogenesis

We present genetic evidence that integrins regulate epithelial–mesenchymal interactions during organogenesis. Mice with a mutation in the α8 gene do not express the integrin α8β1 and exhibit profound deficits in kidney morphogenesis. In wild-type animals, inductive interactions between the ureteric epithelium and metanephric mesenchyme are essential for kidney morphogenesis. In α8 mutant homozygotes, growth and branching of the ureteric bud and recruitment of mesenchymal cells into epithelial structures are defective. Consistent with these phenotypes, α8 expression is induced in mesenchymal cells upon contact with the ureter. Since none of its previously identified ligands appears likely to mediate the essential functions of α8β1 in kidney morphogenesis, we have used an α8β1–alkaline phosphatase chimera to localize novel ligand(s) in the growing ureter. The distribution of these ligand(s) makes them strong candidates for regulators of kidney morphogenesis.

Activin/Nodal signalling maintains pluripotency by controlling Nanog expression

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.

An Olfactory Sensory Map Develops in the Absence of Normal Projection Neurons or GABAergic Interneurons

Olfactory sensory neurons expressing a given odorant receptor project to two topographically fixed glomeruli in the olfactory bulb. We have examined the contribution of different cell types in the olfactory bulb to the establishment of this topographic map. Mice with a homozygous deficiency in Tbr-1 lack most projection neurons, whereas mice with a homozygous deficiency in Dlx-1 and Dlx-2 lack most GABAergic interneurons. Mice bearing a P2-IRES-tau-lacZ allele and deficient in either Tbr-1 or Dlx-1/Dlx-2 reveal the convergence of axons to one medial and one lateral site at positions analogous to those observed in wild-type mice. These observations suggest that the establishment of a topographic map is not dependent upon cues provided by, or synapse formation with, the major neuronal cell types in the olfactory bulb.

Stem cell medicine encounters the immune system

Recent progress in deriving human embryonic stem (hES) cells and defining their capacity to differentiate has inspired hope that they could become a source of replacement cells for damaged or diseased tissues. We review the immunological barriers to transplanting hES cells and consider several potential solutions, including stem-cell banking, modification of the immunogenicity of donor cells and induction of tolerance to the graft. We evaluate the probable efficacy of these approaches with a view to facilitating the use of hES cells in clinical practice.