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The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue, but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus, fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies, but that of BOECs was lower. In terms of invasiveness of biopsy sampling, biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs, but where non-invasive procedures are required (e.g. for children and minors) dental pulp cells from milk teeth represent a valuable alternative.

TSC-22 is a highly conserved member of a novel family of transcription factors, that is a direct target of transforming growth factor-β (TGF-β) in osteoblastic cells. We have investigated the expression of TSC-22 in detail during mouse development using in situ hybridization. We detected strong expression of TSC-22 in the embryo proper first at embryonic day 8.5 (E8.5), in the primitive heart, intermediate mesoderm and the neural tube. The dynamics of the TSC-22 distribution in the neural tube was particularly striking, with ubiquitous expression rostrally and restriction to neural tissue nearer the floor plate more caudally; between E8.5 and E9.5 the zone of restricted expression extended rostrally. At later stages of development, TSC-22 was detected in the mesenchymal compartment of many tissues and organs, including the lung, trachea, kidney, stomach, intestine, tooth buds, and in precartilage condensations. Furthermore, TSC-22 was highly expressed in the floor plate itself and notochord, and the endothelium lining the blood vessels, in particular the major arteries. Many of these sites have been proposed previously as possible TGF-β target tissues; the results imply that TSC-22 may also be a direct TGF-β target gene during mouse embryogenesis. Experiments on TSC-22 expression in embryoid bodies of embryonic stem (ES) cells expressing dominant negative TGF-β binding receptors initially supported this hypothesis. However, examination of somatic chimeras derived from these same mutant ES cells at nominal E9.5 showed that TSC-22 expression in the heart and neural tube was still detectable despite obvious phenotypic abnormalities. We therefore propose that although TSC-22 may be a direct target of TGF-β in late development, other factors are likely to be major regulators of expression at earlier stages. © 2000 Wiley-Liss, Inc.

We describe isolation and characterization of the bovine ortholog of POU5F1(bPOU5F1) encoding octamer-binding transcription factor-4 (Oct-4). The organization of bPOU5F1is similar to its human and murine orthologs, and it shares 90.6% and 81.7% overall identity at the protein level, respectively. Transient transfection of luciferase reporter constructs in murine P19 embryonal carcinoma cells demonstrated that bPOU5F1has a functional promoter and contains two enhancer elements, of which one is repressed by retinoic acid. bPOU5F1was mapped to the major histocompatibility complex on chromosome 23. bPOU5F1mRNA was detected by nested reverse transcription-polymerase chain reaction in immature oocytes and in in vitro-produced preattachment-stage embryos. Oct-4 in oocytes and in vitro-produced preattachment-stage embryos was demonstrated by indirect immunofluorescence. Confocal laser scanning microscopy revealed Oct-4 in both the inner cell mass and trophoblast cells of the blastocyst until Day 10 of development. Immunofluorescence performed on the outgrowths formed at Day 13 postfertilization from in vitro-produced Day 8 blastocysts showed Oct-4 staining in all cells. This expression pattern suggests that bPOU5F1acts early in bovine embryonic development but that its expression is not restricted to pluripotent cells of the blastocyst.

We have analysed the function of transforming growth factor beta (TGF-beta) in yolk sac development in mice by generating somatic chimaeras in which the extraembryonic mesoderm, which gives rise to the endothelial and haematopoietic cells of the yolk sac vasculature, is derived from embryonic stem (ES) cells. The ES cells were stably transfected and express either the full-length type II binding receptor or a kinase-deficient mutant of this receptor. Examination of yolk sacs from chimaeras between E8.5 and 9.5, and analysis of marker expression in embryoid bodies from these mutant ES cell lines in prolonged suspension culture demonstrated that (1) a major function of TGF-beta in yolk sac mesoderm is to regulate production and deposition of fibronectin in the extracellular matrix that maintains yolk sac integrity, (2) TGF-beta signalling is not required for differentiation of extraembryonic mesoderm into endothelial cells but is necessary for their subsequent organisation into robust vessels, and (3) TGF-beta signalling must be tightly regulated for the differentiation of primitive haematopoietic cells to take place normally. Together, these results show that defective TGF-beta signalling in the extraembryonic mesoderm alone is sufficient to account for the extraembryonic phenotype reported previously in TGF-beta1(−/−) mice (Dickson, M. C., Martin, J. S., Cousins, F. M., Kulkarni, A. B., Karlsson, S. and Akhurst, R. J. (1995) Development 121, 1845–1854).

FGFs have been implicated in the induction of mesoderm in amphibian development and are present in the mouse embryo at stages that would be appropriate for a similar function in mammals. Primitive ectoderm would then he the target tissue. We have now characterized changes in the expression of receptors for FGFs during the differentiation of embryonal carcinoma (EC) and embryonic stem (ES) cells from the mouse. These cells resemble those of the inner cell mass and later primitive ectoderm. On Northern blots of mRNA from undifferentiated cells, transcripts for FGF R1, R2 and R3 are expressed. All are unregulated during differentiation of ES cells and are unregulated or remain constant as EC cells differentiate. FGF R4 is only expressed after differentiation to derivatives resembling parietal endoderm. By contrast in human EC cells, FGF R2 is downregulated during differentiation, FGF R1 and FGF R3 are unchanged and FGF R4 is expressed before and after differentiation. In both human and mouse EC cells three members of the FGF family (a FGF, b FGF and k FGF, also known as FGFs 1,2 and 4) are mitogenic in serum-free medium and one (KGF or FGF 7) appears to have no effect on growth although cellular morphology is altered. Differences between human and mouse cells are primarily in the effects of heparin on the FGF-induced response.

Gastrulation in rodents is associated with an increase in the rate of growth and with the start of differentiation within the embryo proper. In an effort to understand the role played by the cell cycle control in these processes, expression of cyclin D1, D2, and D3—three major positive regulators of the G1/S transition—has been investigated by in situ hybrization and RT-PCR. Cyclin D1 and D2 transcripts are first detected in the epiblast at gastrulation, when a proliferative burst occurs, and subsequently in its differentiated derivatives within the embryo proper, indicating that activation of their expression takes place prior to the differentiation of epiblast progenitors. In contrast, cyclin D3 transcript is undetectable in the epiblast itself and its expression is activated exclusively in extraembryonic tissues of both epiblast and trophoblast origin. During neurulation, expression of each cyclin D RNA is dynamically regulated along the anterior-posterior axis. In the hindbrain, cyclin D1 and D2 show distinct segment-specific restricted expression and this pattern is conserved between mouse and chick. These results strongly suggest that D-type cyclins act as developmental regulators. Dev. Dyn. 1998;212:49–62. © 1998 Wiley-Liss, Inc.

Abstract.: In total, 82 ESTs were generated from 51 unique clones randomly selected from a cattle ovary cDNA library. Among these clones, 22 (42.1%) had 5′ and/or 3′ ends that matched with known human or other mammalian coding sequences, 18 (35.3%) matched human or other ESTs, and 11 (21.6%) represented novel transcripts with no significant match to any sequence in the databases. The relatively high frequency of ESTs with no matches in GenBank or dbEST indicates that bovine ovary may be a source of novel candidate genes for loci affecting cattle reproduction traits. Primers were designed for 11 ESTs that had human orthologs in GenBank. These ESTs were mapped to 10 bovine autosomes by PCR screening of a somatic cell hybrid panel. Among these 11 ESTs, 4 corresponded to genes previously mapped in humans and had chromosome assignments on the bovine map that were consistent with available comparative mapping data. Although the human orthologs of the remaining 7 mapped bovine ESTs have not been mapped, the human map location could be predicted on the basis of existing comparative mapping data. Because of the general utility of our approach for comparative genome analysis, we have termed it comparative mapping by annotation and sequence similarity (COMPASS). With the cost of large-scale EST sequencing becoming more affordable, and the rapid expansion of DNA databases, it is likely that COMPASS will be a preferred strategy for high throughput comparative mapping.

During the development of the human fetal germ cells, the first trimester human gonads include different subpopulation of germ cells because of the gradual and unsynchronized development. To elucidate human fetal germ cells development more precisely, human germ cells in the first trimester (9.0–12.5 weeks) were comprehensively analyzed by double and triple whole-mount immunostaining. Stage specific embryonic antigen 1 (SSEA1) has been commonly used for identifying mouse and human germ cells in vivoand in vitro, however, triple whole-mount immunostaining with SSEA1/cKIT/OCT4 clearly showed that both cKIT and OCT4 were specifically expressed in human germ cells in the gonads, however, SSEA1 expression pattern in human germ cells were very weak and speckled, importantly, not specific for human germ cells during the first trimester. By co-localization of cKIT with VASA in the first trimester human germ cells, we could identify several different subpopulations of immature human germ cells. This suggests that there is a transition from immature germ cells expressing cKIT to germ cells expressing VASA during the first trimester. Intriguingly, we observed cytoplasmic localization of VASA followed by prominent and transient Golgi localization of cKIT in human germ cells. Moreover, we observed drastic changes of nuclear morphology as the chromosomes begin to decondense in the human germ cells. This process of chromosome decondensation is manifested as signs of epigenetic reprogramming in human germ cells. One of the epigenetic features of female germ cell development is X chromosome reactivation. To further investigate the precise timing of X chromosome reactivation in human female germ cells, we examined tri-methylation of histone H3 lysine 27 (H3K27me3) accumulations on inactive X chromosome (Xi) in human female germ cells. Here we showed that dynamic decrease of H3K27me3 accumulation on Xi was observed according with increased cytoplasmic VASA expression in human female germ cells. These data provide the first evidence for epigenetic reprogramming and X chromosome reactivation in human fetal germ cells during the first trimester of development.