ES cells derived from cloned embryos in monkey--a jump toward human therapeutic cloning

Therapeutic cloning refers to the derivation of embryonic stem cells （ntESC） from embryos derived from somatic cell nuclear transfer （SCNT） also known as cloning. Cloning involves transplanting a differentiated cell into an oocyte that has had its nucleus （DNA） removed. The reconstructed oocyte can be activated to divide and develop into an embryo. The process that allows this to happen is termed nuclear reprogramming, and is defined as the mechanism through which a differentiated cell de-differentiates or returns to a totipotent state （capable of giving rise to any cell type, including extra-embryonic） and directs embryonic development [1]. Cells from blastocyst stage cloned embryos can be used to generate ntESC lines. Such cell lines can differentiate into any adult cell type, and have tremendous potential for patient-specific disease therapy [2].     

Functional transplantation of salivary gland cells differentiated from mouse early ES cells in vitro

Atrophy or hypofunction of the salivary gland because of aging or disease causes hyposalivation and has an effect on the quality of life of patients, for example not only dry mouth but deterioration in mastication/deglutition disorder and the status of oral hygiene. Currently conducted therapies for atrophy or hypofunction of the salivary gland in clinical practice are only symptomatic treatments with drugs and artificial saliva, and therefore it is preferable to establish a radical therapy. At this time, as a fundamental investigation, by co-culturing mouse early ES (mEES-6) cells with human salivary gland-derived fibroblasts (hSG-fibro), differentiation of mEES-6 cells to salivary gland cells has been attempted. Also, the possibility of cell engraftment was examined. After identifying the cells which were co-cultured with GFP-transfected mEES-6 cells and hSG-fibro, the cells were transplanted into the submandibular gland of SCID mice, and the degree of differentiation into tissues was examined. The possibility of tissue functional reconstitution from co-cultured cells in a three-dimensional culture system was examined. Our results confirmed that the co-cultured cells expressed salivary gland-related markers and had an ability to generate neo-tissues by transplantation in vivo. Moreover, the cells could reconstitute gland structures in a three-dimensional culture system. By co-culture with hSG-fibro, mEES-6 cells were successfully differentiated into salivary gland cells which were transplantable and have tissue neogenetic ability.     

Differential expression of the Wnt and Frizzled genes in Flk1+ cells derived from mouse ES cells

Embryonic stem (ES) cells have the potential to develop into various cell lineages including hemangioblasts (Flk1+), a common progenitor for hematopoietic and vascular endothelial cells. Previous studies indicate that Flk1+ cells, a marker for hemangioblast, can be derived from ES cell and that Flk1+ can be differentiated into hematopoietic or endothelial cells depending on culture conditions. We developed an improved in vitro system to generate Flk1+-enriched cultures from mouse ES cells and used this in vitro system to study the role of Wnt signalling in early endothelial progenitor cells. We determined the expression of the Wnt and Frizzled genes in Flk1+ cells derived from mouse ES cells. RT-PCR analyses identified significantly higher expression of non-canonical Wnt5a and Wnt11 genes in Flk1+ cells compared to Flk1- cells. In contrast, expression of canonical Wnt3a gene was reduced in Flk1+ cells. In addition, Frizzled2, Frizzled5 and Frizzled7 genes were also expressed at a higher level in Flk1+ cells. The differential expression of Wnt and Frizzled genes in Flk1+ cells provides a novel insight into the role of non-canonical Wnt signalling in vascular endothelial fate determination.     

Extra-embryonic endoderm cells derived from ES cells induced by GATA Factors acquire the character of XEN cells

Background  

Three types of cell lines have been established from mouse blastocysts: embryonic stem (ES) cells, trophoblast stem (TS) cells, and extra-embryonic endoderm (XEN) cells, which have the potential to differentiate into their respective cognate lineages. ES cells can differentiate in vitro not only into somatic cell lineages but into extra-embryonic lineages, including trophectoderm and extra-embryonic endoderm (ExEn) as well. TS cells can be established from ES cells by the artificial repression of Oct3/4 or the upregulation of Cdx2 in the presence of FGF4 on feeder cells. The relationship between these embryo-derived XEN cells and ES cell-derived ExEn cell lines remains unclear, although we have previously reported that overexpression of Gata4 or Gata6 induces differentiation of mouse ES cells into extra-embryonic endoderm in vitro.     

Genome‐wide screen of promoter methylation analysis of ES cells and ES derived epidermal‐like cells

Embryonic stem cells (ESCs) are a population of pluripotent cells which can differentiate into different cell types. However, there are few reports with regard to differentiate ESCs into epidermal cells in vitro. In this study, we aimed to investigate differentially methylated promoters involved in process of differentiation from ESCs into epidermal‐like cells (ELCs) induced by human amnion. We successfully induced ESCs into ELCs, which expressed the surface markers of CK19, CK15 and β1‐integrin. With MeDIP‐chip arrays, we identified 3435 gene promoters to be differentially methylated, involving 894 HCP (high CpG‐containing promoter), 974 ICP (intermediate CpG‐containing promoter) and 1567 LCP (low CpG‐containing promoter) among all the 17 500 DNA methylation regions of gene promoters in both ESCs and ELCs. Gene oncology and pathway analysis demonstrated that these genes were involved in all the three categories of GO enrichment analysis, including biological process, molecular function and cellular component. All these data suggested that embryonic stem cells can differentiate into epidermal‐like cells and promoter methylation is of great importance in this process. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro osteogenic differentiation of human ES cells

Since their isolation in 1998, human embryonic stem (hES) cells have been shown to be capable of adopting various cell fates in vitro. Here, we present in vitro data demonstrating the directed commitment of human embryonic stem cells to the osteogenic lineage. Human ES cells are shown to respond to factors that promote osteogenesis, leading to activation of the osteogenic markers osteocalcin, parathyroid hormone receptor, bone sialoprotein, osteopontin, cbfa1, and collagen 1. Moreover, the mineralized nodules obtained are composed of hydroxyapatite, further establishing the similarity of osteoblasts in culture to bone. These results show that osteoblasts can be derived from human ES cultures in vitro and provide the basis for comparison of adult and embryonic-derived osteogenesis, and for an investigation of potential applications for hES cells in orthopaedic tissue repair.     

Production of hematopoietic cells from ES cells

Murine embryonic stem (ES) cells are cell lines established from blastocyst which can contribute to all adult tissues, including the germ-cell lineage, after reincorporation into the normal embryo. ES cell pluripotentiality is preserved in culture in the presence of LIF. LIF withdrawal induces ES cell differentiation to nervous, myocardial, endothelial and hematopoietic tissues. The model of murine ES cell hematopoietic differentiation is of major interest because ES cells are non transformed cell lines and the consequences of genomic manipulations of these cells are directly measurable on a hierarchy of synchronized in vitro ES cell-derived hematopoietic cell populations. These include the putative hemangioblast (which represents the emergence of both hematopoietic and endothelial tissues during development), myeloid progenitors and mature stages of myeloid lineages. Human ES cell lines have been recently derived from human blastocyst in the USA. Their manipulation in vitro should be authorized in France in a near future with the possibility of developing a model of human hematopoietic differentiation. This allows to envisage in the future the use of ES cells as a source of human hematopoietic cells.     

Connections between descending visual interneurons and metathoracic motoneurons in the locust

Summary Connections between the four DMD neurons and metathoracic motoneurons in the locustSchistocerca were examined by recording extracellularly from the interneurons in the pro-mesothoracic connectives and intracellularly from seventeen motoneurons. A DIMD or DCMD spike causes an EPSP in the fast extensor tibiae motoneuron, which can be modified by changing the membrane potential. The EPSP always follows spikes at frequencies up to 200 Hz and with a latency of 0.9 ms, suggesting that the connections are monosynaptic and chemically mediated. EPSPs from the DIMD or DCMD arrive at the same time, their axons having the same conduction velocity, and appear simultaneously in the fast extensor tibiae motoneurons on both sides of the ganglion. There is spatial and temporal summation between the inputs but on no occasion did the motoneurons spike. Three inhibitory neurons are depolarized by DMD inputs and may on occasion spike, but it is not known whether these connections are direct. Similarly the slow excitatory motoneuron to the anterior coxal adductor muscle is hyperpolarized by DMD input. Other leg, flight or ventilatory motoneurons examined received no inputs from the DMD neurons. The connections shown are consistent with the hypothesis that the DMD neurons are in some way involved with initiation of a jump, but to achieve this must act synergistically with other inputs. This work was supported in part by USPHS grant No. NS 09404-03 to C.H.F.R. Dr. Rowell wishes to thank Dr. J. Phillipson for the use of facilities in the Oxford Department of Zoology during sabbatical leave.     

Functional spermatid-like cells derived from the ground-state embryonic stem cells in vitro

正Embryonic stem cells(ESCs)are pluripotent and can self-assemble to form cell clusters or embryoid bodies(EBs),which can then differentiate into all cell types of the three germ layers,as among which one is the primordial germ cell(PGCs)(Daley,2007).In vivo PGCs are the gamete founder cells(the ooctyes and sperms),which transmit genetic information from one generation to the next generation to maintain mammalian life cycles.About 15%of couples in China are infertile;some of these infertility cases are     

Murine myeloid cell lines derived by in vitro infection with recombinant c-myb retroviruses express myb from rearranged vector proviruses.

To date, cellular transformation in vitro by the myb oncogene has been described for avian haemopoietic cells only. In order to exploit the well-characterized murine haemopoietic system to study transformation by myb, we have infected fetal liver cells with retroviral vectors carrying cDNAs that encode either complete or carboxy-terminally truncated c-myb proteins. We describe four cell lines which, despite our ability to efficiently infect haemopoietic target cells, were generated at low frequency. This was due, as least in part, to the requirement for a rearrangement within the vector that allowed expression of myb sequences. Three of the lines express a truncated myb protein while the fourth apparently expresses a normal c-myb protein, and thus constitutes an exception to the general association of truncation with transformation by myb. All four cell lines resemble immature cells of the myelomonocytic lineage and are dependent on colony-stimulating factors (CSFs) for their growth in vitro. One representative line could be converted to CSF-independence by infection with either Abelson murine leukaemia virus or a recombinant granulocyte-macrophage-CSF-encoding retrovirus; unlike the parental line, the resultant sublines were highly tumorigenic when injected into syngeneic mice.     

In vitro enhanced differentiation of neural networks in ES gut-like organ from mouse ES cells by a 5-HT4-receptor activation

Using an embryoid body (EB) culture system, we developed a functional organ-like cluster, a “gut”, from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited various types of spontaneous movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (ICC) (c-kit, a transmembrane receptor that has tyrosine kinase activity, positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified, but enteric neural networks were not identified. In the present study, we succeeded in forming dense enteric neural networks by a 5-HT₄-receptor (SR4) agonist, mosapride citrate (1–10 μM) added only during EB formation. Addition of an SR4-antagonist, GR113808 (10 μM) abolished the SR4-agonist-induced formation of enteric neural networks. The SR4-agonist (1 μM) up-regulated the expression of mRNA of SR4 and the SR4-antagonist abolished this upregulation. 5-HT per se exerted similar effects to those of SR4-agonist, though less potent. These results suggest SR4-agonist differentiated enteric neural networks, mediated via activation of SR4 in the ES gut.