Infertility in male transgenic mice: disruption of sperm development by HSV-tk expression in postmeiotic germ cells

Previous experiments revealed that male transgenic mice bearing a cosmid that included the Class II E alpha gene, about 35 kb of 5' flanking DNA, and the cosmid vector sequences were sterile. To ascertain the cause of the sterility, various subfragments of the cosmid were tested in transgenic mice. Only those pieces of DNA that included some of the E alpha flanking chromosomal DNA and the herpes simplex virus (HSV)-thymidine kinase (tk) gene that was in the vector resulted in male sterility. Histological analysis revealed abnormalities in nuclear morphology of elongating spermatids and retention of mature spermatids within the seminiferous epithelium. Immunocytochemical studies showed that the HSV-tk gene was expressed at low levels in postmeiotic round spermatids and at higher levels in more mature elongating spermatids. To determine whether expression of HSV-tk in spermatids might be responsible for the sterility, the protamine gene promoter was used to direct the expression of HSV-tk to postmeiotic germ cells. Since the mice so treated were also sterile, the data suggest that expression of this enzyme in spermatids is responsible for the sterility phenotype.     

Polony analysis of gene expression in ES cells and blastocysts

Expression profiling of stem cells is challenging due to their small numbers and heterogeneity. The PCR colony (polony) approach has theoretical advantages as an assay for stem cells but has not been applied to small numbers of cells. An assay has been developed that is sensitive enough to detect mRNAs from small numbers of ES cells and from fractions of a single mouse blastocyst. Genes assayed include Oct3, Rex1, Nanog, Cdx2 and GLUT-1. The assay is highly sensitive so that multiple mRNAs from a single blastocyst were easily detected in the same assay. In its present version, the assay is an attractive alternative to conventional RT–PCR for profiling small populations of stem cells. The assay is also amenable to improvements that will increase its sensitivity and ability to analyze many cDNAs simultaneously.     

Differentiation of primate ES cells into retinal cells induced by ES cell-derived pigmented cells

Purpose: Photoreceptors cannot regenerate and recover their functions once disordered. Transplantation of retinal pigment epithelium (RPE) has recently become a possible therapeutic approach for retinal degeneration. In the present study, we investigated the induction of photoreceptors by coculturing primate embryonic stem cells (ESCs) with ESC-derived RPE cells. Methods: RPE cells were derived by coculturing ESCs and Sertoli cells. Photoreceptors were then induced by using ESC-derived RPE cells and retinoic acid (RA) Results: RPE cell generation was confirmed by morphological analysis, which revealed highly pigmented polygonal cells with a compact cell-cell arrangement. After coculturing ESCs and RPE cells, some ESC derivatives became immunopositive for rhodopsin. RT-PCR analysis demonstrated the expression of retina-related gene markers such as Pax6, CRX, IRBP, rhodopsin, rhodopsin kinase, and Muschx10A. When RA was added, a distinct increase in the expression of photoreceptor-specific proteins and genes was found. In addition, the differentiation of bipolar horizontal cells was demonstrated by protein and gene expression. The ESCs that were cocultured with RPE cells and treated with RA were transplanted into the renal capsule or intra-vitreal space of nude mice. Grafted ESC derivatives demonstrated extensive rhodopsin expression, and they survived and organized into recipient tissues, although they formed teratomas. Conclusion: These results indicate that coculturing ESCs with ESC-derived RPE cells is a useful and efficient method for inducing photoreceptors and providing an insight into the use of ESCs for retina regeneration.     

Estimating the quality of reprogrammed cells using ES cell differentiation expression patterns

Somatic cells can be reprogrammed to a pluripotent state by over-expression of defined factors, and pluripotency has been confirmed by the tetraploid complementation assay. However, especially in human cells, estimating the quality of Induced Pluripotent Stem Cell(iPSC) is still difficult. Here, we present a novel supervised method for the assessment of the quality of iPSCs by estimating the gene expression profile using a 2-D "Differentiation-index coordinate", which consists of two "developing lines" that reflects the directions of ES cell differentiation and the changes of cell states during differentiation. By applying a novel liner model to describe the differentiation trajectory, we transformed the ES cell differentiation time-course expression profiles to linear "developing lines"; and use these lines to construct the 2-D "Differentiation-index coordinate" of mouse and human. We compared the published gene expression profiles of iPSCs, ESCs and fibroblasts in mouse and human "Differentiation-index coordinate". Moreover, we defined the Distance index to indicate the qualities of iPS cells, which based on the projection distance of iPSCs-ESCs and iPSCs-fibroblasts. The results indicated that the "Differentiation-index coordinate" can distinguish differentiation states of the different cells types. Furthermore, by applying this method to the analysis of expression profiles in the tetraploid complementation assay, we showed that the Distance index which reflected spatial distributions correlated the pluripotency of iPSCs. We also analyzed the significantly changed gene sets of "developing lines". The results suggest that the method presented here is not only suitable for the estimation of the quality of iPS cells based on expression profiles, but also is a new approach to analyze time-resolved experimental data.     

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.     

Differential expression of genes involved in cGMP-dependent nitric oxide signaling in murine embryonic stem (ES) cells and ES cell-derived cardiomyocytes.

Nitric oxide (NO) performs multiple physiological roles as a biological signaling molecule. The role of NO and cGMP signaling in embryonic stem (ES) cell-derived cardiomyocytes (CM) has been investigated but many questions remain. In this study, we examined the expression of the NO signaling pathway components nitric oxide synthase (NOS-1, 2, 3), soluble guanylyl cyclase (sGCalpha(1) and beta(1)) and protein kinase G (PKG) genes and sGC activity in murine ES cells subjected to differentiation by embryoid body (EB) formation. We found that in undifferentiated ES cells, NOS-1, NOS-3, and sGCbeta(1) were detected while NOS-2, sGCalpha(1), and PKG were very low or undetectable. When ES cells were subjected to differentiation, NOS-1 abruptly decreased within one day, NOS-2 mRNA became detectable after several days, and NOS-3 increased after 7-10 days. Levels of sGCalpha(1), sGCbeta(1), and PKG all increased gradually over a several day time course of differentiation in EB outgrowths. Analysis of sGC activity in cell lysates derived from undifferentiated ES cells revealed that NO could not stimulate cGMP. However, lysates from differentiated EB outgrowths produced abundant cGMP levels after NO stimulation. Purification of ES-cell derived CM revealed that mRNA expression of all the NOS isoforms was very low to absent while sGCalpha(1) and beta(1) subunit mRNAs were abundant and sGC-mediated cGMP production was apparent in this population of cells. These data suggest that cGMP-mediated NO signaling may play a minor role, if any, in undifferentiated ES cells but could be involved in the early differentiation events or physiological processes of ES cells or ES cell-derived lineages.     

Effect of mitotic inducers and retinoic acid blocker on expression of pluripotent genes in ES cells derived from early stage in vitro-produced embryos in buffalo

So far, it has been difficult to generate embryonic stem (ES) cell from early stage preimplantation embryos of buffalo. These ES cells will be more helpful for efficient embryo cloning and generation of body cells as they are more primitive than inner cell mass (ICM)-derived ES cells. The present study was conducted to find the effect of lipopolysaccharide (LPS), melatonin (N-acetyl-5-methoxytryptamine, a pineal gland product), and citral (3,7-dimethyl-2,6-octadienal and a retinoic acid synthesis blocker) on establishment of primary ES cell colonies, the comparative size of the ES cell colonies, and expression of pluripotent genes during extended period of culture in buffalo. Zona-free eight-cell stage in vitro fertilization (IVF) embryos were cultured in ES cell medium supplemented with none (media I as control), LPS (media II), citral melatonin (media III), or melatonin (media IV). The multiplication of blastomere leading to ES cell colony formation and expression of pluripotent genes were assessed up to day 20 of culture. The primary colony formation, the comparative size of the ES cell colonies, and expression of pluripotent genes in these colonies were better in the medium supplemented with melatonin in all days of culture. Within melatonin supplementation, the colony size was comparatively larger on day 8 and day 12 of culture. Further, with this supplementation, the Oct-4 and Nanog expression was comparatively higher on all days of culture. The results indicated that supplementation of melatonin helped in the formation of better primary ES cell colony as well as in the maintenance of pluripotency. The results also indicated that primary colonies developed on day 8 to day 12 of culture may be better for passaging them for establishment of ES cell line from early stage preimplantation IVF embryos of in buffalo.     

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.     

Controlled-rate freezing of human ES cells

A significant obstacle to using human embryonic stem cells (hESCs) arises from extremely poor survival associated with freezing, typically in the range of 1%. This report describes a slow controlled-rate freezing technique commonly used for mammalian embryo cryopreservation. Using a combination of surviving colony number and colony diameter; survival was determined relative to untreated hESCs. Using a dimethyl sulfoxide (DMSO) cryoprotectant and either a homemade controlled-rate freezing device or a commercial freezing device, survival rates of 20%-80% were obtained. To achieve the highest levels of survival, the critical factors were an ice crystal seed (at -7 degrees to -10 degrees C), a freeze rate between 0.3 degrees and 1.8 degrees C/min, and a rapid thaw rate using room temperature water. Slow controlled-rate cooling allows a rapid, simple, and reproducible means of cryopreserving hESCs.