Isolation of embryonic stem-like cells from equine blastocysts and their differentiation in vitro

Embryonic stem (ES) cells are pluripotent cells with the potential capacity to generate any type of cell. We describe here the isolation of pluripotent ES-like cells from equine blastocysts that have been frozen and thawed. Our two lines of ES-like cells (E-1 and E-2) appear to maintain a normal diploid karyotype indefinitely in culture in vitro and to express markers that are characteristic of ES cells from mice, namely, alkaline phosphatase, stage-specific embryonic antigen-1, STAT-3 and Oct 4. After culture of equine ES-like cells in vitro for more than 17 passages, some ES-like cells differentiated to neural precursor cells in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor and platelet-derived growth factor. We also developed a protocol that resulted in the differentiation of ES-like cells in vitro to hematopoietic and endothelial cell lineages in response to bFGF, stem cell factor and oncostatin M. Our observations set the stage for future developments that may allow the use of equine ES-like cells for the treatment of neurological and hematopoietic disorders.     

Generation of cloned calves and transgenic chimeric embryos from bovine embryonic stem-like cells

Bovine embryonic stem-like cells (ES-like cells) appear to maintain a normal diploid karyotype indefinitely during culture in vitro and to express marker proteins that are characteristic of ES cells from mice, namely, alkaline phosphatase (AP), stage-specific embryonic antigen-1 (SSEA-1), STAT-3, and Oct 4. After proliferation of undifferentiated ES-like cells in vitro, some bovine ES-like cells differentiated to neural precursor cells, which were cultured in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF). In addition, calves were successfully cloned using ES-like cells and the frequency of term pregnancies for blastocysts derived from ES-like cells was higher than those of early pregnancies and maintained pregnancies after nuclear transplantation (NT) with bovine somatic cells. Successful cloning from bovine ES-like cells should allow the introduction into cattle of specific genetic characteristics of biomedical and/or agricultural importance.     

On the isolation of embryonic stem cells: Comparative behavior of murine, porcine and ovine embryos

The efficiency of isolation and the characteristics of embryo-derived cell lines from murine, porcine, and ovine embryos cultured on STO feeders or homologous embryonic fibroblasts (HEF) feeders were compared. While murine isolated ICM or intact embryos plated on STO or HEF feeders gave rise to cell lines with embryonic stem cell-like (ES-like) morphology, ovine embryos did not. Cell lines with ES-like morphology were isolated from porcine intact embryos and isolated ICM when plated on STO feeders but not when plated on HEF. Neither murine nor porcine ES-like cell lines expressed cytokeratin 18 or vimentin. Unlike murine ES-like cell lines, porcine ES-like cells did not undergo observable differentiation in vitro or in vivo. Cell lines with epithelial-like morphology were isolated from porcine and ovine embryos. Both porcine and ovine epithelial-like cell kines expressed cytokeratin 18. When induced to differentiate in vitro, porcine and ovine epithelial-like cell lines formed vesicular structures. Electron microscopy revealed that the porcine vesicles were composed of polarized epithelial cells, each with a basally-located nucleus and an apical border containing numerous microvilli with a well organized microfilament core. The results of this study show that conditions which allow isolation of ES cells from murine embryos allow the isolation of porcine embryo-derived cell lines sharing some, but not all, the characteristics of murine ES cells.     

Isolation and culture of pluripotent cells from in vitro produced porcine embryos

The present study was designed to examine whether in vitro produced porcine embryos can be used to establish an embryonic stem (ES) cell line. Porcine embryos were produced by in vitro maturation and in vitro fertilization. Embryos at the 4-cell to blastocyst stages were cultured in an ES medium containing 16% fetal bovine serum with mouse embryonic fibroblasts as a feeder layer. It was found that ES-like colonies were derived only from blastocysts. When these ES-like colonies were separated in 0.25% trypsin-0.02% EDTA solution and cultured again, ES-like colonies were further observed in the subsequent culture until the fourth passage. The cells from ES-like colonies showed positive alkaline phosphatase activity. Some cells from the colonies differentiated into several types of cells in vitro when they were cultured in the medium without feeder layers and leukemin inhibitory factor. Embryoid bodies were also formed when the cells were cultured in a suspension status. These results indicate that porcine ES-like cells can be derived from in vitro produced porcine blastocysts and these ES-like cells are pluripotent. The culture system used in the present study is useful to isolate and culture ES cells from in vitro produced porcine embryos.     

Factors derived from mouse embryonic stem cells promote self-renewal of goat embryonic stem-like cells

Goat embryonic stem (ES)-like cells could be isolated from primary materials-inner cell masses (ICMs) and remain undifferentiated for eight passages in a new culture system containing mouse ES cell conditioned medium (ESCCM) and on a feeder layer of mouse embryo fibroblasts (MEFs). However, when cultured in medium without mouse ESCCM, goat ES-like cells could not survive for more than three passages. In addition, no ES-like cells could be obtained when ICMs were cultured on goat embryo fibroblasts or the primary materials-whole goat blastocysts were cultured on MEFs. Goat ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ES-like cells was confirmed by differentiation into neural cells and fibroblast-like cells in vitro. These results suggest that mouse ES cells might secrete factors playing important roles in promoting goat ES-like cells' self-renewal, moreover, the feeder layers and primary materials could also influence the successful isolation of goat ES-like cells.     

Generation of pluripotent stem cells from neonatal mouse testis

Although germline cells can form multipotential embryonic stem (ES)/embryonic germ (EG) cells, these cells can be derived only from embryonic tissues, and such multipotent cells have not been available from neonatal gonads. Here we report the successful establishment of ES-like cells from neonatal mouse testis. These ES-like cells were phenotypically similar to ES/EG cells except in their genomic imprinting pattern. They differentiated into various types of somatic cells in vitro under conditions used to induce the differentiation of ES cells and produced teratomas after inoculation into mice. Furthermore, these ES-like cells formed germline chimeras when injected into blastocysts. Thus, the capacity to form multipotent cells persists in neonatal testis. The ability to derive multipotential stem cells from the neonatal testis has important implications for germ cell biology and opens the possibility of using these cells for biotechnology and medicine.     

Isolation and characterization of embryonic stem-like cells derived from in vivo-produced cat blastocysts

Embryonic stem (ES)-like cells were isolated from in vivo-produced cat embryos. Total of 101 blastocysts were collected from female cats. The inner cell mass (ICM) were mechanically isolated and cultured on mitomycin-C-treated cat embryonic fibroblast feeder layers in medium supplemented with knockouttrade mark Serum Replacement (KSR-medium) or fetal bovine serum (FBS-medium). Putative ES-like cell colonies developed in both KSR- and FBS-medium conditions, but formed domed and flat colonies, respectively. ICM cell attachment and ES-like cell colony formation were significantly higher in KSR-medium, but subsequent cell proliferation was significantly lower than in FBS-medium. For passaging, 32 and 18 colonies in KSR- and FBS-medium were separated by enzymatic dissociation or mechanical disaggregation. Enzymatic dissociation resulted in cell differentiation; however, mechanical disaggregation generated cells that remained undifferentiated over more than four passages and yielded two cat ES-like cell lines that continued to grow for up to eight passages in FBS-medium. These cells had typical stem cell morphology, expressed high levels of alkaline phosphatase activity, and were positive for the ES cell-markers Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-3, and SSEA-4. These cells formed embryoid bodies (EBs) in suspension culture after extended suspension culture. When simple EBs were cultured on tissue culture plates, they differentiated into several cell types, including epithelium-like and neuron-like cells. In addition, EBs were positive for mesoderm marker, desmin. After prolonged in vitro culture, some colonies spontaneously differentiated into beating myocardiocytes, and were positive for alpha-actinin. These observations indicate that cat ES-like cells were successfully isolated and characterized from in vivo-produced blastocysts.     

A combination of Buffalo rat liver cell-conditioned medium, forskolin and membrane-bound stem cell factor stimulates rapid proliferation of mouse primordial germ cells in vitro similar to that in vivo

Recent studies have shown that stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and the enhancement of cAMP levels increase proliferation and survival of mouse primordial germ cells (PGC) in vitro . Even after the addition of these factors, however, it is still not possible to obtain proliferation of PGC at a rapid rate similar to that in vivo , suggesting the presenge of other growth factor(s) in vivo . We previously reported that tumor necrosis factor-α stimulates proliferation of PGC at earlier migration stages. We now show that the use of SI/SI4-m220 feeder cells and the addition of a medium conditioned with Buffalo rat liver cells and forskolin to the culture medium stimulate PGC obtained from 8.5 days post coitum embryos to proliferate in culture at a rate comparable to that in vivo . Under such conditions, proliferation of PGC continued several days past the timing of growth arrest in vivo ; however, it did stop afterwards. Such proliferating PGC continue to express c-kit and Oct-3 proteins. The characteristics of the culture medium and the requirement of feeder cells were different from those for embryonic stem (ES) cells, suggesting that these rapidly proliferated PGC are not transformed into ES-like EG cells.     

Isolation and culture of embryonic stem-like cells from pig nuclear transfer blastocysts of different days

This study was conducted to establish pig embryonic stem (ES)-like cell lines from nuclear transfer blastocysts. A green fluorescent protein (GFP)-expressing cell line was used as the source of donor cells injected into the enucleated oocytes. Blastocysts were collected at D5 (the fifth day), D7 (the seventh day) and D9 (the ninth day). Differential staining was used to assay the viability and development of blastocysts from the 3 days. The number of inner cell mass (ICM) cells increased from 1.83 ± 0.8 (D5) to 5.37 ± 1.2 (D7) to 7.56 ± 1.5 (D9). The expression profiles of embryonic stem (ES) cell factors (OCT4, SOX2, KLF4 and c-MYC) correlated best with the undifferentiated ES state and were identified by qPCR. The expression of the four factors was increased from D5 to D7, whereas the expression decreased from D7 to D9. We tried to isolate ES-like cells from these embryos. However, ES-like cells from the D7 blastocysts grew slowly and expressed alkaline phosphatase. The cells from the D9 blastocysts grew rapidly but did not express alkaline phosphatase. ES-like cells were not isolated from the D5 blastocysts. These results show that the cells from the D7 embryos are pluripotent but grow slowly. The cells from the D9 embryos grow rapidly but start to lose pluripotency.     

Production of live calves derived from embryonic stem-like cells aggregated with tetraploid embryos

To date, cloned farm animals have been produced by nuclear transfer from embryonic, fetal, and adult cell types. However, mice completely derived from embryonic stem (ES) cells have been produced by aggregation with tetraploid embryos. The objective of the present study was to generate offspring completely derived from bovine ES-like cells. ES-like cells isolated from the inner cell mass of in vitro-produced embryos were aggregated with tetraploid bovine embryos generated by electrofusion at the 2-cell stage. A total of 77 embryo aggregates produced by coculture of two 8-cell-stage tetraploid embryos and a clump of ES-like cells were cultured in vitro. Twenty-eight of the aggregates developed to the blastocyst stage, and 12 of these were transferred to recipient cows. Six calves representing 2 singletons and 2 sets of twins were produced from the transfer of the chimeric embryos. Microsatellite analysis for the 6 calves demonstrated that one calf was chimeric in the hair roots and the another was chimeric in the liver. However, unfortunately, both of these calves died shortly after birth. Two of the placentae from the remaining pregnancies were also chimeric. These results indicate that the bovine ES-like cells used in these studies were able to contribute to development.     

Mitochondria structural reorganization during mouse embryonic stem cell derivation

Mouse embryonic stem (ES) cells are widely used in developmental biology and transgenic research. Despite numerous studies, ultrastructural reorganization of inner cell mass (ICM) cells during in vitro culture has not yet been described in detail. Here, we for the first time performed comparative morphological and morphometric analyses of three ES cell lines during their derivation in vitro. We compared morphological characteristics of blastocyst ICM cells at 3.5 and 4.5 days post coitum on feeder cells (day 6, passage 0) with those of ES cells at different passages (day 19, passage 2; day 25, passage 4; and passage 15). At passage 0, there were 23–36% of ES-like cells with various values of the medium cross-sectional area and nucleocytoplasmic parameters, 55% of fibroblast-like (probably trophoblast derivatives), and ~?19% of dying cells. ES-like cells at passage 0 contained autolysosomes and enlarged mitochondria with reduced numerical density per cell. There were three types of mitochondria that differed in matrix density and cristae width. For the first time, we revealed cells that had two and sometimes three morphologically distinct mitochondria types in the cytoplasm. At passage 2, there were mostly ES cells with a high nucleocytoplasmic ratio and a cytoplasm depleted of organelles. At passage 4, ES cell morphology and morphometric parameters were mostly stable with little heterogeneity. According to our data, cellular structures of ICM cells undergo destabilization during derivation of an ES cell line with subsequent reorganization into the structures typical for ES cells. On the basis of ultrastructural analysis of mitochondria, we believe that the functional activity of these organelles changes during early stages of ES cell formation from the ICM.     

A novel strategy to derive iPS cells from porcine fibroblasts

Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors. This technology has created an interest in deriving iPS cells from domesticated animals such as pigs, sheep and cattle. Moloney murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS cells. However, this retrovirus system infects only mouse and rat cells, which limits its use in establishing iPS cells from other mammals. In our study, we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic fibroblasts. We transfected four human reprogramming factors (Oct4, Sox2, Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 cells. We established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with bFGF. Colonies showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat colonies. Porcine iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.     

Deriving multipotent stem cells from mouse spermatogonial stem cells: a new tool for developmental and clinical research

In recent years, embryonic stem (ES) cell-like cells have been obtained from cultured mouse spermatogonial stem cells (SSCs). These advances have shown that SSCs can transition from being the stem cell-producing cells of spermatogenesis to being multipotent cells that can differentiate into derivatives of all three germ layers. As such, they offer new possibilities for studying the mechanisms that regulate stem cell differentiation. The extension of these findings to human SSCs offers a route to obtaining personalized ES-like or differentiated cells for use in regenerative medicine. Here, we compare the different approaches used to derive ES-like cells from SSCs and discuss their importance to clinical and developmental research.     

Retrospective on reverse genetics in mice around the world and in Japan

The 2007 Nobel Prize for Physiology or Medicine was awarded to Mario R. Capecchi, Martin J. Evans and Oliver Smithies for their contribution in generating mutant mice by gene targeting in embryonic stem (ES) cells. Although there are many experimental animals, it is yet only in mouse that one can genetically examine functions of genes at will. It was merely a dream in the early 1980s that genetic studies with mutants would one day become a reality in mammals. The story began with tetratocarcinoma/embryonal carcinoma cells. Now, through the successes of cloning in mammals, somatic cells such as our skin cells will shortly be transformed into ES-like (induced pluripotent stem) cells by the proper activation of endogenous genes such as Oct4 and Sox2 with chemicals. How have times changed?     

Derivation of stable zebrafish ES-like cells in feeder-free culture

Zebrafish offers an excellent opportunity to combine embryological, genetic and molecular analyses of vertebrate development in vivo. Embryonic stem (ES) cells have enormous potential to study developmental potency and differentiation in vitro and thus to complement in vivo approaches. Zebrafish ES-like cells have been produced on a feeder cell layer. Here, we report the derivation of Z428, a zebrafish ES-like cell line, from blastula embryos in feeder-free culture. Fetal bovine serum, fish serum, fish embryo extract, basic fibroblast growth factor, non-essential amino acids and 2-mercaptoethanol were found to be important for Z428 growth. After more than 120 passages and many freezing/thawing cycles over a period of 20 years, Z428 exhibits stable growth and manifests many ES cell features including an ES cell phenotype, high alkaline phosphatase activity and spontaneous differentiation in culture. Most importantly, Z428 was transplantable to blastula hosts and capable of contributing to embryonic tissues and organ systems of the three germ layers. Therefore, Z428 is a stable cell line and contains ES-like cells with pluripotency in vitro and in vivo, and a feeder layer is dispensable for ES-like cell derivation in zebrafish. The derivation and easy maintenance of zebrafish ES-like cells under feeder-free conditions provide a useful extension of the present toolbox for studying development and differentiation in the zebrafish model.     

Initial culture behaviour of rat blastocysts on selected feeder cell lines

To increase our understanding of rat embryos in culture and to attempt the isolation of blastocyst-derived cell lines, we examinated the initial growth behaviour of rat blastocysts from four strains of rat on four different feeder cell layers. The feeders used were a continuous cell line of murine embryonic fibroblasts (STO), primary mouse (MEF) or primary rat (REF) embryonic fibroblasts, and a continuous cell line of rat uterine epithelial cells (RUCs). A medium that gave optimum plating efficiencies for murine ES cells was used in the rat embryo culture. Each culture system allowed hatching and attachment of the blastocysts, that is, the behaviour was similar on each feeder and each strain for the first 2 days in culture. Subsequently, there was a rapid differentiation of the Inner Cell Mass (ICM) cells on fibroblastic feeder cell layers (STO > MEF > REF), and this was generally complete after 3–6 days in primary culture. On RUCs, the ICM was found to increase in size without differentiation up to and including day 4 and in some cases longer. Embryo-derived cells were obtained by disaggregating and passaging ICMs on REF and RUC feeders. Rounded, refractile, and epithelial-like cells were isolated on REF and colonies of ES-like cells on the RUCs. The ES-like cells were positive for expression of alkaline phosphatase and stage-specific embryonic-antigen 1. This is an important first step towards the derivation and culture of pluripotent ES cells from the rat. © 1995 Wiley-Liss, Inc.     

Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos

Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos. In vivo-derived embryos were cultured on goat fetal fibroblast feeders. Embryos either attached to the feeder layer or remained floating and expanded in culture. Embryos that attached showed a prominent inner cell mass (ICM) and those that remained floating formed structures resembling ICM disks surrounded by trophectodermal cells. ICM cells and embryonic disks were isolated mechanically, cultured on feeder cells in the presence of hLIF, and outgrown into ES-like colonies. Two cell lines were cultured for 25 passages and stained positive for alkaline phosphatase, POU5F1, NANOG, SOX2, SSEA-1, and SSEA-4. Embryoid bodies formed in suspension culture without hLIF. One cell line was cultured for 2 years (over 120 passages). This cell line differentiated in vitro into epithelia and neuronal cells, and could be stably transfected and selected for expression of a fluorescent marker. When cells were injected into SCID mice, teratomas were identified 5-6 weeks after transplantation. Expression of known ES cell markers, maintenance in vitro for 2 years in an undifferentiated state, differentiation in vitro, and formation of teratomas in immunodeficient mice provide evidence that the established cell line represents goat ES cells. This also is the first report of teratoma formation from large animal ES cells.