Pluripotent Embryonic Stem Cell Lines Research Articles

Determining the potency of primordial germ cells by injection into early mouse embryos

Primordial germ cells (PGCs) are the earliest precursors of the gametes. During normal development, PGCs only give rise to oocytes or spermatozoa. However, PGCs can acquire pluripotency invitro by forming embryonic germ (EG) cells and invivo during teratocarcinogenesis. Classic embryological experiments directly assessed the potency of PGCs by injection into the pre-implantation embryo. As no contribution to embryos or adult mice was observed, PGCs have been described as unipotent. Here, we demonstrate that PGCs injected into 8-cell embryos can initially survive, divide, and contribute to the developing inner cell mass. Apoptosis-deficient PGCs exhibit improved survival in isolated epiblasts and can form naive pluripotent embryonic stem cell lines. However, contribution to the post-implantation embryo is limited, with no functional incorporation observed. In contrast, PGC-like cells show an extensive contribution to mid-gestation chimeras. We thus propose that PGC formation invivo establishes a latent form of pluripotency that restricts chimera contribution.

Functional assessment of donated human embryos for the generation of pluripotent embryonic stem cell lines

Research questionCan discarded embryos at blastocyst stage, donated to research because of genetic abnormalities and poor morphological quality, become a reliable source of human embryonic stem cell (HESC) lines? DesignThis study was consecutively conducted with 23 discarded embryos that were donated to research between February 2020 and April 2021. All embryos, except one, were morphologically evaluated and underwent trophectoderm biopsy for preimplantation genetic testing using next-generation sequencing (NGS), and then vitrified. After warming, the embryos were placed in appropriate culture conditions for the generation of HESCs, which was functionally assessed with immunofluorescence and flow cytometry for pluripotency capacity and spontaneous in-vitro differentiation. Cytogenetic assessment of the HESC was conducted with multiplex ligation-dependent probe amplification, and micro array comparative genomic hybridization. ResultsFrom the 23 embryos initially included, 17 survived warming, and 16 of them presented viability. Overall, the embryos presented poor morphological quality after warming. Only the previously untested embryo was capable of generating a new HESC line. Further characterization of this line revealed fully functional, euploid HESCs with preserved pluripotency, becoming a useful resource for research into human development and therapeutic investigation. ConclusionsNone of the donated blastocysts with poor morphological quality in association with genetic abnormalities detected by NGS had the capacity for further in-vitro expansion to originate pluripotent HESC lines. This finding seems to provide extra support to genetic counselling on the suitability of this type of embryo for clinical use.

Effect of NANOG overexpression on porcine embryonic development and pluripotent embryonic stem cell formation in vitro.

The efficiency of establishing pig pluripotent embryonic stem cell clones from blastocysts is still low. The transcription factor Nanog plays an important role in maintaining the pluripotency of mouse and human embryonic stem cells. Adequate activation of Nanog has been reported to increase the efficiency of establishing mouse embryonic stem cells from 3.5 day embryos. In mouse, Nanog starts to be strongly expressed as early as the morula stage, whereas in porcine NANOG starts to be strongly expressed by the late blastocyst stage. Therefore, here we investigated both the effect of expressing NANOG on porcine embryos early from the morula stage and the efficiency of porcine pluripotent embryonic stem cell clone formation. Compared with intact porcine embryos, NANOG overexpression induced a lower blastocyst rate, and did not show any advantages for embryo development and pluripotent embryonic stem cell line formation. These results indicated that, although NANOG is important pluripotent factor, NANOG overexpression is unnecessary for the initial formation of porcine pluripotent embryonic stem cell clones in vitro.

Cinquante nuances de pluripotence

Since the derivation of the first pluripotent embryonic stem cell lines in mice in the early 1980s, a plethora of lines has been obtained from various mammalian species including rodents, lagomorphs and primates. These lines are distinguished by their molecular and functional characteristics and correspond to the different pluripotency states observed in the developing embryo between the "blastocyst" and "gastrula" stages. These cell lines are positioned along a gradient, or continuum of pluripotency, the ends of which are epitomized by the naïve and primed states, respectively. Conventional human pluripotent stem cells self-renew in the primed state of pluripotency (ie, at the bottom of the gradient), a position that is undoubtedly the cause of their natural instability. Recent studies aim to generate naive human pluripotent stem cells (at the top of the gradient). The importance of this research in the perspective of medical applications will be discussed.

Nuclear Treatment and Cell Cycle Synchronization for the Purpose of Mammalian and Primate Somatic Cell Nuclear Transfer (SCNT).

Mammalian somatic cell nuclear transfer (SCNT) is a technically and biologically challenging procedure inducing rapid reprogramming of the nucleus from the differentiated into the totipotent state in a few hours. This procedure was initially successfully accomplished in farm animals, then in rodents, and more recently in primates and in humans. Though ethical concerns regarding SCNT still exist, this procedure can be utilized to generate patient and disease-specific pluripotent embryonic stem cell lines, which carry a great promise in improving our understanding of major disease conditions and a hope for better therapies and regenerative medicine. In this section, we will survey the existing literature and describe how mouse SCNT is performed and the importance of donor cell treatment and cycle synchronization prior to SCNT.

The Immunoglobulin Superfamily Protein Differentiation of Embryonic Stem Cells 1 (Dies1) Has a Regulatory Role in Preadipocyte to Adipocyte Conversion

Differentiation of Embryonic Stem Cells 1 (Dies1) was recently identified as a novel type I immunoglobulin (IgG) domain-containing plasma membrane protein important for effective differentiation of a murine pluripotent embryonic stem cell line. In this setting, Dies1 enhances bone morphogenetic protein 4 (BMP4) signaling. Here we show Dies1 transcript expression is induced ∼225-fold during in vitro adipogenesis of 3T3-L1 murine preadipocytes. Immunocytochemical imaging using ectopic expression of Flag-tagged Dies1 in 3T3-L1 adipocytes revealed localization to the adipocyte plasma membrane. Modulation of adipocyte phenotype with with tumor necrosis factor-α (TNFα) treatment or by siRNA knockdown of the master pro-adipogenic transcription factor peroxisome proliferator activated receptor gamma (PPARγ) resulted in a 90% and 60% reduction of Dies1 transcript levels, respectively. Moreover, siRNA-mediated Dies1 knockdown in 3T3-L1 preadipocytes inhibited adipogenic conversion. Such cultures had a 35% decrease in lipid content and a 45%–65% reduction in expression of key adipocyte transcripts, including that for PPARγ. The standard protocol for full in vitro adipogenic conversion of committed preadipocytes, such as 3T3-L1, does not include BMP4 treatment. Thus we posit the positive role of Dies1 in adipogenesis, unlike that for Dies1 in differentiation of embryonic stem cells, does not include its pro-BMP4 effects. In support of this idea, 3T3-L1 adipocytes knocked down for Dies1 did not evidence decreased phospho-Smad1 levels upon BMP4 exposure. qPCR analysis of Dies1 transcript in multiple murine and human tissues reveals high enrichment in white adipose tissue (WAT). Interestingly, we observed a 10-fold induction of Dies1 transcript in WAT of fasted vs. fed mice, suggesting a role for Dies1 in nutritional response of mature fat cells in vivo. Together our data identify Dies1 as a new differentiation-dependent adipocyte plasma membrane protein whose expression is required for effective adipogenesis and that may also play a role in regard to nutritional status in WAT.

Mouse cloning and somatic cell reprogramming using electrofused blastomeres

Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

Derivation, Characterization, and In Vitro Differentiation of Canine Embryonic Stem Cells.

Embryonic stem (ES) cells have created considerable excitement in the last few years due to their unlimited potential to produce cells for tissue repair and replacement. However, a large animal pre-clinical model is necessary to establish the safety and efficacy of ES cell-derived tissue replacement therapy. The canine model has long been used in medical research, has been well established to study adult stem cell transplantation and has been highly predictive of clinical outcomes in humans, more so than rodent models. Given the documented record for extrapolating from dog to man, we hypothesize that the dog would serve as an ideal pre-clinical in vivo model for studying the clinical applications of ESC derived tissue. Eleven putative ES cell lines were initiated from canine blastocysts harvested from natural matings. One line described here, FHDO-7, has been maintained through 34 passages and has many characteristics of ES cells from other species. FHDO-7 cells are alkaline phosphatase positive and express both message and protein for the Oct4 transcription factor. They also express message for Nanog and do not express message for Cdx2 which is associated with trophectoderm. Furthermore, they express a cluster of pluripotency-associated microRNAs (miR-302b, miR-302c and miR-367) that have been found to be characteristic of human and mouse ES cells. The FHDO-7 cells grow on feeder layers of modified mouse embryonic fibroblasts (MEF) as flat colonies that resemble ES cells from mink, a close phylogenetic relative of dog. When cultured in nonadherent plates without feeders the cells form embryoid bodies (EB). Under various culture conditions the EBs give rise to ectoderm-derived neuronal cells expressing β3-tubulin, mesoderm-derived osteocytes producing bone, and endoderm-derived cells expressing alpha feto protein or Clara cell specific protein. These results indicate that FHDO-7 is a pluripotent embryonic stem cell line.

Nuclear reprogramming and pluripotency of embryonic cells: Application to the isolation of embryonic stem cells in farm animals

Despite their biological and biotechnological interest, pluripotent embryonic stem cell lines (ES cells) have been isolated from cultured embryos only in a very limited number of mammalian species. Here we review the main molecular mechanisms that have been shown in mouse or primates to regulate the maintenance of pluripotency in vitro. We describe the main signaling pathways that participate in the self-renewal of ES cells and provide an outlook on the epigenetic associated mechanisms. We also propose a practical approach to stem cell differentiation that examines the relationships between the genotype of embryos and their culture conditions and consider nuclear reprogramming as a valuable approach in ES cell derivation in farm animals.

Immunocytochemical analysis of vimentin expression patterns in porcine embryos suggests mesodermal differentiation from day 9 after conception.

The expression of the intermediate filament proteins vimentin and keratin in porcine embryos was studied by whole-mount immunocytochemistry between day 7 and day 11 after conception. Expression of vimentin was first detected in the inner cell mass of about 50% of the 9-day-old embryos. In elongated 11-day-old embryos, cells expressing vimentin were observed in the epiblast (after disappearance of Rauber's membrane) and in cells migrating from the epiblast between the trophoblast and the underlying hypoblast layer. A keratin-positive response was observed in trophectoderm cells at all stages. These findings suggest that inner cell mass cells in the pig start differentiating into mesodermal cells not later than day 9 after conception. While the delamination of the mesodermal germ layer is known to correlate with the loss of pluripotency of the inner cell mass cells, the early onset of mesodermal differentiation in the porcine embryo, characterized by vimentin expression and in contrast to the mouse, could in part be responsible for the lack of success in establishing pluripotent embryonic stem cell lines in this species. Our results suggest that further attempts to isolate inner cell mass-derived pluripotent cells should be attempted well before day 9 after conception.

Embryonic stem cells as an in vitro model for mutagenicity, cytotoxicity and embryotoxicity studies: present state and future prospects

Primary cultures or established cell lines of vertebrates are commonly used to analyse the mutagenic, embryotoxic or teratogenic potential of environmental factors, drugs and xenobiotics in vitro. However, these cellular systems do not include developmental processes from early embryonic stages up to terminally differentiated cell types. An alternative approach has been offered by permanent lines of pluripotent stem cells of embryonic origin, such as embryonic carcinoma (EC), embryonic stem (ES) and embryonic germ (EG) cells. The undifferentiated stem cell lines are characterized by nearly unlimited self-renewal capacity and have been shown to differentiate in vitro into cells of all three primary germ layers. Pluripotent embryonic stem cell lines recapitulate cellular developmental processes and gene expression patterns of early embryogenesis during in vitro differentiation, data which are summarized in this review. In addition, recent studies are presented which investigated mutagenic, cytotoxic and embryotoxic effects of chemical substances using in vitro systems of pluripotent embryonic stem cells. Furthermore, an outlook is given on future molecular technologies using embryonic stem cells in developmental toxicology and embryotoxicology.

Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI.

Human embryonic stem (ES) cells are predicted to be a valuable source for producing ES-derived therapeutic spare tissues to treat diseases by controlling their growth and differentiation. To understand the regulative mechanisms of their differentiation in vivo and in vitro, ES cells derived from nonhuman primates could be a powerful tool. We established four ES cell lines from cynomolgus monkey (Macaca fascicularis) blastocysts produced by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The ES cells were characterized by the expression of specific markers such as alkaline phosphatase and stage-specific embryonic antigen-4. They were successfully maintained in an undifferentiated state and with a normal karyotype even after more than 6 months of culture. Pluripotential competence was confirmed by the formation of teratomas containing ectoderm-, mesoderm-, and endoderm- derivatives after subcutaneous injection into SCID mice. Differentiation to a variety of tissues was identified by immunohistochemical analyses using tissue-specific antibodies. Therefore, we established pluripotent ES cell lines derived from monkeys that are widely used as experimental animals. These lines could be a useful resource for preclinical stem cell research, including allogenic transplantation into monkey models of disease.

The small heat shock protein hsp25 is accumulated in P19 embryonal carcinoma cells and embryonic stem cells of line BLC6 during differentiation

Abstract Murine embryonal carcinoma and embryonic stem cell lines were investigated with regard to the occurrence of the small heat shock protein hsp25 during cell growth and differentiation. In the embryonal carcinoma cell line F9 considerable constitutive levels of hsp25 were observed which could be slightly increased by treatment with retinoic acid. No hsp25 was found, however, in the embryonal carcinoma cell line PCC4. When analyzing the pluripotent embryonal carcinoma cell line P19 and the pluripotent embryonic stem cell line BLC6, both characterized by high differentiation capacity, no hsp25 was observed under cell culture conditions maintaining the undifferentiated state. Induction of differentiation caused by prolonged cell culture, retinoic acid treatment, or embryoid body formation, however, resulted in an increase of the level of hsp25. The finding that hsp25 is accumulated in a differentiation-dependent manner suggests that this protein is associated with processes involved in differentiation. Therefore, hsp25 can be regarded as a marker of differentiation in the investigated embryonal carcinoma cell line P19 and the embryonic stem cell line BLC6

Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor (LIF)

The isolation of pluripotent murine embryonic stem (ES) cells has previously been achieved by coculturing the ES cells with fibroblast feeder cells. In this report we demonstrate that ES cell lines can be isolated from murine 129/Sv He blastocysts in the absence of feeder cells in culture medium supplemented with recombinant leukemia inhibitory factor (LIF). Three of the ES cell lines (MBL-1, MBL-2, and MBL-3) were isolated by directly explanting blastocysts, whilst two ES cell lines (MBL-4 and MBL-5) were isolated from blastocysts pretreated by immunosurgery. Three of the ES cell lines contained the Y chromosome (MBL-1, MBL-2, and MBL-5) with a high proportion of the cells displaying a normal diploid karyotype with a modal chromosome number of 40. All of the ES cell lines tested expressed the stem cell markers ECMA-7 and alkaline phosphatase, which were lost on removal of LIF when the ES cells differentiated into a variety of cell types. The full developmental potential of the ES cells was determined by injecting cells from two of the independently derived ES cell lines, MBL-1 and MBL-5, into C57BL/6J blastocysts. A high proportion of the pups born were chimeric as judged by coat pigmentation. Subsequent breeding established that the ES cells had contributed to the germ line. These results demonstrate that feeder cells are not essential for the isolation of pluripotent ES cell lines.

Production of a mutation in mouse En-2 gene by homologous recombination in embryonic stem cells

A full understanding of the function of genes that control developmental events can be obtained only by a combination of molecular and mutational analysis. One putative developmental gene is the mouse engrailed-like gene En-2, which was isolated by virtue of its extensive homology to Drosophila engrailed, which contributes to the control of segmentation in the developing insect. Our hybridization analysis in situ has revealed that expression of En-2 is restricted to a specific domain of the developing central nervous system from 8 days of development on, indicating a role for the gene in establishing spatial domains in the brain. Unfortunately no En-2 mutations are available to elucidate further its function in development. To this end, we report here the isolation of three pluripotent embryonic stem cell lines in which one copy of the homoeobox-containing gene, En-2, has been altered by homologous recombination.

Pluripotent embryonic stem cell lines can be derived from tw5/tw5 blastocysts.

Mouse embryos homozygous for tw5, a recessive lethal mutation in the t complex located on chromosome 17, develop normally until the elongated egg cylinder stage, approximately 6.5 days after fertilization. At this time, the endoderm is morphologically abnormal and the embryonic ectoderm begins to show signs of pyknosis. Death of the embryo usually occurs within the next 2 days (ref. 1). A serious difficulty in the study of lethal t-mutant gene expression during embryogenesis has been to obtain appropriate experimental material, particularly during the period immediately following implantation. Recently, a method involving the use of teratocarcinoma-conditioned medium was devised for establishing pluripotent stem cell cultures directly from the inner cell mass (ICM) of a normal mouse blastocyst2. Using this method, we have established three embryonic stem cell lines derived from embryos carrying tw5. We report here that one of the cell lines is homozygous for the mutation (tw5/tw5), whereas the other two are heterozygous (+/tw5). When injected into athymic mice, each cell line is capable of forming tumours that contain differentiated derivatives of all three primary germ layers.