Establishment Of Embryonic Stem Cells Research Articles

Stem cell factor's role in enhancing the quality of fertilized and cloned porcine embryos for improved embryonic stem cell derivation.

Stem cell factor (SCF), a cytokine growth factor, is expressed in various tissues of the male and female reproductive organs, including the testis, ovary, and endometrium. Its primary function involves cell survival, differentiation, and proliferation, achieved through its binding to the c-kit receptor. This study aimed to scrutinize the effects of SCF treatment during in vitro culture (IVC) on both the developmental potential and the efficiency of establishing embryonic stem cells (ESCs) from fertilized and cloned porcine embryos. The rates of cleavage and blastocyst formation exhibited no significant differences between fertilized and cloned embryos, even with the addition of SCF. However, it's worth noting that embryos cloned with Cloud eGFP as donor cells demonstrated notably increased rates of hatched blastocysts when treated with SCF, and this increase was statistically significant (p < 0.05). Furthermore, following the complete dissection of the blastocysts, although there was no significant difference in the SCF-treated group, the area of expansion was significantly reduced (p < 0.01) in the group treated with the antagonistic blocker (ACK2) compared to both the control and SCF-treated groups. These outcomes suggest that the SCF/c-kit signaling pathway might play a pivotal role in embryo implantation. As anticipated, the efficiency of deriving ESCs was significantly higher (p < 0.01) in the group subjected to SCF treatment (12.82 ± 1.02%) compared to the control group (5.41 ± 2.25%). In conclusion, this study highlights the crucial role of SCF in enhancing the quality of porcine embryos, a vital step in obtaining high-quality ESCs.

Japanese Laws and the Current Status of Regenerative Medicine in the Tohoku Region.

The aim of this study was to review Japanese laws regarding regenerative medicine and the current status of clinical application of regenerative medicine, to learn about the advantages and problems, and to thereby serve as a reference for measures necessary for the development of regenerative medicine. Regenerative medicine started in 1957 with the transplantation of hematopoietic stem cells, followed by the establishment of embryonic stem cells in 1981 and induced pluripotent stem cells in 2006, and continues to evolve progressively. At the same time, however, problems have emerged due to lax legal regulations, such as the use of treatments that lack scientific evidence. The Japanese government enacted two laws to regulate regenerative medicine: the Law to Ensure the Safety of Regenerative Medicine and the Amend the Pharmaceutical Affairs Law in 2013. These laws were enacted with the aim of providing safe regenerative medicine promptly and smoothly and developing many regenerative medicine products. In these laws, regenerative medicine is defined as medical treatment that restores lost functions of damaged organs and tissues with the help of cellular and tissue-based products. Nowadays, there are two major methods of regenerative medicine. One representative method involves the transplantation of devices that activates self-regenerative ability by introducing living cells into patients' body. The other method is the activation and differentiation of endogenous stem cells with cell growth and differentiation factors. The current status of regenerative medicine in the Tohoku region after the enactment of these laws is described in detail. This clarified the advantages and disadvantages associated with regenerative medicine as it is currently practiced in Japan. Development of regenerative medicine in dentistry will be advanced by learning about its clinical application in medicine.

High glucose impairs osteogenic differentiation of embryonic stem cells via early diversion of beta-catenin from Forkhead box O to T cell factor interaction.

Diabetes, which is characterized by an increase in blood glucose concentration, is accompanied by low bone turnover, increased fracture risk, and the formation of embryonic skeletal malformations. Yet, there are few studies elucidating the underlying alterations in signaling pathways leading to these osteogenic defects. We hypothesized here that bone formation deficiencies in a high glucose environment result from altered activity of beta-catenin (CTNNB1), a key contributor to osteogenic differentiation, dysregulation of which has also been implicated in the development of diabetes. To test this hypothesis, we used a previously established embryonic stem cell (ESC) model of differentiation that mimics the diabetic environment of the developing embryo. We differentiated murine ESCs within osteogenic-inducing media containing either high (diabetic) or low (physiological) levels of D-glucose and performed time course analyses to study the influence of high glucose on early and late bone cell differentiation. Endpoint measures for osteogenic differentiation were reduced in a glucose-dependent manner and expression of precursor-specific markers altered at multiple time points. Furthermore, transcriptional activity of the lymphoid enhancer factor (LEF)/T cell factor (TCF) transcription factors during precursor formation stages was significantly elevated while levels of CTNNB1 complexed with Forkhead box O 3a (FOXO3a) declined. Modulation of AKT, a known upstream regulator of both LEF/TCF and FOXO3a, as well as CTNNB1 rescued some of the reductions in osteogenic output seen in the high glucose condition. Within our in vitro model, we found a clear involvement of LEF/TCF and FOXO3a signaling pathways in the regulation of osteogenic differentiation, which may account for the skeletal deficiencies found in newborns of diabetic mothers.

Blastomere aggregation using phytohemagglutinin-L improves the establishment efficiency of porcine parthenogenesis-derived embryonic stem-like cell lines.

Aggregation of blastomeres is a promising method to improve the developmental competence of blastocysts and may be useful for the production of chimeric animals and the establishment of embryonic stem cell lines by increasing inner cell masses. Here, we determined the optimal conditions for blastomere aggregation using phytohemagglutinin-L (PHA-L) and examined PHA-L efficiency by comparing it with Well of the Well (WOW), a general blastomere aggregation method. As a result, we confirmed that treatment with 15 μg/ml PHA-L for 144 h was effective for blastomere aggregation and embryonic development of three zona-free 2-cell stage embryos (TZ2Es) after parthenogenetic activation (PA). The TZ2Es cultured with PHA-L showed a significantly (p < 0.05) higher blastomere aggregation rate than the WOW method (93.5 ± 1.9% vs. 78.0 ± 8.5%). In addition, our results demonstrated that TZ2Es aggregation through PHA-L improved the quality of PA-derived blastocysts and improved porcine embryonic stem-like cell (pESLCs) seeding efficiency and quality of colonies. It was also observed that PHA-L-derived pESLC could remain undifferentiated and exhibit typical embryonic stem cell pluripotency markers, embryoid body (EB)-forming ability, and differentiation into cell lineages of three germ layers. Pig blastomere aggregation technology is expected to improve embryo quality and the efficiency of embryonic stem cell establishment and embryoid-body formation. It can also be used in blastocyst complementation systems and in the production of chimeric animals.

Effects of LPA on the development of sheep in vitro fertilized embryos and attempt to establish sheep embryonic stem cells

Lysophosphatidic acid (LPA) is a small molecule glycerophospholipid, which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development. In this study, sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment. At first, the maturation medium containing estrus female sheep serum and synthetic oviduct fluid (SOF) were optimized for sheep IVF, and then the effects of LPA were investigated. From 0.1 to 10 μmol L–1, LPA had no significant effect on the cleavage rate (P>0.05), but the maturation rate and blastocyst rate increased dependently with LPA concentration (P<0.05), and the blastocyst morphology was normal. When the LPA concentration was 15 μmol L–1, the maturation rate, cleavage rate and blastocyst rate decreased significantly (P<0.05), and the blastocyst exhibited abnormal morphology and could not develop into high-quality blastocyst. Besides, the exogenous LPA increases the expression of LPAR2, LPAR4, TE-related gene CDX-2 and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10 μmol L–1. The expression of LPAR2, LPAR4, CDX-2 and OCT-4 from the LPA-0.1 μmol L–1 to LPA-10 μmol L–1 groups in early embryos were extremely significant (P<0.05), while the expression of these genes significantly decreased in 15 μmol L–1 LPA-treated embryos compared with LPA-10 μmol L–1 group (P<0.05). The inner cell mass in 15 μmol L–1 LPA-treated embryos was also disturbed, and the blastocysts formation was abnormal. Secondly, the sheep IVF blastocysts were applied to establish embryonic stem cells. The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells. They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10 μmol L–1, while 15 μmol L–1 LPA decreased OCT-4 and CDX-2 expression in the derived cells. The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1 μmol L–1 group and LPA-10 μmol L–1 group extremely significantly increased (P<0.05), but there was significant decrease in LPA-15 μmol L–1 group compared with LPA-10 μmol L–1 group (P<0.05). Meanwhile, the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10 μmol L–1 concentration. This study references the IVF embryo production and embryonic stem cell research of domestic animals.

Heterozygous loss of Zbtb38 leads to early embryonic lethality via the suppression of Nanog and Sox2 expression.

ObjectivesMammalian DNA methyltransferases are essential to re‐establish global DNA methylation patterns during implantation, which is critical for transmitting epigenetic information to the next generation. In contrast, the significance of methyl‐CpG binding proteins (MBPs) that bind methylated CpG remains almost unknown at this stage. We previously demonstrated that Zbtb38 (also known as CIBZ)—a zinc finger type of MBP—is required for mouse embryonic stem (ES) cell proliferation by positively regulating Nanog expression. However, the physiological function of Zbtb38 in vivo remains unclear.Materials and MethodsThis study used the Cre‐loxP system to generate conditional Zbtb38 knockout mice. Cell proliferation and apoptosis were studied by immunofluorescence staining. Quantitative real‐time PCR, immunoblotting and immunofluorescence were performed to investigate the molecular mechanisms.ResultsGermline loss of the Zbtb38 single allele resulted in decreased epiblast cell proliferation and increased apoptosis shortly after implantation, leading to early embryonic lethality. Heterozygous loss of Zbtb38 reduced the expression of Nanog, Sox2, and the genes responsible for epiblast proliferation, differentiation, and cell viability. Although this early lethal phenotype, Zbtb38 is dispensable for ES cell establishment and identity.ConclusionsThese findings indicate that Zbtb38 is essential for early embryonic development via the suppression of Nanog and Sox2 expression.

Cis-regulatory architecture of human ESC-derived hypothalamic neuron differentiation aids in variant-to-gene mapping of relevant complex traits

The hypothalamus regulates metabolic homeostasis by influencing behavior and endocrine systems. Given its role governing key traits, such as body weight and reproductive timing, understanding the genetic regulation of hypothalamic development and function could yield insights into disease pathogenesis. However, given its inaccessibility, studying human hypothalamic gene regulation has proven challenging. To address this gap, we generate a high-resolution chromatin architecture atlas of an established embryonic stem cell derived hypothalamic-like neuron model across three stages of in vitro differentiation. We profile accessible chromatin and identify physical contacts between gene promoters and putative cis-regulatory elements to characterize global regulatory landscape changes during hypothalamic differentiation. Next, we integrate these data with GWAS loci for various complex traits, identifying multiple candidate effector genes. Our results reveal common target genes for these traits, potentially affecting core developmental pathways. Our atlas will enable future efforts to determine hypothalamic mechanisms influencing disease susceptibility.

Synthetic modified Fezf2 mRNA (modRNA) with concurrent small molecule SIRT1 inhibition enhances refinement of cortical subcerebral/corticospinal neuron identity from mouse embryonic stem cells.

During late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed “cortical” here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and that its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro.

25th ANNIVERSARY OF CLONING BY SOMATIC CELL NUCLEAR TRANSFER: Generation of genetically engineered livestock using somatic cell nuclear transfer.

Genetic engineering (GE) of livestock initially has been accomplished primarily using pronuclear microinjection into zygotes (1985-1996). The applications of the technology were limited due to low integration efficiency, aberrant transgene expression resulting from random integration and the presence of genetic mosaicism in transgenic founder animals. Despite enormous efforts to established embryonic stem cells (ESCs) for domestic species, the ESC GE technology does not exist for livestock. Development of somatic cell nuclear transfer (SCNT) has bypassed the need in livestock ESCs and revolutionized the field of livestock transgenesis by offering the first cell-based platform for precise genetic manipulation in farm animals. For nearly two decades since the birth of Dolly (1996-2013), SCNT was the only method used for the generation of knockout and knockin livestock. Arrival of CRISPRS/Cas9 system, a new generation of gene-editing technology, gave us an ability to introduce precise genome modifications easily and efficiently. This technological advancement accelerated production of GE livestock by SCNT and reinstated zygote micromanipulation as an important GE approach. The primary advantage of the SCNT technology is the ability to confirm in vitro that the desired genetic modification is present in the somatic cells prior to animal production. The edited cells could also be tested for potential off-target mutations. Additionally, this method eliminates the risk of genetic mosaicism frequently observed following zygote micromanipulation. Despite its low efficiency, SCNT is a well-established procedure in numerous laboratories around the world and will continue to play an important role in the GE livestock field.

Effects of in vitro Culture Period of Reconstructed Embryos and Genetic Background of Feeder Cells on Establishment of Embryonic Stem Cells Derived from Somatic Cell Nuclear Transfer Blastocysts in Pigs

Effects of <i>in</i> vitro Culture Period of Reconstructed Embryos and Genetic Background of Feeder Cells on Establishment of Embryonic Stem Cells Derived from Somatic Cell Nuclear Transfer Blastocysts in Pigs

Identification and Characterization of the OCT4 Upstream Regulatory Region in Sus scrofa.

OCT4 plays pivotal roles in maintaining pluripotency during early mammalian embryonic development and in embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region to study pluripotency. However, there is still a lack of information about the porcine OCT4 upstream reporter system. To improve our understanding of the porcine OCT4 regulatory region, we identified conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. The similarity of nucleotide sequences in the 5′ upstream region was low among mammalian species. However, the OCT4 promoter and four regulatory regions, including distal and proximal enhancer elements, had high similarity. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay results indicated that the porcine OCT4 distal enhancer and proximal enhancer were highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively. A comparison analysis of naïve and primed state marker gene expression in a dual-reporter assay showed that the expression levels of naïve and primed markers differed in fluorescence signal between high-expressing cells and low-expressing cells. Similar to OCT4 upstream-based reporter systems derived from other species, the porcine OCT4 upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work provides basic information about the porcine OCT4 upstream region and various porcine OCT4 fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and the establishment of embryonic stem cells in pigs.

Co-option of the PRDM14–CBFA2T complex from motor neurons to pluripotent cells during vertebrate evolution

Gene regulatory networks underlying cellular pluripotency are controlled by a core circuitry of transcription factors in mammals, including POU5F1. However, the evolutionary origin and transformation of pluripotency-related transcriptional networks have not been elucidated in deuterostomes. PR domain-containing protein 14 (PRDM14) is specifically expressed in pluripotent cells and germ cells, and is required for establishing embryonic stem cells (ESCs) and primordial germ cells in mice. Here, we compared the functions and expression patterns of PRDM14 orthologues within deuterostomes. Amphioxus PRDM14 and zebrafish PRDM14, but not sea urchin PRDM14, compensated for mouse PRDM14 function in maintaining mouse ESC pluripotency. Interestingly, sea urchin PRDM14 together with sea urchin CBFA2T, an essential partner of PRDM14 in mouse ESCs, complemented the self-renewal defect in mouse Prdm14 KO ESCs. Contrary to the Prdm14 expression pattern in mouse embryos, Prdm14 was expressed in motor neurons of amphioxus embryos, as observed in zebrafish embryos. Thus, Prdm14 expression in motor neurons was conserved in non-tetrapod deuterostomes and the co-option of the PRDM14-CBFA2T complex from motor neurons into pluripotent cells may have maintained the transcriptional network for pluripotency during vertebrate evolution.This article has an associated 'The people behind the papers' interview.

124 Functional analysis of porcine OCT4 transcriptional regulatory region-based reporter system

Gene OCT4 plays pivotal roles in maintaining pluripotency of early mammalian embryonic development and embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region for the study of pluripotency. However, there is still a lack of sufficient information about the porcine OCT4 upstream reporter system. To improve our understanding of the porcine OCT4 regulatory region, first, we conducted an investigation to find conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. A similarity of nucleotide sequences of the 5′ upstream region was low among mammalian species. However, the OCT4 promoter and 4 regulatory regions including distal and proximal enhancer elements have a high similarity. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay indicated that the porcine OCT4 distal enhancer and proximal enhancer are highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively (n=3). Comparison analysis of naïve (Tbx3, Nr0b1, Rex1, Esrrb, Nanog, Klf2) or primed (Gata6, Mixl1, Fgf5, Otx2) state marker gene expression in a dual-reporter assay using pOCT4-DE-eGFP and pOCT4-PE-DsRed2 showed that expression of naïve and primed markers were up-regulated in cells with high green fluorescent protein and red fluorescent protein expression, respectively (n=3). Porcine OCT4-upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work could provide basic information for the porcine OCT4 upstream region and the various porcine OCT4-fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and for the establishment of embryonic stem cells in pigs. This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Development of High Value-Added Food Technology Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA, 118042-03-1-HD020).

Distinct requirements for the maintenance and establishment of mouse embryonic stem cells

Mouse embryonic stem cells (ESCs) that maintain a sustainable pluripotent state are derived from the inner cell mass (ICM) of blastocysts, in which pluripotency is lost during differentiation in vivo. It is unclear when and how the ability to maintain pluripotency is acquired during the derivation of ESCs. We analyzed the required culture condition for the maintenance and establishment of ESCs in detail. Even at low concentration of the GSK3β inhibitor and LIF (LowGiL), the expression levels of pluripotency markers and the chimera-producing ability of the cells were comparable with those of ESCs cultured in the presence of both inhibitors and LIF (2iL). However, blastocysts underwent spontaneous differentiation, and ESCs were not established under LowGiL condition. Time-course analysis showed that 2iL condition for three days from the initiation of culture was sufficient for the acquisition of permanent pluripotency. Although X chromosome-linked pluripotent genes were significantly up-regulated during the culture of both male and female blastocysts in 2iL condition, no such up-regulation was observed in LowGiL condition. In conclusion, 2iL-dependent activation of these X-linked genes at the earliest phase of ESC derivation is one of the molecular bases for the acquisition of permanent pluripotency.

Nuclear envelope structural proteins facilitate nuclear shape changes accompanying embryonic differentiation and fidelity of gene expression

BackgroundNuclear size and shape are specific to a cell type, function, and location, and can serve as indicators of disease and development. We previously found that lamin A/C and associated nuclear envelope structural proteins were upregulated when murine embryonic stem (ES) cells differentiated to primitive endoderm cells. Here we further investigated the morphological changes of nuclei that accompany this differentiation.ResultsThe nuclei of undifferentiated wild type cells were found shaped as flattened, irregular ovals, whereas nuclei of Gata4-positive endoderm cells were more spherical, less flattened, and with a slightly reduced volume. The morphological change was confirmed in the trophectoderm and primitive endoderm lineages of E4.5 blastocysts, compared to larger and more irregularly shaped of the nuclei of the inner cell mass. We established ES cells genetically null for the nuclear lamina proteins lamin A/C or the inner nuclear envelope protein emerin, or compound mutant for both lamin A/C and emerin. ES cells deficient in lamin A/C differentiated to endoderm but less efficiently, and the nuclei remained flattened and failed to condense. The size and shape of emerin-deficient nuclei also remained uncondensed after treatment with RA. The emerin/lamin A/C double knockout ES cells failed to differentiate to endoderm cells, though the nuclei condensed but retained a generally flattened ellipsoid shape. Additionally, ES cells deficient for lamin A/C and/or emerin had compromised ability to undergo endoderm differentiation, where the differentiating cells often exhibited coexpression of pluripotent and differentiation markers, such as Oct3/4 and Gata4, respectively, indicating an infidelity of gene regulation.ConclusionsThe results suggest that changes in nuclear size and shape, which are mediated by nuclear envelope structural proteins lamin A/C and/or emerin, also impact gene regulation and lineage differentiation in early embryos. Nevertheless, mice lacking both lamin A/C and emerin were born at the expected frequency, indicating their embryonic development is completed despite the observed protein deficiency.

Derivation and characterization of putative embryonic stem cells from cloned rabbit embryos

The present study aimed to establish embryonic stem (ES) cell lines, i.e., ntES cells, using rabbit blastocyst stage embryos cloned by somatic cell nuclear transfer. First, we investigated the development of cloned rabbit embryos reconstructed with normal fibroblasts and fibroblasts transfected with enhanced green fluorescence protein (eGFP). Blastocyst rates were 27.4% and 23.9%, respectively, for the embryos reconstructed with normal fibroblasts and fibroblasts transfected with eGFP compared with that from the parthenogenetic group (43.1%). One ntES cell line was established from embryos reconstructed with eGFP-transfected fibroblasts (1 of 17, 5.9%), and three ntES cell lines were derived from those with normal fibroblasts (3 of 17, 17.6%). All the ntES cell lines retained alkaline phosphatase activity and expressed ES cell–specific markers SSEA-4, Oct-4, TRA-1-60, and TRA-1-81. The pluripotency was further confirmed by reverse transcription–polymerase chain reaction analyses of Oct-4, Nanog, and Sox-2 expressions in ntES cell lines. The differentiation capacity of ntES cells was also examined in vitro and in vivo, by which these ntES cell lines were able to differentiate into all three germ layers through embryoid bodies and teratomas. In conclusion, it is apparent that the efficiency of ntES cells derived using eGFP-transfected donor cells is lower than that with nontransfected, normal fibroblasts donor cells. Similar to those from parthenogenetic embryos, all ntES cell lines derived from cloned rabbit embryos are able to express pluripotency markers and retain their capability to differentiate into various cell lineages both in vitro and in vivo.

Therapeutic approaches for treating hemophilia A using embryonic stem cells

Hemophilia A is an X-linked rescessive bleeding disorder that results from F8 gene aberrations. Previously, we established embryonic stem (ES) cells (tet-226aa/N6-Ainv18) that secrete human factor VIII (hFVIII) by introducing the human F8 gene in mouse Ainv18 ES cells. Here, we explored the potential of cell transplantation therapy for hemophilia A using the ES cells. Transplant tet-226aa/N6-Ainv18 ES cells were injected into the spleens of severe combined immunodeficiency (SCID) mice, carbon tetrachloride (CCl4)-pretreated wild-type mice, and CCl4-pretreated hemophilia A mice. F8 expression was induced by doxycycline in drinking water, and hFVIII-antigen production was assessed in all cell transplantation experiments. Injecting the ES cells into SCID mice resulted in an enhanced expression of the hFVIII antigen; however, teratoma generation was confirmed in the spleen. Transplantation of ES cells into wild-type mice after CCl4-induced liver injury facilitated survival and engraftment of transplanted cells without teratoma formation, resulting in hFVIII production in the plasma. Although CCl4 was lethal to most hemophilia A mice, therapeutic levels of FVIII activity, as well as the hFVIII antigen, were detected in surviving hemophilia A mice after cell transplantation. Immunolocalization results for hFVIII suggested that transplanted ES cells might be engrafted at the periportal area in the liver. Although the development of a safer induction method for liver regeneration is required, our results suggested the potential for developing an effective ES-cell transplantation therapeutic model for treating hemophilia A in the future.

Cellular reprogramming in farm animals: an overview of iPSC generation in the mammalian farm animal species.

Establishment of embryonic stem cell (ESC) lines has been successful in mouse and human, but not in farm animals. Development of direct reprogramming technology offers an alternative approach for generation of pluripotent stem cells, applicable also in farm animals. Induced pluripotent stem cells (iPSCs) represent practically limitless, ethically acceptable, individuum-specific source of pluripotent cells that can be generated from different types of somatic cells. iPSCs can differentiate to all cell types of an organism’s body and have a tremendous potential for numerous applications in medicine, agriculture, and biotechnology. However, molecular mechanisms behind the reprogramming process remain largely unknown and hamper generation of bona fide iPSCs and their use in human clinical practice. Large animal models are essential to expand the knowledge obtained on rodents and facilitate development and validation of transplantation therapies in preclinical studies. Additionally, transgenic animals with special traits could be generated from genetically modified pluripotent cells, using advanced reproduction techniques. Despite their applicative potential, it seems that iPSCs in farm animals haven’t received the deserved attention. The aim of this review was to provide a systematic overview on iPSC generation in the most important mammalian farm animal species (cattle, pig, horse, sheep, goat, and rabbit), compare protein sequence similarity of pluripotency-related transcription factors in different species, and discuss potential uses of farm animal iPSCs. Literature mining revealed 32 studies, describing iPSC generation in pig (13 studies), cattle (5), horse (5), sheep (4), goat (3), and rabbit (2) that are summarized in a concise, tabular format.

Expression pattern of OCT 4, NANOG and HSP 90 β gene in oocytes and various stages of in vitro produced sheep embryos

The study was conducted to assess the expression pattern of pluripotent genes (OCT 4 and NANOG) and stress response gene HSP 90 ß by quantitative real time polymerase chain reaction (qRT-PCR) in immature and mature oocytes and 2 cell, 4 cell, morula stages of in vitro fertilized (IVF) and parthenogenetically activated (PA) preimplantation ovine embryos to investigate the similarity between them in turn for the establishment of embryonic stem cells (ESCs) from parthenote embryos. The pluripotent related genes showed almost similar pattern of expression between IVF and PA embryos at all the developmental stages which indicated that PA embryos could be used as an alternate source for the production of embryonic stem cells. The stress response gene showed increased levels after in vitro maturation and non significant differences between IVF and PA embryos, thus, indicating no significant differences between the quantum of stress experienced by the 2 groups of embryos which would not affect the development of embryo quality and in turn affect the parthenote embryonic stem cells (PESCs).

Establishment of gynogenetic haploid embryonic stem cells and generation of cloned oocytes in mice

The establishment of embryonic stem cells (ESCs) with one set of maternal genome will be able to provide the valuable cell source for the genetic analysis of female genome, the production of genetically modified animals, and the regenerative medicine. However, available information about these ESCs is very limited. The aim of this study was to establish gynogenetic haploid ES cells (GHESCs) with maternal haploid genome in mice. In addition, we performed the generation of cloned oocytes by using nuclear transfer with GHESCs.