Embryonic Stem Cell-like Cells Research Articles

Developmental potential of artificially produced chimeric embryos following transfer to surrogate Goats

An experiment was conducted to study the developmental potential of goat chimeric embryos. A total of 8393 mature oocytes were randomly divided into two groups. Gr 1: oocytes (n=5222) were activated with 5 μM calcium ionophore for 5 to 7 min followed by treatment with 2.0 mM DMAP (dimethylaminopurine) for 4 h in mCR2 aa (modified Charles Rosenkrans 2 amino acid) medium. Gr 2: oocytes (n=3171) were used for in vitro fertilization (IVF). Inner cell mass from hatched blastocysts of parthenogenetic activated embryos were used to produce embryonic stem cell-like cells while 2 cell embryos obtained from IVF were utilized to produce tetraploid embryos. Aggregation efficiency, compact morula and blastocyst formations were 84.93±8.13, 80.53±10.55 and 7.05±3.08%, respectively. A total of 47 chimeric goat embryos were transferred into 17 Sirohi female recipients, resulted in in vivo development of foetus in two recipients. In first recipient, foetus looked like a very thick and hyperechoic amniotic ring until 49 days; thereafter, it was completely resorbed. In second recipient, foetus and amniotic ring were visible as hypoechoic structure from 50 days onwards; thereafter it was completely resorbed at 58 days of embryo transfer. In conclusion, the study indicated that foetal resorption was observed at various stages of pregnancy after transfer of chimeric embryos into recipients. It appears that placental atrophy may be the principal cause of the loss of foetuses.

Identification of microRNAs related with neural germ layer lineage-specific progenitors during reprogramming.

Differentiated cells can be reprogrammed to embryonic stem cell-like cells called induced pluripotent stem cells (iPSCs), in which the natural developmental differentiation process is reversed. It is unclear whether the multi-lineage cells can be isolated and identified during reprogramming. In the current study, we detected the expression of lineage markers, isolated neural lineages, and identified the related microRNAs during iPSC formation. Our results demonstrated that a neuroectoderm appeared earlier than mesoderm and definitive endoderm before forming colonies when mouse embryonic fibroblasts were subjected to iPSC formation using transcription factors (TFs). On day 3, the cells expressed Sox1 and Nestin and had ultrastructure consistent with the transition to identity neural germ layer lineage. Fluorescence-activated cell sorting analysis revealed a peak (40%) in neural progenitor marker-positive cells. When subsequently cultured in a neural precursor cell medium, these cells proliferated slowly, became round and aggregated, generating into neurons and glia. Genome-wide microRNA (miRNA) analysis identified 45 differentially regulated miRNAs. Molecular network analysis demonstrated that these miRNAs validated 6,047 experimental mRNA targets. The GO functional annotation analysis of mRNA targets revealed that most genes were related to neurogenesis, such as growth cone, neuronal cell body, neuron projection, and cell junction synapse. The network of protein-protein interactions was observed, which demonstrated that key nodes of neural lineage reprogramming-associated targets were Sall1, Foxa2, Nf2, Ctnnb1, Shh, and Bmpr1a. Therefore, these data suggested that TFs can drive the reprogramming of somatic cells towards a pluripotent state via neuroectoderm. Moreover, the neural lineage reprogramming system can address how miRNAs influence their target sites.

Activation of Wnt/β-Catenin Signaling Pathway Enhances the Derivation of Buffalo (Bubalus bubalis) Embryonic Stem Cell-Like Cells.

Wnt/β-Catenin signaling pathway plays an important role in maintaining self-renewal and pluripotency of human and mouse embryonic stem cells (ESCs). Activation of Wnt/β-Catenin signaling pathway by glycogen synthase kinase-3 (GSK3) inhibitor, the Wnt signaling agonist, could maintain the pluripotency of human and mouse ESCs in the presence of serum. However, the role of signaling pathway in the derivation of buffalo ESCs remains unclear. In this study, we used GSK3 inhibitors (6-bromoindirubin-3'-oxime [BIO] and CHIR99021) and investigated the effect of Wnt/β-Catenin activation on colony formation, proliferation, self-renewal, and pluripotency of Chinese swamp buffalo (buffalo) embryonic stem cell-like cells (ES-like cells), which were isolated from blastocysts. The results showed that buffalo ES-like cells displayed typical morphological characteristics of pluripotent stem cells: positive for alkaline phosphatase staining, expression of pluripotent markers, including OCT4, SOX2, SSEA-1, SSEA-4, LIN28, CH1, NANOG, and the proliferative markers, PCNA and C-MYC. Furthermore, activation of Wnt/β-Catenin signaling pathway by GSK3 inhibitors could promote colony formation and proliferation of buffalo ES-like cells and maintain their undifferentiated state, and upregulate the expression levels of pluripotent-related genes and proliferation-related genes. These results indicated that Wnt/β-Catenin signaling pathway plays an important role in the derivation and pluripotency of buffalo ES-like cells.

The regulation of Oct4 in human gingival fibroblasts stimulated by cyclosporine A: Preliminary observations

Background/purposeOct4, a key transcription factor, could reprogram human somatic fibroblasts into embryonic stem cell-like pluripotent cells. The exact mechanism of cyclosporine A (CsA)-induced gingival overgrowth is still unclear. The aim of this study was to investigate the effects of CsA on the expression of Oct4 in cultured human gingival fibroblasts (HGFs) in vitro. Materials and methodsThe effects of CsA on HGFs were used to elucidate whether Oct4 expression could be induced by CsA by using quantitative real-time reverse transcription-polymerase chain reaction and western blot. Cell growth in CsA-treated HGFs with Oct4 lentiviral-mediated shRNAi knockdown was evaluated by tetrazolium bromide reduction assay. ResultsCsA was found to upregulate Oct4 transcript in a dose-dependent manner (p < 0.05). CsA also dose-dependently increased Oct4 protein expression (p < 0.05). The lentivirus expressing sh-Oct4 successfully prevented the CsA-induced Oct4 mRNA and protein in HGFs (p < 0.05). However, knockdown of Oct4 was insufficient to inhibit CsA-stimulated cell growth in HGFs. Furthermore, double knockdown with pluripotency-associated transcription factor Nanog showed that the down-regulation of Oct4/Nanog by lentiviral infection significantly inhibited CsA-stimulated cell growth (p < 0.05). ConclusionTaken together, CsA was first found to upregulate Oct4 mRNA and protein expression in HGFs. The silencing Oct4 could not suppress cell growth unless Nanog was repressed simultaneously.

Pluripotency Potential of Embryonic Stem Cell-Like Cells Derived from Mouse Testis.

Objective During the cultivation of spermatogonial stem cells (SSCs) and their conversion into embryonic stem-like (ES-like) cells, transitional ES-like colonies and epiblast-like cells were observable. In the present experimental study, we aimed to analyze the efficiency of the multipotency or pluripotency potential of ES-like cells, transitional colonies and epiblast-like cells. Materials and Methods In this experimental study, SSCs were isolated from transgenic octamer-binding transcription factor 4 (Oct4)-green fluorescent protein (GFP)-reporter mice. During cell culture ES-like, transitional and epiblast- like colonies developed spontaneously. The mRNA and protein expression of pluripotency markers were analyzed by Fluidigm real-time polymerase chain reaction (RT-PCR) and immunocytochemistry, respectively. Efficiency to produce chimera mice was evaluated after injection of ES and ES-like cells into blastocysts. Results Microscopic analyses demonstrated that the expression of Oct4-GFP in ES-like cells was very strong, in epiblast-like cells was not detectable, and was only partial in transitional colonies. Fluidigm RT-PCR showed a higher expression of the germ cell markers Stra-8 and Gpr-125 in ES-like cells and the pluripotency genes Dppa5, Lin28, Klf4, Gdf3 and Tdgf1 in ES-like colonies and embryonic stem cells (ESCs) compared to the epiblast-like and transitional colonies. No significant expression of Oct-4, Nanog, Sox2 and c-Myc was observed in the different groups. We showed a high expression level of Nanog and Klf4 in ES-like, while only a partial expression was observed in transitional colonies. We generated chimeric mice after blastocystic injection from ES and ES-like cells, but not from transitional colonies. We observed that the efficiency to produce chimeric mice in ES cells was more efficient (59%) in comparison to ES-like cells (22%). ConclusionThis new data provides more information on the pluripotency or multipotency potentials of testis-derived ES-like cells in comparison to transitional colonies and epiblast-like cells.

Patient-derived induced pluripotent stem cells for models of cancer and cancer stem cell research.

Induced pluripotent stem cells (iPSCs) are embryonic stem cell-like cells reprogrammed from somatic cells by four transcription factors, OCT4, SOX2, KLF4 and c-MYC. iPSCs derived from cancer cells (cancer-iPSCs) could be a novel strategy for studying cancer. During cancer cell reprogramming, the epigenetic status of the cancer cell may be altered, such that it acquires stemness and pluripotency. The cellular behavior of the reprogrammed cells exhibits dynamic changes during the different stages of reprogramming. The cells may acquire the properties of cancer stem cells (CSCs) during the process of reprogramming, and lose their carcinogenic properties during reprogramming into a cancer-iPSCs. Differentiation of cancer-iPSCs by teratoma formation or organoid culturing could mimic the process of tumorigenesis. Some of the molecular mechanisms associated with cancer progression could be elucidated using the cancer-iPSC model. Furthermore, cancer-iPSCs could be expanded in culture system or bioreactors, and serve as cell sources for research, and as personal disease models for therapy and drug screening. This article introduces cancer studies that used the cell reprogramming strategy.

Previously claimed male germline stem cells from porcine testis are actually progenitor Leydig cells

BackgroundMale germline stem cells (mGSCs) offer great promise in regenerative medicine and animal breeding due to their capacity to maintain self-renewal and to transmit genetic information to the next generation following spermatogenesis. Human testis-derived embryonic stem cell-like cells have been shown to possess potential of mesenchymal progenitors, but there remains confusion about the characteristics and origin of porcine testis-derived stem cells.MethodsPorcine testis-derived stem cells were obtained from primary testicular cultures of 5-day old piglets, and selectively expanded using culture conditions for long-term culture and induction differentiation. The stem cell properties of porcine testis-derived stem cells were subsequently assessed by determining the expression of pluripotency-associated markers, alkaline phosphatase (AP) activity, and capacity for sperm and multilineage differentiation in vitro. The gene expression profile was compared via microarray analysis.ResultsWe identified two different types of testis-derived stem cells (termed as C1 and C2 here) during porcine testicular cell culture. The gene expression microarray analysis showed that the transcriptome profile of C1 and C2 differed significantly from each other. The C1 appeared to be morphologically similar to the previously described mouse mGSCs, expressed pluripotency- and germ cell-associated markers, maintained the paternal imprinted pattern of H19, displayed alkaline phosphatase activity, and could differentiate into sperm. Together, these data suggest that C1 represent the porcine mGSC population. Conversely, the C2 appeared similar to the previously described porcine mGSCs with three-dimensional morphology, abundantly expressed Leydig cell lineage and mesenchymal cell-specific markers, and could differentiate into testosterone-producing Leydig cells, suggesting that they are progenitor Leydig cells (PLCs).ConclusionCollectively, we have established the expected characteristics and markers of authentic porcine mGSCs (C1). We found for the first time that, the C2, equivalent to previously claimed porcine mGSCs, are actually progenitor Leydig cells (PLCs). These findings provide new insights into the discrepancies among previous reports and future identification and analyses of testis-derived stem cells.

Optimization of caprine embryo production in different media for generation of embryonic stem cell-like cells

The aim of the present study was to optimize the production of blastocyst for obtaining caprine embryonic stem cell-like cells. A total of 4372 cumulus oocyte complexes (COCs) were recovered by slicing the 1187 caprine ovaries and were matured in maturation media for 27 h in humidified atmosphere at 38.5°C with 5% CO2 in CO2 incubator. After 27 h of maturation, oocytes were denuded and were co-incubated with buck semen in fertilization medium (TALP medium + 8 mg/ml fatty acid free BSA and 50 μg/ml heparin) for 18 h. Good quality zygotes (2483) were selected and randomly divided into 2 groups (experiment 1), viz. Group 1 (1312) wherein the presumptive zygotes were cultured in RVCL while in Group 2 (1171) the presumptive zygotes were cultured in mCR2aa medium. The cleavage rate, blastocyst and hatched blastocyst production was significantly higher in Gr 1 (47.45±2.93, 10.13±1.31 and 3.90±1.13%) than Gr 2 (37.75±2.46, 4.20±0.93 and 1.66±0.72%). In experiment 2, after in-vitro fertilization, morula stage embryos and inner cell mass (ICM) from blastocyst and hatched blastocyst were used to isolate ES cell-like cells. Thus the results indicated that the RVCL medium is the best medium as far as the embryonic development up to blastocyst stage in comparison to mCR2aa media. Furthermore, the formation of putative embryonic stem cell colonies were higher from hatched blastocysts (91.6%) as compared to that of blastocysts (82.1%) and it was significantly higher than that from morulas (34.3%).

Alternative dominance of the parental genomes in hybrid cells generated through the fusion of mouse embryonic stem cells with fibroblasts

For the first time, two types of hybrid cells with embryonic stem (ES) cell-like and fibroblast-like phenotypes were produced through the fusion of mouse ES cells with fibroblasts. Transcriptome analysis of 2,848 genes differentially expressed in the parental cells demonstrated that 34–43% of these genes are expressed in hybrid cells, consistent with their phenotypes; 25–29% of these genes display intermediate levels of expression, and 12–16% of these genes maintained expression at the parental cell level, inconsistent with the phenotype of the hybrid cell. Approximately 20% of the analyzed genes displayed unexpected expression patterns that differ from both parents. An unusual phenomenon was observed, namely, the illegitimate activation of Xist expression and the inactivation of one of two X-chromosomes in the near-tetraploid fibroblast-like hybrid cells, whereas both Xs were active before and after in vitro differentiation of the ES cell-like hybrid cells. These results and previous data obtained on heterokaryons suggest that the appearance of hybrid cells with a fibroblast-like phenotype reflects the reprogramming, rather than the induced differentiation, of the ES cell genome under the influence of a somatic partner.

Integrating piggyBac Transposon Transgenes into Mouse Fibroblasts by Electroporation.

Mouse embryonic fibroblasts can be reprogrammed to embryonic stem (ES) cell-like pluripotent stem cells by the forced expression of four transcription factors-OCT4, SOX2, KLF4, and c-MYC. The piggyBac transposon system has proven effective as a vehicle for the delivery of transgenes into fibroblasts and for successful reprogramming to induced pluripotent stem (iPS) cells. This protocol is designed for use with the Neon electroporation system. It can be adapted to other types of electroporation systems.

Basic fibroblast growth factor is critical to reprogramming buffalo (Bubalus bubalis) primordial germ cells into embryonic germ stem cell-like cells

Primordial germ cells (PGCs) are destined to form gametes in vivo, and they can be reprogrammed into pluripotent embryonic germ (EG) cells in vitro. Buffalo PGC have been reported to be reprogrammed into EG-like cells, but the identities of the major signaling pathways and culture media involved in this derivation remain unclear. Here, the effects of basic fibroblast growth factor (bFGF) and downstream signaling pathways on the reprogramming of buffalo PGCs into EG-like cells were investigated. Results showed bFGF to be critical to buffalo PGCs to dedifferentiate into EG-like cells (20 ng/mL is optimal) with many characteristics of pluripotent stem cells, including alkaline phosphatase (AP) activity, expression of pluripotency marker genes such as OCT4, NANOG, SOX2, SSEA-1, CDH1, and TRA-1-81, and the capacity to differentiate into all three embryonic germ layers. After chemically inhibiting pathways or components downstream of bFGF, data showed that inhibition of the PI3K/AKT pathway led to significantly lower EG cell derivation, while inhibition of P53 activity resulted in an efficiency of EG cell derivation comparable to that in the presence of bFGF. These results suggest that the role of bFGF in PGC-derived EG-like cell generation is mainly due to the activation of the PI3K/AKT/P53 pathway, in particular, the inhibition of P53 function.

Pluripotency and differentiation of cells from human testicular sperm extraction: An investigation of cell stemness.

Human male germ-line stem cells (hmGSCs) and human testis-derived embryonic stem cell-like (htESC-like) cells are claimed to be in vitro pluripotent counterparts of spermatogonial stem cells (SSCs), but the origin and pluripotency of human testis-derived cell cultures are still under debate. The aim of this study was to generate putative pluripotent stem cells in vitro from human testicular sperm-extracted (TESE) samples of infertile men, and to assess their pluripotency and capacity to differentiate. TESE samples were minced, enzymatically disaggregated and dispersed into single-cell or cluster suspensions, and then cultured. Initially, cell clusters resembled those described for hmGSCs and htESC-like cells, and were positive for markers such as OCT4/POU5F1, NANOG, and TRA-2-54. Prolonged propagation of cell clusters expressing pluripotency markers did not thrive; instead, the cells that emerged possessed characteristics of mesenchymal stromal cells (MSCs) such as STRO-1, CD105/EGLN1, CD13/ANPEP, SOX9, vimentin, and fibronectin. KIT, SOX2, and CD44 were not expressed by these MSCs. The multipotential differentiation capacity of these cells was confirmed using Oil Red-O and Alizarin Red staining after induction with specific culture conditions. It is therefore concluded that pluripotent stem cells could not be derived using the conditions previously reported to be successful for TESE samples.

Activation and Inhibition of The Wnt3A Signaling Pathway in Buffalo (Bubalus bubalis) Embryonic Stem Cells: Effects of WNT3A, Bio and Dkk1.

This research studies the effects of activation and inhibition of Wnt3A signaling pathway in buffalo (Bubalus bubalis) embryonic stem (ES) cell-like cells. To carry on this experimental study, the effects of activation and inhibition of Wnt3A signaling in buffalo ES cell-like cells were examined using Bio (0.5 mM) combined with WNT3A (200 ng/ml), as an activator, and Dickkopf-1 (Dkk1, 250 ng/ml), as an inhibitor, of the pathway. ES cells were cultured up to three weeks in ES cell medium without fibroblast growth factor-2 (FGF-2) and leukemia inhibitory factor (LIF), but in the presence of Bio, WNT3A, Bio+WNT3A and Dkk1. The effects of these supplements were measured on the mean area of ES cell colonies and on the expression levels of a number of important genes related to pluripotency (Oct4, Nanog, Sox2 and c-Myc) and the Wnt pathway (β-catenin). ES cell colonies cultured in ES cell medium that contained optimized quantities of LIF and FGF-2 were used as the control. Data were collected for week-1 and week-3 treated cultures. In addition, WNT3A-transfected ES cells were compared with the respective mock-transfected colonies, either alone or in combination with Dkk1 for expression of β-catenin and the pluripotency-related genes. Data were analyzed by ANOVA, and statistical significance was accepted at P<0.05. Among various examined concentrations of Bio (0.5-5 mM), the optimum effect was observed at the 0.5 mM dose as indicated by colony area and expressions of pluripotency-related genes at both weeks-1 and -3 culture periods. At this concentration,the expressions of Nanog, Oct3/4, Sox2, c-Myc and β-catenin genes were nonsignificantly higher compared to the controls. Expressions of these genes were highest in the Bio+WNT3A treated group, followed by the WNT3A and Bio-supplemented groups, and lowest in the Dkk1-treated group. The WNT-transfected colonies showed higher expressions compared to both mock and Dkk1-treated mock transfected colonies. WNT3A functions to maintain the pluripotency of ES cell-like cells both as an exogenous growth factor as well as an endogenously expressed gene. It complements the absence of FGF-2 and LIF, otherwise propounded essential for buffalo ES cell culture. WNT3A antagonizes the inhibitory effects of Dkk1 and acts in combination with its activator, Bio, to activate the Wnt signaling pathway.

Spermatogonial stem cells: Current biotechnological advances in reproduction and regenerative medicine.

Spermatogonial stem cells (SSCs) are the germ stem cells of the seminiferous epithelium in the testis. Through the process of spermatogenesis, they produce sperm while concomitantly keeping their cellular pool constant through self-renewal. SSC biology offers important applications for animal reproduction and overcoming human disease through regenerative therapies. To this end, several techniques involving SSCs have been developed and will be covered in this article. SSCs convey genetic information to the next generation, a property that can be exploited for gene targeting. Additionally, SSCs can be induced to become embryonic stem cell-like pluripotent cells in vitro. Updates on SSC transplantation techniques with related applications, such as fertility restoration and preservation of endangered species, are also covered on this article. SSC suspensions can be transplanted to the testis of an animal and this has given the basis for SSC functional assays. This procedure has proven technically demanding in large animals and men. In parallel, testis tissue xenografting, another transplantation technique, was developed and resulted in sperm production in testis explants grafted into ectopical locations in foreign species. Since SSC culture holds a pivotal role in SSC biotechnologies, current advances are overviewed. Finally, spermatogenesis in vitro, already demonstrated in mice, offers great promises to cope with reproductive issues in the farm animal industry and human clinical applications.

From skin to other cell types of the body.

Regenerative medicine allows for the customization of tissues and organs which may bring hope to patients with incurable diseases and severe injuries. Therefore, reliable and safe methods for the generation of specific cell types must be established. Recently, different strategies have emerged to convert somatic cells into differentiated cells of interest. One of these strategies is cellular reprogramming, which converts somatic cells into induced pluripotent stem cells (iPSCs). These iPSCs are embryonic stem cell-like cells with almost unlimited developmental potential and can be differentiated into specific lineages. Alternatively, the method of transdifferentiation can be used to directly convert one terminally differentiated cell into another cell type. Both of these methods have proven to have the potential to push the field of cell replacement therapy forward. In this context, the skin is of particular interest because it represents an ideal source of somatic cells for reprogramming to pluripotency as well as transdifferentiation. In this review, we briefly compare both above-mentioned strategies and summarize the latest advances in this highly dynamic field of research.

Motor neuron derivation from human embryonic and induced pluripotent stem cells: experimental approaches and clinical perspectives.

Motor neurons are cells located in specific areas of the central nervous system, such as brain cortex (upper motor neurons), brain stem, and spinal cord (lower motor neurons), which maintain control over voluntary actions. Motor neurons are affected primarily by a wide spectrum of neurological disorders, generally indicated as motor neuron diseases (MNDs): these disorders share symptoms related to muscular atrophy and paralysis leading to death. No effective treatments are currently available. Stem cell-derived motor neurons represent a promising research tool in disease modeling, drug screening, and development of therapeutic approaches for MNDs and spinal cord injuries. Directed differentiation of human pluripotent stem cells - human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) - toward specific lineages is the first crucial step in order to extensively employ these cells in early human development investigation and potential clinical applications. Induced pluripotent stem cells (iPSCs) can be generated from patients’ own somatic cells (for example, fibroblasts) by reprogramming them with specific factors. They can be considered embryonic stem cell-like cells, which express stem cell markers and have the ability to give rise to all three germ layers, bypassing the ethical concerns. Thus, hiPSCs constitute an appealing alternative source of motor neurons. These motor neurons might be a great research tool, creating a model for investigating the cellular and molecular interactions underlying early human brain development and pathologies during neurodegeneration. Patient-specific iPSCs may also provide the premises for autologous cell replacement therapies without related risks of immune rejection. Here, we review the most recent reported methods by which hESCs or iPSCs can be differentiated toward functional motor neurons with an overview on the potential clinical applications.

Induction of dedifferentiated male mouse adipose stromal vascular fraction cells to primordial germ cell-like cells.

The adipose stromal vascular fraction (SVF) contains abundant mesenchymal stem cell populations that have a limited ability to self-renew and differentiate. Male mouse adipose SVF cells were dedifferentiated by reprogramming factors (c-Myc, Oct4, Sox2, and Klf4) to form embryonic stem cell-like cells (ESCLCs), which upgraded their limited differentiation potential. The ESCLCs were induced to differentiate toward epiblast-like cells (EpiLCs) and primordial germ cell-like cells (PGCLCs) by culturing in media supplied with activin A and BMP-4, respectively. The derived ESCLCs possess embryonic stem cell features and can automatically form embryonic bodies. After culture in EpiLC induction medium for 2-3 days, ESCLCs formed flattened epithelial structures that were different from their original water drop-like colonies, and the expression of pluripotency-related genes decreased. When the cells that had been cultured in EpiLC induction medium for 2 days were isolated and cultured in PGCLC induction medium for 4-6 days, they formed typical water drop-like colonies again. Moreover, expression of the pluripotency-related genes and the primordial germ cell (PGC) specification-related genes increased. During progression from ESCLCs toward EpiLCs and PGCLCs, the levels of histone methylases H3K9me2 and H3K27me3 kept changing, which resembled those seen in PGC specification. The derived PGCLCs expressed SSEA-1, Blimp-1, and Stella. Furthermore, methylation of Igf2r and Snrpn was retained, but H19 and Kcnq1ot1 methylation levels were slightly reduced compared to non-PGCLCs, suggesting that the derived PGCLCs may have initiated the process of imprint erasure.

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro.

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

Generation of induced pluripotent stem cells from mouse adipose tissue.

The discovery that embryonic stem (ES) cell-like cells can be generated by simply over-expressing four key genes in adult somatic cells has changed the face of regenerative medicine. These induced pluripotent stem (iPS) cells have a wide range of potential uses from drug testing and in vitro disease modeling to personalized cell therapies for patients. However, prior to the realization of their potential, many issues need to be considered. One of these is the low-efficiency formation of iPSC. It has been extensively demonstrated that the somatic cell type can greatly influence reprogramming outcomes. We have shown that adipose tissue-derived cells (ADCs) can be easily isolated from adult animals and can be reprogrammed to a pluripotent state with high efficiency. Here, we describe a protocol for the high-efficiency derivation of ADCs and their subsequent use to generate mouse iPSC using Oct4, Sox2, Klf4, and cMyc retroviral vectors.

Induced pluripotent stem cells: origins, applications, and future perspectives

Embryonic stem (ES) cells are widely used for different purposes, including gene targeting, cell therapy, tissue repair, organ regeneration, and so on. However, studies and applications of ES cells are hindered by ethical issues regarding cell sources. To circumvent ethical disputes, great efforts have been taken to generate ES cell-like cells, which are not derived from the inner cell mass of blastocyst-stage embryos. In 2006, Yamanaka et al. first reprogrammed mouse embryonic fibroblasts into ES cell-like cells called induced pluripotent stem (iPS) cells. About one year later, Yamanaka et al. and Thomson et al. independently reprogrammed human somatic cells into iPS cells. Since the first generation of iPS cells, they have now been derived from quite a few different kinds of cell types. In particular, the use of peripheral blood facilitates research on iPS cells because of safety, easy availability, and plenty of cell sources. Now iPS cells have been used for cell therapy, disease modeling, and drug discovery. In this review, we describe the generations, applications, potential issues, and future perspectives of iPS cells.