Hengxi Wei

Mouse undifferentiated spermatogonial stem cells cultured as aggregates under simulated microgravity

Dynamic simulated microgravity (SMG) culture systems provide environments that stimulate stem cell proliferation and differentiation. However, the effect of SMG on spermatogonial stem cells (SSCs) remains unclear. Here, we used a rotating cell culture system (RCCS) to determine its effect on mouse SSC proliferation and differentiation. SSCs were enriched from mouse pub testis and cocultured with Sertoli cell feeders pre‐treated with mitomycin C on fibrin scaffolds in a rotary bioreactor for 14 days. Our results show that mouse SSCs cultured in a rotary bioreactor exhibited enhanced proliferation surpassing those cultured in static conditions, although SSC cultures in SMG underwent a growth lag at initial 3 days. After 14 days, mouse SSCs and feeders grew into cell aggregates with average diameters of 242.63 ± 16.53 μm compared with those in conventional static culture (49.51 ± 15.64 μm). Related detection revealed that proliferating SSCs in SMG remained undifferentiated, maintained clone‐forming capacity and were capable of differentiation into round spermatids with flagella. The growth characteristics of mouse SSCs in RCCS suggest that the resulting aggregates are similar to native in vivo cells. Rotary bioreactors that create SMG environments may be an alternative to conventional systems for the clinical application of SSCs.

Efficient production of chimeric mice from embryonic stem cells injected into 4- to 8-cell and blastocyst embryos

BackgroundProduction of chimeric mice is a useful tool for the elucidation of gene function. After successful isolation of embryonic stem (ES) cell lines, there are many methods for producing chimeras, including co-culture with the embryos, microinjection of the ES cells into pre-implantation embryos, and use of tetraploid embryos to generate the full ES-derived transgenic mice. Here, we aimed to generate the transgenic ES cell line, compare the production efficiency of chimeric mice and its proportion to yield the male chimeric mice by microinjected ES cells into 4- to 8-cell and blastocysts embryos with the application of Piezo-Micromanipulator (PMM), and trace the fate of the injected ES cells.ResultsWe successfully generated a transgenic ES cell line and proved that this cell line still maintained pluripotency. Although we achieved a satisfactory chimeric mice rate, there was no significant difference in the production of chimeric mice using the two different methods, but the proportion of the male chimeric mice in the 4- to 8-cell group was higher than in the blastocyst group. We also found that there was no tendency for ES cells to aggregate into the inner cell mass using in vitro culture of the chimeric embryos, indicating that they aggregated randomly.ConclusionsThese results showed that the PMM method is a convenient way to generate chimeric mice and microinjection of ES cells into 4- to 8-cell embryos can increase the chance of yielding male chimeras compared to the blastocyst injection. These results provide useful data in transgenic research mediated by ES cells.

Generation of porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene by somatic cell nuclear transfer

Cas9 endonuclease, from so-called clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems of Streptococcus pyogenes, type II functions as an RNA-guided endonuclease and edits the genomes of prokaryotic and eukaryotic organisms, including deletion and insertion by DNA double-stranded break repair mechanisms. In previous studies, it was observed that Cas9, with a genome-scale lentiviral single-guide RNA library, could be applied to a loss-of-function genetic screen, although the loss-of-function genes have yet to be verified in vitro and this approach has not been used in porcine cells. Based on these observations, lentiviral Cas9 was used to infect porcine primary fibroblasts to achieve cell colonies carrying Cas9 endonuclease. Subsequently, porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene were generated by somatic cell nuclear transfer, and three 30 day transgenic porcine fetal fibroblasts (PFFs) were obtained. Polymerase chain reaction (PCR), reverse transcription-PCR and western blot analysis indicated that the PFFs were Cas9-positive. In addition, one of the three integrations was located near to known functional genes in the PFF1 cell line, whereas neither of the integrations was located in the PFF1 or PFF2 cell lines. It was hypothesized that these transgenic PFFs may be useful for conditional genomic editing in pigs, and for generating ideal modified porcine models.

Analysis of imprinted gene expression and implantation in haploid androgenetic mouse embryos

The successful development of mammalian embryos requires both parental genomes. Nuclear transfer techniques have been adapted to generate uniparental embryos, which possess two sets of paternal or maternal genomes. These embryos fail to develop to term because of abnormal imprinted gene expression, which is not regulated by Mendelian inheritance. Uniparental embryos provide us with an important model to investigate imprinted gene function and ontogenesis. To evaluate the pre‐ and post‐developmental ability of haploid androgenetic mouse embryos, and to analyse the expression of imprinted genes Igf2r, Asb4 and Mest in haploid androgenetic/gynogenetic blastocysts, we produced the haploid mouse embryos using the enucleation technique, examined their development at 6.5 dpc and quantified gene expression by quantitative real‐time PCR. The results demonstrated that the developmental potential of haploid embryos was severely impaired and revealed that the haploid androgenones could induce the deciduas reaction, but failed to retain a live foetus at 6.5 dpc. Expression of imprinted genes Igf2r and Asb4 was unregulated in haploid androgenetic/gynogenetic blastocysts.

Characteristics of spermatogonial stem cells derived from neonatal porcine testis

The aim of this study was to isolate and characterise porcine spermatogonial stem cells (PSSCs). The putative porcine germline stem cells from testis were isolated successfully by an improving way of enrichment with lymphocyte separation medium (LSM). Results from RT‐PCR analyses showed that PSSCs were positive for OCT4, SOX2, NANOG, PGP9.5, c‐MYC, KEL4 and PRDM‐14 which are multipotent stem cell markers. At the protein level, the results of immunofluorescence analyses showed that PSSCs were positive for OCT4, PGP9.5, SOX2 and CD29. We successfully differentiated these PSSCs into adipocytes and muscle cells and then defined their characteristics, including morphology, surface stem cell markers, and mechanical properties. But the experiment of teratoma formation was negative. The results indicated the PSSCs could be multipotent. Atomic force microscopy was used to characterise the morphological and mechanical properties of undifferentiated PSSCs, as well as the differentiated adipocytes and muscle cells, which could be potentially useful for distinguishing PSSCs from differentiated cells.

Functional verification of the diphtheria toxin A gene in a recombinant system

Diphtheria toxin A (DTA), a segment of the diphtheria toxin (tox), inhibits protein synthesis in cells. When released from a cell, DTA is nontoxic and cannot enter other cells independently without the help of diphtheria toxin B. In this study, we artificially synthesized the DTA gene sequence and cloned it into pEGFP-N1 to generate the recombinant vector pEGFP-N1-DTA. This recombinant vector was then transfected into 293T cells to observe the effect of DTA protein expression on enhanced green fluorescent protein (EGFP) protein expression and the proliferation of 293T cells. After 48 h, high levels of EGFP expression were seen in control pEGFP-N1-transfected cells, whereas very low levels were seen in cells transfected with pEGFP-N1-DTA. Reverse transcription polymerase chain reaction confirmed the expression of the DTA gene in cells transfected with pEGFP-N1-DTA. Further, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed a significant difference in cell proliferation between the control group and the pEGFP-N1-DTA-transfected group. Using the expression of EGFP expression as an indicator, this study revealed that DTA expression can inhibit intracellular protein synthesis and cell proliferation.

Differential gene expression in mouse spermatogonial stem cells and embryonic stem cells

Mouse spermatogonial stem cells (mSSCs) may be reprogrammed to become pluripotent stem cells under in vitro culture conditions, due to epigenetic modifications, which are closely associated with the expression of transcription factors and epigenetic factors. Thus, this study was conducted to compare the gene expression of transcription factors and epigenetic factors in mSSCs and mouse embryonic stem cells (mESCs). Firstly, the freshly isolated mSSCs [mSSCs (f)] were enriched by magnetic-activated cell sorting with Thy1.2 (CD90.2) microbeads, and the typical morphological characteristics were maintained under in vitro culture conditions for over 5 months to form long-term propagated mSSCs [mSSCs (l)]. These mSSCs (l) expressed pluripotency-associated genes and were induced to differentiate into sperm. Our findings indicated that the mSSCs (l) expressed high levels of the transcription factors, Lin28 and Prmt5, and the epigenetic factors, Tet3, Parp1, Max, Tert and Trf1, in comparison with the mESCs, with the levels of Prmt5, Tet3, Parp1 and Tert significantly higher than those in the mESCs. There was no significant difference in Kdm2b expression between mSSCs (l) and mESCs. Furthermore, the gene expression of N-Myc, Dppa2, Tbx3, Nr5a2, Prmt5, Tet3, Parp1, Max, Tert and Trf1 in the mSSCs (l) was markedly higher in comparison to that in the mSSCs (f). Collectively, our results suggest that the mSSCs and the mESCs displayed differential gene expression profiles, and the mSSCs possessed the potential to acquire pluripotency based on the high expression of transcription factors and epigenetic factors. These data may provide novel insights into the reprogramming mechanism of mSSCs.

Development and imprinted gene expression in uniparental preimplantation mouse embryos in vitro

Increasing numbers of reports show that imprinted genes play a crucial role in fetal development, and uniparental embryos, which possess two paternally or two maternally derived pronuclei, are excellent tools for investigating the biological significance of imprinted genes. In the present study, to examine the in vitro developmental ability and expression pattern of eight imprinted genes in uniparental embryos, we produced androgenones, gynogenones, and parthenogenones using enucleation. Our data confirmed the previously observed restriction in haploid androgenetic development potential and first indicated that diploid androgenetic embryos were arrested in the 3/4-cell stage. Some imprinted genes were expressed in androgenetic, gynogenetic, and parthenogenetic blastocysts, suggesting that they were unable to maintain their imprinted expression status in uniparental embryos and that both paternal and maternal alleles are required for the specific expression of some imprinted genes.

The effect of microRNAs on the regulatory network of pluripotency in embryonic stem cells: The effect of microRNAs on the regulatory network of pluripotency in embryonic stem cells

Embryonic stem cells (ESCs) are pluripotent stem cells characterized by their ability to self-renew and their pluripotency to differentiate into all cell types. MicroRNA (miRNA) is a small non-coding RNA molecule which can regulate transcriptional and post-transcriptional gene expression, and may also play significant roles in regulating proliferation and differentiation of ESCs. The maintenance of pluripotency in ESCs may involve a regulatory network of many factors and pathways regulated by miRNA, which includes ESCs transcription factors, cell cycle regulation, epigenetic modifications as well as intracelluar signal transduction. This review mainly elaborates the biogenesis of miRNA, the miRNA families regulating the pluripotency of ESCs, and the effect of miRNA on the regulatory network of pluripotency in ESCs.

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.