Ming-Tao Zhao

Tracing the Stemness of Porcine Skin-Derived Progenitors (pSKP) Back to Specific Marker Gene Expression

Multipotent skin-derived progenitors (SKP) can produce both neural and mesodermal progeny in vitro, sharing the characteristics of embryonic neural crest stem cells. However, the molecular basis for the property of multiple lineage potential and neural crest origin of SKPs is still elusive. Here we report the cooperative expression of pluripotency related genes (POU5F1, SOX2, NANOG, STAT3) and neural crest marker genes (p75NTR, TWIST1, PAX3, SNAI2, SOX9, SOX10) in GFP-transgenic porcine skin-derived progenitors (pSKP). The proportion of cells positive for POU5F1, nestin, fibronectin, and vimentin were 12.3%, 15.1%, 67.9% and 53.7%, showing the heterogeneity of pSKP spheres. Moreover, pSKP cells can generate both neural (neurons and glia) and mesodermal cell types (smooth muscle cells and adipocytes) in vitro, indicating the multiple lineage potency. Four transcription factors (POU5F1, SNAI2, SOX9, and PAX3) were identified that were sensitive to mitogen (FBS) and/or growth factors (EGF and bFGF). We infer that POU5F1, SNAI2, SOX9, and PAX3 may be the key players for maintaining the neural crest derived multipotency of SKP cells in vitro. This study has provided new insight into the molecular mechanism of stemness for somatic-derived stem cells at the level of transcriptional regulation.

Locus-Specific DNA Methylation Reprogramming During Early Porcine Embryogenesis

ABSTRACT During early mammalian embryogenesis, there is a wave of DNA demethylation postfertilization and de novo methylation around implantation. The paternal genome undergoes active DNA demethylation, whereas the maternal genome is passively demethylated after fertilization in most mammals except for sheep and rabbits. However, the emerging genome-wide DNA methylation landscape has revealed a regulatory and locus-specific DNA methylation reprogramming pattern in mammalian preimplantation embryos. Here we optimized a bisulfite sequencing protocol to draw base-resolution DNA methylation profiles of several selected genes in gametes, early embryos, and somatic tissue. We observed locus-specific DNA methylation reprogramming in early porcine embryos. First, some pluripotency genes (POU5F1 and NANOG) followed a typical wave of DNA demethylation and remethylation, whereas CpG-rich regions of SOX2 and CDX2 loci were hypomethylated throughout development. Second, a differentially methylated region of an imprint control region in the IGF2/H19 locus exhibited differential DNA methylation which was maintained in porcine early embryos. Third, a centromeric repeat element retained a moderate DNA methylation level in gametes, early embryos, and somatic tissue. The diverse DNA methylation reprogramming during early embryogenesis is thought to be possibly associated with the multiple functions of DNA methylation in transcriptional regulation, genome stability and genomic imprinting. The latest technology such as oxidative bisulfite sequencing to identify 5-hydroxymethylcytosine will further clarify the DNA methylation reprogramming during porcine embryonic development.

Centrosome Abnormalities During Porcine Oocyte Aging

Centrosomes are critically important for maintaining meiotic spindle integrity in the meiosis II (MII) stage where oocytes are arrested in most mammalian species before fertilization takes place. In women of advanced ages or during in vitro fertilization (IVF) procedures, aneuploidy is frequently seen as a result of oocyte aging, which is strongly related to centrosome instability. Abnormal distribution of centrosomes and microtubules has been reported in aging human and mouse oocytes. This study reports the dynamic changes of centrosomes and the microtubule cytoskeleton in porcine oocytes during aging and treatment by caffeine to restore spindle integrity in aging oocytes. We tested the effects of caffeine on the MII spindle with focus on microtubules and on the centrosome proteins γ‐tubulin and NuMA (nuclear mitotic apparatus protein). The results revealed that in porcine oocytes aged for 48 hr, centrosomes were absent and spindles became abnormal and disorganized; however, caffeine could prevent these changes or restore centrosome integrity in the meiotic spindle poles and displayed similar MII spindles as those seen in fresh oocytes. Environ. Mol. Mutagen., 2009.

Oxamflatin Treatment Enhances Cloned Porcine Embryo Development and Nuclear Reprogramming

Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low cloning efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro-fertilized (IVF) embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the cloned embryos, indicating these genes were successfully reprogrammed. However, compared to the lack of methylation of XIST in day-7 IVF embryos, a higher methylation level in day-7 cloned embryos was observed, implying that X chromosomes were activated in day-7 IVF blastocysts, but were not fully activated in cloned embryos, i.e., reprogramming of XIST was delayed. A time-course analysis of XIST DNA methylation on day-13, -15, -17, and -19 in vivo embryos revealed that XIST methylation initiated at about day 13 and was not completed by day 19. The methylation of the XIST gene in day-19 control cloned embryos was delayed again when compared to in vivo embryos. However, methylation of XIST in Oxamflatin-treated embryos was comparable with in vivo embryos, which further demonstrated that Oxamflatin could accelerate the delayed reprogramming of XIST gene and thus might improve cloning efficiency.

Xenopus Egg Extract Treatment Reduced Global DNA Methylation of Donor Cells and Enhanced Somatic Cell Nuclear Transfer Embryo Development in Pigs

Abstract The efficiency to produce offspring by somatic cell nuclear transfer (SCNT) is low. It has been showed that treatment of donor cells with Xenopus oocyte extract increased live births in ovine and handmade cloned embryo development in pigs. Scriptaid treatment after oocyte activation is another approach to improve SCNT efficiency. The present study was carried out to investigate (a) the effects of treatment of donor cells with Xenopus egg extract on donor cell DNA methylation at days 0 and 4 with two digitonin permeabilization concentrations (10 and 15 μg/mL), (b) the effects of treatment of donor cells with Xenopus egg extract on early development of cloned embryos, and (c) the effects of combined treatments, treating donor cells with extract before nuclear transfer and treatment of cloned embryos with scriptaid after oocyte activation, on embryo development. Compared to the control, a decrease of DNA methylation in donor cells was observed at 2.5 h after extract treatment. However, this effect was not observed after the cells were cultured for four more days. More embryos developed into blastocysts in the Xenopus egg extract-treated group than in the control (13.4±1.9% vs. 9.1±1.9%, p=0.01). Furthermore, scriptaid treatment of cloned embryos further increased the frequency of development to blastocyst, compared to the control reconstructed with the same extract-treated cells (22.5±0.9% vs. 15.3±0.9%, p<0.01). In addition, egg extract treatments increased the cell number in the blastocysts. This study demonstrated that Xenopus egg extract treatment reduced donor cell DNA methylation and enhanced the SCNT embryo development. Moreover, the combined treatments of donor cells with egg extract before nuclear transfer and of cloned embryos with scriptaid could improve cloned embryo development additively.

Effects of griseofulvin on in vitro porcine oocyte maturation and embryo development

Griseofulvin is an orally administered antifungal drug that affects microtubule formation in vitro and interferes with microtubule dynamics in vivo as clearly shown for mitotic cells in several cell systems. This article reports the effects of griseofulvin on in vitro maturation of porcine oocytes and subsequent effects on embryo development. Our results revealed a concentration‐dependent effect on meiotic spindles with 20–40 μM griseofulvin affecting oocyte maturation, and 40 μM affecting fertilization and embryo development. These concentrations of griseofulvin did not affect mitochondrial and cortical granule distribution that also depend on microtubule and cytoskeletal functions during oocyte maturation. Specific effects on the meiotic spindle included spindle disorganization and aberrant chromosome separation displayed as prominent chromosome clusters in oocytes treated with 40 μM griseofulvin. These results strongly suggested that griseofulvin affected porcine oocyte in vitro maturation and following embryo development by disturbing microtubule dynamics. Environ. Mol. Mutagen. 2012.

The in vivo developmental potential of porcine skin-derived progenitors and neural stem cells.

Multipotent skin-derived progenitors (SKPs) can be traced back to embryonic neural crest cells and are able to differentiate into both neural and mesodermal progeny in vitro. Neural stem cells (NSCs) are capable of self-renewing and can contribute to neuron and glia in the nervous system. Recently, we derived porcine SKPs and NSCs from the same enhanced green fluorescent protein (EGFP) transgenic fetuses and demonstrated that SKPs could contribute to neural and mesodermal lineages in vivo. However, it remains unclear whether porcine SKPs and NSCs can generate ectoderm and mesoderm lineages or other germ layers in vivo. Embryonic chimeras are a well-established tool for investigating cell lineage determination and cell potency through normal embryonic development. Thus, the purpose of this study was to investigate the in vivo developmental potential of porcine SKPs and fetal brain-derived NSCs by chimera production. Porcine SKPs, NSCs, and fibroblasts were injected into precompact in vitro fertilized embryos (IVF) and then transferred into corresponding surrogates 24 h postinjection. We found that porcine SKPs could incorporate into the early embryos and contribute to various somatic tissues of the 3 germ layers in postnatal chimera, and especially have an endodermal potency. However, this developmental potential is compromised when they differentiate into fibroblasts. In addition, porcine NSCs fail to incorporate into host embryos and contribute to chimeric piglets. Therefore, neural crest-derived SKPs may represent a more primitive state than their counterpart neural stem cells in terms of their contributions to multiple cell lineages.

The multi-potentiality of skin-derived stem cells in pigs

Multipotent skin-derived stem cells represent neural-crest derived precursors which have neural and mesodermal potency and can generate neurons, glias, smooth muscle cells, and adipocytes. Transcriptional profiling studies show that both intrinsic programs and extrinsic signaling pathways mediate their neural and mesodermal potency. In addition, recent progress implies that skin-derived stem cells may have a broader developmental potency than previously expected, of which is their potential to generate germline cells in vitro. In this review, we discuss the transcriptional profiling of multipotency and neural crest-derived characteristics of skin-derived stem cells, and argue for their potential germ-line competency in the view of nuclear and cellular reprogramming.

Deciphering the Mesodermal Potency of Porcine Skin-Derived Progenitors (SKP) by Microarray Analysis

Skin stem cells have an essential role in maintaining tissue homeostasis by dynamically replenishing those constantly lost during tissue turnover or following injury. Multipotent skin derived progenitors (SKP) can generate both neural and mesodermal progeny, representing neural crest-derived progenitors during embryogenesis through adulthood. SKP cells develop into spheres in suspension and can differentiate into fibroblast-like cells (SFC) in adhesive culture with serum. Concomitantly they gradually lose the neural potential but retain certain mesodermal potential. However, little is known about the molecular mechanism of the transition of SKP spheres into SFC in vitro. Here we characterized the transcriptional profiles of porcine SKP spheres and SFC by microarray analysis. We found 305 upregulated and 96 downregulated genes, respectively. The downregulated genes are mostly involved in intrinsic programs like the Dicer pathway and asymmetric cell division, whereas upregulated genes are likely to participate in extrinsic signaling pathways such as ErbB signaling, MAPK signaling, ECM-receptor reaction, Wnt signaling, cell communication, and tumor growth factor (TGF)-β signaling pathways. These intrinsic programs and extrinsic signaling pathways collaborate to mediate the transcription-state transition between SKP spheres and SFC. We speculate that these potential signaling pathways may play an important role in regulating the cell fate transition between SKP spheres and SFC in vitro.

Contribution to neural and mesodermal lineages by porcine skin-derived progenitors (SKPs) in vivo

Multipotent skin-derived progenitors (SKPs) are neural crest derived and can be isolated from both embryonic and adult skin in humans, rodents and pigs1,2. The SKPs are capable of generating both neural and mesodermal progeny in vitro: neurons, Schwann cells, adipocytes, osteocytes, and chondrocytes, thus exhibiting properties similar to embryonic neural crest stem cells3. However, SKPs show distinct transcriptional profiles when compared to neural stem cells in the central nervous system and skin derived fibroblast, indicating a novel type of multipotent stem cells derived from skin. The SKPs are derived from hair follicle progenitors and exhibit adult dermal stem cell properties, contributing to dermal maintenance, would-healing, and hair follicle morphogenesis4. Transplantation experiments demonstrate that labeled murine SKP spheres can differentiate into dorsal root ganglia and autonomic ganglia when integrated into chick embryos, showing their neural potential in vivo5. However, it is still unclear whether SKPs can differentiate into neural and mesodermal lineages in developing mammalian embryos. Isolated porcine SKPs from embryonic and adult porcine skin have demonstrated their neural and mesodermal potency in vitro1,6. Since swine are an important model for human medicine and are a potential resource of tissue for xenotransplantation7 we addressed the in vivo differentiation of SKPs by injecting enhanced green fluorescent protein (eGFP)-tagged8 porcine SKP cells into early embryos. Porcine SKP spheres were derived from the back skin of day 45 eGFP transgenic fetuses and dispersed into single cells by using Accumax solution (Sigma). Then 10-15 SKP cells were injected into the center of a peri-morula stage embryo. The injected embryos were cultured in PZM3 medium overnight at 38.5 °C in 5% CO2 in air and the next day transferred to the oviduct of a surrogate on day 4 of her estrous cycle8. The integration of SKP cells within the embryos were visualized by eGFP fluorescence before embryos transfer (Figure 1A). In total, we injected 416 embryos and performed 6 embryos transfers and produced two fetuses on day 45 of gestation (Supplemental Table 1). Various tissues (placenta, brain, skin, liver, trunk, kidneys and genital ridge) were collected and parts of tissues were stabilized immediately in RNAlater RNA Stabilization Reagent (Qiagen). Genomic DNA was extracted by using an AllPrep DNA/RNA mini kit (Qiagen). PCR was performed by using GoTaq Green Master Mix (Promega). To exclude any potential false positive PCR results, two sets of eGFP primers (Supplemental Table 2) were used. The two eGFP bands were found in genomic DNA from both brain and kidney (Figure 1I). It may also appear in genital ridge because kidney and genital ridge are bound together at this developmental stage and a clear demarcation could not be determined. Moreover, no eGFP bands appeared in wild-type porcine genomic DNA (Figure 1I). Next we performed immunohistochemistry to confirm the eGFP expression in these tissues. Additional fetal tissues were fixed, embedded, sectioned and placed on plus charge slides. Sample slides were incubated with primary anti-eGFP antibody (1:400, Abcam) for 1 h, washed, and then with MACH 2 HPR-polymer secondary antibody (1:1000, Biocare Medical) for 30 min. Romulin Red (Biocare Medical) was used as chromogen for 10 min. Slides were then counterstained in CAT Hematoxylin for 5 min, washed, dehydrated and coverslipped. We found that the eGFP positive cells were dispersed in brain, kidney and genital ridges (Figure 1B-H). These results show that porcine SKP cells can develop into neural (brain) and mesodermal lineages (kidney and genital ridge) in vivo. Chimera production is widely used to test the pluripotency of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. Unlike ES or iPS cells, multipotent neural stem cells could not participate in development to form chimeric embryos in rodents9. However, a few reports indicate that multipotent stem cells could give rise to cells of multiple germ layers10-11. For instance, adult neural stem cells can contribute to the formation of chimeric chick and mouse embryos and generate cells of all three germ layers. Since SKP cells have been widely demonstrated to be multipotent and can differentiate into neural and mesodermal progeny in vitro and in transplantation experiments, it raises the question on the integration and differentiation of SKPs into embryonic tissues in vivo. Our study shows that porcine SKP cells can also contribute to neural (brain) and mesodermal (kidney) progeny in vivo, implying that the developmental potential of SKPs may not be as limited as expected before. SKPs are considered to have neural crest origin and share similar characteristics with neural crest stem cells in the peripheral nervous system; whereas we found that SKPs could also produce neural cells in the brain which belongs to the central nervous system. Although the early embryo may reprogram SKPs into a more pluripotent state, our data at least suggest the feasibility of converting skin into neural cells in brain and kidney cells. Together with other in vivo and in vitro findings, our data will strengthen the possibility of SKPs being used as a therapeutic resource for disease modeling and regenerative medicine in the future. In addition, SKP cells may potentially integrate into germ cell lines as eGFP positive SKP cells appeared in the developing kidney and genital ridge. Although it is too early to conclude that SKP cells can produce live chimeric pigs, it is encouraging to reprogram skin into kidney and brain in developing porcine embryos, implying a broad developmental potency of porcine SKPs. Figure 1 Contribution of porcine SKPs into neural and mesodermal lineages. (A) eGFP expression in injected day 5 IVF embryos 24 h post injection. (B) Positive control for anti-eGFP antibody: the section was from a uterus where placenta tissue was GFP positive ...