Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada.
Stem Cells Dev. 2011 May;20(5):809-19. doi: 10.1089/scd.2010.0395. Epub 2011 Jan 9.
We previously reported the differentiation of cells derived from porcine female fetal skin into cells resembling germ cells and oocytes. A subpopulation of these cells expressed germ cell markers and formed aggregates resembling cumulus-oocyte complexes. Some of these aggregates extruded large oocyte-like cells (OLCs) that expressed markers consistent with those of oocytes. The objective of the current study was to further characterize OLCs differentiated from porcine skin-derived stem cells. Reverse transcriptase (RT)-polymerase chain reaction and Western blot revealed the expression of connexin37 and connexin43, both of which are characteristic of ovarian follicles. The expression of meiosis markers DMC1 and synaptonemal complex protein, but not STRA8 and REC8, was detected in the OLC cultures. Immunofluorescence with an antibody against synaptonemal complex protein on chromosome spreads revealed a very small subpopulation of stained OLCs that had a similar pattern to leptotene, zytotene, or pachytene nuclei during prophase I of meiosis. Sodium bisulfite sequencing of the differentially methylated region of H19 indicated that this region is almost completely demethylated in OLCs, similar to in vivo-derived oocytes. We also investigated the differentiation potential of male skin-derived stem cells in the same differentiation medium. Large cells with oocyte morphology were generated in the male stem cell differentiation cultures. These OLCs expressed oocyte genes such as octamer-binding transcription factor 4 (OCT4), growth differentiation factor-9b (GDF9B), deleted in azoospermia-like (DAZL), VASA, zona pellucida B (ZPB), and zona pellucida C (ZPC). It was concluded that skin-derived stem cells from both male and female porcine fetuses are capable of entering an oocyte differentiation pathway, but the culture system currently in place is inadequate to support the complete development of competent oocytes.
我们之前曾报道过从猪胎儿皮肤中分离出来的细胞分化为类似生殖细胞和卵母细胞的细胞。这些细胞的一个亚群表达生殖细胞标记物，并形成类似于卵丘-卵母细胞复合物的聚集物。这些聚集物中的一些会排出大型卵母细胞样细胞（OLC），这些细胞表达的标记物与卵母细胞一致。本研究的目的是进一步表征从猪皮肤来源的干细胞中分化而来的 OLC。逆转录（RT）-聚合酶链反应和 Western blot 显示，连接蛋白 37 和连接蛋白 43 的表达，这两种蛋白都是卵巢卵泡的特征。在 OLC 培养物中检测到减数分裂标记物 DMC1 和联会复合体蛋白的表达，但没有 STRA8 和 REC8 的表达。在染色体铺片中用抗联会复合体蛋白的抗体进行免疫荧光染色显示，一小部分染色的 OLC 具有类似于减数分裂 I 前期细线期、粗线期或双线期核的类似模式。H19 的差异甲基化区域的亚硫酸氢盐测序表明，该区域在 OLC 中几乎完全去甲基化，与体内来源的卵母细胞相似。我们还研究了相同分化培养基中雄性皮肤来源干细胞的分化潜能。在雄性干细胞分化培养物中产生了具有卵母细胞形态的大细胞。这些 OLC 表达卵母细胞基因，如八聚体结合转录因子 4（OCT4）、生长分化因子 9b（GDF9B）、缺失在无精子症样（DAZL）、VASA、透明带 B（ZPB）和透明带 C（ZPC）。结论是，来自雄性和雌性猪胎儿的皮肤来源干细胞都能够进入卵母细胞分化途径，但目前使用的培养系统不足以支持有能力的卵母细胞的完全发育。
