卵巢早衰患者来源特异干细胞分化为单倍体细胞的研究

Study of differentiation of premature ovarian failure patients-specific induced pluripotent stem cells into haploid cells

目的探索卵巢早衰患者来源的诱导多能干细胞(POF-iPSC)分化为单倍体细胞的可能。方法以卵巢早衰患者外周血单个核细胞进行核转染自行建系POF-iPSC,培养基中添加激活素A、糖原合酶激酶-3抑制剂CHIR-99021共2 d,随后添加骨形态生成蛋白4及表皮生长因子5 d,再添加视黄酸7 d,设基线(D0)、培养7 d(D7)、培养14 d(D14)组及无诱导的对照组。应用实时定量PCR及免疫荧光染色分别检测原始生殖细胞或减数分裂相关基因的mRNA及蛋白表达水平;荧光原位杂交技术(FISH)检测细胞内X染色体单体;流式细胞仪检测单倍体细胞比例。结果 POF-iPSC在诱导过程中,D0组细胞形态为长梭形纤维样,D7组细胞体积变大,但胞质稀薄,具有多个触角,D14组细胞核及核仁变得明显。在mRNA相对表达水平上,NANOG随分化而下降,BLIMP1逐步上升,STELLA、OCT4、DDX4、γH2AX、MLH1于D7组最高,DAZL、SYCP3、PRDM9于D7组及D14组逐步提高(P均<0.01)。在蛋白表达上,D0组细胞中DAZL、PRDM9、SCP3蛋白均呈阴性表达,D7组细胞中DAZL及PRDM9蛋白呈阳性表达,D14组细胞中DAZL、PRDM9、SCP3蛋白均呈阳性表达。针对X染色体着丝粒的FISH检测显示,对照组细胞中均未见单个X染色体显色的细胞;D14组的部分细胞核中有单个X染色体显色,提示其单倍体的可能。D0组、D7组、D14组及对照组细胞中均可见发生分裂状态的染色体。其中D14组单倍体细胞比例高于D0组、D7组及对照组(P均<0.01)。结论 POF-iPSC体外培养后能分化为类原始生殖细胞,部分细胞可完成减数分裂为单倍体细胞。

Objective To explore the possibility of premature ovarian failure patients specificinduced pluripotent stem cells(POF-iPSCs) differentiating into haploid cells. Methods The monocytes in the peripheral blood of POF patients were nuclear stained and reprogrammed into iPSCs to establish POFiPSCs, and subsequently cultured with activin A and glycogen synthase kinase-3(GSK-3) inhibitor(CHIR-99021) for 2 d, supplemented with bone morphogenetic protein 4(BMP 4) and epidermal growth factor(EGF) for 5 d, and retinoic acid(RA) for 7 d. The baseline(D0), 7 d-culture(D7) and 14 d-culture(D14) and control groups were established. qPCR and immunofluorescent staining were performed to detect the expression levels of mRNA and protein of primordial germ cells and the meiosis-related genes. FISH was adopted to detect the intracellular X chromosome segregation. Flow cytometry was conducted used to determine the proportion of haploid cells. Results During the induction process of POF-iPSCs, the fusiform fiber-like cells were observed in the D0 group, the cell body was enlarged with thin cytoplasm and multiple antennae in the D7 group, and the cell nucleus and nucleolus became evident in the D14 group. The relative expression level of NANOG mRNA was down-regulated, whereas that of BLIMP1 was gradually up-regulated along the differentiation. STELLA,OCT4, DDX4, γH2 AX and MLH1 mRNA levels were the highest in the D7 group, and DAZL, SYCP3 and PRDM9 mRNA levels were gradually up-regulated from the D7 to D14 groups(all P < 0.01). DAZL, PRDM9 and SCP3 proteins were not expressed in the D0 group. DAZL and PRDM9 proteins were positively expressed in the D7 group. DAZL, PRDM9 and SCP3 proteins were expressed in the D14 group. FISH demonstrated that no X chromosome segregation was detected in the control group. X chromosome segregation was observed in partial cell nucleus in the D14 group, prompting the possibility of haploid cells. The chromosomes during meiosis were observed in all groups. In the D14 group, the proportion of haploid cells was significantly higher compared with those in the remaining three groups(all P < 0.01). Conclusion POF-iPSCs can differentiate into primordial germ cells after in vitro culture. Some cells can complete meiosis and differentiate into haploid cells.