稳定表达E-cadherin的TW2R细胞增强人胚胎干细胞生长

E-cadherin-expressing TW2R feed cells enhance growth of undifferentiated human embryonic stem cells

目的构建一种可高效支持人胚胎干细胞生长的人源饲养层细胞系。方法首先通过在TW2R细胞系稳定表达人E-钙黏素(E-cadherin)基因构建永生化人源饲养层细胞系TWE3R,并比较人胚胎干细胞在TWE3R及其亲本细胞上的克隆形成数。将H9人胚胎干细胞在TWE3R细胞上经过10代连续传代培养后,进行人胚胎干细胞特异性标志物分析、拟胚体形成分析和畸胎瘤形成检测等实验。结果本研究成功构建了永生化的人源饲养层细胞系TWE3R,TWE3R细胞可比其亲本细胞更高效地支持人胚胎干细胞生长,并且TWE3R饲养层细胞还具有支持低密度接种条件下(每平方厘米5个)人胚胎干细胞生长的特性。此外,H9人胚胎干细胞在TWE3R细胞上长期传代培养后,仍可保持所有人胚胎干细胞的未分化特性、正常细胞核型以及在体内外均可分化形成所有3个胚层衍生物的潜能。结论本研究所建立的永生化人源饲养层细胞系TWE3R具有高效支持胚胎干细胞生长的特性。

Objective To establish an immortalized human feeder cell line that efficiently supports the growth of undifferentiated human embryonic stem(hES) cells. Methods The immortalized TWE3 R cell line was established by stably expressing the human E-cadherin gene in the TW2 R cell line. The numbers of hESC colonies on TWE3 R feeder cells were compared with those on its parental cell lines. After H9 hES cells were cultured on TWE3 R cells for 10 consecutive passages, pluripotency marker analysis, embryoid body(EB) formation assays and teratoma formation assays were conducted. Results We succeeded in establishing the TWE3 R human feeder cell line that showed higher efficiency to support the growth of hES cells than its parental cell line. TWE3 R feeder cells supported the growth of H9 ESCs at low density(5 cells/cm;). Additionally, after long-term culture on TWE3 R feeders, H9 ESCs retained all undifferentiated characteristics, a normal karyotype and the potential to differentiate into derivatives of all three embryonic germ layers. Conclusions The immortalized TWE3 R human feeder cell line is capable of supporting efficient growth of human ESCs.