Laboratorio de Biología del Desarrollo Celular, LIAN, FLENI, Ruta 9, Km 52,5, Escobar, B1625XAF, Buenos Aires, Argentina.
Apoptosis. 2012 Feb;17(2):132-42. doi: 10.1007/s10495-011-0668-z.
Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate to a wide range of specialized cells and hold great promise as models for human development and disease, as well as for drug discovery and cell-replacement therapies. Group B Coxsackie viruses (CVBs) produce acute myocarditis, pancreatitis, non-septic meningitis and encephalitis in neonates, children and young adults. Moreover, CVBs can produce spontaneous miscarriage after early embryo infection. It was reported that hESCs express CVBs receptors and are susceptible to CVB3 infection. Apoptosis is one of the hallmarks of CVBs infection although details regarding CVB3 involvement in the apoptotic processes remain elusive. In order to evaluate the mechanisms of cell death induced by CVB3 in these pluripotent cells, we infected HUES-5 (H5) and WA01 (H1) hESC lines with CVB3. After validating the maintenance of stemness in these hESC lines when grown as confluent monolayers in feeder-free conditions, we analysed several aspects of programmed cell death triggered by CVB3. In all cases, we detected chromatin condensation, DNA fragmentation and caspase-9 and 3 cleavages. Moreover, we observed the presence of cleaved PARP product which was preceded by the appearance of p17, the catalytically active fragment of caspase-3. Mitochondrial function assays revealed a MOI dependent decrease in cell viability at 24 h post-infection (pi). No appreciable modifications in Bcl-2, Bcl-X(L) and Bax protein levels were observed upon CVB3 infection during 5-24 h observation period. However, a marked decrease in pro-apoptotic Bad abundance was detected without changes in its mRNA levels. In this study we found that the hESCs are highly susceptible to CVB3 infection and display elevated apoptosis rates, thus emerging as suitable human non-transformed in vitro models to study CVB3-induced apoptosis and resulting relevant to understand CVBs pathogenesis.
人类胚胎干细胞（hESCs）是自我更新的多能细胞，可分化为广泛的特化细胞，并在人类发育和疾病模型、药物发现和细胞替代疗法方面具有巨大的应用潜力。B 组柯萨奇病毒（CVBs）可导致新生儿、儿童和年轻成人发生急性心肌炎、胰腺炎、非化脓性脑膜炎和脑炎。此外，CVB 还可导致早期胚胎感染后自然流产。据报道，hESCs 表达 CVBs 受体，易受 CVB3 感染。凋亡是 CVB 感染的特征之一，尽管 CVB3 如何参与凋亡过程的细节仍不清楚。为了评估 CVB3 在这些多能细胞中诱导细胞死亡的机制，我们用 CVB3 感染了 HUES-5（H5）和 WA01（H1）hESC 系。在证实这些 hESC 系在无饲养层条件下以融合单层生长时维持干细胞特性后，我们分析了 CVB3 触发的程序性细胞死亡的几个方面。在所有情况下，我们都检测到染色质浓缩、DNA 片段化以及 caspase-9 和 3 的切割。此外，我们还观察到 cleaved PARP 产物的存在，其前体是 caspase-3 的催化活性片段 p17。线粒体功能测定显示，感染后 24 小时（pi）细胞活力随 MOI 依赖性降低。在 5-24 小时的观察期内，CVB3 感染时 Bcl-2、Bcl-X（L）和 Bax 蛋白水平没有明显变化。然而，我们发现促凋亡 Bad 的丰度明显降低，但其 mRNA 水平没有变化。在这项研究中，我们发现 hESCs 极易感染 CVB3，并显示出较高的凋亡率，因此成为研究 CVB3 诱导的凋亡和相关的 CVBs 发病机制的合适的人类非转化体外模型。
