含白血病抑制因子条件培养液对于小鼠iPS细胞多能性维持的影响

Effect of LIF-conditional Medium on the Pluripotency Maintaining of Mouse Induced Pluripotent Stem Cells

白血病抑制因子(Leukemia inhibitory factor,LIF)在维持小鼠胚胎干细胞(Embryonic stem cells,ES cells)和诱导多能干细胞(induced pluripotent stem cells,iPS cells)体外长期培养和多能性方面具有重要作用,但在专利权的保护下,商品化的LIF始终维持较高的价格,给干细胞相关研究带来了高昂经费支出.因此,寻找商品LIF的替代品用于小鼠iPS细胞培养,将对于降低研发成本具有重要意义.本研究使用一种可分泌小鼠LIF的永生化细胞,利用其条件培养液进行iPS细胞的培养,探讨了不同稀释比对于小鼠iPS细胞体外培养及多能性的影响.将条件培养液分别稀释10倍、20倍、40倍和80倍加入培养基中培养iPS细胞,以商品化LIF作为对照组.结果显示,在不同稀释比的培养条件下,iPS细胞均可以保持典型的胚胎干细胞样克隆,且均能在稀释10倍、20倍、40倍、80倍条件下稳定传代20代以上,其中在稀释40倍条件下iPS细胞的克隆形态最好,细胞克隆较大,呈典型的隆起状,克隆周边折光较好;免疫荧光染色显示,各实验组细胞均表达多能性蛋白,Oct4、Sox2、Nanog、SSEA1.定量PCR分析显示,与在商品化LIF培养液中培养的iPS细胞相比,在40倍稀释的条件培养液内培养的细胞,在第12代的多能基因Oct4、Sox2、Nanog、AKP和CD31的表达量均显著高于对照组.此外,在10、20和80倍稀释培养条件下,Nanog的表达量高于对照,差异显著.体外分化结果显示,不同浓度LIF条件培养液和商品化LIF培养的iPS细胞均能形成类胚体,并分化为三个胚层的细胞.当将细胞注入免疫缺陷小鼠皮下,仅40倍稀释和商品化LIF培养的iPS细胞能够形成畸胎瘤,并分化为外、中、内三胚层组织.本研究优化获得了一个能够在体外长期维持iPS细胞多能性的LIF条件培养系统.

Leukemia inhibitory factor(LIF)plays an important role in maintaining long-term culture and pluripotency of mouse embryonic stem cells(ES cells)and induced pluripotent stem cells(iPS cells)in vitro.However,under the protection of patent rights,the commercialized LIF products are always at high prices,which brings high expenditures to stem cell related research.Therefore,a substitute for commercialized LIF will be of great significance for reducing the cost of research in this field.In this research,an immortalized cell line that secreted mouse LIF,was used to prepare conditioned medium,and pluripotency of mouse iPS cells was investigated in the medium with different dilution ratios.The conditioned medium was diluted 10,20,40 and 80 folds,respectively,and their effects were compared with commercialized LIF.The results showed that iPS cells could maintain typical embryonic stem cell-like colonies and could be stably passaged to more than 20 passages in different diluted medium.In 40-fold diluted conditioned medium,mouse iPS cells had the best morphology of colonies,that the cell colonies were large and showed a typical dome-shaped.Immunofluorescence staining showed that pluripotent protein,Oct4,Sox2,Nanog and SSEA1 were positive.Q-PCR analysis showed that compared to control group,40-fold diluted conditioned medium cultured iPS cells expressed high-level of Oct4,Sox2,Nanog,AKP and CD31 at 12 passages.In addition,in 10,20 and 80-fold dilution culture conditions,the expression level of Nanog was higher than that in control group.iPS cells cultured in different medium could differentiate into three germ layer cells.When injected subcutaneously into immunodeficient mice,only iPS cells cultured in 40-fold dilution and commercial LIF formed teratomas and differentiated into tissue of three germ layers.In conclusion,optimized LIF conditioned culture system could maintain the pluripotency of mouse iPS cells in vitro during a long term.