APA微囊微环境影响胚胎干细胞增殖分化的体外研究

Microenvironment effect of APA microcapsule on embryonic stem cell proliferation and differentiation

以小鼠胚胎干细胞(embryonic stem cell,ESC)为模型,在生理条件下对ESC进行微囊化包封、培养,并利用免疫组织化学技术及RT-PCR方法检测其生长及未分化状态,以期建立微囊化ESC这一体外培养模型,同时明确海藻酸钠-聚赖氨酸-海藻酸钠(alginate-poly-lysine-alginate,APA)微囊微环境对:ESC增殖及分化潜能的影响。结果表明:ESC能够在微囊(包括液化型及非液化型)或微球(海藻酸钙胶珠)内生长良好,但因生长环境存在差异,其表现的生长行为各具特征。比较其它类型,ESC在液化型APA微囊内的存活期限最长。经体外维持培养3周以上,仍能持续表达胚胎源未分化干细胞的标志性蛋白AP,SSEA-1及转录因子Pct-4。为进一步明确微囊内增殖的ESC是否仍具有多向分化的干细胞潜能,应用机械破囊法释放微囊内ESC团,并在体外进行定向诱导。经过近3周的条件诱导,其结果为:细胞团DTZ染色阳性; anti-insulin免疫荧光检测阳性;且特异性表达Pdx-1,Ins-1基因。上述结果证明:APA微囊为ESC维持未分化状态的增殖提供了特殊的微环境,APA微囊内所形成的ESC团仍具有多向分化的干细胞潜能。

We undertook a series of studies to evaluate the role of microenvironment during embryonic stem cell （ESC） proliferation and differentiation. In this paper, cell microencapsulation technology was employed, which allows the free exchange of nutrients, oxygen and biologically active products between the entrapped cell and culture medium. We analyzed the feasibility of mouse ESCs in microcapsules and evaluated the growth, metabolic activity and differentiation of ESCs once enclosed in alginate-Ca2＋ microbead, solid or liquefied core alginate-poly-lysine-alginate （APA） microcapsule, respectively. We found that ESCs grew gradually in both types of microcapsules, but the appearance of cells was distinctive for each type of capsule. In the case of unliquefied microcapsules, cells created multiple spherical or lens-shaped aggregates. In contrast, the liquefied alginate core allowed the enclosed ESCs to grow together in a clump at the periphery of the capsule. Combined with cell viability and activity of glucose/lactic acid metabolism, the liquefied core of APA might provide more suitable culture conditions for the ESC growth in comparison with the unliquefied type or alginate-Ca2~. For better evaluating the nature of ESC growth in APA microcapsules in vitro （that is whether or not encapsulated ESCs maintained undifferentiated state while they kept the ability for proliferation）, the expression of the typical markers for undifferentiated, dividing ESCs, such as the stage specific embryonic antigen （SSEA-1） and alkaline phosphatase （AP）, was detected by immunochemistry and immunofluorescence staining. The results showed that cell aggregates formed in the microcapsule still expressed the marker proteins at a higher level on day 22 in vitro. The expression of gene Oct-4, a transcription factor necessary for maintaining ESCs in an undifferentiated state, was also detected when RT-PCR assay was employed （on day 22 in vitro）. In addition, cell aggregates were released from the microcapsules by mechanical disruption and induced into insulin-producing ceils. These findings further indicate that most of the ESCs in APA microcapsule maintain their multi-potential even though the culture time prolonged as long as 22 d in vitro. Taken together, APA microcapsule provides a suitable microenvironment that promotes ESCs to maintain their stemness. Therefore, the microenvironment plays an important role in the process of ESC proliferation and differentiation.