Stem Cell Research Laboratory, GROW Laboratory, Narayana Nethralaya Foundation, Bangalore, India; School of Biosciences and Technology, VIT University, Vellore, India.
Stem Cell Research Laboratory, GROW Laboratory, Narayana Nethralaya Foundation, Bangalore, India; Post Graduate Department of Biotechnology, Jamal Mohamed College (Autonomous), Tiruchirappalli, India.
Cytotherapy. 2016 Apr;18(4):546-61. doi: 10.1016/j.jcyt.2016.01.003.
Autologous transplantation of ex vivo cultured cells the treatment of choice for patients with limbal stem cell deficiency. The most commonly used cell sources for transplantation limbal, conjunctival or oral mucosal tissue. Protocols vary for culturing each tissue type, and there are no comparative studies on transplantation outcomes using these different culture techniques. To overcome this limitation, we devised a simple protocol that can uniformly promote growth and differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage.
Biopsies were cultured as explants on de-epithelialized human amniotic membrane in the presence of recombinant epidermal growth factor and insulin. Cultured cells were characterized using immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction for stem/progenitor markers (ABCG2 and P63α) and differentiation markers (CK3, CK12, CK4, CK13, CK15 and CONNEXIN 43). Fluorescence-activated cell sorter analysis was performed for ABCG2.
The results revealed that cells of all three biopsies differentiated into the corneal lineage. Positivity of CK3/12, CK4, CK12 and CONNEXIN 43 immunostaining and the relative mRNA expression of CK3, CK4, CK12, CK13, CK15 and CONNEXIN 43 could be detected in the cultured biopsies.
Unlike tissue-specific protocols, our protocol can unequivocally promote differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage. This simple standardized protocol can be adapted for ocular surface reconstruction using stem cell transplantation.
自体细胞体外培养移植是治疗角膜缘干细胞缺陷患者的首选方法。最常用于移植的细胞来源是角膜缘、结膜或口腔黏膜组织。每种组织类型的培养方案都不同，并且没有关于使用这些不同培养技术进行移植结果的比较研究。为了克服这一限制，我们设计了一种简单的方案，可以统一促进来自角膜缘、结膜或口腔黏膜活检的细胞向角膜谱系生长和分化。
将活检组织作为外植体在去上皮化的人羊膜上培养，同时存在重组表皮生长因子和胰岛素。使用免疫组织化学和定量逆转录聚合酶链反应来鉴定干细胞/祖细胞标志物（ABCG2 和 P63α）和分化标志物（CK3、CK12、CK4、CK13、CK15 和 CONNEXIN 43）来鉴定培养的细胞。通过荧光激活细胞分选术分析 ABCG2。
结果表明，三种活检组织的细胞均分化为角膜谱系。培养活检组织中可检测到 CK3/12、CK4、CK12 和 CONNEXIN 43 免疫染色阳性，以及 CK3、CK4、CK12、CK13、CK15 和 CONNEXIN 43 的相对 mRNA 表达。
与组织特异性方案不同，我们的方案可以明确促进来自角膜缘、结膜或口腔黏膜活检的细胞向角膜谱系分化。这种简单的标准化方案可适用于使用干细胞移植进行眼表面重建。
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