Faculty of Medicine and Health Technology, Tampere University, Arvo Ylpön katu 34, 33520, Tampere, Finland.
Tampere Center for Child Health Research, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.
Stem Cell Res Ther. 2019 Aug 5;10(1):236. doi: 10.1186/s13287-019-1354-2.
The differentiation of corneal limbal stem cells (LSCs) from human pluripotent stem cells (hPSCs) has great power as a novel treatment for ocular surface reconstruction and for modeling corneal epithelial renewal. However, the lack of profound understanding of the true LSC population identity and the regulation of LSC homeostasis is hindering the full therapeutic potential of hPSC-derived LSCs as well as primary LSCs.
The differentiation trajectory of two distinct hPSC lines towards LSCs was characterized extensively using immunofluorescence labeling against pluripotency, putative LSC, and mature corneal epithelium markers. Cell counting, flow cytometry, and qRT-PCR were used to quantify the differences between distinct populations observed at day 11 and day 24 time points. Initial differentiation conditions were thereafter modified to support the maintenance and expansion of the earlier population expressing ABCG2. Immunofluorescence, qRT-PCR, population doubling analyses, and transplantation into an ex vivo porcine cornea model were used to analyze the phenotype and functionality of the cell populations cultured in different conditions.
The detailed characterization of the hPSC differentiation towards LSCs revealed only transient expression of a cell population marked by the universal stemness marker and proposed LSC marker ABCG2. Within the ABCG2-positive population, we further identified two distinct subpopulations of quiescent ∆Np63α-negative and proliferative ∆Np63α-positive cells, the latter of which also expressed the acknowledged intestinal stem cell marker and suggested LSC marker LGR5. These populations that appeared early during the differentiation process had stem cell phenotypes distinct from the later arising ABCG2-negative, ∆Np63α-positive third cell population. Importantly, novel culture conditions modulating the Wnt and BMP signaling pathways allowed efficient maintenance and expansion of the ABCG2-positive populations. In comparison to ∆Np63α-positive hPSC-LSCs cultured in the initial culture conditions, ABCG2-positive hPSC-LSCs in the novel maintenance condition contained quiescent stem cells marked by p27, demonstrated notably higher population doubling capabilities and clonal growth in an in vitro colony-forming assay, and increased regenerative potential in the ex vivo transplantation model.
The distinct cell populations identified during the hPSC-LSC differentiation and ABCG2-positive LSC maintenance may represent functionally different limbal stem/progenitor cells with implications for regenerative efficacy.
人多能干细胞（hPSCs）来源的角膜缘干细胞（LSCs）分化具有强大的潜力，可用于重建眼表和模拟角膜上皮更新。然而，由于对真正的 LSC 群体特性和 LSC 稳态调控缺乏深入了解，阻碍了 hPSC 来源的 LSCs 以及原代 LSCs 的充分治疗潜能。
使用针对多能性、假定 LSC 和成熟角膜上皮标志物的免疫荧光标记，广泛表征两种不同 hPSC 系向 LSCs 的分化轨迹。使用细胞计数、流式细胞术和 qRT-PCR 来量化第 11 天和第 24 天时间点观察到的不同群体之间的差异。此后，对初始分化条件进行了修改，以支持表达 ABCG2 的早期群体的维持和扩增。使用免疫荧光、qRT-PCR、群体倍增分析和体外猪角膜模型移植来分析在不同条件下培养的细胞群体的表型和功能。
对 hPSC 向 LSCs 分化的详细表征表明，只有短暂表达了一个由普遍的干性标志物和假定的 LSC 标志物 ABCG2 标记的细胞群体。在 ABCG2 阳性群体中，我们进一步鉴定了两个不同的亚群，静止的 ∆Np63α-阴性和增殖的 ∆Np63α-阳性细胞，后者还表达了公认的肠干细胞标志物和建议的 LSC 标志物 LGR5。这些在分化过程中早期出现的群体具有不同于后来出现的 ABCG2-阴性、∆Np63α-阳性第三细胞群体的干细胞表型。重要的是，调节 Wnt 和 BMP 信号通路的新型培养条件允许高效维持和扩增 ABCG2 阳性群体。与在初始培养条件下培养的 ∆Np63α-阳性 hPSC-LSCs 相比，在新型维持条件下的 ABCG2 阳性 hPSC-LSCs 含有静止的干性标志物 p27，在体外集落形成测定中表现出显著更高的群体倍增能力和克隆生长能力，并在体外移植模型中提高了再生潜力。
在 hPSC-LSC 分化过程中鉴定的不同细胞群体和 ABCG2 阳性 LSC 的维持可能代表具有不同功能的角膜缘干细胞/祖细胞，这对再生功效具有重要意义。
