Abstract
BackgroundThe 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.MethodsThe 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.ResultsThe 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.ConclusionsThe 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.
Highlights
The differentiation of corneal limbal stem cells (LSCs) from human pluripotent stem cells has great power as a novel treatment for ocular surface reconstruction and for modeling corneal epithelial renewal
The distinct cell populations identified during the Human pluripotent stem cell (hPSC)-LSC differentiation and ATP-binding cassette super-family G member 2 (ABCG2)-positive LSC maintenance may represent functionally different limbal stem/progenitor cells with implications for regenerative efficacy
The study consisted of two main parts, the first being the detailed characterization of hPSC differentiation process towards LSCs (Fig. 1a), and the second being establishing novel culture conditions for the maintenance of an ABCG2-positive LSC phenotype and further characterization of the stemness and functionality of the distinct populations observed in indicated time points and culture conditions (Fig. 1b)
Summary
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. Disturbances in the renewal process due to LSC dysfunction or loss manifest as a clinical condition called limbal stem cell deficiency (LSCD) that may in unilateral cases be treated with autologous cultured limbal epithelial transplantation (CLET) [2]. One of the most critical current challenges in the field of LSC therapies, with both primary and hPSC-derived cells, is the identification and maintenance of the clinically relevant LSC population. The tumor protein p63 is currently acknowledged as the classical identifier of colony-forming LSCs with proven clinical relevance and serves as a hallmark of high-quality CLET transplants [6, 7]. Studies in mice have pointed to ABCG2 being a universal marker of stemness in several tissues [14] and describing a slow-cycling subpopulation of colony-forming primary LSCs in humans [15, 16]. ABCG2 has been widely acknowledged as a marker for putative LSCs in vivo [5]
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