Corneal epithelial differentiation of human pluripotent stem cells generates ABCB5+ and ∆Np63α+ cells with limbal cell characteristics and high wound healing capacity

Abstract

Background Differentiation of functional limbal stem cells (LSCs) from human pluripotent stem cells (hPSCs) is an important objective which can provide novel treatment solutions for patients suffering from limbal stem cell deficiency (LSCD). Yet, further characterization is needed to better evaluate their immunogenicity and regenerative potential before clinical applications. Methods Human PSCs were differentiated towards corneal fate and cryopreserved using a clinically applicable protocol. Resulting hPSC-LSC populations were examined at days 10–11 and 24–25 during differentiation as well as at passage 1 post-thaw. Expression of cornea-associated markers including PAX6, ABCG2, ∆Np63α, CK15, CK14, CK12 and ABCB5 as well as human leukocyte antigens (HLAs) was analyzed using immunofluorescence and flow cytometry. Wound healing properties of the post-thaw hPSC-LSCs were assessed via calcium imaging and scratch assay. Human and porcine tissue-derived cultured LSCs were used as controls for marker expression analysis and scratch assays at passage 1. Results The day 24–25 and post-thaw hPSC-LSCs displayed a similar marker profile with the tissue-derived LSCs, showing abundant expression of PAX6, ∆Np63α, CK15, CK14 and ABCB5 and low expression of ABCG2. In contrast, day 10–11 hPSC-LSCs had lower expression of ABCB5 and ∆Np63α, but high expression of ABCG2. A small portion of the day 10–11 cells coexpressed ABCG2 and ABCB5. The expression of class I HLAs increased during hPSC-LSCs differentiation and was uniform in post-thaw hPSC-LSCs, however the intensity was lower in comparison to tissue-derived LSCs. The calcium imaging revealed that the post-thaw hPSC-LSCs generated a robust response towards epithelial wound healing signaling mediator ATP. Further, scratch assay revealed that post-thaw hPSC-LSCs had higher wound healing capacity in comparison to tissue-derived LSCs. Conclusions Clinically relevant LSC-like cells can be efficiently differentiated from hPSCs. The post-thaw hPSC-LSCs possess functional potency in calcium responses towards injury associated signals and in wound closure. The developmental trajectory observed during hPSC-LSC differentiation, giving rise to ABCG2+ population and further to ABCB5+ and ∆Np63α+ cells with limbal characteristics, indicates hPSC-derived cells can be utilized as a valuable cell source for the treatment of patients afflicted corneal blindness due to LSCD. Graphical Abstract

Transcriptomic Landscape and Functional Characterization of Human Induced Pluripotent Stem Cell-Derived Limbal Epithelial Progenitor Cells

“…Our data show that hiPSC-derived CD200 − ITGB4 + SSEA4 + cells have similar wound healing capacity, melanosome uptake, the ability to form colonies and expression of LEPC markers (CK15, CK19, P-cadherin, CK14, and p63) as T-LEPC [ 14 , 42 , 46 ]. Our data strongly suggest that hiPSC-LEPC are more similar to progenitor and transient amplifying cells than terminally differentiated corneal epithelium and their potential to regenerate the corneal epithelium is similar to the capacity observed in vivo [ 46 , 49 , 50 , 51 ].…”

Toward Corneal Limbus In Vitro Model: Regulation of hPSC‐LSC Phenotype by Matrix Stiffness and Topography During Cell Differentiation Process

“…Potentially, this cell type could have high regenerative capacity in corneal diseases with stiffened corneal matrix and in wound healing conditions. Indeed, our group has previously demonstrated excellent wound healing capacity of d24 hPSC-LSCs on plastic, [9] which supports the statement that the increased wound healing potential of d24 hPSC-LSCs is dependent of a stiff matrix.…”

“…In our previous studies, we have demonstrated the capability to produce high‐quality hPSC‐LSCs in xeno‐free conditions. [ 9,10 ]…”

“…In our previous studies, we have demonstrated the capability to produce high-quality hPSC-LSCs in xeno-free conditions. [9,10] However, currently used standard two dimensional (2D) in vitro culture substrates with unphysiological stiffness are not sufficient to mimic native limbal environment and pass over the important regulatory effect of the physical niche. Recent studies have highlighted the importance of the surrounding mechanical environment in LSC regulation, mediated by the YES-associated protein (YAP) signaling both in human and murine corneas.…”

Multi‐parametric surface plasmon resonance‐based intake quantification of label‐free light‐activated nanoparticles by therapeutic limbal stem cells for corneal blindness

“…Given the high level of interest in NP based carriers, it was surprising to realize that there are, to our knowledge, no reports describing methods for studying the real-time intake of label-free NP by LSCs and especially with LSCs differentiated from human pluripotent stem cells (hPSC-LSCs) providing novel and highly scalable source of LSCs for regenerative applications. [16][17][18]…”