Differentiation of human embryonic stem cells into corneal epithelial progenitor cells under defined conditions

Abstract

The development of cell-based therapies using stem cells represents a significant breakthrough in the treatment of limbal stem cell deficiency (LSCD). The aim of this study was to develop a novel protocol to differentiate human embryonic stem cells (hESCs) into corneal epithelial progenitor cells (CEPCs), with similar features to primary cultured human limbal stem cells (LSCs), using a medium composed of DMEM/F12 and defined keratinocyte serum-free medium (KSFM) (1:1) under different carbon dioxide (CO2) levels in culture. The differentiated cells exhibited a similar morphology to limbal stem cells under 5%, 7%, and 9% CO2 and expressed the LSC markers ABCG-2 and p63; however, CK14 was only expressed in the cells cultured under 7% and 9% CO2. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis indicated that the ABCG2, p63, and CK14 levels in the 7% CO2 and 9% CO2 groups were higher than those in the 5% CO2 group and in undifferentiated hESCs (p<0.05). The highest expression of ABCG2 and p63 was exhibited in the cells cultured under 7% CO2 at day 6 of differentiation. Western blotting indicated that the ABCG2 and p63 levels were higher at day 6 than the other time points in the 7% CO2 and 9% CO2 groups. The highest protein expression of ABCG2 and p63 was identified in the 7% CO2 group. The neural cell-specific marker tubulin β3 and the epidermal marker K1/10 were also detected in the differentiated cells via immunofluorescent staining; thus, cell sorting was performed via fluorescence-activated cell sorting (FACS), and ABCG2-positive cells were isolated as CEPCs. The sorted cells formed three to four layers of epithelioid cells by airlifting culture and expressed ABCG2, p63, CK14, and CK3. In conclusion, the novel induction system conditioned by 7% CO2 in this study may be an effective and feasible method for CEPC differentiation.

Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method

“…Most previously published studies to generate corneal epithelial cells from hPSCs have relied on the use of undefined, xenogeneic factors such as conditioned medium, PA6 feeder cells, Bowman’s, or amniotic membranes [ 16 , 19 , 47 , 48 ]. A recent, defined protocol to differentiate a single hESC line to corneal epithelial progenitor cells utilized simple keratinocyte serum-free medium (KSFM) mixed with DMEM/F12 for increased calcium concentration, together with a simple col IV matrix [ 30 ]. ABCG2- and p63-positive populations were produced in just 6 days but differentiation required elevated CO 2 levels.…”

“…ABCG2- and p63-positive populations were produced in just 6 days but differentiation required elevated CO 2 levels. The mechanism behind these differentiation conditions was not discussed [ 30 ].…”

“…Recently, strict regulatory demands and efforts toward safer cell therapy products have prompted the search for more standardized and xenogeneic-free culture and differentiation protocols. In response, some xeno-free protocols for RPE differentiation [ 24 – 28 ], retinal organoid culture [ 29 ], and corneal epithelial progenitor cell differentiation [ 30 ] have been introduced within the past 2 years. However, they tend to be complex and difficult to standardize across cell lines and laboratories.…”

Universal Corneal Epithelial-Like Cells Derived from Human Embryonic Stem Cells for Cellularization of a Corneal Scaffold

“… 28 In addition, CEC derived from hESC in DMEM/F12 mixed at 1:1 with keratinocyte serum-free medium under 7% O 2 are seeded on decellularized porcine limbal matrix followed by air-lift culture to induce epithelial stratification. 29 Although human DC is a better choice compared to the animal counterparts, as they possess human corneal properties and will not cause xenogeneic immune response, 30 the limited availability of the source still imposes a major hurdle to their use.…”

Corneal cell therapy: with iPSCs, it is no more a far-sight

“…In case of differentiation of the iPSCs towards CE fate, collagen IV [ 47 ] has been demonstrated to be an ideal substrate. Interestingly, Zhang et al demonstrated that slightly elevated CO 2 was conductive to the differentiation of CE progenitors from hESCs [ 49 ]. However, the underlying mechanism remained unclear which points to the need for further standardization of the crucial parameters such as the compatibility of the substrate, nature of the pluripotent cells, and the media used for culturing the cells apart from the oxygen modulation.…”