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

<div><p>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.</p></div

A Novel Peptide from Polypedates megacephalus Promotes Wound Healing in Mice

Amphibian skin contains wound-healing peptides, antimicrobial peptides, and insulin-releasing peptides, which give their skin a strong regeneration ability to adapt to a complex and harsh living environment. In the current research, a novel wound-healing promoting peptide, PM-7, was identified from the skin secretions of Polypedates megacephalus, which has an amino acid sequence of FLNWRRILFLKVVR and shares no structural similarity with any peptides described before. It displays the activity of promoting wound healing in mice. Moreover, PM-7 exhibits the function of enhancing proliferation and migration in HUVEC and HSF cells by affecting the MAPK signaling pathway. Considering its favorable traits as a novel peptide that significantly promotes wound healing, PM-7 can be a potential candidate in the development of novel wound-repairing drugs

Protein expression of CEPC and hESC markers at different time points and in different culture conditions.

<p>(A-B) ABCG2, p63, CK14, CK3, and OCT4 protein expression in the 7% CO₂ (A) and 9% CO₂ (B) groups at different time points (3, 6, and 9 days). (C) Protein level of ABCG2, p63, CK14, CK3, and OCT4 at 5%, 7%, and 9% CO₂ at day 6 of differentiation. The undifferentiated hESCs were used as the control group.</p

Time course of ABCG-2, p63, CK14, and CK3 during hESC differentiation culture (3, 6, 9, and 12 days).

<p>Data are representative of three experiments and presented as the mean ± SD.*p < 0.05, **p < 0.01.</p

Immunofluorescence images of the differentiated cells.

<p>Expression of ABCG2, p63, and CK14 at day 6 and CK3 at day 12 of differentiation in the cells cultured under 5% CO₂ (A), 7% CO₂ (B), and 9% CO₂ (C). Scale bar, 20 μm.</p

Number of cells counted using a hemocytometer.

<p>Data represent mean ± SD of three independent experiments with triplicate dishes.</p

Immunostaining of non-corneal epithelial differentiation markers and isolation of CEPCs derived from hESCs by FACS.

<p>(A) Immunofluorescent staining of tubulin β3, CK1/10, and MITF. (B) FACS analysis of ABCG2-positive cells in different culture conditions at day 6 of differentiation. (C) Immunofluorescent staining of the sorted cells. ABCG2, p63, and CK14 were strongly expressed in the sorted cells. CK3, tubulin β3, and CK1/10 were not detected. Scale bar, 20 μm.</p

Primary cultured hLSCs and morphology of cells from hESCs at different stages of differentiation.

<p>(A) Microscopic image of hESCs and the expression of OCT4 and SSEA-3. (B) Morphology of hLSCs and immunofluorescent staining of ABCG2, p63, and CK14. (C) Representative images of the differentiated cells at days 3, 6, 9, and 12 of differentiation. Scale bar, 20 μm.</p

Characterization of epitheloid cell sheet on APLM.

<p>(A) Epitheloid cell sheets under airlifting culture. (B) HE staining of epitheloid cell sheet. (C-G) Immunofluorescent staining of ABCG2 (C), p63 (D), CK14 (E), CK3 (F), tubulin β3 (G), and CK1/10 (H) in the cells cultured on APLM. Scale bar, 20 μm. (I-J) Gene (I) and protein (J) expression of ABCG2, p63, CK14, and CK3 in epitheloid cell sheets. hESCs seeded on APLM were used as the control. Data are representative of three experiments and presented as the mean ± SD.*p < 0.05, **p < 0.01.</p