Fig 1.
Mouse keratinocyte culture in different conditions.
Bright-field microscopic images of (A) cells grown for 3 or 10 days in different media, and (B) colonies formed when cells were grown in KSFM-Ca2+ medium for an extended period of time. The relative expression of (C) the basal keratinocyte gene Krt14 and (E) epithelial marker genes, i.e. Krt8, Krt15 and Krt18, in cultured keratinocytes and fibroblasts compared to those in mESCs. (D) Cells were subjected to immunofluorescence staining with anti-KRT14; photographs were taken under fluorescent microscope. Results represent data of at least three duplicate experiments +/-SD. The scale bars in microscope image correspond to 100 μm.
Fig 2.
The growth properties of long-term cultured keratinocytes derived in KSFM-Ca2+ medium.
The average doubling time of representative cell lines at (A) the early passages, and (B) at different passages was calculated based on the cell numbers plated, harvested and days in culture. (C) Comparison of the average doubling time +/-SD of cells at <10, 11–20, 21–30 and >30 passages. Cells of medium and high passages grew significantly faster than those of low passages. The primary murine keratinocytes grown in DK-SFM (p0) and the KSFM-Ca2+ cells at different passages were examined for (D) p16INK4a mRNA, (E) The relative telomere length, in comparison to levels in mESCs, set as 1, and (F) chromosome numbers and results represent data from 12–15 metaphase cells examined.
Fig 3.
Formation of cell-cell junctions depends on extracellular calcium.
(A) Primary keratinocytes grown in DKSFM and cell lines grown in KSFM-Ca2+ with or without switching to DKSFM and KSFM+Ca2+ media for 48 h were examined (A) by immunofluorescence staining using anti-E-Cad and anti-ZO-1, and (B) by qPCR for mRNA of E-Cadherin (Cdh1) compared to the level in mESCs as 1. Data represent at least triplet samples +/-SD. Scale bar represents 20 μm.
Fig 4.
Characterization of epithelial cells adapted in DKSFM media.
Cells adapted to grow in DKSFM were (A) examined with microscope and bright-field images captured, (B) examined for growth rate through the calculation of doubling time at different passages, (C) subjected to immunofluorescence staining using antibodies for E-Cad and ZO-1, and images were captured with Zeiss fluorescent microscope, and (D) measured for E-Cad (Cdh1) expression by qPCR and expression was compared to that in mESCs as 1. Results represent duplicate data of at least 3 biological samples.
Fig 5.
Global gene expression and RNA-seq analysis.
(A) Of the significant genes detected in the KSFM-Ca2+ and DKSFM cells, 16% were significantly up-regulated in the KSFM-Ca2+ cells whereas 20% were significantly up-regulated in the DKSFM cells. Enrichment analysis reveals the top functions up-regulated in (B) KSFM-Ca2+ cell and (C) DKSFM cells.
Fig 6.
Differential functional pathways in KSFM-Ca2+ and DKSFM cells.
Differential gene expression in KSFM-Ca2+ and DKSFM cells were validated with qPCR, on (A) epithelial differentiation markers, including Krt1, Krt8, Krt10, Krt14 and Lor, (B) inflammatory genes Ccl2 and Pai-1, and (D) Pcna and Ki67 implicated in proliferation. The expression at protein levels of (C) PAI-1 and pIκBα was examined by Western blotting and β-Actin was used as a loading control. The expression of PCNA, Ki67 and pH3 (E) was examined by immunofluorescent staining and (F) the percentage of staining positive cells were quantified based on biological triplicates. Arrowheads pointed at typical staining positive cells. (G) The doubling time of the KSFM-Ca2+ cells and the DKSFM cells was calculated and results represent the average of >20 data sets. Statistical analyses were performed with Student’s t-test.
Fig 7.
Stem and differentiation characteristics of the KSFM-Ca2+ and DKSFM cells.
(A) RNA-seq revealed common and unique stemness gene expression signatures of KSFM-Ca2+ and DKSFM cells. (B) The expression of selective stem genes, i.e. Cd34, Sca-1, Krt 15, Krt18, were validated by qPCR. (C) Photographs of colonies formed with KSFM-Ca2+ and DKSFM cells and the numbers of colonies were counted and CFE calculated. (D) Organoids in 3D BME culture in different culture media were photographed under microscopy in brightfields. Arrows point at the representative organoids. The DKSFM cell-derived organoids grown in DKSFM media plus 1 mM Ca2+ were (E) subjected to histological analyses with H&E staining and immunostaining using antibodies indicated, and (F) examined by qPCR for expression of keratinocyte terminal differentiation genes, i.e. Krt1, Krt10, Ivl, and Lor. The scale bars in microscope images correspond to 100 μm. Statistical analyses were performed with Student’s t-test using data derived from at least triplicate biological samples.