Participation of Adult Mouse Bone Marrow Cells in Reconstitution of Skin

Results of recent studies have indicated that bone marrow cells can differentiate into various cells of ectodermal, mesodermal, and endodermal origins when transplanted into the body. However, the problems associated with those experiments such as the long latent period, rareness of the event, and difficulty in controlling the processes have hampered detailed mechanistic studies. In the present study, we examined the potency of mouse bone marrow cells to differentiate into cells comprising skin tissues using a skin reconstitution assay. Bone marrow cells from adult green fluorescent protein (GFP)-transgenic mice were transplanted in a mixture of embryonic mouse skin cells (17.5 days post-coitus) onto skin defects made on the backs of nude mice. Within 3 weeks, fully differentiated skin with hair was reconstituted. GFP-positive cells were found in the epidermis, hair follicles, sebaceous glands, and dermis. The localization and morphology of the cells, results of immunohistochemistry, and results of specific staining confirmed that the bone marrow cells had differentiated into epidermal keratinocytes, sebaceous gland cells, follicular epithelial cells, dendritic cells, and endothelial cells under the present conditions. These results indicate that this system is suitable for molecular and cellular mechanistic studies on differentiation of stem cells to various epidermal and dermal cells

Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium.

Induced pluripotent stem (iPS) cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF)-derived iPS cells (253G1) and human adult corneal limbal epithelial cells (HLEC)-derived iPS cells (L1B41). We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA) differentiation method, as Pax6(+)/K12(+) corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later) in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells

Gene expression analysis for corneal epithelium-related markers in iPS cells at various time points during differentiation by the SDIA method.

<p>While L1B41 began expressing significant levels of K12 after 6 weeks, other iPS cells began exhibiting K12 expression at approximately 10 weeks or later, and K12 expression in L1B41 was significantly higher than 253G1 at 6–12 weeks. Significant K3 expression levels were detected in L1B41 cells after 8 weeks in SDIA culture. Pax6 and delta-N p63 began to be expressed at 2 weeks and kept a similar expression pattern for several weeks in all iPS cells. K14 was highly expressed in both L1B41 and L1C51. The graph shows the mean ± S.E. of 3–7 independent samples. *p<0.05 (L1B41 vs. 253G1, Mann-Whitney rank-sum test), N.S. = not significant.</p

iPS cells were established from human corneal limbal epithelial cells (HLECs).

<p>(A) Three iPS-like cell colonies (L1B41, L1C51, and L1B34) were cloned after reprogramming of HLEC using Yamanaka 4 factors. (B) Immunofluorescent analysis showed that all 3 isolated iPS cell lines expressed the pluripotent stem-cell markers Nanog, Oct3/4, Sox2, and SSEA4. (C) Karyotype analysis showed no obvious aberration in the 3 iPS cell clones derived from HLEC. (D) Global expression analysis among iPS cells by microarray showed that the 3 HLEC-derived iPS cells exhibited similar expression to HDF-derived iPS cells. (E) HLEC-derived L1B41 was able to form a teratoma that contained the tissue originating from the 3 germ layers in the testis of SCID mice. Scale bar: (A) 200 µm, (B) 100 µm.</p