[Experimental study on the differentiation of human induced pluripotent stem cells into epidermal-like stem cells].

OBJECTIVE: To investigate the feasibility of differentiation of human induced pluripotent stem cells (iPSCs) into epidermal-like stem cells. METHODS: (1) Human strain of iPSCs were plated on-to trophoblast of inactivated Fb strain of mouse embryos and cultured in complete medium of embryonic stem cells, iPSCs were subcultured by collagenase IV digestion method. The morphology and growth of iPSCs were observed under inverted phase contrast microscope, and the cells were stained with alkaline phosphatase (AKP). iPSCs were cultured in incomplete medium of embryonic stem cells to observe the ability of embryoid body formation. (2) Human iPSCs were inoculated onto 6-well plate covered with human amniotic membrane to culture as induction group. Other iPSCs were cultured on 6-well plate without human amniotic membrane as control group. Morphological changes in iPSCs in two groups were observed. Expressions of integrin beta1 and CK19 of iPSCs in two groups were determined by immunocytochemical staining. RESULTS: Human iPSCs showed a typical stem cell clone-like growth with a clear boundary, and they proliferated vigorously in complete medium of embryonic stem cells. These cells were AKP-positive. iPSCs formed embryoid body in trophoblast-free and suspension culture conditions. After 4 days of co-culture, stem cell clones were formed on the surface of amniotic membrane in induction group, and part of the cells were integrin beta1 and CK19 positive. Most of the cells died, and no integrin beta1 and CK19 positive cells were found in control group. CONCLUSIONS: Human iPSCs can be differentiated into epidermal-like stem cells by amniotic membrane induction, and it lays an experimental basis for providing new source of seed cells of skin tissue engineering.

[The frequency and function of FoxP3+ regulatory T cell in patients with acute hepatitis B].

AIM: To investigate the dynamic variety of frequency and function of FoxP3+ regulatory T cells in patients with acute hepatitis B (AHB). METHODS: Peripheral blood mononuclear cells (PBMCs) from 15 AHB patients at acute phase (week 1 of illness), convalescent phase (primary occurrence of both ALT level normalization and HBsAg negative conversion), resolved phase (at least 8 weeks after both ALT normalization and HBsAg seroconversion, and 15 health subjects were analyzed for FoxP3 (Forkhead/winged helix transcription factor) mRNA expression in MACS magnetic beads-purified CD4+ T cells by real-time RT-PCR assay. The effects of Treg cells on the proliferation of CD4+ CD25- T cells were examined by a 3H-thymidine incorporation assay. RESULTS: AHB patients presented a significantly higher FoxP3 mRNA expression at convalescent phase than acute phase (t= -6.04, P<0.01) and resolved phase (t=4.45, P<0.01), and healthy controls (t=3.44, P<0.01). We also observed that the suppression efficiency of Treg cells on proliferation of CD4+ CD25- T cells was lower at acute phase than convalescent phase (t= -5.30, P<0.01) and resolved phase (t= -3.20, P<0.05), but there was no significant difference between healthy controls and any phase of AHB. CONCLUSION: AHB patients presented lower circulating Treg frequency and suppression function at acute phase, and both of them are increase at convalescent phase, and then return to normal level along with disease resolved. This follow-up study furthers our understanding of Treg's role in immunopathogenesis of hepatitis B.