Collagen complexes increase the efficiency of iPS cells generated using fibroblasts from adult mice

Abstract

Different interventions are being tested for restoration of the youthfulness of adult mouse-derived fibroblasts. However, fundamental issues, such as the decline of adult mouse-derived fibroblast activity with age, remain unresolved. Therefore, in this study, we examined whether treatment with collagen complexes has beneficial effects on the rejuvenation or reprogramming of adult mouse-derived fibroblasts. Further, we investigated the mechanisms of rejuvenation of adult mouse-derived fibroblasts during treatment with total collagen complexes. We isolated total collagen complexes from the tails of young mice and cultured adult mouse-derived fibroblasts with or without the collagen complexes. When compared with fibroblasts cultured without collagen complexes, adult-derived fibroblasts cultured with collagen complexes over five consecutive passages showed a more youthful state, expanded at a higher rate, and exhibited reduced spontaneous cell death. The fibroblasts cultured in the presence of collagen complexes also showed extensive demethylation in the promoter regions of cell cycle-related genes such as PCNA, increased proliferation, and decreased senescence. In addition, the efficiency of reprogramming of fibroblasts to become induced pluripotent stem (iPS) cells was significantly higher in young- and adult-derived fibroblasts cultured with collagen complexes than in adult-derived fibroblasts cultured alone. Furthermore, mechanistic evidence shows that genes involved in anti-proliferative pathways, including Ink4a/Arf locus genes and p53, were downregulated in fibroblasts exposed to collagen complexes. Interestingly, our results suggest that the rejuvenation process was mediated via the α2β1 integrin-dependent Bmi-1 pathway. Thus, collagen complexes both stimulate proliferation and inhibit cell death and growth arrest in fibroblasts, which appears to be a promising approach for improving the efficiency of reprogramming.

Fig. 1.

Construction of the retroviral vector for generation of transgenic mice. (A) The retrovirus vector was designed to express the four stemness factors by tetracycline-mediated induction. For further details, see the Methods section. (B) Confirmation of transgenic mice using four-factor PCR. Red dotted lines indicate transgenic mice. (C) Western blotting analysis of the four stemness factors with tetracycline-mediated induction in control and transgenic mouse-derived fibroblasts. Fibroblasts were isolated and cultured by adding doxycycline for 10 days and then subjected to Western blot analysis using each antibody.

Fig. 2.

Effects on both of senescence and apoptosis of collagen complexes. A) Senescence-associated β-galactosidase staining (blue) in Y-, Ac-, and A-fibroblasts. Magnification, × 200. B) Cell proliferation assay. Data are means ± SEM of three experiments performed in triplicate with p values < 0.05. C) Senescence-associated β-galactosidase staining in A-fibroblasts according to collagen types. D) Determination of apoptotic Y-, Ac-, and A- fibroblasts by FACS analysis. (down) Data were obtained from three independent experiments (means ± SD; lower panel; P < 0.05). Groups marked with different letters (a–c) are significantly different from each other at P < 0.05.

Fig. 3.

α2β1 integrin-dependent Bmi-1 regulation analysis in adult fibroblasts cultured with collagen complexes. (A) Western blot analysis in Y-, Ac-, and A-fibroblasts. (B) Western blot analysis of cell cycle regulatory proteins in Y-, Ac-, and A-fibroblasts. (C) RT-qPCR analysis of expression of Ink4/Arf tumor suppressor locus genes in Y-, Ac-, and A-fibroblasts. RT-qPCR was normalized to GAPDH levels. Groups marked with different letters (a–c) are significantly different from each other at P < 0.05. (D) Schema for the rejuvenation in aged fibroblasts on dishes coated with collagen complexes.

Fig. 4.

Real-time RT-qPCR and flow cytometry analysis of AKT/mTOR-related gene expression. A) Akt, Src, Map2K1, and mROR mRNA expression in Y-, Ac-, and A-derived fibroblasts. Gene expression was normalized using a GAPDH housekeeping gene. B) Comparison of p-Akt and p-mTOR expression using flow cytometry in Y-, Ac-, and A-derived fibroblasts.

Fig. 5.

Generation of iPS cells. A) Immunostaining analysis using pluripotency markers such as c-myc, Nanog, Oct3/4, Sox2, and SSEA-1. Nuclei were stained with DAPI. B) Pluripotency marker expression. Primers used for Oct4, Sox2, Klf4, c-Myc, Nanog, Utf1, Rex1, Fbx15, Dax, e-Ras, and Cripto specifically detected the mRNA transcripts.

Fig. 6.

Differentiation of iPS cells in vitro and in vivo. A) RT-PCR analysis of differentiation markers for the three germ layers (endoderm, GATA4 and α-fetoprotein; mesoderm, brachyury and GATA2; and ectoderm, NESTIN and βIII-tubulin). Of note, a faint signal of BMP4 RT-PCR product in distilled water may be caused by flowing during application of electrophoresis to an RT-PCR product derived from A-derived iPS cells. Y, Ac, A and DW indicate Y-, Ac-, A-derived iPS cells, and distilled water, respectively. B) Immunocytochemical analysis of representative iPS cell lines during in vitro differentiation using three germ layer markers (ectoderm, tubulin; mesoderm, brachyury; endoderm, HNF3b). C) Teratoma formation analysis. Histological sections were examined with H&E staining and immunohistochemistry. Endodermal derivatives, epithelia, expressed a high level of beta-catenin. Mesodermal derivatives were exemplified by striated muscle and smooth muscle (middle). Tissues of ectodermal lineage, including the neural epithelium, expressed glial fibrillary acidic protein (GFAP).