ヒト iPS サイボウ カラ チュウハイヨウ ト シンケイ ジョウヒ オ カイシタ モンヨウケイ カンサイボウ ノ ジュリツ ト チリョウ コウカ ノ ケントウ

熊本大

Characterization of PSP- and RA-Pα-derived mesenchymal stem cells.

<p>(A): Imaging of CFU-F formation in the PSP- and RA-Pα-derived MSCs. (B): CFU-F numbers for the PSP- and RA-Pα-derived MSCs. 300 cells were spread in each 6-well dish. Colony number was calculated on day 14 (n = 3, mean ± SD). **P < 0.01. (C): Analysis of marker expressions in PSP- and RA-Pα-derived MSCs by flow cytometer. MSC-related markers: CD49d, CD73, CD90, CD105, CD140α, CD140β, CD271 and STRO-1; hematopoietic markers: CD34 and CD45. (D): Analysis of paracrine factor expressions in PSP- and RA-Pα-derived MSCs by qPCR. TGFB1: tumor growth factor beta 1, HGF: hepatocyte growth factor, BMP2: bone morphogenetic protein 2, VEGF-A: vascular endothelial growth factor A, EGF: epidermal growth factor, bFGF: basic fibroblast growth factor, and PDGFB: platelet-derived growth factor beta. Data are means ± SDs of three independent experiments. *P < 0.05, **P < 0.01, compared between PSP- and RA-Pα-derived MSCs (t test).</p

DNA microarray analysis of PSP-MSCs and RA-Pα-MSCs.

<p>(A-C): Hierarchical clustering of gene sets signatures, pluripotent markers (A), MSC markers (B) and paracrine factors (C), in various MSCs and N1-12 iPSCs. The datasets of all genes investigated were clustered according to Euclidean distance metrics. The labels represent the following cells: N1-12; N1-12 iPS cells, BM-MSC; BM-MSC (PRC-010) purchased from Bay bioscience Co.; N1-12 PSP; PSP-MSC derived from N1-12, 201B7 PSP; PSP-MSC derived from 201B7, N1-12 RA-Pα; RA-Pα-MSC derived from N1-12, 201B7 RA-Pα; RA-Pa-MSC derived from 201B7. (D): Principal component analysis. All datasets were classified into three principal components, PC2 (25.43%), PC3 (15.34%), and PC4 (7.81%), and were simplified into three-dimensional scores. (E, F): Gene ontology (GO) analysis of 286 commonly upregulated data sets for PSP-MSC (E) and 359 data sets for RA-Pα-MSC (F). The top-ten GO terms are listed. GO terms were detected with a cutoff P-value of 0.1. Values are–log10 corrected P-value. Red color indicates different GO terms between (E) and (F).</p

Treatments with PSP-MSC and RA-Pα-MSC on an in vivo skin-injury model of wound healing.

<p>(A, B): Representative photograph (A) and size of wound area (B) during the wound healing from day 0 to day 14 treated with PBS alone (n = 6), PSP-MSC (n = 6), and RA-Pα-MSC (n = 6). All experiments were conducted twice and representative data are shown. Data are means ± SDs. *P < 0.05, **P < 0.01, compared with PBS alone (t test). The divisions of scale are 1 mm (A). (C): Histological analysis of the wound edges in the day-14 mice treated with PSP-MSC and RA-Pα-MSC. Left panels are hematoxylin and eosin staining and right panels represent immunostaining with anti-human HLA antibodies. The arrows indicate the nest of PSP-MSC and RA-Pα-MSC stained with anti-human HLA antibodies (green) and DAPI for nuclei (blue). Scale bar = 40 μm.</p

Differentiation potential of PSP- and RA-Pα-derived MSCs in vitro.

<p>(A-D): Differentiation of PSP- (A and B) and RA-Pα-derived (C and D) MSCs into adipocytes, chondrocytes, and osteocytes. Representative staining of adipocytes (Oil red O staining, left panels in A and C), chondrocytes (alcian blue staining, middle panels in A and C) and osteocytes (Alizarin red staining, right panels in A and C). Scale bar = 40 μm, Relative expression levels of lineage-specific markers by qPCR (B and D); FABP4 and PPARγ for adipocyte, SOX9 and AGGRECAN for chondrocyte, and OPN and RUNX2 for osteocyte. Each experiment was conducted in triplicate (mean ± SD). **P < 0.01, compared with undifferentiated PSP-MSCs (B) or RA-Pα-MSCs (D) (t test). Symbols: PSP; undifferentiated PSP-MSCs, RA-Pα; undifferentiated RA-Pα-MSCs, adipo; day 28 under adipocyte differentiation, chondro; day 28 under chondrocyte differentiation, osteo; day 28 under osteocyte differentiation.</p

Treatments with PSP-MSC and RA-Pα-MSC on an in vivo pressure-induced skin ulcer model of wound healing.

<p>(A): Experimental design to generate the pressure-induced skin ulcer in mice. (B, C): Representative photograph (B) and size of wound area (C) during the wound healing from day 5 to day 20 treated with PBS alone (n = 8), PSP-MSC (n = 8), and RA-Pα-MSC (n = 8). All experiments were conducted twice and representative data are shown. Data are means ± SDs. *P < 0.05, **P < 0.01, compared with PBS alone (t test). The divisions of scale are 1 mm (B). (D): Histological analysis of the wound edges on day 20 from mice treated with PSP-MSC and RA-Pα-MSC. Left panels are hematoxylin and eosin staining and right panels represent immunostaining with anti-human HLA antibodies. The arrows indicate the nest of PSP-MSC and RA-Pα-MSC stained with anti-human HLA antibodies (green) and DAPI for nucleus (blue). Scale bar = 40 μm.</p

Induction of hiPSC-derived MSCs under mesodermal and neuroepithelial differentiation conditions.

<p>(A, B): The proportions of PDGFRα and VEGFR2 expression in differentiated N1-12 cells on day 2, 4, 6 and 8 under the mesodermal differentiation condition. Representative data (A) and graph (B). Number indicates the percentage of each population (A). Experiments were conducted three times (mean ± SD). (C): Quantitative PCR (qPCR) analysis of the relative mRNA levels of NANOG and OCT3/4 as multipotent markers, VIMENTIN and PDGFRβ for mesenchyme, BRACHYURY for mesoderm, and SOX1 for neuroepithelium during the mesodermal (left) and neuroepithelial (right) differentiations. Symbols: d-number indicates day-number of differentiation, PSP; immediately after sorting on day 6 under the mesoderm differentiation, RA-Pα: immediately after sorting on day 10 under the neuroepithelial differentiation, P3 or P6; passage 3 or 6 after sorting. Each value represents the mean fold compared to day-0 undifferentiated iPSCs. Each experiment was conducted three times (mean ± SD). (D): Bright-light image of PSP-MSC and RA-Pα cells on day 21 after sorting. Scale bar: 200 μm. (E): The proportion of PDGFRα and VEGFR2 expressions in day-10 differentiated N1-12 under the neuroepithelial differentiation condition; number indicates the percentage of each population.</p

PARK2-mediated mitophagy is involved in regulation of HBEC senescence in COPD pathogenesis

<div><p>Cigarette smoke (CS)-induced mitochondrial damage with increased reactive oxygen species (ROS) production has been implicated in COPD pathogenesis by accelerating senescence. Mitophagy may play a pivotal role for removal of CS-induced damaged mitochondria, and the PINK1 (PTEN-induced putative kinase 1)-PARK2 pathway has been proposed as a crucial mechanism for mitophagic degradation. Therefore, we sought to investigate to determine if PINK1-PARK2-mediated mitophagy is involved in the regulation of CS extract (CSE)-induced cell senescence and in COPD pathogenesis. Mitochondrial damage, ROS production, and cell senescence were evaluated in primary human bronchial epithelial cells (HBEC). Mitophagy was assessed in BEAS-2B cells stably expressing EGFP-LC3B, using confocal microscopy to measure colocalization between TOMM20-stained mitochondria and EGFP-LC3B dots as a representation of autophagosome formation. To elucidate the involvement of PINK1 and PARK2 in mitophagy, knockdown and overexpression experiments were performed. PINK1 and PARK2 protein levels in lungs from patients were evaluated by means of lung homogenate and immunohistochemistry. We demonstrated that CSE-induced mitochondrial damage was accompanied by increased ROS production and HBEC senescence. CSE-induced mitophagy was inhibited by <i>PINK1</i> and <i>PARK2</i> knockdown, resulting in enhanced mitochondrial ROS production and cellular senescence in HBEC. Evaluation of protein levels demonstrated decreased PARK2 in COPD lungs compared with non-COPD lungs. These results suggest that PINK1-PARK2 pathway-mediated mitophagy plays a key regulatory role in CSE-induced mitochondrial ROS production and cellular senescence in HBEC. Reduced PARK2 expression levels in COPD lung suggest that insufficient mitophagy is a part of the pathogenic sequence of COPD.</p></div