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, 13 (7), e0200790
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Mesenchymal Stem Cells Derived From Human iPS Cells via Mesoderm and Neuroepithelium Have Different Features and Therapeutic Potentials

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Mesenchymal Stem Cells Derived From Human iPS Cells via Mesoderm and Neuroepithelium Have Different Features and Therapeutic Potentials

Shinya Eto et al. PLoS One.

Abstract

Mesenchymal stem cells (MSCs) isolated from adult human tissues are capable of proliferating in vitro and maintaining their multipotency, making them attractive cell sources for regenerative medicine. However, the availability and capability of self-renewal under current preparation regimes are limited. Induced pluripotent stem cells (iPSCs) now offer an alternative, similar cell source to MSCs. Herein, we established new methods for differentiating hiPSCs into MSCs via mesoderm-like and neuroepithelium-like cells. Both derived MSC populations exhibited self-renewal and multipotency, as well as therapeutic potential in mouse models of skin wounds, pressure ulcers, and osteoarthritis. Interestingly, the therapeutic effects differ between the two types of MSCs in the disease models, suggesting that the therapeutic effect depends on the cell origin. Our results provide valuable basic insights for the clinical application of such cells.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Induction of hiPSC-derived MSCs under mesodermal and neuroepithelial differentiation conditions.
(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.
Fig 2
Fig 2. Characterization of PSP- and RA-Pα-derived mesenchymal stem cells.
(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).
Fig 3
Fig 3. Differentiation potential of PSP- and RA-Pα-derived MSCs in vitro.
(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.
Fig 4
Fig 4. DNA microarray analysis of PSP-MSCs and RA-Pα-MSCs.
(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).
Fig 5
Fig 5. Treatments with PSP-MSC and RA-Pα-MSC on an in vivo skin-injury model of wound healing.
(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.
Fig 6
Fig 6. Treatments with PSP-MSC and RA-Pα-MSC on an in vivo pressure-induced skin ulcer model of wound healing.
(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.
Fig 7
Fig 7. Suppressing degeneration of knee cartilage in the OA model mice treated with PSP-MSC and RA-Pα-MSC.
(A): Experimental design to treat OA model mice with PSP-MSC and RA-Pα-MSC. On day 0, the anterior cruciate ligament (ACL) from each animal was transected, and then the medial meniscus was removed from the lower-limb joints. Two weeks later, PSP-MSC and RA-Pα-MSC were injected into the knee joint. On day 28, the treated knees were analyzed. (B): Histological analysis of knees in the OA model mice treated with HA alone, PSP-MSC, and RA-Pα-MSC on day 28 in sections stained with Safranin O. Scale bar = 20 μm. (C): Effect was evaluated by Modified Mankin scores in the OA model mice treated with HA (n = 9), PSP-MSC (n = 9), and RA-Pα-MSC (n = 9). Modified Mankin scores were measured by dyeability of the paracellular and intercellular regions, and the alignment of chondrocytes. The score is from 0 to 8 point. Data are means ± SDs. *P < 0.05, **P < 0.01, compared with HA alone (t test).

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Grant support

This study was supported, in part, by grants from Japan Society for the Promotion Science (JSPS, URLs: http://www.jsps.go.jp/english/) program on Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation (Grant Number S2803), the Ministry of Health, Labor, and Welfare of Japan (URLs: http://www.mhlw.go.jp/english/), the Japan Agency for Medical Research and Development (A-MED, URLs: https://www.amed.go.jp/en/), Core Research for Evolutional Science and Technology (CREST, URLs: https://www.jst.go.jp/kisoken/crest/en/), Grant-in-Aid for Scientific Research (KAKENHI C, 25505002 and 18K06264, URLs: https://www.jsps.go.jp/english/e-grants/) and the Japan Science and Technology Agency (JST, URLs: https://www.jst.go.jp/EN/). T.E. received all of above grants. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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