Ips Cells Can Be Efficiently Differentiated Back To Mscs Using A Short Exposure To Tgfβ

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1 Ips Cells Can Be Efficiently Differentiated Back To Mscs Using A Short Exposure To Tgfβ Dmitriy Sheyn, PhD 1, Shiran Ben-David, BSc 1, Sandra Ann De Mel, BSc 1, Loren Ornelas, BSc 1, Anais Sahabian, MSc 1, Dhruv Sareen, PhD 1, Xiaoyu Da, MSc 1, Wafa Tawackoli, PhD 1, Dan Gazit, DMD, PhD 1,2, Zulma Gazit, Ph.D. 1,2. 1 Cedars-Sinai Medical Center, Los Angeles, CA, USA, 2 Hebrew University of Jerusalem, Jerusalem, Israel. Disclosures: D. Sheyn: None. S. Ben-David: None. S.A. De Mel: None. L. Ornelas: None. A. Sahabian: None. D. Sareen: None. X. Da: None. W. Tawackoli: None. D. Gazit: None. Z. Gazit: None. Introduction: Mesenchymal stem cells (MSCs) are to date the ultimate source for skeletal tissue engineering and regeneration of various skeletal conditions. However, their availability and self-renewal ability is limited. In case of osteoporotic patients, MSCs are in decreased numbers and are often dysfunctional. The recent discoveries of induced pluripotent cells (ipsc) reprogrammed from terminally differentiated cells opened new horizons in stem cell therapy and may be implemented to overcome these challenges. We hypothesized that ipscs differentiated to MSC-like cells, using short exposure to TGFb1, will have similarities to human bone marrow-derived MSCs (BM-MSCs) and will form bone in vivo. Methods: Healthy control dermal fibroblasts were reprogrammed with nucleofection of plasmid vectors (Addgene) encoding for episomal plasmid expression of six factors: OCT4, SOX2, KLF4, L-MYC, LIN28, and p53 shrna. IPSCs formed embryoid bodies (EBs) and those were cultured for 7 days, followed by shortterm (2 days) exposure to TGFβ1. During this process (Fig. 1A), two populations were identified: cells that migrated out of the EB in early stages (attached or aimscs) and cells that left after the EB transfer to another plate (transferred or timscs, Fig. 1A). Cell proliferation was assessed by seeding at 5 10^3cells/cm^2 density and grown for 4-6 days, trypsinized, and counted. This process was repeated until p6 and number of doublings per day was calculated. Cell tumorigenic potential was tested using soft agar assay. The transition of the cells towards MSC stage was monitored using flow cytometry of MSC surface markers (CD105, CD44, CD90). Gene expression profile of both aimsc and timsc was studied using RNA sequencing and compared to BM-MSCs. Three pairwise comparisons were done to compare gene expression between the three cell populations. For each comparison significantly upregulated/downregulated genes were defined at a log2(fold change)>2/-2 and a P-value<0.05. The functional annotation clustering analysis was performed using DAVID database. Additionally, gene expression profiles from 120 normal tissues and cell lines associated with different diseases were normalized. The pairwise global correlations among aimscs, timscs, BM-MSCs and others were calculated. To adjust for multiple measures in statistical test, a small P = was used for detecting the real correlations among the cell lines. Differentiation potential of the imscs towards adipogenic and osteogenic lineages was evaluated in vitro. Osteogenic potential in vivo was assessed using nucleofection with BMP6 encoding plasmid, ectopic implantation and µct imaging. Results: The proliferation analysis of both aimscs and timscs showed increased proliferation rate during the first passages, peaking at 1.5 doublings per day at passage 5, compared to BM-MSCs that doubled 0.5/day. However, the proliferation decreased towards later passages and equalised with BM-MCSs to the value of ~0.7 doublings/day (Fig 1B). In order to exclude the presence of pluripotent cells among imscs, soft agar assay was performed to dismiss potential tumorigenesis. BM-MSCs showed the highest

2 ability to survive and proliferate in soft gel environment compared to both imsc cell types (Fig 1C). In order to monitor the transition of the imscs to MSC-like phenotype, surface MSC markers were tested on passage 4 of the imscs and compared to BM-MSCs and original ips cells. All cell types expressed CD44; CD90 was expressed by all but timscs and CD105 was expressed by BM-MSC and aimscs, but not by ipscs and timscs. Longitudinal analysis from p1 to p6 comparing aimsc and timscs to BM-MSCs (Fig 1D) showed that after few passages imscs express CD44 and CD90 similarly to BM-MSCs, but only aimscs express the CD105 marker early after reprogramming. When the gene expression profiles of aimscs and timscs were compared to BM-MSCs, the correlations between the three were very high (R>0.92, Fig. 2B). The strongest correlation was between aimscs and timscs. The correlation between aimscs and BM-MSCS was slightly stronger than timscs and BM-MSCs. All three cell types had no or very weak correlation (R<0.4) with the 112 cell lines tested, including cancer cell lines and primary tissues. The closest correlation was found to bone and connective tissue s transcriptomes. The GO analysis showed that aimscs upregulated MSC related genes whereas timscs upregulated inflammatory-related genes comparing to BM-MSCs (Fig. 2C). Both types of imscs differentiated in vitro under osteogenic or adipogenic conditions and the results showed that both cell types retain their multidifferentiation potential (Fig. 3A-B), although aimscs showed higher differentiation in vitro than timscs and BM-MSCs. All three cell types over-expressing BMP6 after nucleofection were injected ectopically in mice, and a significant bone formation by aimscs was detected after 2 and 4 weeks, and higher than BM-MSC-formed bone on week 4. timsc resulted in no or very little bone formation, (Fig. 3C). Discussion: We found that the MSCs can be derived from ips cells, with high proliferative and no tumorigenic capability. They lose their pluripotent markers acquiring MSC markers by 4-6 weeks post differentiation. When compared to BM-MSCs, aimscs present higher resemblance than timsc in markers expression, transcriptomic profile, differentiation potential in vitro and bone tissue formation. Significance: These results of these study will pave the way to novel treatments for as of today untreatable disorders like senile osteoporosis and will provide new source of MSCs that can be rejuvenated from any type of cells.

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5 ORS 2015 Annual Meeting Poster No: 0580