Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Fibromodulin Reprogrammed Cells-based Bone Regeneration
Zhong Zheng, PhD1, Tara Aghaloo, DDS, MD, PhD1, Chen-shuang Li, DDS1, Pu Yang, DDS, MD, PhD1, Soonchul Lee, MD, PhD2, Jin-Hee Kwak, DDS, MS1, Ali Zarringhalam, BS1, Maxwell Murphy, MS1, Kang Ting, DMD, DMedSci1, Chia Soo, MD1.
1UCLA, Los Angeles, CA, USA, 2CHA Univeristy, Gyeongghi-do, Korea, Republic of.

PURPOSE: Bone injuries comprise 25-50% of all musculoskeletal pathologies. Unfortunately, de novo bone formation is inadequate for critical-sized defects due to the lack of sufficient endogenous progenitor cells. Additionally, current cell-based restorative strategies for tissue generation are hindered by numerous obstacles such as inadequate cell availability, painful and invasive cell-harvesting procedures, and tumorigenesis. We successfully reprogrammed human dermal fibroblasts to a multipotent state with elevated pluripotency marker expression and multiple lineage differentiation potentials without tumorigenicity. In this study, we further described FReP cell-based bone regeneration in detail.
METHODS: Human newborn foreskin BJ fibroblasts were reprogrammed by fibromodulin (FMOD) under serum-free conditions. Robust FMOD reprogrammed (FReP) cell osteogenic differentiation was quantified by gene profile targeting pluripotent, mesenchymal, and osteogenic genes. Immunostaining against bone specific markers and mineralization further verified the phenotypes of osteogenic differentiation. Furthermore, we implanted FReP cells that had undergone a short-term (3-days) in vitro osteogenic differentiation into critical-sized SCID mouse calvarial defects to evaluate in vivo bone regeneration of FReP cells.
RESULTS: After a 4-week cultivation in osteogenic medium in vitro, Alizarin Red staining and von Kossa staining demonstrated the mineralization of FReP cells, which was also confirmed by immunostaining against the major osteogenic markers ALP, BSPII, and OCN. Moreover, gene profile revealed three stages of insight into FReP cell osteogenic differentiation in vitro: In stage 1 (week 1), the elevated expression of essential transcription factors required for pluripotent state maintenance and pluripotent markers were dramatically reduced, while expression of mesodermal differentiation marker BMP4 was markedly elevated in FReP cells. Meanwhile, FReP cells with elevated autocrine FGF2 and IGF2 expression were driven to the osteochondroprogenitor lineage. In stage 2 (week 2-3), pro-osteogenic growth factors BMP2, BMP4, FGF2, and IGF2 kept increasing in FReP cells. Thus, FReP cells differentiated into pre-osteoblasts, expressing RUNX2, OSX, OPN, and ALP, but not SOX9 (which indicates chondrogenesis) or TWIST1 (which inhibits terminal osteogenic differentiation). In stage 3 (week 4), FReP cell-derived pre-osteoblasts further differentiated into osteoblasts, with high levels of RUNX2, OCN, and IGF1 (another stimulator for terminal osteogenesis). As proof of concept in a clinically relevant model, after 3-day in vitro osteogenic initiation, FReP cells resulted in significantly more bone formation than BJ fibroblasts did in critical-sized SCID mouse calvarial defects at week 8 post-implantation. Moreover, the spatial co-localization of human nuclei and MHC Class I staining with osteogenic markers confirmed the engraftment and differentiation of FReP cells into new osteogenic regions within the defect, while BJ fibroblasts were only detectable in the fibrosis regions.
CONCLUSIONS: We have generated novel multipotent FReP cells by exposing dermal fibroblasts to FMOD, which could potentially shift the paradigm of reprogramming autologous cells for tissue reconstruction to a safer protein-based process. In this study, we have provided extended evidence that FMOD reprogramming technology has significant osteogenic potential both in vitro and in vivo and that FReP cells are a promising candidate for in situ bone tissue regeneration.


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