Skeletal Muscle Regeneration by Fibromodulin Reprogrammed Cells without Tumorigenic Risks
Pin Ha, MD, DDS, MS, Chenshuang Li, DDS, PhD, Jong Kil Kim, BS, Wenlu Jiang, DDS, PhD, Grace Xinlian Chang, BS, SukMin Yoon, DDS, Eric Chen, DDS, Xinli Zhang, PhD, Kang Ting, DMD, DMEDSC, Chia Soo, MD, FACS, Zhong Zheng, PhD.
UCLA, Los Angeles, CA, USA.
PURPOSE: Skeletal muscle, one of the most commonly injured tissue, is easily lost by severe injuries from car accidents, natural disasters, or salvage surgeries for tumors. Unfortunately, the skeletal muscle mass has limited repair capacity. Direct transplantation of committed myoblasts is hindered by inadequate cell availability, limited cell spreading, and poor survivability of implanted cells. In addition, using mesenchymal stem cells for tissue regeneration is always accompanied by the painful, invasive procedures (i.e., tissue biopsy, bone marrow aspiration, and liposuction) that potentially cause severe complications or fatal outcomes. The tumorigenic risk of pluripotent cells also remains as the major concern for clinical application, and intramuscular injection is one of the most common routes for teratoma formation that validates the pluripotency in vivo in skeletal muscle regeneration. Previously, we have established a novel platform technology using a single molecule, fibromodulin (FMOD), to reprogram human dermal fibroblast into a multipotent state while circumventing oncogene usage and genome integrating. The yielded FMOD ReProgrammed (FReP) cells hold significant potential for myogenic differentiation both in vitro and in vivo. Our current study focused on its tumorigenic risk assessment.
METHODS: RNA-seq was performed to compare global gene expression of FReP cells and induced pluripotent stem cells (iPSCs) which holds the high tumorigenic risks. Differential mRNA expressions were identified by TopHat-Cufflinks package, functionally annotated via DAVID Bioinformatics Resource, and aligned with human proto-oncogenes and tumor suppressor genes listed in the UniProt database. Soft agar colony formation assay, the standard tumorigenicity test, was used to examine the cellular survival ability in an anchorage-independent manner under low nutritional and oxygen concentration microenvironment in vitro. On top of intramuscular injection, intratesticular injection was also carried out to further evaluate the tumorigenic potential of FReP cells, as intratesticular stromal cells produce more supportive environment that fosters implanted cells in comparison with subcutaneous and intramuscular microenvironment.
RESULTS: Functional analysis of more than 2300 differential genes between FReP cells and iPSCs by KEGG pathways revealed enrichment of genes involved in the ‘Pathways in cancer' with significant similarity of term overlap (Kappa value = 1.0). Notably, FReP cells showed lower expression of more proto-oncogenes but higher
expression of more tumor suppressor genes when compared to iPSCs. Unlike iPSCs, FReP cell neither proliferated nor formed colonies in soft agar after 14-day cultivation. Furthermore, in intramuscular injection, 2 of 8 iPSC-implanted animals (25%) ended up with tumor formation instead of skeletal muscle generation, while none of FReP cell-implanted animals presented tumor formation. Intratesticular injection of iPSCs resulted in 100% (10/10) teratoma formation, but FReP cells showed 0% (0/10) tumor formation in 4 months.
CONCLUTIONS: Our in vitro and in vivo studies collectively showed that FReP cells are less likely to generate tumors in vivo, which suggested that FReP cells is a safe cell source for skeletal muscle regeneration.
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