Non-viral, Tumor-free Induction of Transient Cell Reprogramming in Mouse Skeletal Muscle to Enhance Tissue Regeneration

Abstract

Overexpression of Oct3/4, Klf4, Sox2, and c-Myc (OKSM) transcription factors can de-differentiate adult cells in vivo. While sustained OKSM expression triggers tumorigenesis through uncontrolled proliferation of toti-and pluripotent cells, transient reprogramming induces pluripotency-like features and proliferation only temporarily, without teratomas. We sought to transiently reprogram cells within mouse skeletal muscle with a localized injection of plasmid DNA encoding OKSM (pOKSM), and we hypothesized that the generation of proliferative intermediates would enhance tissue regeneration after injury. Intramuscular pOKSM administration rapidly upregulated pluripotency (Nanog, Ecat1, and Rex1) and early myogenesis genes (Pax3) in the healthy gastrocnemius of various strains. Mononucleated cells expressing such markers appeared in clusters among myofibers, proliferated only transiently, and did not lead to dysplasia or tumorigenesis for at least 120 days. Nanog was also upregulated in the gastrocnemius when pOKSM was administered 7 days after surgically sectioning its medial head. Enhanced tissue regeneration after reprogramming was manifested by the accelerated appearance of centronucleated myofibers and reduced fibrosis. These results suggest that transient in vivo reprogramming could develop into a novel strategy toward the acceleration of tissue regeneration after injury, based on the induction of transiently proliferative, pluripotent-like cells in situ. Further research to achieve clinically meaningful functional regeneration is warranted.

Epigenetic changes during aging and their reprogramming potential

“…The direct injection of OSKM plasmids into adult mice induces pluripotent stem cells markers in liver within a couple of days (Yilmazer et al 2013). Local injection of OSKM plasmids into skeletal muscle of adult mice induces pluripotency markers and improves skeletal muscle regeneration with less fibrosis and more myofibers (de Lázaro et al 2019), but the long-term safety of these treatments is not known beyond 120 days.…”

Cellular Plasticity: A Route to Senescence Exit and Tumorigenesis

“…The interplay between cellular reprogramming and senescence has drastic implications for tumor development and progression [ 30 , 31 , 32 , 33 , 34 , 35 ]. In this context, malignant cells can undergo EMT-mediated reprogramming to acquire features of plasticity and stemness that help these cells overcome the tumor suppressor action of senescence and continue proliferating [ 36 , 37 , 38 , 39 , 40 , 41 , 42 ].…”

Transient reprogramming of postnatal cardiomyocytes to a dedifferentiated state

“…Some of us have previously demonstrated that somatic cells can be transiently reprogrammed in vivo through non-integrating vector mediated expression of OSKM without leading to teratoma or tumorigenesis [ 53 – 57 ]. Ourselves and others have also demonstrated that transient in vivo reprogramming can be used to enhance the regeneration of injured tissue [ 56 , 58 , 59 ] and rejuvenate aged tissues [ 60 ] without leading to teratoma generation. The present study demonstrates that transient OSKM expression in cardiomyocytes in vitro enables a temporary passage through a proliferative de-differentiated state which is encouraging for the potential applications of this approach in the heart.…”

“…Given the similarities between OSKM induced transient reprogramming presented here and the endogenous regenerative mechanisms observed in model organisms [6,10,11], transient OSKM expression could be a novel strategy to stimulate proliferation of cells in the injured myocardium in vivo. Some of us have previously demonstrated that somatic cells can be transiently reprogrammed in vivo through non-integrating vector mediated expression of OSKM without leading to teratoma or tumorigenesis [53][54][55][56][57]. Ourselves and others have also demonstrated that transient in vivo reprogramming can be used to enhance the regeneration of injured tissue [56,58,59] and rejuvenate aged tissues [60] without leading to teratoma generation.…”