(2gs) Unleashing the Therapeutic Potential of Cells: Cellular Reprogramming and Tissue Engineering for Enhanced Function and Healing

Cell-based therapies have emerged as highly promising tools in modern medicine for regenerative applications. Throughout my PhD research, I have dedicated my efforts to manipulating cell and tissue biology to enhance their in-vivo function. Specifically, my work has revolved around three crucial aspects of cell and tissue engineering: stem cell bioengineering for improved functionality, and tissue engineering approaches for promoting healthy aging in skeletal muscle, and in-vivo muscle reprogramming using transcription factors to enhance outcomes after peripheral nerve injuries (PNI).

My first project focused on improving the multipotency of human skin derived stem cells for treating neurodegenerative diseases. In our lab, we isolated neural crest stem cells (NCSCs) from neonatal and adult human skin [1, 2]. These cells resemble embryonic NCSCs and differentiated into neurons, schwann cells, melanocytes and smooth muscle cells, serving as a promising patient-specific cell source for regenerative medicine applications [2, 3]. However, NCSCs quickly lost their multipotency in culture, significantly limiting their clinical applications. To overcome this hurdle, we mimicked embryonic signaling events in-vitro and treated the cells with chemicals that upregulated the Wnt and BMP signaling pathways. Through extensive RNA sequencing and cellular assays, we showcased how this treatment upregulated key genes associated with neural crest multipotency, such as Sox10, while also enhancing their differentiation ability. Intriguingly, we also discovered that this treatment rewired the metabolic landscape of the cells, making them more glycolytic and less reliant on oxidative phosphorylation for their energy needs. In summary, our research introduced a virus-free and cost-effective approach to bolster the biological function of adult tissue-derived stem cells [4]. Presently, we are working on implanting these enhanced cells in a mouse model of demyelination, aiming to explore their potential in treating demyelinating disorders like Parkinson's disease.

Addressing the issue of skeletal muscle aging, or sarcopenia, has been another vital aspect of my research. This condition is characterized by the age-associated loss of skeletal muscle mass and strength. One of the prominent hallmarks of skeletal muscle senescence is the accumulation of fat or myosteatosis, leading to impaired fatty acid metabolism. Our investigations identified the loss of the lactate receptor GPR81 in aging skeletal muscle, which correlated with myosteatosis. Loss of GPR81 is accompanied by increased expression of key senescence hallmarks such as SaβGal, γH2AX, ROS accumulation, mitochondrial dysfunction and impaired ability to differentiate to myotubes. We hypothesized that chemical agonists of GPR81 could reverse hallmarks of aging in human skeletal muscle cells. To this end, we treated aged myoblasts with the GPR81 agonist- 3-chloro-5-hydroxy BA (CHBA) for 10 days. Indeed, CHBA treatment of human myoblasts in-vitro could enhance mitochondrial function, repair DNA damage, and reverse ROS accumulation. Next, to confirm our findings in-vivo, we used the progeric mouse model of aging, and administered CHBA to old mice for 1 month. Surprisingly, CHBA administration significantly enhanced isometric muscle force production and grip strength, accompanied by improvement in overall health of the mice. In conclusion, our study underlines the significance of the lactate receptor GPR81 for maintaining skeletal muscle function and suggests GPR81 agonists as potential therapeutic candidates for mitigating skeletal muscle aging.

Furthermore, I explored in-vivo bioengineering of tissues to improve outcomes for debilitating diseases. PNIs affect more than 20 million Americans and severely impact the quality of life by causing long-term disability. These injuries disproportionately affect young individuals and soldiers who fail to regain normal movement and demonstrate persistent weakness. Most current interventions to treat PNI target accelerated nerve growth. However, since the speed of axon regeneration post injury is very slow (1-2mm/day), the skeletal muscle becomes atrophied by the time the nerves regenerate, and is incapable of “accepting” innervation. To this end, we hypothesize that reprogramming the skeletal muscle to an embryonic-like state may preserve its reinnervation capability following PNI. We used a transgenic mouse model to express the transcription factor NANOG in the mouse skeletal muscle. NANOG expression de-differentiated the muscle to an embryonic state, evidenced by increased eMYHC expression, and prevented skeletal muscle atrophy. In a nerve injury model, NANOG expression in the muscle enhanced reinnervation, as seen by enhanced neuromuscular junction formation, improved electromyography (EMG) recordings, and enhanced muscle force. In summary, while all current approaches to treat nerve injuries focus on neural regeneration, we show for the first time that reprogramming the denervated tissues could be crucial to improve functional outcomes after nerve injuries.

In conclusion, my research sheds light on the critical role of understanding embryonic signaling events and cellular bioenergetics in enhancing cellular functionality for regenerative medicine applications [5, 6]. The combination of bioengineering tools, transgenic models, and tissue engineering approaches has the potential to unlock novel cures for debilitating conditions, including demyelinating disorders and nerve injuries. By bridging the gap between fundamental cellular mechanisms and practical clinical applications, my work strives to pave the way for transformative advancements in regenerative medicine.

1. Mehrotra, P., et al., Adult tissue–derived neural crest-like stem cells: Sources, regulatory networks, and translational potential. Stem cells translational medicine, 2020. 9(3): p. 328-341.
2. Moghadasi Boroujeni, S., et al., Neural crest stem cells from human epidermis of aged donors maintain their multipotency in vitro and in vivo. Scientific reports, 2019. 9(1): p. 9750.
3. Bajpai, V.K., et al., Reprogramming postnatal human epidermal keratinocytes toward functional neural crest fates. Stem Cells, 2017. 35(5): p. 1402-1415.
4. Mehrotra, P., et al., Wnt/BMP Mediated Metabolic Reprogramming Preserves Multipotency of Neural Crest-Like Stem Cells. Stem Cells, 2023. 41(3): p. 287-305.
5. Rajabian, N., et al., Reversine ameliorates hallmarks of cellular senescence in human skeletal myoblasts via reactivation of autophagy. Aging Cell, 2023. 22(3): p. e13764.
6. Choudhury, D., et al., Inhibition of glutaminolysis restores mitochondrial function in senescent stem cells. Cell reports, 2022. 41(9).