Abstract
Transdifferentiation of human non-muscle cells directly into myogenic cells by forced expression of MyoD represents one route to obtain highly desirable human myogenic cells. However, functional properties of the tissue constructs derived from these transdifferentiated cells have been rarely studied. Here, we report that three-dimensional (3D) tissue constructs engineered with iMyoD-hTERT-NHDFs, normal human dermal fibroblasts transduced with genes encoding human telomerase reverse transcriptase and doxycycline-inducible MyoD, generate detectable contractile forces in response to electrical stimuli upon MyoD expression. Withdrawal of doxycycline in the middle of 3D culture results in 3.05 and 2.28 times increases in twitch and tetanic forces, respectively, suggesting that temporally-controlled MyoD expression benefits functional myogenic differentiation of transdifferentiated myoblast-like cells. Treatment with CHIR99021, a Wnt activator, and DAPT, a Notch inhibitor, leads to further enhanced contractile forces. The ability of these abundant and potentially patient-specific and disease-specific cells to develop into functional skeletal muscle constructs makes them highly valuable for many applications, such as disease modeling.
Highlights
Teratoma formation associated with pluripotent stem c ells[16]
An immortalized normal human dermal fibroblasts (NHDFs) cell line was generated by transducing primary NHDFs with a vector encoding hTERT to overcome the Hayflick limit in primary cell expansion[18], the hTERT-NHDFs were transduced with the inducible MyoD (iMyoD) vector to obtain iMyoD-hTERT-NHDFs
Even after 7-day DOX induction, the myotubes were still short and the myogenic index[19] was low (Figure S2A), indicative of retarded myogenic differentiation. Cells at this stage were proliferative and could be passaged or cryopreserved with their proliferative and myogenic capacities retained. These results suggest that DOX-induced MyoD expression can guide iMyoD-hTERT-NHDFs and primary iMyoD-NHDFs to become myoblast-like cells, but it is insufficient to efficiently drive terminal myogenic differentiation of these myoblast-like cells
Summary
Teratoma formation associated with pluripotent stem c ells[16]. the efficiency of myogenic transdifferentiation needs to be improved, and literature information on functional evaluation, such as contractile force generation, of these cells is scarce. We established a cell line through viral transduction of primary normal human dermal fibroblasts (NHDFs) with genes encoding hTERT and doxycycline (DOX) inducible MyoD (iMyoD). We examined the abilities of these cells to proliferate and to transdifferentiate into myogenic cells upon DOX-induced MyoD expression. We investigated the roles of two small molecules CHIR99021 (a Wnt signaling activator) and DAPT (a Notch signaling inhibitor) in enhancing functional myogenic differentiation of these iMyoD-converted fibroblasts. We further engineered three-dimensional (3D) tissue constructs with these cells and examined their ability to generate contractile forces in response to electrical stimuli. The effects of temporal modulation of MyoD expression and treatment with CHIR99021 and DAPT on contractile force generation of these 3D constructs were investigated
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