Functional Skeletal Muscle Constructs from Transdifferentiated Human Fibroblasts

www.nature.com/scientificreports OPEN Functional skeletal muscle constructs from transdiferentiated human fbroblasts Bin Xu1, Allison Siehr1 & Wei Shen1,2,3* Transdiferentiation 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 transdiferentiated cells have been rarely studied. Here, we report that three-dimensional (3D) tissue constructs engineered with iMyoD-hTERT- NHDFs, normal human dermal fbroblasts 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 benefts functional myogenic diferentiation of transdiferentiated 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-specifc and disease-specifc cells to develop into functional skeletal muscle constructs makes them highly valuable for many applications, such as disease modeling. Even though normal skeletal muscle has self-regenerative ability, it has remained a major challenge to treat genetic muscle diseases and volumetric muscle loss 1. Using human myogenic cells to develop cell-based thera- pies to repair or restore diseased or lost muscle tissue represents an important strategy to address this challenge. Human myogenic cells are also highly desirable for creating muscle tissue models or disease models, which can be used for fundamental studies of muscle physiology and pathology and for drug screening and validation. Many studies have used animal myogenic cells, such as the C2C12 cell line and myoblasts isolated from animal models of muscular dystrophy, to create muscle tissue models or disease models. Tese studies have made signifcant contributions to the research area, but striking interspecies diferences make animal cells unable to provide reli- able models for many human genetic diseases2–5. Despite their importance, studies using human myogenic cells have been hampered by insufcient cell quan- tity. Traditionally, human myogenic cells have been obtained from muscle biopsies, which have limited availability, and the cells have poor expansion capacity6. Generation of immortalized human myoblasts is technically challenging. Transduction of human myoblasts with human telomerase reverse transcriptase (hTERT) is not sufcient to immortalize these cells 7. Even though overexpression of cyclin-dependent kinase 4 (CDK4) or cyclin D1 with hTERT could result in myoblasts capable of extensive proliferation, these cells have compromised myogenic diferentiation potential 7,8. Recent advances in deriving myogenic cells from induced human pluripotent stem cells (hiPSCs) may provide opportunities to address the limited availability of human myogenic cells 9–12. Alternatively, transdiferentiation, which is direct conversion of one somatic cell type to another without going through the pluripotent state13, may ofer another approach to obtaining human myogenic cells. In both methods, somatic cells are used as the starting cells, making it possible to obtain abundant, patient-specifc, and disease- specifc human myogenic cells for therapeutic or disease modeling purposes. Direct conversion of fbroblasts into myogenic cells by forced expression of the transcription factor MyoD was frst demonstrated about three decades ago 14, representing the earliest example of transdiferentiation. Since then, it has been shown that MyoD-induced transdiferentiation can convert many other cell types into myogenic cells15, though fbroblasts remain the most commonly used starting cell source. Human myogenic cells obtained through transdiferentiation have several advantages over those derived from hiPSCs: direct conversion of one somatic cell type to the target lineage is substantially quicker than reprograming the starting cell type to hiPSCs and then further guiding diferentiation toward the target lineage; transdiferentiated cells avoid the risk of 1Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA. 2Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA. 3Institute for Engineering in Medicine, University of Minnesota, Minneapolis, MN 55455, USA. *email: [email protected] Scientifc Reports | (2020) 10:22047 | https://doi.org/10.1038/s41598-020-78987-8 1 Vol.:(0123456789) www.nature.com/scientificreports/ teratoma formation associated with pluripotent stem cells16. However, the efciency of myogenic transdiferentiation needs to be improved, and literature information on functional evaluation, such as contractile force generation, of these cells is scarce. In this study, we established a cell line through viral transduction of primary normal human dermal fbroblasts (NHDFs) with genes encoding hTERT and doxycycline (DOX) inducible MyoD (iMyoD). Te hTERT gene was introduced into the cells to enhance their expansion capacity. NHDFs were chosen because they can be obtained through a minimally invasive procedure. We examined the abilities of these cells to proliferate and to transdif- ferentiate into myogenic cells upon DOX-induced MyoD expression. We investigated the roles of two small mol- ecules CHIR99021 (a Wnt signaling activator) and DAPT (a Notch signaling inhibitor) in enhancing functional myogenic diferentiation of these iMyoD-converted fbroblasts. We further engineered three-dimensional (3D) tissue constructs with these cells and examined their ability to generate contractile forces in response to electrical stimuli. Te efects of temporal modulation of MyoD expression and treatment with CHIR99021 and DAPT on contractile force generation of these 3D constructs were also investigated. Results and discussion Generate iMyoD-hTERT-NHDFs and primary iMyoD-NHDFs. We set out to generate iMyoD- NHDFs by lentivirally transducing primary NHDFs with a Tet-On VP64-MyoD (iMyoD) vector as previously reported (Fig. 1A)17. Tis vector allows constitutive co-expression of a puromycin resistance gene for selection of transduced cells and a reverse tetracycline transactivator for doxycycline (DOX) inducible expression of MyoD and a red fuorescent reporter DsRed; the transcriptional activator VP64 fused to MyoD enhances chromatin remodeling and benefts transdiferentiation17. To address the limited proliferative capacity of primary iMyoD- NHDFs, we further generated an immortalized iMyoD-hTERT-NHDF cell line (Fig. 1A). An immortalized NHDF cell line (hTERT-NHDFs) was generated by transducing primary NHDFs with a vector encoding hTERT to overcome the Hayfick limit in primary cell expansion18, then the hTERT-NHDFs were transduced with the iMyoD vector to obtain iMyoD-hTERT-NHDFs. Te presence of the transduced genes in the genomic DNA of these cells was confrmed by PCR analysis (Figure S1). To confrm the robustness of the transdiferentiation method, primary NHDFs from two donors were used, and the conclusions in this paper are supported by the cell lines derived from both primary NHDFs. Before DOX induction, both iMyoD-hTERT-NHDFs and primary iMyoD-NHDFs displayed normal fbroblast morphology. To test their proliferation capacity, the cells were seeded in 48-well plates (2 × 104 cells in each well) and cultured in an NHDF culture medium, and the population doubling as a function of time was measured. Te iMyoD-hTERT-NHDFs exhibited prolonged and stable proliferation capacity, with an almost unchanged growth rate even when the population doubling value was greater than 60 (Fig. 1B). In contrast, primary iMyoD-NHDFs showed gradually reduced growth rates, and the growth became very slow when the population doubling value reached approximately 30 (Fig. 1B). Tese results confrm that hTERT transduction allows the cells to overcome their expansion limit, providing an abundant source of human myogenic cells. The myogenic diferentiation efciency upon DOX induction is low. When iMyoD-hTERT- NHDFs and primary iMyoD-NHDFs were seeded at approximately 20% confuency and cultured in the NHDF culture medium supplemented with DOX for 3 days, both cell types exhibited similar myogenic diferentiation tendency, as indicated by elongated cell morphology and positive immunofuorescence staining with the MF20 antibody for total myosin heavy chains (total MHC), a characteristic marker for skeletal muscle (Fig. 1C). How- ever, even afer 7-day DOX induction, the myotubes were still short and the myogenic index (defned as the ratio of the nuclei number in the MF20 + cells containing three or more nuclei to the total number of nuclei)19 was low (Figure S2A), indicative of retarded myogenic diferentiation. Cells at this stage were proliferative and could be passaged or cryopreserved with their proliferative and myogenic capacities retained. Tese results sug- gest that DOX-induced MyoD expression can guide iMyoD-hTERT-NHDFs and primary iMyoD-NHDFs to become myoblast-like cells, but it is insufcient to efciently drive terminal myogenic diferentiation of these myoblast-like cells. It is known that normal myogenesis involves myogenic lineage commitment and terminal diferentiation 20, so we reasoned that conversion of iMyoD-hTERT-NHDFs or primary iMyoD-NHDFs

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