Direct reprogramming of urine-derived cells with inducible MyoD for modeling human muscle disease

Abstract

BackgroundCellular models of muscle disease are taking on increasing importance with the large number of genes and mutations implicated in causing myopathies and the concomitant need to test personalized therapies. Developing cell models relies on having an easily obtained source of cells, and if the cells are not derived from muscle itself, a robust reprogramming process is needed. Fibroblasts are a human cell source that works well for the generation of induced pluripotent stem cells, which can then be differentiated into cardiomyocyte lineages, and with less efficiency, skeletal muscle-like lineages. Alternatively, direct reprogramming with the transcription factor MyoD has been used to generate myotubes from cultured human fibroblasts. Although useful, fibroblasts require a skin biopsy to obtain and this can limit their access, especially from pediatric populations.ResultsWe now demonstrate that direct reprogramming of urine-derived cells is a highly efficient and reproducible process that can be used to establish human myogenic cells. We show that this method can be applied to urine cells derived from normal individuals as well as those with muscle diseases. Furthermore, we show that urine-derived cells can be edited using CRISPR/Cas9 technology.ConclusionsWith progress in understanding the molecular etiology of human muscle diseases, having a readily available, noninvasive source of cells from which to generate muscle-like cells is highly useful.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0103-9) contains supplementary material, which is available to authorized users.

Efficient exon skipping of SGCG mutations mediated by phosphorodiamidate morpholino oligomers

“…Previously, we showed the ability to directly reprogram urine-derived cells (UDC) into the myogenic lineage through the overexpression of MyoD (38). To further analyze this SGCG exon-skipping strategy, we obtained UDCs from an LGMD 2C patient harboring a frameshift mutation caused by the deletion of SGCG exon 6 (ex6del).…”

“…Primary fibroblasts from a healthy control subject were obtained from the American Type Culture Collection (ATCC, CCD-1127Sk). Primary UDCs were isolated from the urine of deidentified healthy volunteers and an LGMD 2C patient harboring a deletion of SGCG exon 6, as previously described (38). Primary fibroblasts were cultured in proliferation media consisting of DMEM (Thermo Fisher Scientific, 11955-065) supplemented with 15% FBS (Thermo Fisher Scientific, 26140-079).…”

“…Primary fibroblasts were cultured in proliferation media consisting of DMEM (Thermo Fisher Scientific, 11955-065) supplemented with 15% FBS (Thermo Fisher Scientific, 26140-079). Primary UDCs were cultured in UDC proliferation media as described (38).…”

“…Total RNA was isolated in 1 ml of TRIzol (Thermo Fisher Scientific, 15596018) according to the manufacturer's instructions and included the Reprogrammed UDCs. Primary UDCs were transduced with the iMyoD lentivirus as previously described (38). Myogenic reprogramming of iMyoD-transduced urine cells was performed as previously described with minor modifications.…”

Generation of a Urine-Derived Ips Cell Line from a Patient with a Ventricular Septal Defect and Heart Failure and the Robust Differentiation of These Cells to Cardiomyocytes via Small Molecules

“…Human iPSCs have been successfully established from different tissues, particularly blood cells [6], extra-embryonic tissues [7], and skin fibroblasts [8,9]. Human urine-derived cells (UCs) are an ideal source for iPSC generation because they are simply and noninvasively obtained [10,11]. Particularly in the pediatric population, parents are more receptive to this method of cell collection.…”