Duchenne Muscular Dystrophy Myogenic Cells from Urine-Derived Stem Cells Recapitulate the Dystrophin Genotype and Phenotype

Abstract

A ready source of autologous myogenic cells is of vital importance for drug screening and functional genetic studies in Duchenne muscular dystrophy (DMD), a rare disease caused by a variety of dystrophin gene mutations. As stem cells (SCs) can be easily and noninvasively obtained from urine specimens, we set out to determine whether they could be myogenically induced and useful in DMD research. To this end, we isolated stem cells from the urine of two healthy donors and from one patient with DMD, and performed surface marker characterization, myogenic differentiation (MyoD), and then transfection with antisense oligoribonucleotides to test for exon skipping and protein restoration. We demonstrated that native urine-derived stem cells express the full-length dystrophin transcript, and that the dystrophin mutation was retained in the cells of the patient with DMD, although the dystrophin protein was detected solely in control cells after myogenic transformation according to the phenotype. Notably, we also showed that treatment with antisense oligoribonucleotide against dystrophin exon 44 induced skipping in both native and MyoD-transformed urine-derived stem cells in DMD, with a therapeutic transcript-reframing effect, as well as visible protein restoration in the latter. Hence MyoD-transformed cells may be a good myogenic model for studying dystrophin gene expression, and native urine stem cells could be used to study the dystrophin transcript, and both diagnostic procedures and splicing modulation therapies in both patients and control subjects, without invasive and costly collection methods. New, bankable bioproducts from urine stem cells, useful for prescreening studies and therapeutic applications alike, are also foreseeable after further, more in-depth characterization.

Urine-Derived Stem Cells: Applications in Regenerative and Predictive Medicine

“…Regarding challenges and open questions, UDSC biological characterizations need to be deeper explored, with a special focus on donor age effects on UDSC properties, as previously mentioned. Moreover, in UDSC, the expression levels of some characteristic surface markers, analyzed by flow cytometry, show discrepancy among different studies [20,21,29,32,38,46,68,69,73,84,95,[98][99][100]. These discrepancies can be attributed to different culture conditions, such as the composition of the culture medium, or can be a native characteristic of the isolated cells that can be heterogeneous within the same individual or between different individuals.…”

“…Furthermore, CRISPR/Cas9-edited USC produced the most frequent mutation responsible for limb-girdle muscular dystrophy type 2C [94]. Additionally, the antisense oligonucleotide strategy was used to correct the defect of dystrophin exon 44, by exon skipping, in UDSC differentiated into myogenic cells from Duchenne muscular dystrophy patients and healthy controls [95].…”

EMQN best practice guidelines for genetic testing in dystrophinopathies

“…Although pathogenic variants have been identified in DMD mRNA from blood [ 29 , 30 ], only low level illegitimate DMD transcription occurs in lymphocytes, so analysis is technically challenging and any aberrant splicing may not reflect splicing in muscle. Recently, alternative sources of DMD transcripts have emerged such as stem cells [ 31 ] or extracellular RNA [ 32 ] from urine that could help overcome the difficulty of obtaining muscle biopsies in some patients.…”

Phenotype predictions for exon deletions/duplications: A user guide for professionals and clinicians using Becker and Duchenne muscular dystrophy as examples

“…For transcript analysis, a muscle biopsy is required, which is an invasive and painful procedure. Nowadays, alternatives are available, for example, analyzing blood (usually expressing low levels of dystrophin RNA; Chelly, Kaplan, Maire, Gautron, & Kahn, ), converting fibroblasts or stem cells derived from urine into myogenic cells with viral myoD gene transduction (Aartsma‐Rus et al, ; Falzarano et al, ) or by generating iPS cells and differentiation into myogenic lineages (Young et al, ). This is however quite labor intensive, time consuming, and expensive.…”