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Dystrophin-deficient cardiomyocytes derived from human urine: new biologic reagents for drug discovery.

ABSTRACT: The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs). USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.

SUBMITTER: Guan X

PROVIDER: S-EPMC3966181 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Dystrophin-deficient cardiomyocytes derived from human urine: new biologic reagents for drug discovery.

Guan Xuan X Mack David L DL Moreno Claudia M CM Strande Jennifer L JL Mathieu Julie J Shi Yingai Y Markert Chad D CD Wang Zejing Z Liu Guihua G Lawlor Michael W MW Moorefield Emily C EC Jones Tara N TN Fugate James A JA Furth Mark E ME Murry Charles E CE Ruohola-Baker Hannele H Zhang Yuanyuan Y Santana Luis F LF Childers Martin K MK

Stem cell research 20131223 2

The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-deriv ...[more]

PMID: 24434629

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Project description:BACKGROUND:We are developing a novel therapy for Duchenne muscular dystrophy (DMD), involving the transplantation of autologous, skeletal muscle-derived stem cells that have been genetically corrected to express dystrophin. Dystrophin is normally expressed in activated satellite cells and in differentiated muscle fibres. However, in past preclinical validation studies, dystrophin transgenes have generally been driven by constitutive promoters that would be active at every stage of the myogenic differentiation process, including in proliferating muscle stem cells. It is not known whether artificial dystrophin expression would affect the properties of these cells. AIMS:Our aims are to determine if mini-dystrophin expression affects the proliferation or myogenic differentiation of DMD skeletal muscle-derived cells. METHODS:Skeletal muscle-derived cells from a DMD patient were transduced with lentivirus coding for mini-dystrophins (R3-R13 spectrin-like repeats (?R3R13) or hinge2 to spectrin-like repeats R23 (?H2R23)) with EGFP (enhanced green fluorescence protein) fused to the C-terminus, driven by a constitutive promoter, spleen focus-forming virus (SFFV). Transduced cells were purified on the basis of GFP expression. Their proliferation and myogenic differentiation were quantified by ethynyl deoxyuridine (EdU) incorporation and fusion index. Furthermore, dystrophin small interfering ribonucleic acids (siRNAs) were transfected to the cells to reverse the effects of the mini-dystrophin. Finally, a phospho-mitogen-activated protein kinase (MAPK) array assay was performed to investigate signalling pathway changes caused by dystrophin expression. RESULTS:Cell proliferation was not affected in cells transduced with ?R3R13, but was significantly increased in cells transduced with ?H2R23. The fusion index of myotubes derived from both ?R3R13- and ?H2R23 -expressing cells was significantly compromised in comparison to myotubes derived from non-transduced cells. Dystrophin siRNA transfection restored the differentiation of ?H2R23-expressing cells. The Erk1/2- signalling pathway is altered in cells transduced with mini-dystrophin constructs. CONCLUSIONS:Ectopic expression of dystrophin in cultured human skeletal muscle-derived cells may affect their proliferation and differentiation capacity. Caution should be taken when considering genetic correction of autologous stem cells to express dystrophin driven by a constitutive promoter.

Dataset Information

Dystrophin-deficient cardiomyocytes derived from human urine: new biologic reagents for drug discovery.

Publications

Dystrophin-deficient cardiomyocytes derived from human urine: new biologic reagents for drug discovery.

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