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.

Use of adeno-associated virus to enrich cardiomyocytes derived from human stem cells

“…The derivation and 3 maintenance of iPSCs was described previously 6 This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.…”

“…Human iPS cell line UC3-4 6 were differentiated into cardiac lineage as described 3 previously 7 . Spontaneously contracting cell clusters could be observed between day 10 to 4 14, and the beating area gradually increased over time.…”

Induced pluripotent stem cells for modeling neurological disorders

“…Modeling DMD in vitro will help disclose the neurological mechanism of this disease and even allow to correct the dystrophin deficit in the muscle. To date, cardiomyoblast cells, muscle cells and neurons have been generated from iPSC cells [79][80][81][82] . The first group to reprogram cells from DMD patients was Park et al [66] in 2008, followed by other groups modeling DMD in vitro and whose primary objective was to correct the dystrophin in muscle cells.…”

Use of Adeno-Associated Virus to Enrich Cardiomyocytes Derived from Human Stem Cells

“…The derivation and maintenance of iPSCs was described previously. 6 Briefly, undifferentiated iPSCs were maintained under feederfree condition with daily change of mTeSR-1 medium (Cat No. 05850; Stemcell Technologies, Vancouver, BC, Canada), following manufacturer's instructions.…”

“…Human iPS cell line UC3-4 6 was differentiated into cardiac lineage as described previously. 7 Spontaneously contracting cell clusters could be observed between days 10 and 14, and the beating area gradually increased over time.…”