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In vitro and in vivo differentiation of induced pluripotent stem cells generated from urine-derived cells into cardiomyocytes.

ABSTRACT: Generation of human cardiomyocytes from cells derived from various sources, including skin biopsy, has been made possible by breakthrough advances in stem cell research. However, it is attractive to build up a negligibly invasive way to create induced pluripotent stem (iPS) cells. In this study, we created iPS cells from human urine-derived epithelial cells by gene transduction using lentiviral vectors in a totally noninvasive manner. Then, we induced the differentiation of iPS cells into functional cardiomyocytes both in vitro and in vivo Action potentials were recorded in putative cardiomyocytes and spontaneous beating cells were observed. Our results offered an alternative method to generate cardiomyocytes in a totally noninvasive manner from an easily accessible source. The availability of urine and its potent reprogramming characteristics will provide opportunities for the use of cells with specific genotypes to study the pathogenesis and molecular mechanisms of disease in vitro.

SUBMITTER: Jiang YF

PROVIDER: S-EPMC5829497 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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<i>In vitro</i> and <i>in vivo</i> differentiation of induced pluripotent stem cells generated from urine-derived cells into cardiomyocytes.

Jiang Yu-Feng YF Chen Min M Zhang Nan-Nan NN Yang Hua-Jia HJ Rui Qing Q Zhou Ya-Feng YF

Biology open 20180108 1

Generation of human cardiomyocytes from cells derived from various sources, including skin biopsy, has been made possible by breakthrough advances in stem cell research. However, it is attractive to build up a negligibly invasive way to create induced pluripotent stem (iPS) cells. In this study, we created iPS cells from human urine-derived epithelial cells by gene transduction using lentiviral vectors in a totally noninvasive manner. Then, we induced the differentiation of iPS cells into functi ...[more]

PMID: 29212797

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Project description:Despite preclinical studies demonstrating the functional benefit of transplanting human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) into damaged myocardium, the ability of these immature cells to adopt a more adult-like cardiomyocyte (CM) phenotype remains uncertain. To address this issue, we tested the hypothesis that prolonged in vitro culture of human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived CMs would result in the maturation of their structural and contractile properties to a more adult-like phenotype. Compared to their early-stage counterparts (PSC-CMs after 20-40 days of in vitro differentiation and culture), late-stage hESC-CMs and hiPSC-CMs (80-120 days) showed dramatic differences in morphology, including increased cell size and anisotropy, greater myofibril density and alignment, sarcomeres visible by bright-field microscopy, and a 10-fold increase in the fraction of multinucleated CMs. Ultrastructural analysis confirmed improvements in the myofibrillar density, alignment, and morphology. We measured the contractile performance of late-stage hESC-CMs and hiPSC-CMs and noted a doubling in shortening magnitude with slowed contraction kinetics compared to the early-stage cells. We then examined changes in the calcium-handling properties of these matured CMs and found an increase in calcium release and reuptake rates with no change in the maximum amplitude. Finally, we performed electrophysiological assessments in hESC-CMs and found that late-stage myocytes have hyperpolarized maximum diastolic potentials, increased action potential amplitudes, and faster upstroke velocities. To correlate these functional changes with gene expression, we performed qPCR and found a robust induction of the key cardiac structural markers, including ?-myosin heavy chain and connexin-43, in late-stage hESC-CMs and hiPSC-CMs. These findings suggest that PSC-CMs are capable of slowly maturing to more closely resemble the phenotype of adult CMs and may eventually possess the potential to regenerate the lost myocardium with robust de novo force-producing tissue.

Project description:BACKGROUND:Organoids play an important role in basic research, drug screening, and regenerative medicine. Here, we aimed to develop a novel kind of three-dimensional (3D) organoids generated from urine-derived stem cells (USCs) and to explore whether kidney-specific extracellular matrix (kECM) could enable such organoids for renal function in vitro. METHODS:USCs were isolated from human urine samples and cultured with kECM extraction to generate 3D organoids in vitro. Eight densities from 1000 to 8000 cells per organoids were prepared, and both ATP assay and Live/Dead staining were used to determine the optimal USC density in forming organoids and kECM additive concentration. The morphology and histology of as-made organoids were evaluated by hematoxylin and eosin (H.E.) staining, immunofluorescence staining and whole mount staining. Additionally, RT-qPCR was implemented to detect renal-related gene expression. Drug toxicity test was conducted to evaluate the potential application for drug screening. The renal organoids generated from whole adult kidney cells were used as a positive control in multiple assessments. RESULTS:The optimized cell density to generate ideal USC-derived organoids (USC-organoids) was 5000 cells/well, which was set as applying density in the following experiments. Besides, the optimal concentration of kECM was revealed to be 10%. On this condition, Live/Dead staining showed that USC-organoids were well self-organized without significant cell death. Moreover, H.E. staining showed that compact and viable organoids were generated without obvious necrosis inside organoids, which were very close to renal organoids morphologically. Furthermore, specific proximal tubule marker Aquaporin-1 (AQP1), kidney endocrine product erythropoietin (EPO), kidney glomerular markers Podocin and Synaptopodin were detected positively in USC-organoids with kECM. Nephrotoxicity testing showed that aspirin, penicillin G, and cisplatin could exert drug-induced toxicity on USC-organoids with kECM. CONCLUSIONS:USC-organoids could be developed from USCs via an optimal procedure. Combining culture with kECM, USC-organoid properties including morphology, histology, and specific gene expression were identified to be similar with real renal organoids. Additionally, USC-organoids posed kECM in vitro showed the potential to be a drug screening tool which might take the place of renal organoids to some extent in the future.

Dataset Information

In vitro and in vivo differentiation of induced pluripotent stem cells generated from urine-derived cells into cardiomyocytes.

Publications

<i>In vitro</i> and <i>in vivo</i> differentiation of induced pluripotent stem cells generated from urine-derived cells into cardiomyocytes.

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