RESUMEN
Background: Poor adherence to daily human growth hormone (hGH) treatment has been shown to be associated with poor clinical outcomes for growth hormone deficiency (GHD) patients. However, few studies have examined the perception of adherence to hGH treatment among both physicians and caregivers in Japan. Objective: The aim of this study is to examine the perception of adherence for daily hGH treatment among physicians and caregivers of pediatric and adolescent patients treated with GH in Japan. Moreover, we explore reasons for skipping treatment and the potential impact of a once-weekly treatment on adherence. Methods: A cross-sectional survey was conducted in Japan among physicians that prescribe daily hGH treatment and caregivers that have administered daily hGH treatment to children/adolescents for 3 months or longer. The Morisky Medication Adherence Scale (MMAS-8) was used to gauge perceived adherence for both physician and caregiver groups. Caregivers were also questioned regarding reasons for missing injections. Moreover, both groups were asked about the impact of a once-weekly treatment on adherence. Results: Responses were collected from 123 physicians and 112 caregivers. Physicians reported that 18.1% of patients have poor adherence based on the MMAS-8 instrument. In contrast, 32.1% of the caregivers reported poor adherence. "Simply forgetting", "Patient refused/resisted", and being "Busy with school activities, etc" were the most commonly selected reasons by caregivers for missing an injection. Physicians felt that a once-weekly injection could improve adherence for 64.5% of patients with poor adherence. Moreover, 56.9% of the caregivers that reported an experience of missed injections felt that a once-weekly injection would improve their adherence. Conclusion: Approaches to improve adherence to hGH treatment in Japan are continuously needed. While further research is needed to understand factors most likely to improve adherence, availability of a once-weekly treatment is expected to help improve adherence.
RESUMEN
Patient-derived disease-specific induced pluripotent stem cells (iPSCs) have opened the door to recreating pathological conditions in vitro using differentiation into diseased cells corresponding to each target tissue. To investigate muscular disease, we have established a myogenic differentiation protocol mediated by inducible MYOD1 expression that drives human iPSCs into myocytes. This highly reproducible differentiation protocol yields a homogenous skeletal muscle cell population, reaching efficiencies as high as 70-90%. Such high efficiency enables us to evaluate the efficacy of exon skipping in disease-specific myocytes. These disease-specific iPSC-derived myocytes can be applied not only for the validation of therapeutic efficacy of specific antisense oligonucleotide but also for the screening of exon skipping chemicals combined with the multiwell differentiation system.
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Diferenciación Celular/genética , Exones , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Musculares/citología , Células Musculares/metabolismo , Empalme del ARN , Técnicas de Cultivo de Célula , Reprogramación Celular/genética , Técnicas de Reprogramación Celular , Células Nutrientes , Regulación del Desarrollo de la Expresión Génica , Humanos , Desarrollo de Músculos/genética , Proteína MioD/genética , Oligonucleótidos Antisentido/genética , Transducción GenéticaRESUMEN
Patient-derived induced pluripotent stem cells (iPSCs) have opened the door to recreating pathological conditions in vitro using differentiation into diseased cells corresponding to each target tissue. Yet for muscular diseases, a method for reproducible and efficient myogenic differentiation from human iPSCs is required for in vitro modeling. Here, we introduce a myogenic differentiation protocol mediated by inducible transcription factor expression that reproducibly and efficiently drives human iPSCs into myocytes. Delivering a tetracycline-inducible, myogenic differentiation 1 (MYOD1) piggyBac (PB) vector to human iPSCs enables the derivation of iPSCs that undergo uniform myogenic differentiation in a short period of time. This differentiation protocol yields a homogenous skeletal muscle cell population, reproducibly reaching efficiencies as high as 70-90 %. MYOD1-induced myocytes demonstrate characteristics of mature myocytes such as cell fusion and cell twitching in response to electric stimulation within 14 days of differentiation. This differentiation protocol can be applied widely in various types of patient-derived human iPSCs and has great prospects in disease modeling particularly with inherited diseases that require studies of early pathogenesis and drug screening.
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Reprogramación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteína MioD/genética , Transfección/métodos , Transgenes , Animales , Diferenciación Celular , Dermis/citología , Dermis/metabolismo , Doxiciclina/farmacología , Estimulación Eléctrica , Células Nutrientes/citología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Lípidos/química , Ratones , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Proteína MioD/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle degenerating disease caused by a dystrophin deficiency. Effective suppression of the primary pathology observed in DMD is critical for treatment. Patient-derived human induced pluripotent stem cells (hiPSCs) are a promising tool for drug discovery. Here, we report an in vitro evaluation system for a DMD therapy using hiPSCs that recapitulate the primary pathology and can be used for DMD drug screening. Skeletal myotubes generated from hiPSCs are intact, which allows them to be used to model the initial pathology of DMD in vitro. Induced control and DMD myotubes were morphologically and physiologically comparable. However, electric stimulation of these myotubes for in vitro contraction caused pronounced calcium ion (Ca(2+)) influx only in DMD myocytes. Restoration of dystrophin by the exon-skipping technique suppressed this Ca(2+) overflow and reduced the secretion of creatine kinase (CK) in DMD myotubes. These results suggest that the early pathogenesis of DMD can be effectively modelled in skeletal myotubes induced from patient-derived iPSCs, thereby enabling the development and evaluation of novel drugs.
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Células Madre Pluripotentes Inducidas/patología , Modelos Biológicos , Distrofia Muscular de Duchenne/etiología , Distrofia Muscular de Duchenne/patología , Adulto , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Distrofina/metabolismo , Estimulación Eléctrica , Exones/genética , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Lactante , Masculino , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteína MioD/metabolismo , Oligonucleótidos Antisentido/farmacología , Tetraciclina/farmacología , TransfecciónRESUMEN
The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70-90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.
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Diferenciación Celular/efectos de los fármacos , Miopatías Distales/genética , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de la Membrana/genética , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Atrofia Muscular/genética , Proteína MioD/genética , Animales , Biomarcadores/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Miopatías Distales/metabolismo , Miopatías Distales/patología , Doxiciclina/farmacología , Disferlina , Estimulación Eléctrica , Expresión Génica , Perfilación de la Expresión Génica , Vectores Genéticos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones , Ratones SCID , Modelos Biológicos , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Proteínas Musculares/metabolismo , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Proteína MioD/metabolismo , TransfecciónRESUMEN
Induced pluripotent stem (iPS) cells are generated from adult somatic cells by transduction of defined factors. Given their unlimited proliferation and differentiation potential, iPS cells represent promising sources for cell therapy and tools for research and drug discovery. However, systems for the directional differentiation of iPS cells toward paraxial mesodermal lineages have not been reported. In the present study, we established a protocol for the differentiation of mouse iPS cells into paraxial mesodermal lineages in serum-free culture. The protocol was dependent on Activin signaling in addition to BMP and Wnt signaling which were previously shown to be effective for mouse ES cell differentiation. Independently of the cell origin, the number of transgenes, or the type of vectors used to generate iPS cells, the use of serum-free monolayer culture stimulated with a combination of BMP4, Activin A, and LiCl enabled preferential promotion of mouse iPS cells to a PDGFR-α(+)/Flk-1(-) population, which represents a paraxial mesodermal lineage. The mouse iPS cell-derived paraxial mesodermal cells exhibited differentiation potential into osteogenic, chondrogenic, and myogenic cells both in vitro and in vivo and contributed to muscle regeneration. Moreover, purification of the PDGFR-α(+)/KDR(-) population after differentiation allowed enrichment of human iPS cell populations with paraxial mesodermal characteristics. The resultant PDGFR-α(+)/KDR(-) population derived from human iPS cells specifically exhibited osteogenic, chondrogenic, and myogenic differentiation potential in vitro, implying generation of paraxial mesodermal progenitors similar to mouse iPS cell-derived progenitors. These findings highlight the potential of protocols based on the serum-free, stepwise induction and purification of paraxial mesodermal cell lineages for use in stem cell therapies to treat diseased bone, cartilage, and muscle.