RESUMEN
BACKGROUND: Regenerative medicine strategies employing nephron progenitor cells (NPCs) are a viable approach that is worthy of substantial consideration as a promising cell source for kidney diseases. However, the generation of induced nephron progenitor-like cells (iNPCs) from human somatic cells remains a major challenge. Here, we describe a novel method for generating NPCs from human urine-derived cells (UCs) that can undergo long-term expansion in a serum-free condition. RESULTS: Here, we generated iNPCs from human urine-derived cells by forced expression of the transcription factors OCT4, SOX2, KLF4, c-MYC, and SLUG, followed by exposure to a cocktail of defined small molecules. These iNPCs resembled human embryonic stem cell-derived NPCs in terms of their morphology, biological characteristics, differentiation potential, and global gene expression and underwent a long-term expansion in serum-free conditions. CONCLUSION: This study demonstrates that human iNPCs can be readily generated and expanded, which will facilitate their broad applicability in a rapid, efficient, and patient-specific manner, particularly holding the potential as a transplantable cell source for patients with kidney disease.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Nefronas/metabolismo , Diferenciación Celular/genética , Reprogramación Celular/genética , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Humanos , Células Madre Pluripotentes Inducidas/citología , Nefronas/crecimiento & desarrollo , Nefronas/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma/genética , Orina/citologíaRESUMEN
BACKGROUND: Human keratinocytes and derived products are crucial for skin repair and regeneration. Despite substantial advances in engineered skin equivalents, their poor availability and immunorejection remain major challenges in skin grafting. METHODS: Induced keratinocyte-like cells (iKCs) were directly reprogrammed from human urine cells by retroviral transduction of two lineage-specific transcription factors BMI1 and â³NP63α (BN). Expression of keratinocyte stem cell or their differentiation markers were assessed by PCR, immunofluorescence and RNA-Sequencing. Regeneration capacity of iKCs were assessed by reconstitution of a human skin equivalent under air-interface condition. RESULTS: BN-driven iKCs were similar to primary keratinocytes (pKCs) in terms of their morphology, protein expression, differentiation potential, and global gene expression. Moreover, BN-iKCs self-assembled to form stratified skin equivalents in vitro. CONCLUSIONS: This study demonstrated an approach to generate human iKCs that could be directly reprogrammed from human somatic cells and extensively expanded in serum- and feeder cell-free systems, which will facilitate their broad applicability in an efficient and patient-specific manner.
Asunto(s)
Reprogramación Celular/fisiología , Queratinocitos/fisiología , Células Cultivadas/fisiología , Técnicas de Reprogramación Celular , Femenino , Humanos , Técnicas In Vitro , Masculino , Fenómenos Fisiológicos de la PielRESUMEN
Reprogramming of 'adult' differentiated somatic cells to 'embryonic' pluripotent stem cells accompanied by increased rate of glycolysis. Conversely, glycolysis triggers accumulation of advanced glycation end products (AGEs), a potential causative factor in aging, by promoting methylglyoxal production. Therefore, it is reasonable that pluripotent stem cells (PSCs) would specifically regulate glycolysis to maintain their embryonic features. In this study, we focused on glycine decarboxylase (GLDC), a key enzyme in the glycine cleavage system that regulates glycolysis and methylglyoxal production in cancer. GLDC was exclusively expressed in PSCs, and inhibition of this enzyme induced alterations of metabolome and AGE accumulation, thereby suppressing the embryonic pluripotent state. Surprisingly, the level of accumulated AGEs in somatic cells gradually decreased during reprogramming, ultimately disappearing in iPSCs. In addition, ectopic expression of GLDC or treatment with the AGE inhibitor LR-90 promoted reprogramming. Together, these findings suggest that GLDC-mediated regulation of glycolysis and controlling AGE accumulation is related to maintenance and induction of pluripotency.
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Reprogramación Celular , Regulación Enzimológica de la Expresión Génica , Productos Finales de Glicación Avanzada/metabolismo , Glicina-Deshidrogenasa (Descarboxilante)/biosíntesis , Glucólisis , Células Madre Pluripotentes Inducidas/enzimología , Butiratos/farmacología , Línea Celular , Productos Finales de Glicación Avanzada/genética , Glicina/genética , Glicina/metabolismo , Glicina-Deshidrogenasa (Descarboxilante)/genética , Humanos , Células Madre Pluripotentes Inducidas/citologíaRESUMEN
Control of adipogenesis in mesenchymal stem cells (MSCs) offers enormous potential for management of obesity- and aging-related diseases. Celastrol, the traditional Chinese medicine extracted from Tripterygium wilfordi, exhibits anti-obesity effects in in vitro and in vivo murine models. This study describes how celastrol affects multilineage differentiation potential of human adipose-derived stem cells (hADSCs). We performed in vitro adipogenic differentiation of hADSCs and investigated how celastrol-induced lipid accumulation and expression of adipocyte differentiation markers varied with dose, duration, and donor age. In addition, we assessed the effect of celastrol on osteogenic and chondrogenic differentiation of hADSCs. During adipogenic induction of hADSCs, the inhibitory effect of celastrol on lipid accumulation and adipogenesis depended on dose, duration, time of administration, and individual donor. Inhibition was mediated by proliferator-activated receptor-γ (PPARG) and CCAAT/enhancer-binding protein alpha (CEBPA). Celastrol also suppressed differentiation of hADSCs into the osteogenic and chondrogenic lineages. Celastrol plays a regulatory role in multilineage differentiation of human MSCs. Our findings provide important insights regarding management of obesity and stem cell therapy.
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Adipogénesis/efectos de los fármacos , Tejido Adiposo/citología , Células Madre/citología , Triterpenos/farmacología , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Biomarcadores/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Persona de Mediana Edad , Triterpenos Pentacíclicos , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Triterpenos/administración & dosificación , Adulto JovenRESUMEN
The generation of induced neural stem cells (iNSCs) from somatic cells using defined factors provides new avenues for basic research and cell therapies for various neurological diseases, such as Parkinson's disease, Huntington's disease, and spinal cord injuries. However, the transcription factors used for direct reprogramming have the potential to cause unexpected genetic modifications, which limits their potential application in cell therapies. Here, we show that a combination of four chemical compounds resulted in cells directly acquiring a NSC identity; we termed these cells chemically-induced NSCs (ciNSCs). ciNSCs expressed NSC markers (Pax6, PLZF, Nestin, Sox2, and Sox1) and resembled NSCs in terms of their morphology, self-renewal, gene expression profile, and electrophysiological function when differentiated into the neuronal lineage. Moreover, ciNSCs could differentiate into several types of mature neurons (dopaminergic, GABAergic, and cholinergic) as well as astrocytes and oligodendrocytes in vitro. Taken together, our results suggest that stably expandable and functional ciNSCs can be directly reprogrammed from mouse fibroblasts using a combination of small molecules without any genetic manipulation, and will provide a new source of cells for cellular replacement therapy of neurodegenerative diseases.
Asunto(s)
Técnicas de Reprogramación Celular/métodos , Reprogramación Celular/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/citología , Animales , Diferenciación Celular , Línea Celular , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Células-Madre Neurales/metabolismo , Neurogénesis , Neuronas/citología , Neuronas/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
In a previous study, we isolated human amniotic fluid (AF)-derived mesenchymal stem cells (AF-MSCs) and utilized normoxic conditioned medium (AF-MSC-norCM) which has been shown to accelerate cutaneous wound healing. Because hypoxia enhances the wound healing function of mesenchymal stem cell-conditioned medium (MSC-CM), it is interesting to explore the mechanism responsible for the enhancement of wound healing function. In this work, hypoxia not only increased the proliferation of AF-MSCs but also maintained their constitutive characteristics (surface marker expression and differentiation potentials). Notably, more paracrine factors, VEGF and TGF-ß1, were secreted into hypoxic conditioned medium from AF-MSCs (AF-MSC-hypoCM) compared to AF-MSC-norCM. Moreover, AF-MSC-hypoCM enhanced the proliferation and migration of human dermal fibroblasts in vitro, and wound closure in a skin injury model, as compared to AF-MSC-norCM. However, the enhancement of migration of fibroblasts accelerated by AF-MSC-hypoCM was inhibited by SB505124 and LY294002, inhibitors of TGF-ß/SMAD2 and PI3K/AKT, suggesting that AF-MSC-hypoCM-enhanced wound healing is mediated by the activation of TGF-ß/SMAD2 and PI3K/AKT. Therefore, AF-MSC-hypoCM enhances wound healing through the increase of hypoxia-induced paracrine factors via activation of TGF-ß/SMAD2 and PI3K/AKT pathways.
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Hipoxia de la Célula , Medios de Cultivo Condicionados/farmacología , Transducción de Señal/efectos de los fármacos , Piel/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Líquido Amniótico/citología , Animales , Benzodioxoles/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cromonas/farmacología , Femenino , Fibroblastos/citología , Humanos , Imidazoles/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos ICR , Morfolinas/farmacología , Piridinas/farmacología , Piel/patología , Proteína Smad2/antagonistas & inhibidores , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Oct4-Sox2-Nanog transcriptional networks are critical for the maintenance of embryonic stem (ES) cell self-renewal and induction of pluripotency. However, in transcription factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), Nanog is initially dispensable and Oct4 remains the sole factor that could not be substituted/omitted. Here, we show that mouse fibroblasts could be reprogrammed into iPSCs by Nanog and Bmi1, which replaces Sox2, Klf4, and c-Myc, in the absence of Oct4. Furthermore, we show that in the presence of shh agonists (oxysterol and purmophamine), which replaces the function of Bmi1, a single transcription factor, Nanog is sufficient to reprogram mouse fibroblasts into iPSCs. Nanog-induced iPSCs resemble mESCs in terms of morphology, global gene expression profiles, epigenetic status and pluripotency both in vitro and in vivo. These findings support that Nanog can replace the Oct4 for the somatic cell reprogramming and underlie the mechanisms of Nanog in reprogramming process.
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Reprogramación Celular , Fibroblastos/citología , Proteínas de Homeodominio/metabolismo , Células Madre Pluripotentes Inducidas/citología , Animales , Células Cultivadas , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas de Homeodominio/genética , Factor 4 Similar a Kruppel , Ratones , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
Spinal cord injury (SCI) is a clinical condition that leads to permanent and/or progressive disabilities of sensory, motor, and autonomic functions. Unfortunately, no medical standard of care for SCI exists to reverse the damage. Here, we assessed the effects of induced neural stem cells (iNSCs) directly converted from human urine cells (UCs) in SCI rat models. We successfully generated iNSCs from human UCs, commercial fibroblasts, and patient-derived fibroblasts. These iNSCs expressed various neural stem cell markers and differentiated into diverse neuronal and glial cell types. When transplanted into injured spinal cords, UC-derived iNSCs survived, engrafted, and expressed neuronal and glial markers. Large numbers of axons extended from grafts over long distances, leading to connections between host and graft neurons at 8 weeks post-transplantation with significant improvement of locomotor function. This study suggests that iNSCs have biomedical applications for disease modeling and constitute an alternative transplantation strategy as a personalized cell source for neural regeneration in several spinal cord diseases.
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Células-Madre Neurales , Traumatismos de la Médula Espinal , Humanos , Ratas , Animales , Células-Madre Neurales/metabolismo , Traumatismos de la Médula Espinal/terapia , Traumatismos de la Médula Espinal/metabolismo , Neuronas/metabolismo , Axones , Médula Espinal , Diferenciación Celular/fisiologíaRESUMEN
X-linked Adrenoleukodystrophy (X-ALD) is a neuro-metabolic disorder that is caused by malfunction of a peroxisomal transporter protein, adenosine ATP-binding cassette transporter superfamily D member 1 (ABCD1). We established an induced pluripotent stem cell (iPSC) line from a 42-year-old male X-ALD patient-derived dermal fibroblasts with Sendai virus-mediated reprogramming. Established iPSCs stably expanded, expressed genes of pluripotency, and maintained normal karyotype. In vitro differentiation assay revealed the characteristics of all three germ layers.
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Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP/genética , Adrenoleucodistrofia/genética , Línea Celular , Células Madre Pluripotentes Inducidas , Adulto , Diferenciación Celular , Técnicas de Reprogramación Celular , Humanos , Mutación INDEL , Cariotipo , MasculinoRESUMEN
Human neural stem cells (NSCs) hold enormous promise for neurological disorders, typically requiring their expandable and differentiable properties for regeneration of damaged neural tissues. Despite the therapeutic potential of induced NSCs (iNSCs), a major challenge for clinical feasibility is the presence of integrated transgenes in the host genome, contributing to the risk for undesired genotoxicity and tumorigenesis. Here, we describe the advanced transgene-free generation of iNSCs from human urine-derived cells (HUCs) by combining a cocktail of defined small molecules with self-replicable mRNA delivery. The established iNSCs were completely transgene-free in their cytosol and genome and further resembled human embryonic stem cell-derived NSCs in the morphology, biological characteristics, global gene expression, and potential to differentiate into functional neurons, astrocytes, and oligodendrocytes. Moreover, iNSC colonies were observed within eight days under optimized conditions, and no teratomas formed in vivo, implying the absence of pluripotent cells. This study proposes an approach to generate transplantable iNSCs that can be broadly applied for neurological disorders in a safe, efficient, and patient-specific manner.
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Técnicas de Reprogramación Celular/métodos , Reprogramación Celular , Células-Madre Neurales/citología , ARN Mensajero/metabolismo , Orina/citología , Adulto , Animales , Células Cultivadas , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células-Madre Neurales/metabolismo , ARN Mensajero/genética , TransgenesRESUMEN
Charcot-Marie-Tooth disease (CMTX) is inherited neurological disorder caused by gap junction beta 1 gene (GJB1) mutation. We generated induced pluripotent stem cell (iPSC) line from 36-year-old CMTX disease patient by electroporation of skin fibroblasts with episomal vectors encoding OCT4, SOX2, KLF4, L-MYC, LIN28 and shRNA-p53. Established iPSCs expressed various pluripotency markers, had differentiation potential of three germ layers in vitro, had normal karyotype and retained GJB1 mutation. This CMT patient-derived iPSC line could be useful in vitro tool for CMTX research as disease modeling and drug development.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Conexinas , Fibroblastos , Células Madre Pluripotentes Inducidas , Mutación , Piel , Adulto , Línea Celular , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Enfermedad de Charcot-Marie-Tooth/patología , Conexinas/genética , Conexinas/metabolismo , Electroporación , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Factor 4 Similar a Kruppel , Masculino , Piel/metabolismo , Piel/patología , Factores de Transcripción/sangre , Factores de Transcripción/genética , Proteína beta1 de Unión ComunicanteRESUMEN
Charcot-Marie-Tooth disease (CMT1B) is an inherited neurological disorder caused by mutation of the myelin protein zero (MPZ) gene. We generated an induced pluripotent stem cell (iPSC) line from an 81-year-old patient with CMT1B by electroporating of lymphoblastoid cell lines with episomal plasmids encoding OCT4, SOX2, KLF4, L-MYC, LIN28, and p53-targeting shRNA. The established iPSCs expressed various pluripotency markers, demonstrated the potential to differentiate into cells of the three germ layers in vitro, had a normal karyotype and retained the MPZ mutation.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Células Madre Pluripotentes Inducidas/metabolismo , Proteína P0 de la Mielina/genética , Factores de Transcripción/metabolismo , Anciano de 80 o más Años , Diferenciación Celular , Línea Celular , Humanos , Factor 4 Similar a Kruppel , Masculino , Mutación , Proteína P0 de la Mielina/metabolismoRESUMEN
X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder caused by a mutation in the ATP-binding cassette transporter subfamily D member 1 (ABCD1) gene. We generated two induced pluripotent stem cell (iPSC) lines from X-ALD patients with adrenomyeloneuropathy (AMN) by Sendai virus containing OCT4, SOX2, KLF4 and c-MYC. Established iPSC lines expressed various pluripotency markers, had differentiation potential of three germ layers in vitro, had normal karyotype and retained ABCD1 mutation.
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Adrenoleucodistrofia/metabolismo , Células Madre Pluripotentes Inducidas/citología , Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP/metabolismo , Células Cultivadas , Citometría de Flujo , Humanos , Inmunohistoquímica , Cariotipo , Factor 4 Similar a Kruppel , Mutación/genética , FenotipoRESUMEN
X-linked Adrenoleukodystrophy (X-ALD) is a genetic disease that caused by mutations in adenosine triphosphate [ATP]-binding-cassette transporter superfamily D member 1 (ABCD1) gene. We generated an induced pluripotent stem cell (iPSC) line from a 21-year-old male X-ALD patient-derived fibroblasts by Sendai virus mediated reprogramming. Established iPSCs stably expanded while maintaining immunoreactivity for various pluripotency markers and alkaline phosphatase, as well as normal 44+XY karyotype. Under the differentiation condition, the cells gave rise to cells of three germ layers.
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Adrenoleucodistrofia/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Células Madre Pluripotentes Inducidas/citología , Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP/genética , Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP/metabolismo , Adrenoleucodistrofia/metabolismo , Diferenciación Celular , Línea Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , MutaciónRESUMEN
Somatic cells can be reprogrammed to generate induced pluripotent stem cells (iPSCs) by overexpression of four transcription factors, Oct4, Klf4, Sox2, and c-Myc. However, exogenous expression of pluripotency factors raised concerns for clinical applications. Here, we show that iPS-like cells (iPSLCs) were generated from mouse somatic cells in two steps with small molecule compounds. In the first step, stable intermediate cells were generated from mouse astrocytes by Bmi1. These cells called induced epiblast stem cell (EpiSC)-like cells (iEpiSCLCs) are similar to EpiSCs in terms of expression of specific markers, epigenetic state, and ability to differentiate into three germ layers. In the second step, treatment with MEK/ERK and GSK3 pathway inhibitors in the presence of leukemia inhibitory factor resulted in conversion of iEpiSCLCs into iPSLCs that were similar to mESCs, suggesting that Bmi1 is sufficient to reprogram astrocytes to partially reprogrammed pluripotency. Next, Bmi1 function was replaced with Shh activators (oxysterol and purmorphamine), which demonstrating that combinations of small molecules can compensate for reprogramming factors and are sufficient to directly reprogram mouse somatic cells into iPSLCs. The chemically induced pluripotent stem cell-like cells (ciPSLCs) showed similar gene expression profiles, epigenetic status, and differentiation potentials to mESCs.