RESUMEN

Recently, we and others generated induced tissue-specific stem/progenitor (iTS/iTP) cells. The advantages of iTS/iTP cells compared with induced pluripotent stem (iPS) cells are (1) easier generation, (2) efficient differentiation, and (3) no teratomas formation. In this study, we generated mouse induced pancreatic stem cells (iTS-P cells) by the plasmid vector expressing Yes-associated protein 1 (YAP). The iTS-P YAP9 cells expressed Foxa2 (endoderm marker) and Pdx1 (pancreatic marker) while the expressions of Oct3/4 and Nanog (marker of embryonic stem [ES] cells) in iTS-P YAP9 cells was significantly lower compared with those in ES cells. The iTS-P YAP9 cells efficiently differentiated into insulin-expressing cells compared with ES cells. The ability to generate autologous iTS cells may be applied to diverse applications of regenerative medicine.

Asunto(s)

Diferenciación Celular , Células Madre Pluripotentes Inducidas , Proteínas Señalizadoras YAP , Animales , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Páncreas/citología , Páncreas/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Transactivadores/metabolismo , Transactivadores/genética

RESUMEN

Previous studies have demonstrated that extracellular vesicles (EVs) from dental pulp stem cells (DPSCs), which release abundant hepatocyte growth factor (HGF) and transforming growth factor-ß1 (TGF-ß1), contribute to the pathogenesis of Sjögren's syndrome (SS). However, depending on the condition of DPSCs, this effect is often not achieved. In this study, we established induced pluripotent stem (iPS) cells highly capable of releasing HGF and TGF-ß1 and iPS cells barely capable of releasing them, and administered each EV to SS model mice to see if there was a difference in therapeutic effect. EVs were collected from each iPS cell and their characteristics and shapes were examined. When they were administered to SS model mice, the EVs from iPS cells with higher concentrations of HGF and TGF-ß1 showed significantly reduced inflammatory cell infiltration in salivary gland tissues, increased saliva volume, and decreased anti-SS-A and anti-SS-B antibodies. A comprehensive search of microRNA arrays for differences among those EVs revealed that EVs from iPS cells with higher concentrations of HGF and TGF-ß1 contained more of the let-7 family. Thereafter, we examined the expression of toll-like receptors (TLRs), which are said to be regulated by the let-7 family, by qPCR, and found decreased TLR4 expression. Focusing on MAPK, a downstream signaling pathway, we examined cytokine concentrations in mouse macrophage culture supernatants and Western blotting of murine splenic tissues and found higher concentrations of anti-inflammatory cytokines in the EVs-treated group and decreased TLR4, NF-κB and phosphorylation (p)-p-38 MAPK expression by Western blotting. Alternatively, p-Smad2/3 was upregulated in the EVs-treated group. Our findings suggest that the let-7 family in EVs may suppress the expression of TLR4 and NF-κB, which may be involved in the suppression of MAPK-mediated pro-inflammatory cytokine production.

Asunto(s)

Vesículas Extracelulares , Células Madre Pluripotentes Inducidas , Síndrome de Sjögren , Animales , Ratones , Vesículas Extracelulares/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Inmunidad Innata , Células Madre Pluripotentes Inducidas/metabolismo , FN-kappa B/metabolismo , Síndrome de Sjögren/metabolismo , Síndrome de Sjögren/patología , Receptor Toll-Like 4/metabolismo , Factor de Crecimiento Transformador beta1

RESUMEN

Mitochondria are the powerhouse of the cell and dynamically control fundamental biological processes including cell reprogramming, pluripotency, and lineage specification. Although remarkable progress in induced pluripotent stem cell (iPSC)-derived cell therapies has been made, very little is known about the role of mitochondria and the mechanisms involved in somatic cell reprogramming into iPSC and directed reprogramming of iPSCs in terminally differentiated cells. Reprogramming requires changes in cellular characteristics, genomic and epigenetic regulation, as well as major mitochondrial metabolic changes to sustain iPSC self-renewal, pluripotency, and proliferation. Differentiation of autologous iPSC into terminally differentiated ß-like cells requires further metabolic adaptation. Many studies have characterized these alterations in signaling pathways required for the generation and differentiation of iPSC; however, very little is known regarding the metabolic shifts that govern pluripotency transition to tissue-specific lineage differentiation. Understanding such metabolic transitions and how to modulate them is essential for the optimization of differentiation processes to ensure safe iPSC-derived cell therapies. In this review, we summarize the current understanding of mitochondrial metabolism during somatic cell reprogramming to iPSCs and the metabolic shift that occurs during directed differentiation into pancreatic ß-like cells.

Asunto(s)

Epigénesis Genética , Células Madre Pluripotentes , Humanos , Diferenciación Celular , Reprogramación Celular , Células Madre Pluripotentes/metabolismo , Mitocondrias/metabolismo

RESUMEN

The following protocol describes the generation of microglia cells from human-induced pluripotent stem cells (hiPSCs) using commercially available kits by StemCell Technologies. This protocol consists of three major steps: (1) Differentiation of hematopoietic precursor cells, (2) Microglia differentiation, and (3) Microglia maturation. Assays are described to characterize hematopoietic precursor cells and mature microglia.

Asunto(s)

Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Humanos , Microglía , Células Madre Embrionarias , Células Madre Hematopoyéticas , Diferenciación Celular

RESUMEN

Induced pluripotent stem cell (iPSC) techniques have had considerable breakthroughs in modeling human neurological diseases. Multiple protocols inducing neurons, astrocytes, microglia, oligodendrocytes, and endothelial cells have been well-established thus far. However, these protocols have limitations, including the long time period to get cells of interest or the challenge of culturing more than one cell type simultaneously. Protocols for handling multiple cell types within a shorter time period are still being established. Here we describe a simple and reliable co-culture system to study interactions between neurons and oligodendrocyte precursor cells (OPC) in health and in disease.

Asunto(s)

Células Madre Pluripotentes Inducidas , Humanos , Técnicas de Cocultivo , Células Endoteliales , Neuronas/metabolismo , Oligodendroglía , Diferenciación Celular

RESUMEN

Pluripotent stem cells have a multitude of potential applications in the areas of disease modeling, drug screening, and cell-based therapies for genetic diseases, including muscular dystrophies. The advent of induced pluripotent stem cell technology allows for the facile derivation of disease-specific pluripotent stem cells for any given patient. Targeted in vitro differentiation of pluripotent stem cells into the muscle lineage is a key step to enable all these applications. Transgene-based differentiation using conditional expression of the transcription factor PAX7 leads to the efficient derivation of an expandable and homogeneous population of myogenic progenitors suitable for both in vitro and in vivo applications. Here, we describe an optimized protocol for the derivation and expansion of myogenic progenitors from pluripotent stem cells using conditional expression of PAX7. Importantly, we further describe an optimized procedure for the terminal differentiation of myogenic progenitors into more mature myotubes, which are better suited for in vitro disease modeling and drug screening studies.

Asunto(s)

Células Madre Pluripotentes Inducidas , Distrofias Musculares , Células Madre Pluripotentes , Humanos , Fibras Musculares Esqueléticas , Distrofias Musculares/metabolismo , Diferenciación Celular , Desarrollo de Músculos/genética

RESUMEN

To master the technology of reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs), which will lay a good foundation for setting up a technology platform on reprogramming human cancer cells into iPSCs. Mouse iPSCs (i.e., Oct4-GFP miPSCs) was successfully generated from mouse embryonic fibroblasts (MEFs) harboring Oct4-EGFP transgene by introducing four factors, Oct4, Sox2, c-Myc and Klf4, under mESC (Murine embryonic stem cells) culture conditions. Oct4-GFP miPSCs were similar to mESCs in morphology, proliferation, mESC-specific surface antigens and gene expression. Additionally, Oct4-GFP miPSCs could be cultured in suspension to form embryoid bodies (EBs) and differentiate into cell types of the three germ layers in vitro. Moreover, Oct4-GFP miPSCs could develop to teratoma and chimera in vivo. Unlike cell cycle distribution of MEFs, Oct4-GFP miPSCs are similar to mESCs in the cell cycle structure which consists of higher S phase and lower G1 phase. More importantly, our data demonstrated that MEFs harboring Oct4-EGFP transgene did not express GFP, until they were reprogrammed to the pluripotent stage (iPSCs), while the GFP expression was progressively lost when these pluripotent Oct4-GFP miPSCs exposed to EB-mediated differentiation conditions, suggesting the pluripotency of Oct4-GFP miPSCs can be real-time monitored over long periods of time via GFP assay. Altogether, our findings demonstrate that Oct4-GFP miPSC line is successfully established, which will lay a solid foundation for setting up a technology platform on reprogramming cancer cells into iPSCs. Furthermore, this pluripotency reporter system permits the long-term real-time monitoring of pluripotency changes in a live single-cell, and its progeny.

Asunto(s)

Células Madre Pluripotentes Inducidas , Animales , Diferenciación Celular , Células Cultivadas , Reprogramación Celular/genética , Células Madre Embrionarias , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Ratones

RESUMEN

Corneal epithelium maintains visual acuity and is regenerated by the proliferation and differentiation of limbal progenitor cells. Transplantation of human limbal progenitor cells could restore the integrity and functionality of the corneal surface in patients with limbal stem cell deficiency. However, multiple protocols are employed to differentiate human induced pluripotent stem (iPS) cells into corneal epithelium or limbal progenitor cells. The aim of this study was to optimize a protocol that uses bone morphogenetic protein 4 (BMP4) and limbal cell-specific medium. Human dermal fibroblast-derived iPS cells were differentiated into limbal progenitor cells using limbal cell-specific (PI) medium and varying doses (1, 10, and 50 ng/mL) and durations (1, 3, and 10 days) of BMP4 treatment. Differentiated human iPS cells were analyzed by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemical studies at 2 or 4 weeks after BMP4 treatment. Culturing human dermal fibroblast-derived iPS cells in limbal cell-specific medium and BMP4 gave rise to limbal progenitor and corneal epithelial-like cells. The optimal protocol of 10 ng/mL and three days of BMP4 treatment elicited significantly higher limbal progenitor marker (ABCG2, ∆Np63α) expression and less corneal epithelial cell marker (CK3, CK12) expression than the other combinations of BMP4 dose and duration. In conclusion, this study identified a successful reprogramming strategy to induce limbal progenitor cells from human iPS cells using limbal cell-specific medium and BMP4. Additionally, our experiments indicate that the optimal BMP4 dose and duration favor limbal progenitor cell differentiation over corneal epithelial cells and maintain the phenotype of limbal stem cells. These findings contribute to the development of therapies for limbal stem cell deficiency disorders.

Asunto(s)

Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Epitelio Corneal/citología , Epitelio Corneal/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Biomarcadores , Línea Celular , Linaje de la Célula/genética , Células Cultivadas , Humanos

RESUMEN

Stem cells play essential roles in the development and tissue homeostasis of animals and are closely associated with carcinogenesis and aging. Also, the somatic cell reprogramming process to induced pluripotent stem (iPS) cells shares several characteristics with carcinogenesis. In this chapter, we focus on iPS cells and the reprogramming process of somatic cells in the naked mole-rat (NMR), the longest-living rodent with remarkable cancer resistance capabilities. NMR somatic cells show resistance to reprogramming induction, and generated NMR-iPS cells have a unique tumor-resistant phenotype. This phenotype is regulated by expressional activation of the tumor suppressor ARF gene and loss-of-function mutation in oncogene ERAS. Notably, it was also found that NMR somatic cells undergo senescence when ARF is suppressed during reprogramming, which would contribute to the resistance to both reprogramming and cancer in NMR somatic cells. Further studies on reprogramming resistance in NMR somatic cells and their concomitant tumor resistance in NMR-iPS cells would contribute to a better understanding of both cancer resistance and delayed aging in NMRs. In addition, NMR-iPS cells can be used as a new and important cell source for advancing research concerning several extraordinary physiological characteristics of NMR. Furthermore, study of NMR-iPS cells could lead to the development of safer regenerative therapies in the future.

Asunto(s)

Células Madre Pluripotentes Inducidas , Neoplasias , Animales , Reprogramación Celular , Ratas Topo/genética , Neoplasias/genética , Oncogenes

RESUMEN

We previously reported that transient overexpression of reprogramming factors can be used to generate induced pluripotent stem (iPS) cells, induced tissue-specific stem (iTS) cells, and fibroblast-like (iF) cells from pancreatic tissue. iF cells have tumorigenic ability and behave similarly to pancreatic cancer cells. In this study, we analyzed gene expression in iF cells and iTS-P cells (iTS cells from pancreatic tissue) via microarray analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The expression levels of the Mybl2 and Lyn genes, which are reported to be oncogenes, were significantly higher in iF cells than in iTS-P cells. The expression level of Nestin, which is expressed in not only pancreatic progenitor cells but also pancreatic ductal adenocarcinomas, was also higher in iF cells than in iTS-P cells. Itgb6 and Fgf13, which are involved in the pathogenesis of diseases such as cancer, exhibited higher expression levels in iF cells than in iTS-P cells. Unexpectedly, the expression levels of genes related to epithelial-mesenchymal transition (EMT), except Bmp4, were lower in iF cells than in iTS-P cells. These data suggest that the Mybl2, Lyn, Nestin, Itgb6, and Fgf13 genes could be important biomarkers to distinguish iTS-P cells from iF cells.

RESUMEN

BACKGROUND: Vivax malaria is associated with significant morbidity and economic loss, and constitutes the bulk of malaria cases in large parts of Asia and South America as well as recent case reports in Africa. The widespread prevalence of vivax is a challenge to global malaria elimination programmes. Vivax malaria control is particularly challenged by existence of dormant liver stage forms that are difficult to treat and are responsible for multiple relapses, growing drug resistance to the asexual blood stages and host-genetic factors that preclude use of specific drugs like primaquine capable of targeting Plasmodium vivax liver stages. Despite an obligatory liver-stage in the Plasmodium life cycle, both the difficulty in obtaining P. vivax sporozoites and the limited availability of robust host cell models permissive to P. vivax infection are responsible for the limited knowledge of hypnozoite formation biology and relapse mechanisms, as well as the limited capability to do drug screening. Although India accounts for about half of vivax malaria cases world-wide, very little is known about the vivax liver stage forms in the context of Indian clinical isolates. METHODS: To address this, methods were established to obtain infective P. vivax sporozoites from an endemic region in India and multiple assay platforms set up to detect and characterize vivax liver stage forms. Different hepatoma cell lines, including the widely used HCO4 cells, primary human hepatocytes as well as hepatocytes obtained from iPSC's generated from vivax patients and healthy donors were tested for infectivity with P. vivax sporozoites. RESULTS: Both large and small forms of vivax liver stage are detected in these assays, although the infectivity obtained in these platforms are low. CONCLUSIONS: This study provides a proof of concept for detecting liver stage P. vivax and provide the first characterization of P. vivax liver stage forms from an endemic region in India.

Asunto(s)

Estadios del Ciclo de Vida , Hígado/parasitología , Malaria Vivax/parasitología , Plasmodium vivax/crecimiento & desarrollo , India , Plasmodium vivax/aislamiento & purificación

RESUMEN

We investigated whether sequential reprogramming via porcine induced pluripotent stem cells (piPSCs) or exposure to oocyte cytoplasm following nuclear transfer could generate nuclear transfer-derived ESCs (piPSCs-ntESCs). Nuclear transfer embryos were reconstructed with piPSCs possessing a ZsGreen fluorescent marker for expression of exogenous Nanog and Lin28. Reconstructed oocytes developed to morphologically normal 8-cell/morulae (35/93, 37.6%) and blastocysts (12/93, 12.9%). Although most green fluorescent protein-positive blastocysts showed efficient outgrowth (8/10, 80%), none formed primary colonies and all cultures degenerated. Conversely, 15% of fluorescent positive 8-cell/morula stage embryos showed outgrowth (6/40), with three forming primary colonies (7.5%). All three were expanded and maintained as piPSC-ntESC lines. These cell lines expressed stem cell marker genes and proteins. Despite inactivation of one X chromosome, all piPSC-ntESC lines formed teratomas comprising derivatives from all three embryonic germ layers. Strong SSEA1, 3, and 4 expression was detected at the 8-cell/morula stage in embryos reconstructed from both piPSCs and porcine embryonic fibroblasts (PEFs). SSEA3 was notably absent from IVF controls at pre-implantation embryo stages. Finally, we attempted to establish ntESCs from 8-cell/morulae reconstructed with PEFs using the same culture conditions as those for piPSC-ntESC derivation. Although eight primary colonies arose from 107 embryos (7.5%), they all degenerated after the first passage culture. Early and sustained expression of key reprogramming regulatory factors may be critical for pluripotent stem cell derivation to derive piPSC-ntESCs from 8-cell/morula stages, while the expression of SSEAs may be involved in the initial stem cell colony formation phases.

Asunto(s)

Embrión de Mamíferos/citología , Células Madre Embrionarias/citología , Células Madre Pluripotentes Inducidas/citología , Técnicas de Transferencia Nuclear , Animales , Técnicas de Cultivo de Célula , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/metabolismo , Femenino , Células Madre Pluripotentes Inducidas/metabolismo , Oocitos/citología , Oocitos/metabolismo , Porcinos

RESUMEN

BACKGROUND: Stem cell transplantation represents a potential therapeutic option for muscular dystrophies (MD). However, to date, most reports have utilized mouse models for recessive types of MD. Here we performed studies to determine whether myotonic dystrophy 1 (DM1), an autosomal dominant type of MD, could benefit from cell transplantation. METHODS: We injected human pluripotent stem (PS) cell-derived myogenic progenitors into the muscles of a novel mouse model combining immunodeficiency and skeletal muscle pathology of DM1 and investigated transplanted mice for engraftment as well as for the presence of RNA foci and alternative splicing pattern. FINDINGS: Engraftment was clearly observed in recipient mice, but unexpectedly, we detected RNA foci in donor-derived engrafted myonuclei. These foci proved to be pathogenic as we observed MBNL1 sequestration and abnormal alternative splicing in donor-derived transcripts. INTERPRETATION: It has been assumed that toxic CUG repeat-containing RNA forms foci in situ in the nucleus in which it is expressed, but these data suggest that CUG repeat-containing RNA may also exit the nucleus and traffic to other nuclei in the syncytial myofiber, where it can exert pathological effects. FUND: This project was supported by funds from the LaBonte/Shawn family and NIH grants R01 AR055299 and AR071439 (R.C.R.P.). R.M-G. was funded by CONACyT-Mexico (#394378).

Asunto(s)

Núcleo Celular/genética , Músculo Esquelético/metabolismo , Distrofia Miotónica/genética , ARN/genética , Empalme Alternativo , Animales , Núcleo Celular/metabolismo , Modelos Animales de Enfermedad , Huésped Inmunocomprometido , Ratones , Células Musculares/citología , Células Musculares/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , ARN/administración & dosificación

RESUMEN

Stem cells hold tremendous promise for replacing or regenerating tissues damaged by injury and disease as well as to study developmental biology and pathomechanisms. The discovery of methods to generate and culture human pluripotent stem cells (hESC and hiPSC) paved the way for producing genetically defined organ and tissue-specific cell types in a controlled laboratory setting. Cell and tissue engineering approaches have proven essential to unlocking the power of human pluripotent stem cells for both disease modeling and regenerative medicine. This editorial summarizes impressive examples of burgeoning research by leading groups that harness cellular and tissue engineering principles to study mechanisms of disease and injury, and in the context of repairing damaged tissue. These studies highlight both the power of these approaches, as well as ongoing challenges in the field.

Asunto(s)

Células Madre Pluripotentes Inducidas/metabolismo , Medicina Regenerativa , Ingeniería de Tejidos , Animales , Humanos , Células Madre Pluripotentes Inducidas/citología

RESUMEN

Cancer stem cells (CSCs) are specific targets for therapeutic applications, but the rarity of CSCs within tumors makes the isolation of CSCs difficult. To overcome these problems, we generated CSCs in vitro using established reprogramming techniques. We transduced four previously established reprogramming factors, Oct3/4, Sox2, Klf4, and L-myc, into the colon cancer cell lines LoVo and OUMS-23, and investigated the biological characteristics of these lines. Tra-1-60+ cells were obtained from reprogrammed induced pluripotent stem (iPS) cell-like colonies and showed CSC properties, including colony formation, maintenance of colonies by repeated passages, and feeder cell dependency, as well as increased expressions of CSC markers such as CD133 and ALDH1. The CSC-like cells showed increased chemoresistance to 5-fluorouracil and elevated tumorigenicity upon transplantation into kidneys of immune-deficient mice. These tumors shifted to a poorly differentiated stage with many atypical cells, cytoplasmic mucin, and focal papillary components, with demonstrated dedifferentiation. The principal component analysis from DNA microarrays showed that though both cell lines moved to iPS cells after reprogramming, they were not completely identical to iPS cells. Significantly elevated gene expression of Decorin and CD90 was observed in CSC-like cells. Together, these results show that reprogramming of cancer cells produced not pluripotent stem cells but CSC-like cells, and these findings will provide biological information about genuine CSCs and help establish new CSC-targeted therapies.

Asunto(s)

Reprogramación Celular , Neoplasias del Colon/patología , Células Madre Neoplásicas/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Células Madre Pluripotentes Inducidas/citología , Factor 4 Similar a Kruppel , Ratones SCID , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Adipose-derived mesenchymal stem cells (ADSCs) have attracted attention due to their potential for use in the treatment of various diseases. However, the self-renewal capacity of ADSCs is restricted and their function diminishes during passage. We previously generated induced tissue-specific stem cells from mouse pancreatic cells using a single synthetic self-replicating Venezuelan Equine Encephalitis (VEE)-reprogramming factor (RF) RNA replicon (SR-RNA) expressing the reprogramming factors POU class 5 homeobox 1 (OCT4), Krueppel-like factor 4 (KLF4), Sex determining region Y-box 2 (SOX2), and Glis Family Zinc Finger 1 (GLIS1). This vector was used to generate induced pluripotent stem (iPS) cells. Here, we applied this SR-RNA vector to generate human iTS cells from aged mesenchymal stem cells (hiTS-M cells) deficient in self-renewal that were derived from adipose tissue. These hiTS-M cells transfected with the SR-RNA vector survived for 15 passages. The hiTS-M cells expressed cell surface markers similar to those of human adipose-derived mesenchymal stem cells (hADSCs) and differentiated into fat cells and osteoblasts. Global gene expression profiling showed that hiTS-M cells were transcriptionally similar to hADSCs. These data suggest that the generation of iTS cells has important implications for the clinical application of autologous stem cell transplantation.

Asunto(s)

Tejido Adiposo/citología , Reprogramación Celular/genética , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , ARN/genética , Adulto , Células Cultivadas , Biología Computacional/métodos , Femenino , Perfilación de la Expresión Génica , Vectores Genéticos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Proteína Homeótica Nanog/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , ARN/síntesis química , Transcriptoma

RESUMEN

Myotonic dystrophy 1 (DM1) is a multisystem disorder primarily affecting the central nervous system, heart and skeletal muscle. It is caused by an expansion of the CTG trinucleotide repeats in the 3' untranslated region of the DMPK gene. Although patient myoblasts have been used for studying the disease in vitro, the invasiveness as well as the low accessibility to muscle biopsies motivate the development of alternative reliable myogenic models. Here, we established two DM1 induced pluripotent stem (iPS) cell lines from patient-derived fibroblasts and, using the PAX7 conditional expression system, differentiated these into myogenic progenitors and, subsequently, terminally differentiated myotubes. Both DM1 myogenic progenitors and myotubes were found to express the intranuclear RNA foci exhibiting sequestration of MBNL1. Moreover, we found the DM1-related mis-splicing, namely BIN1 exon 11 in DM1 myotubes. We used this model to test a specific therapy, antisense oligonucleotide treatment, and found that this efficiently abolished RNA foci and rescued BIN1 mis-splicing in DM1 iPS cell-derived myotubes. Together, our results demonstrate that myotubes derived from DM1 iPS cells recapitulate the critical molecular features of DM1 and are sensitive to antisense oligonucleotide treatment, confirming that these cells can be used for in vitro disease modeling and candidate drug testing or screening.This article has an associated First Person interview with the first author of the paper.

Asunto(s)

Descubrimiento de Drogas , Células Madre Pluripotentes Inducidas/patología , Distrofia Miotónica/tratamiento farmacológico , Distrofia Miotónica/patología , Animales , Huesos/patología , Diferenciación Celular , Reprogramación Celular , Modelos Animales de Enfermedad , Fibroblastos/patología , Humanos , Masculino , Ratones Endogámicos NOD , Ratones SCID , Desarrollo de Músculos , Fibras Musculares Esqueléticas/patología , Oligonucleótidos Antisentido/farmacología , Fenotipo

RESUMEN

Induced pluripotent stem (iPS) cells have significant implications for overcoming most of the ethical issues associated with embryonic stem (ES) cells. The pattern of expressed genes, DNA methylation, and covalent histone modifications in iPS cells are very similar to those in ES cells. However, it has recently been shown that, following the reprogramming of mouse/human iPS cells, epigenetic memory is inherited from the parental cells. These findings suggest that the phenotype of iPS cells may be influenced by their cells of origin and that their skewed differentiation potential may prove useful in the generation of differentiated cell types that are currently difficult to produce from ES/iPS cells for the treatment of human diseases. Our recent study demonstrated the generation of induced tissue-specific stem (iTS) cells by transient overexpression of the reprogramming factors combined with tissue-specific selection. iTS cells are cells that inherit numerous components of epigenetic memory from donor tissue and acquire self-renewal potential. This review describes the "epigenetic memory" phenomenon in iPS and iTS cells and the possible clinical applications of these stem cells.

Asunto(s)

Epigénesis Genética , Células Madre Pluripotentes Inducidas/metabolismo , Especificidad de Órganos , Animales , Diferenciación Celular/genética , Metilación de ADN/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Neoplasias/patología , Especificidad de Órganos/genética

RESUMEN

Induced pluripotent stem (iPS) cell technology lead terminally differentiated cells into the pluripotent stem cells through the expression of defined reprogramming factors. Although, iPS cells have been established in a number of mammalian species, including mouse, human, and monkey, studies on iPS cells in avian species are still very limited. To establish chick iPS cells, six factors were used within the poly-cistronic reprogramming vector (PB-R6F), containing M3O (MyoD derived transactivation domain fused with Oct3/4), Sox2, Klf4, c-Myc, Lin28, and Nanog. The PB-R6F derived iPS cells were alkaline-phosphatase and SSEA-1 positive, which are markers of pluripotency. Elevated levels of endogenous Oct3/4 and Nanog genes were detected in the established iPS cells, suggesting the activation of the FGF signaling pathway is critical for the pluripotent status. Histological analysis of teratoma revealed that the established chick iPS cells have differentiation ability into three-germ-layer derived tissues. This is the first report of establishment of avian derived iPS cells with a single poly-cistronic transposon based expression system. The establishment of avian derived iPS cells could contribute to the genetic conservation and modification of avian species.

Asunto(s)

Reprogramación Celular , Elementos Transponibles de ADN , Fibroblastos/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Transcripción Genética , Activación Transcripcional , Animales , Diferenciación Celular , Células Cultivadas , Técnicas de Reprogramación Celular , Pollos , Técnicas de Cocultivo , Femenino , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Fenotipo , Análisis de Secuencia de ADN , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma , Transfección , Transposasas/genética , Transposasas/metabolismo

RESUMEN

Recent reports show an increase in the incidence of Autism Spectrum Disorders (ASD) to 1 in every 59 children up to 8 years old in 11 states in North America. Induced pluripotent stem cell (iPSC) technology offers a groundbreaking platform for the study of polygenic neurodevelopmental disorders in live cells. Robust inflammation states and immune system dysfunctions are associated with ASD and several cell types participate on triggering and sustaining these processes. In this review, we will examine the contribution of neuroinflammation to the development of autistic features and discuss potential therapeutic approaches. We will review the available tools, emphasizing stem cell modeling as a technology to investigate the various molecular pathways and different cell types involved in the process of neuroinflammation in ASD.