RESUMEN
Expansion of the hexanucleotide repeat (HR) in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in Caucasians. All C9orf72-ALS/FTD patients share a common risk (R) haplotype. To study C9orf72 expression and splicing from the mutant R allele compared to the complementary normal allele in ALS/FTD patients, we initially created a detailed molecular map of the single nucleotide polymorphism (SNP) signature and the HR length of the various C9orf72 haplotypes in Caucasians. We leveraged this map to determine the allelic origin of transcripts per patient, and decipher the effects of pathological and normal HR lengths on C9orf72 expression and splicing. In C9orf72 ALS patients' cells, the HR expanded allele, compared to non-R allele, was associated with decreased levels of a downstream initiated transcript variant and increased levels of transcripts initiated upstream of the HR. HR expanded R alleles correlated with high levels of unspliced intron 1 and activation of cryptic donor splice sites along intron 1. Retention of intron 1 was associated with sequential intron 2 retention. The SNP signature of C9orf72 haplotypes described here enables allele-specific analysis of transcriptional products and may pave the way to allele-specific therapeutic strategies.
Asunto(s)
Alelos , Proteína C9orf72/genética , Susceptibilidad a Enfermedades , Regulación de la Expresión Génica , Haplotipos , Empalme del ARN , Esclerosis Amiotrófica Lateral/diagnóstico , Esclerosis Amiotrófica Lateral/etiología , Demencia Frontotemporal/diagnóstico , Demencia Frontotemporal/etiología , Genotipo , Humanos , Intrones , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Sitios de Empalme de ARNRESUMEN
Congenital human cytomegalovirus (HCMV) infection is associated with neurodevelopmental disabilities. To dissect the earliest events of infection in the developing human brain, we studied HCMV infection during controlled differentiation of human embryonic stem cells (hESC) into neural precursors. We traced a transition from viral restriction in hESC, mediated by a block in viral binding, toward HCMV susceptibility in early hESC-derived neural precursors. We further revealed the role of platelet-derived growth factor receptor alpha (PDGFRα) as a determinant of the developmentally acquired HCMV susceptibility.
Asunto(s)
Diferenciación Celular/fisiología , Infecciones por Citomegalovirus/fisiopatología , Citomegalovirus/fisiología , Células Madre Embrionarias/citología , Células-Madre Neurales/virología , Acoplamiento Viral , Factores de Edad , Infecciones por Citomegalovirus/prevención & control , Células Madre Embrionarias/fisiología , Humanos , Células-Madre Neurales/fisiologíaRESUMEN
In the pMN domain of the spinal cord, Notch signaling regulates the balance between motor neuron differentiation and maintenance of the progenitor state for later oligodendrocyte differentiation. Here, we sought to study the role of Notch signaling in regulation of the switch from the pMN progenitor state to differentiated motor neurons in a human model system. Human embryonic stem cells (hESCs) were directed to differentiate to pMN-like progenitor cells by the inductive action of retinoic acid and a Shh agonist, purmorphamine. We found that the expression of the Notch signaling effector Hes5 was induced in hESC-derived pMN-like progenitors and remained highly expressed when they were cultured under conditions favoring motor neuron differentiation. Inhibition of Notch signaling by a γ-secretase inhibitor in the differentiating pMN-like progenitor cells decreased Hes5 expression and enhanced the differentiation toward motor neurons. Conversely, over-expression of Hes5 in pMN-like progenitor cells during the differentiation interfered with retinoic acid- and purmorphamine-induced motor neuron differentiation and inhibited the emergence of motor neurons. Inhibition of Notch signaling had a permissive rather than an inductive effect on motor neuron differentiation. Our results indicate that Notch signaling has a regulatory role in the switch from the pMN progenitor to the differentiated motor neuron state. Inhibition of Notch signaling can be harnessed to enhance the differentiation of hESCs toward motor neurons.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/metabolismo , Neuronas Motoras/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Madre Embrionarias/citología , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/metabolismo , Humanos , Morfolinas/farmacología , Neuronas Motoras/citología , Purinas/farmacología , Transducción de Señal/efectos de los fármacos , Tretinoina/farmacologíaRESUMEN
Many genes associated with CpG islands undergo de novo methylation in cancer. Studies have suggested that the pattern of this modification may be partially determined by an instructive mechanism that recognizes specifically marked regions of the genome. Using chromatin immunoprecipitation analysis, here we show that genes methylated in cancer cells are specifically packaged with nucleosomes containing histone H3 trimethylated on Lys27. This chromatin mark is established on these unmethylated CpG island genes early in development and then maintained in differentiated cell types by the presence of an EZH2-containing Polycomb complex. In cancer cells, as opposed to normal cells, the presence of this complex brings about the recruitment of DNA methyl transferases, leading to de novo methylation. These results suggest that tumor-specific targeting of de novo methylation is pre-programmed by an established epigenetic system that normally has a role in marking embryonic genes for repression.
Asunto(s)
Metilación de ADN , Histonas/metabolismo , Neoplasias/genética , Células CACO-2 , Proteínas Portadoras , Células Cultivadas , Neoplasias del Colon/genética , Islas de CpG/genética , Epigénesis Genética , Humanos , Lisina/metabolismo , Metilación , Metiltransferasas/metabolismo , Proteínas del Envoltorio ViralRESUMEN
Anticancer drugs are at the frontline of cancer therapy. However, innate resistance to these drugs occurs in one-third to one-half of patients, exposing them to the side effects of these drugs with no meaningful benefit. To identify the genes and pathways that confer resistance to such therapies, we performed a genome-wide screen in haploid human embryonic stem cells (hESCs). These cells possess the advantage of having only one copy of each gene, harbour a normal karyotype, and lack any underlying point mutations. We initially show a close correlation between the potency of anticancer drugs in cancer cell lines to those in hESCs. We then exposed a genome-wide loss-of-function library of mutations in all protein-coding genes to 10 selected anticancer drugs, which represent five different mechanisms of drug therapies. The genetic screening enabled us to identify genes and pathways which can confer resistance to these drugs, demonstrating several common pathways. We validated a few of the resistance-conferring genes, demonstrating a significant shift in the effective drug concentrations to indicate a drug-specific effect to these genes. Strikingly, the p53 signalling pathway seems to induce resistance to a large array of anticancer drugs. The data shows dramatic effects of loss of p53 on resistance to many but not all drugs, calling for clinical evaluation of mutations in this gene prior to anticancer therapy.
Asunto(s)
Antineoplásicos , Células Madre Embrionarias Humanas , Humanos , Células Madre Embrionarias Humanas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Haploidia , Antineoplásicos/farmacología , Antineoplásicos/metabolismo , MutaciónRESUMEN
For use in regenerative medicine, large-scale manufacturing of human pluripotent stem cells (hPSCs) under current good manufacturing practice (cGMPs) is required. Much progress has been made since culturing under static two-dimensional (2D) conditions on feeders, including feeder-free cultures, conditioned and xeno-free media, and three-dimensional (3D) dynamic suspension expansion. With the advent of horizontal-blade and vertical-wheel bioreactors, scale-out for large-scale production of differentiated hPSCs became possible; control of aggregate size, shear stress, fluid hydrodynamics, batch-feeding strategies, and other process parameters became a reality. Moving from substantially manipulated processes (i.e., 2D) to more automated ones allows easer compliance to current good manufacturing practices (cGMPs), and thus easier regulatory approval. Here, we review the current advances in the field of hPSC culturing, advantages, and challenges in bioreactor use, and regulatory areas of concern with respect to these advances. Manufacturing trends to reduce risk and streamline large-scale manufacturing will bring about easier, faster regulatory approval for clinical applications.
Asunto(s)
Técnicas de Cultivo de Célula , Células Madre Pluripotentes , Reactores Biológicos , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Humanos , Medicina RegenerativaRESUMEN
Biological sex is a fundamental trait influencing development, reproduction, pathogenesis, and medical treatment outcomes. Modeling sex differences is challenging because of the masking effect of genetic variability and the hurdle of differentiating chromosomal versus hormonal effects. In this work we developed a cellular model to study sex differences in humans. Somatic cells from a mosaic Klinefelter syndrome patient were reprogrammed to generate isogenic induced pluripotent stem cell (iPSC) lines with different sex chromosome complements: 47,XXY/46,XX/46,XY/45,X0. Transcriptional analysis of the hiPSCs revealed novel and known genes and pathways that are sexually dimorphic in the pluripotent state and during early neural development. Female hiPSCs more closely resembled the naive pluripotent state than their male counterparts. Moreover, the system enabled differentiation between the contributions of X versus Y chromosome to these differences. Taken together, isogenic hiPSCs present a novel platform for studying sex differences in humans and bear potential to promote gender-specific medicine in the future.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Femenino , Masculino , Caracteres Sexuales , Células Cultivadas , Diferenciación Celular/genéticaRESUMEN
In the mammalian embryo, a formative pluripotent phase is proposed to exist at the early post-implantation period, during the transition from the pre-implantation naive-to the post-implantation primed-epiblast. By recapitulating a laminin component of the extracellular matrix niche during embryonic formative transition, and defined culture conditions, we generated cultures highly enriched for self-renewing human pluripotent stem cells (hPSCs), exhibiting properties of early post-implantation epiblast cells. These hPSCs display post-implantation-epiblast gene expression profiles. FGF and TGF-ß signaling maintain their self-renewal for multiple passages. They have inactive canonical Wnt signaling, do not express primitive streak markers, and are competent to initiate differentiation toward germline and somatic fates. hPSCs exhibiting early post-implantation epiblast properties may shed light on human embryonic PSCs development and may serve for initiating somatic and germ cell specification.
Asunto(s)
Estratos Germinativos , Células Madre Pluripotentes , Animales , Humanos , Células Madre Pluripotentes/metabolismo , Embrión de Mamíferos , Línea Primitiva , Diferenciación Celular , Vía de Señalización Wnt , MamíferosRESUMEN
The mechanisms that govern human neural specification are not completely characterized. Here we used human embryonic stem cells (hESCs) to study the role of fibroblast growth factor (FGF)-signaling in early human neural specification. Differentiation was obtained by culturing clusters of hESCs in chemically-defined medium. We show that FGF-signaling, which is endogenously active during early differentiation of hESCs, induces early neural specification, while its blockage inhibits neuralization. The early neuralization effect of FGF-signaling is not mediated by promoting the proliferation of existing neural precursors (NPs) or prevention of their apoptosis. The neural instructive effect of FGF-signaling occurs after an initial FGF-independent differentiation into primitive ectoderm-like fate. We further show that FGF-signaling can induce neuralization by a mechanism which is independent of modulating bone morphogenic protein (BMP)-signaling. Still, FGF-signaling is not essential for hESC neuralization which can occur in the absence of FGF and BMP-signaling. Collectively, our data suggest that human neural induction is instructed by FGF-signaling, though neuralization of hESCs can occur in its absence.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Sistema Nervioso/embriología , Transducción de Señal , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Medios de Cultivo/química , Embrión de Mamíferos , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Factores de TiempoRESUMEN
Replication timing is an important developmentally regulated regional property that is correlated with chromosome structure and gene expression, but little is known about the establishment and maintenance of these patterns. Here we followed the fate of replication timing patterns in cells that undergo reprogramming either through somatic-cell nuclear transplantation or by the generation of induced pluripotential stem cells. We have investigated three different paradigms, stage-specific replication timing, parental allele-specific asynchrony (imprinted regions), and random allelic asynchronous replication. In all cases, somatic replication timing patterns were reset exactly at the appropriate stage in early development and could be properly established upon re-differentiation. Taken together, these results suggest that, unlike DNA methylation, the molecular mechanisms governing replication timing are not only stable but can also be easily reprogrammed.
Asunto(s)
Reprogramación Celular/genética , Momento de Replicación del ADN/genética , Replicación del ADN/genética , Técnicas de Transferencia Nuclear , Células Madre Pluripotentes/metabolismo , Alelos , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Mapeo Cromosómico , Desarrollo Embrionario/genética , Epigénesis Genética , Femenino , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Impresión Genómica/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Células Madre Pluripotentes/citología , Activación TranscripcionalRESUMEN
The Hadassah hESC Research Center's aim is to be a supplier of clinical and research-grade human embryonic stem cell (hESC) lines. In 2012, we derived the first three entirely GMP-compliant and xeno-free, fully-characterised, feeder-dependent (human umbilical cord) hESC lines developed under cleanroom conditions. In 2018, we established four new GMP and xeno-free, feeder-independent MCB hESCs under GMP conditions using commercially available reagents, media and matrix. All cell lines were derived under Israeli Ministry of Health's National Ethics Committee for Genetic Research in Humans and the ethical considerations that guided the development of the hESCs strictly followed Israeli law. Hadassah has provided its clinical-grade hESC lines to commercial entities of which two are already in clinical trials, establishing Hadassah as a key provider of clinical-grade hESC lines.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes/metabolismo , Diferenciación Celular , HumanosRESUMEN
Neuroglial precursor cells (NPC) possess immune-modulatory properties by which they prevent immune-mediated injury in experimental autoimmune encephalomyelitis (EAE). It is unclear whether cell transplantation in a clinical-relevant setup induces ongoing therapeutic effects in a chronic-active model of progressive multiple sclerosis (MS). We examined whether human embryonic stem cell (hESC)-derived NPCs inhibit progressive EAE in Biozzi AB/H mice, manifesting with chronic-active neuroinflammation and demyelinated plaques. hESC-derived NPCs were propagated for 6-8 weeks as spheres enriched for Olig2+ cells to switch from neuronal to glial commitment and to enrich for oligodendrocyte progenitor cells. NPC were transplanted intracerebroventricularly at 30 days post-EAE induction, after the acute relapse. We evaluated effects of cell transplantation on clinical parameters, neuroinflammation, myelination, and axonal loss. Transplanted animals exhibited a significantly milder disease, reduced neuroinflammation, reduced demyelination, and reduced axonal loss as compared to control EAE mice. Toluidine-blue semi-thin staining showed a bystander neuroprotective effect of human precursor cells preventing the loss of myelinated fibers in superficial layer of the cervical dorsal funiculus. Human Olig2+ cells were detected along spinal cord meninges after 65 days of follow-up. In co-cultures in vitro, Olig2+ human precursors inhibited Concanavalin A-induced murine T cell activation and proliferation. To conclude, glial-committed human NPC induce ongoing immune-regulatory and neuroprotective effects, following transplantation into mice with a clinical-relevant model of chronic-active MS and during established disease, entering the chronic phase. These properties highlight the therapeutic potential of human NPC transplantation in chronic MS and their delivery via the cerebrospinal fluid.
Asunto(s)
Esclerosis Múltiple/inmunología , Esclerosis Múltiple/terapia , Células-Madre Neurales/trasplante , Células Precursoras de Oligodendrocitos/citología , Animales , Encefalomielitis Autoinmune Experimental/inmunología , Humanos , Ratones , Vaina de Mielina/inmunología , Neuronas/citología , Factor de Transcripción 2 de los Oligodendrocitos/metabolismo , Trasplante de Células Madre/métodosRESUMEN
The application of human embryonic stem (hES) cells in regenerative medicine will require rigorous quality control measures to ensure the safety of hES cell-derived grafts. During propagation in vitro, hES cells can acquire cytogenetic abnormalities as well as submicroscopic genetic lesions, such as small amplifications or deletions. Many of the genetic abnormalities that arise in hES cell cultures are also implicated in human cancer development. The causes of genetic instability of hES cells in culture are poorly understood, and commonly used cytogenetic methods for detection of abnormal cells are capable only of low-throughput analysis on small numbers of cells. The identification of biomarkers of genetic instability in hES cells would greatly facilitate the development of culture methods that preserve genomic integrity. Here we show that CD30, a member of the tumor necrosis factor receptor superfamily, is expressed on transformed but not normal hES cells, and that CD30 expression protects hES cells against apoptosis.
Asunto(s)
Carcinoma Embrionario/metabolismo , Carcinoma Embrionario/patología , Antígeno Ki-1/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Biomarcadores/análisis , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular Transformada , Supervivencia Celular , Transformación Celular Neoplásica , Células Cultivadas , Humanos , Inmunohistoquímica , CariotipificaciónRESUMEN
Neural precursors have been derived from human embryonic stem cells (hESC) using the bone morphogenetic protein antagonist noggin. These neural precursors can be further differentiated to produce neural cells that express central nervous system (CNS) markers. We have recently shown that naive hESC can be directed to differentiate into peripheral sensory (PS) neuron-like cells and putative neural crest precursors by co-culturing with PA6 stromal cells. In the present study, we examine whether hESC-derived neural precursors (NPC) can differentiate into the peripheral nervous system, as well as CNS cells. As little as 1 week after co-culture with PA6 cells, cells with the molecular characteristics of PS neurons and neural crest are observed in the cultures. With increased time in culture, more PS-like neurons appear, in parallel with a reduction in the neural crest-like cells. These results provide the first evidence that neural precursors derived from hESC have the potential to develop into PS neurons-like as well as CNS-like neuronal cells. About 10% of the cells in NPC-PA6 co-cultures express PS neuron markers after 3 weeks, compared with <1% of hESC cultured on PA6. This enrichment for peripheral neurons makes this an attractive system for generation of peripheral neurons for pathophysiology study and drug development for diseases of the peripheral nervous system such as Familial Dysautonomia and varicella virus infection.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Neuronas Aferentes/citología , Animales , Proteínas Portadoras/metabolismo , Técnicas de Cocultivo , Humanos , Ratones , Nervios Periféricos/citología , Células del Estroma/metabolismoRESUMEN
Genetic modifications of human embryonic stem cells (hESCs) that will efficiently promote stable homogenous gene silencing, and will also allow monitoring of the silencing level, may be invaluable for the study of function of genes in early human embryogenesis, differentiation, and maintenance of pluripotency of hESCs. RNA-mediated interference (RNAi) emerges as a highly efficient tool for specific knockdown of gene expression. Lentiviruses are efficient vectors for the delivery and stable expression of transgenes in hESCs. We sought to develop a lentiviral-RNAi-based system that will efficiently induce homogenous gene silencing and will allow the monitoring of its relative level in hESCs. Dual-promoter lentiviral vectors coexpressing an RNAi cassette and a reporter gene were initially used for efficient and stable induction of heterogeneous levels of gene silencing in polyclonal hESCs. This step was further combined with the isolation of transduced clones with different homogenous levels of gene silencing. The level of silencing in each of the clones correlated and could be monitored by the level of expression of the vector's reporter transgene. Thus, our system allows easy identification of clones with relatively different homogenous levels of gene silencing. Our approach would be valuable for the study of function of genes, in particular those whose role in hESCs biology depends on their level of expression.
Asunto(s)
Células Madre Embrionarias/fisiología , Silenciador del Gen , Lentivirus/genética , Interferencia de ARN , Línea Celular , Clonación Molecular , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes/metabolismo , HumanosRESUMEN
Mammalian somatic cell nuclear transfer (SCNT) is a technically and biologically challenging procedure inducing rapid reprogramming of the nucleus from the differentiated into the totipotent state in a few hours. This procedure was initially successfully accomplished in farm animals, then in rodents, and more recently in primates and in humans. Though ethical concerns regarding SCNT still exist, this procedure can be utilized to generate patient and disease-specific pluripotent embryonic stem cell lines, which carry a great promise in improving our understanding of major disease conditions and a hope for better therapies and regenerative medicine. In this section, we will survey the existing literature and describe how mouse SCNT is performed and the importance of donor cell treatment and cycle synchronization prior to SCNT.
Asunto(s)
Ciclo Celular/fisiología , Técnicas de Transferencia Nuclear , Animales , Ciclo Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Femenino , Humanos , Mamíferos , Ratones , Oocitos/citología , Oocitos/metabolismo , PrimatesRESUMEN
Embryonic stem (ES) cells have the potential to proliferate indefinitely in culture and can differentiate into any cell type. The emergence of ES cell lines from human embryos in the past 5 years has attracted profound public and scientific interest, given the far-reaching potential applications of these cells in regenerative medicine. In the future, it is possible that human ES (hES) cells might serve as an unlimited source of cells for transplantation therapy under conditions that result from cell degeneration or malfunction, and that genetically manipulated hES cells might serve as vectors to carry and express genes in target organs following transplantation in the course of gene therapy. This chapter reviews the properties of hES cells and their potential advantages and limitations for cell-based therapies. We also describe various approaches that might be utilized with hES cells to avoid potential immune rejection after allogeneic transplantation and hence circumvent the need for systemic immune suppression. Up-to-date research in establishing committed tissue-specific progenitors from ES cells and evidence of their function after transplantation in various animal disease models is also reviewed. The chapter concludes that hES cells show great promise for regenerative medicine although significant developments are still required to exploit their potential for cell and gene therapy.
Asunto(s)
Blastocisto/citología , Trasplante de Células Madre , Células Madre , Animales , Enfermedades Cardiovasculares/cirugía , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Clonales , Diabetes Mellitus Tipo 1/cirugía , Humanos , Modelos Animales , Enfermedades Neurodegenerativas/cirugíaRESUMEN
Teratoma tumor formation is an essential criterion in determining the pluripotency of human pluripotent stem cells. However, currently there is no consistent protocol for assessment of teratoma forming ability. Here we present detailed characterization of a teratoma assay that is based on subcutaneous co-transplantation of defined numbers of undifferentiated human embryonic stem cells (hESCs) with mitotically inactivated feeder cells and Matrigel into immunodeficient mice. The assay was highly reproducible and 100% efficient when 100,000 hESCs were transplanted. It was sensitive, promoting teratoma formation after transplantation of 100 hESCs, though larger numbers of animals and longer follow-up were required. The assay could detect residual teratoma forming cells within differentiated hESC populations however its sensitivity was decreased in the presence of differentiated cells. Our data lay the foundation, for standardization of a teratoma assay for pluripotency analysis. The assay can also be used for bio-safety analysis of pluripotent stem cell-derived differentiated progeny.
Asunto(s)
Bioensayo/normas , Células Madre Embrionarias/patología , Células Madre Pluripotentes/patología , Teratoma/patología , Animales , Biomarcadores/metabolismo , Recuento de Células , Diferenciación Celular , Colágeno/administración & dosificación , Combinación de Medicamentos , Células Madre Embrionarias/trasplante , Células Nutrientes/citología , Células Nutrientes/trasplante , Fibroblastos/citología , Fibroblastos/trasplante , Humanos , Inyecciones Subcutáneas , Cariotipificación , Laminina/administración & dosificación , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Pluripotentes/trasplante , Proteoglicanos/administración & dosificación , Sensibilidad y Especificidad , Tasa de Supervivencia , Teratoma/mortalidadRESUMEN
Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Embrionarias/citología , Técnicas de Cultivo de Célula/ética , HumanosRESUMEN
Somatic cell nuclear transfer (SCNT) is a technically and biologically challenging procedure during which a differentiated committed nucleus undergoes rapid reprogramming into the totipotent state in a few hours. SCNT can be utilized to generate patient- and disease-specific embryonic stem cell (ESC) lines, which carry great promise in improving our understanding of major disease conditions and hope for better therapies. In this section, we will describe how mouse SCNT is performed and survey the importance of donor cell cycle synchronization and the methods to perform it.