RESUMO
Induced pluripotent stem cells (iPSCs) with potential for therapeutic applications can be derived from somatic cells via ectopic expression of a set of limited and defined transcription factors. However, due to risks of random integration of the reprogramming transgenes into the host genome, the low efficiency of the process, and the potential risk of virally induced tumorigenicity, alternative methods have been developed to generate pluripotent cells using nonintegrating systems, albeit with limited success. Here, we show that c-KIT+ human first-trimester amniotic fluid stem cells (AFSCs) can be fully reprogrammed to pluripotency without ectopic factors, by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The cells share 82% transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion, they maintain genetic stability, protein level expression of key pluripotency factors, high cell-division kinetics, telomerase activity, repression of X-inactivation, and capacity to differentiate into lineages of the three germ layers, such as definitive endoderm, hepatocytes, bone, fat, cartilage, neurons, and oligodendrocytes. We conclude that AFSC can be utilized for cell banking of patient-specific pluripotent cells for potential applications in allogeneic cellular replacement therapies, pharmaceutical screening, and disease modeling.
Assuntos
Líquido Amniótico/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Ácido Valproico/farmacologia , Líquido Amniótico/citologia , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Genoma Humano , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariotipagem , Cinética , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fenótipo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma , Transgenes , Inativação do Cromossomo X/efeitos dos fármacosRESUMO
The use of genomic DNA rather than cDNA or mini-gene constructs in gene therapy might be advantageous as these contain intronic and long-range control elements vital for accurate expression. For gene therapy of cystic fibrosis though, no bacterial artificial chromosome (BAC), containing the whole CFTR gene is available. We have used Red homologous recombination to add a to a previously described vector to construct a new BAC vector with a 250.3-kb insert containing the whole coding region of the CFTR gene along with 40.1 kb of DNA 5' to the gene and 25 kb 3' to the gene. This includes all the known control elements of the gene. We evaluated expression by RT-PCR in CMT-93 cells and showed that the gene is expressed both from integrated copies of the BAC and also from episomes carrying the oriP/EBNA-1 element. Sequencing of the human CFTR mRNA from one clone showed that the BAC is functional and can generate correctly spliced mRNA in the mouse background. The BAC described here is the only CFTR genomic construct available on a convenient vector that can be readily used for gene expression studies or in vivo studies to test its potential application in gene therapy for cystic fibrosis.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulação da Expressão Gênica , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Eletroforese em Gel de Campo Pulsado , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Humanos , Hibridização in Situ Fluorescente , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação Genética/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Human artificial chromosomes (HACs) can be formed de novo by transfection of large fragments of cloned alphoid DNA into human HT1080 cells in tissue culture. In order to generate HACs carrying a gene of interest, one can either co-transfect the alphoid DNA and the gene of interest, or one can clone both into a single vector prior to transfection. Here we describe linking approximately 70 kb of alphoid DNA onto a 156-kb BAC carrying the human HPRT gene using Red homologous recombination in the EL350 Escherichia coli host [Lee et al., Genomics 73 (2001) 56-65]. A selectable marker and EGFP marker were then added by loxP/Cre recombination using the arabinose inducible cre gene in the EL350 bacteria. The final construct generates minichromosomes in HT1080 cells and the HPRT gene is expressed. The retrofitting vector can be used to add the approximately 70 kb of alphoid DNA to any BAC carrying a gene of interest to generate a HAC vector. The method can also be used to link any unrelated BAC or PAC insert onto another BAC clone. The EL350 bacteria are an excellent host for building up complex vectors by a combination of homologous and loxP/Cre recombination.
Assuntos
Cromossomos Artificiais Humanos/genética , DNA Recombinante/genética , Vetores Genéticos/genética , Linhagem Celular Tumoral , Cromossomos Artificiais Bacterianos/genética , DNA Satélite/genética , Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hibridização in Situ Fluorescente , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
The mutation Met132Val in the ACTA1 gene was identified in a patient with mild nemaline myopathy (NM). We examined actin mRNA and protein from biopsy samples. Sixty-one percent of the mRNA from the biopsy was not cleaved with BstX1, indicating the presence of mutant messenger in vivo. Monomeric actin was extracted from 2.5 mg of mutant muscle and wild type muscle. A proportion of the NM actin did not polymerise in 50 mM KCl, 2.5 mM MgCl2 but all the wild-type actin did. NM actin was fully polymerised by 50 mM KCl, 2.5 mM MgCl2, 150 nM rhodamine-phalloidin. Thin filaments reconstituted with this co-polymer were different from wild-type. The NM actin produces faster sliding of thin filaments at pCa5 and higher relative isometric force. We conclude that the mutant mRNA and protein is expressed and that the mutation reduces polymerisability and alters thin filament function.
Assuntos
Actinas/genética , Músculo Esquelético/fisiopatologia , Mutação/genética , Miopatias da Nemalina/genética , Miopatias da Nemalina/fisiopatologia , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/fisiologia , Actinas/química , Actinas/isolamento & purificação , Substituição de Aminoácidos , Sequência de Bases/genética , Biópsia , Análise Mutacional de DNA , Humanos , Metionina/genética , Modelos Moleculares , Contração Muscular/genética , Músculo Esquelético/química , Músculo Esquelético/patologia , Polímeros/metabolismo , Estrutura Quaternária de Proteína/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Valina/genéticaRESUMO
Osteogenesis imperfecta (OI) is a genetic bone pathology with prenatal onset, characterized by brittle bones in response to abnormal collagen composition. There is presently no cure for OI. We previously showed that human first trimester fetal blood mesenchymal stem cells (MSCs) transplanted into a murine OI model (oim mice) improved the phenotype. However, the clinical use of fetal MSC is constrained by their limited number and low availability. In contrast, human fetal early chorionic stem cells (e-CSC) can be used without ethical restrictions and isolated in high numbers from the placenta during ongoing pregnancy. Here, we show that intraperitoneal injection of e-CSC in oim neonates reduced fractures, increased bone ductility and bone volume (BV), increased the numbers of hypertrophic chondrocytes, and upregulated endogenous genes involved in endochondral and intramembranous ossification. Exogenous cells preferentially homed to long bone epiphyses, expressed osteoblast genes, and produced collagen COL1A2. Together, our data suggest that exogenous cells decrease bone brittleness and BV by directly differentiating to osteoblasts and indirectly stimulating host chondrogenesis and osteogenesis. In conclusion, the placenta is a practical source of stem cells for the treatment of OI.
Assuntos
Córion/citologia , Células-Tronco Fetais/citologia , Células-Tronco Fetais/transplante , Fraturas Ósseas/terapia , Osteogênese Imperfeita/terapia , Placenta/citologia , Animais , Osso e Ossos/anormalidades , Osso e Ossos/metabolismo , Terapia Baseada em Transplante de Células e Tecidos , Condrócitos/citologia , Condrócitos/metabolismo , Condrogênese , Córion/metabolismo , Colágeno Tipo I/agonistas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Feminino , Células-Tronco Fetais/metabolismo , Feto , Fraturas Ósseas/genética , Fraturas Ósseas/metabolismo , Fraturas Ósseas/patologia , Expressão Gênica , Humanos , Injeções Intraperitoneais , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologia , Placenta/metabolismo , Gravidez , Transplante de Células-Tronco , Transplante HeterólogoRESUMO
Human amniotic fluid cells have been used traditionally as a diagnostic tool for genetic anomalies. More recently it has been recognized that amniotic fluid contains populations of stem cells. Mesenchymal stem cells (AFMSC) were first to be described. These cells are able to differentiate towards mesodermal lineages. More recently cells with broader potential, defined as amniotic fluid stem cells (AFSC), were also isolated. They have intermediate characteristics between embryonic and adult stem cells and are able to differentiate into lineages representative of all three germ layers but unlike ES cells they do not form tumours in vivo. Furthermore, AFSC have been reverted to functional pluripotency in a transgene-free approach using an epigenetics modifier. These characteristics, together with absence of ethical issues concerning their employment, have made stem cells from amniotic fluid a promising candidate for cell therapy and tissue engineering.
Assuntos
Líquido Amniótico/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Diferenciação Celular , Humanos , Imunomodulação/imunologia , Células-Tronco Pluripotentes/citologia , Células-Tronco/imunologiaRESUMO
Efficient delivery of large intact vectors into mammalian cells remains problematical. Here we evaluate delivery by bacterial invasion of two large BACs of more than 150 kb in size into various cells. First, we determined the effect of several drugs on bacterial delivery of a small plasmid into different cell lines. Most drugs tested resulted in a marginal increase of the overall efficiency of delivery in only some cell lines, except the lysosomotropic drug chloroquine, which was found to increase the efficiency of delivery by 6-fold in B16F10 cells. Bacterial invasion was found to be significantly advantageous compared with lipofection in delivering large intact BACs into mouse cells, resulting in 100% of clones containing intact DNA. Furthermore, evaluation of expression of the human hypoxanthine phosphoribosyltransferase (HPRT) gene from its genomic locus, which was present in one of the BACs, showed that single copy integrations of the HPRT-containing BAC had occurred in mouse B16F10 cells and that expression of HPRT from each human copy was 0.33 times as much as from each endogenous mouse copy. These data provide new evidence that bacterial delivery is a convenient and efficient method to transfer large intact therapeutic genes into mammalian cells.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Animais , Linhagem Celular , Escherichia coli/genética , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Camundongos , PlasmídeosRESUMO
Stem cells have considerable potential to repair damaged organs and tissues. We previously showed that prenatal transplantation of human first trimester fetal blood mesenchymal stem cells (hfMSCs) in a mouse model of osteogenesis imperfecta (oim mice) led to a phenotypic improvement, with a marked decrease in fracture rate. Donor cells differentiated into mature osteoblasts, producing bone proteins and minerals, including collagen type Iα2, which is absent in nontransplanted mice. This led to modifications of the bone matrix and subsequent decrease of bone brittleness, indicating that grafted cells directly contribute to improvement of bone mechanical properties. Nevertheless, the therapeutic effect was incomplete, attributing to the limited level of engraftment in bone. In this study, we show that although migration of hfMSCs to bone and bone marrow is CXCR4-SDF1 (SDF1 is stromal-derived factor) dependent, only a small number of cells present CXCR4 on the cell surface despite high levels of internal CXCR4. Priming with SDF1, however, upregulates CXCR4 to increase the CXCR4(+) cell fraction, improving chemotaxis in vitro and enhancing engraftment in vivo at least threefold in both oim and wild-type bone and bone marrow. Higher engraftment in oim bones was associated with decreased bone brittleness. This strategy represents a step to improve the therapeutic benefits of fetal cell therapy toward being curative.
Assuntos
Fraturas Ósseas/prevenção & controle , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Osteoblastos/transplante , Osteogênese Imperfeita/cirurgia , Receptores CXCR4/metabolismo , Animais , Matriz Óssea/metabolismo , Diferenciação Celular , Células Cultivadas , Quimiocina CXCL12/metabolismo , Quimiotaxia , Colágeno Tipo I/deficiência , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Feminino , Fraturas Ósseas/metabolismo , Fraturas Ósseas/patologia , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/imunologia , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/imunologia , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologia , Fatores de Tempo , Regulação para CimaRESUMO
Foetal stem cells (FSCs) can be isolated during gestation from many different tissues such as blood, liver and bone marrow as well as from a variety of extraembryonic tissues such as amniotic fluid and placenta. Strong evidence suggests that these cells differ on many biological aspects such as growth kinetics, morphology, immunophenotype, differentiation potential and engraftment capacity in vivo. Despite these differences, FSCs appear to be more primitive and have greater multi-potentiality than their adult counterparts. For example, foetal blood haemopoietic stem cells proliferate more rapidly than those found in cord blood or adult bone marrow. These features have led to FSCs being investigated for pre- and post-natal cell therapy and regenerative medicine applications. The cells have been used in pre-clinical studies to treat a wide range of diseases such as skeletal dysplasia, diaphragmatic hernia and respiratory failure, white matter damage, renal pathologies as well as cancers. Their intermediate state between adult and embryonic stem cells also makes them an ideal candidate for reprogramming to the pluripotent status.
Assuntos
Sangue Fetal/citologia , Células-Tronco Fetais/citologia , Feto/citologia , Medicina Regenerativa , Âmnio/citologia , Líquido Amniótico/citologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Feminino , Células-Tronco Fetais/transplante , Humanos , Camundongos , Fenótipo , Placenta/citologia , Gravidez , Terapêutica/métodosRESUMO
Hemophilia A is caused by mutations in the gene encoding factor VIII (F8) and is an important target for gene therapy. The F8 gene contains 26 exons spread over approximately 186 kb and no work using the intact genomic locus has been carried out. We have constructed a 250-kb BAC carrying all 26 exons, the introns, and more than 40 kb of upstream and 20 kb of downstream DNA. This F8 BAC was further retrofitted with either the oriP/EBNA-1 elements from Epstein-Barr virus, which allow episomal maintenance in mammalian cells, or alphoid DNA, which allows human artificial chromosome formation in some human cell lines. Lipofection of the oriP/EBNA-1-containing version into mouse Hepa1-6 cells resulted in expression of F8 mRNA spanning the F8 gene. The >300-kb BAC carrying alphoid DNA was successfully delivered to 293A and HT1080 cells using bacterial delivery, resulting in greater than endogenous levels of F8 mRNA expression.
Assuntos
Cromossomos Artificiais Bacterianos , DNA/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Fator VIII/genética , RNA Mensageiro/genética , Recombinação Genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Células Cultivadas , DNA/análise , Fator VIII/metabolismo , Fibrossarcoma/genética , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Marcação de Genes , Vetores Genéticos , Humanos , Rim/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Plasmídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
We investigated the effects of two mutations in human cardiac troponin I, Arg(145)-->Gly and Gly(203)-->Ser, that are reported to cause familial hypertrophic cardiomyopathy. Mutant and wild-type troponin I, overexpressed in Escherichia coli, were used to reconstitute troponin complexes in vanadate-treated guinea pig cardiac trabeculae skinned fibres, and thin filaments were reconstituted with human cardiac troponin and tropomyosin along with rabbit skeletal muscle actin for in vitro motility and actomyosin ATPase assays. Troponin containing the Arg(145)-->Gly mutation inhibited force in skinned trabeculae less than did the wild-type, and had almost no inhibitory function in the in vitro motility assay. There was an enhanced inhibitory function with mixtures of 10-30% [Gly(145)]troponin I with the wild-type protein. Skinned trabeculae reconstituted with troponin I containing the Gly(203)-->Ser mutation and troponin C produced less Ca(2+)-activated force (64+/-8% of wild-type) and demonstrated lower Ca(2+) sensitivity [Delta(p)Ca(50) (log of the Ca(2+) concentration that gave 50% of maximal activation) 0.25 unit (P<0.05)] compared with wild-type troponin I, but thin filaments containing [Ser(203)]-troponin I were indistinguishable from those containing the wild-type protein in in vitro motility and ATPase assays. Thus these two mutations each result in hypertrophic cardiomyopathy, but have opposite effects on the overall contractility of the muscle in the systems we investigated, indicating either that we have not yet identified the relevant alteration in contractility for the Gly(203)->Ser mutation, or that the disease does not result directly from any particular alteration in contractility.
Assuntos
Cardiomiopatia Hipertrófica/fisiopatologia , Coração/fisiologia , Contração Miocárdica/fisiologia , Troponina I/genética , Troponina I/fisiologia , Actinas/metabolismo , Substituição de Aminoácidos , Animais , Cálcio/metabolismo , Cardiomiopatia Hipertrófica/genética , Cobaias , Humanos , Fibras Musculares Esqueléticas/fisiologia , Mutação de Sentido Incorreto , Subfragmentos de Miosina/metabolismo , Fenótipo , Tropomiosina/metabolismoRESUMO
We have compared the in vitro regulatory properties of recombinant human cardiac troponin reconstituted using wild type troponin T with troponin containing the DeltaLys-210 troponin T mutant that causes dilated cardiomyopathy (DCM) and the R92Q troponin T known to cause hypertrophic cardiomyopathy (HCM). Troponin containing DeltaLys-210 troponin T inhibited actin-tropomyosin-activated myosin subfragment-1 ATPase activity to the same extent as wild type at pCa8.5 (>80%) but produced substantially less enhancement of ATPase at pCa4.5. The Ca(2+) sensitivity of ATPase activation was increased (DeltapCa(50) = +0.2 pCa units) and cooperativity of Ca(2+) activation was virtually abolished. Equimolar mixtures of wild type and DeltaLys-210 troponin T gave a lower Ca(2+) sensitivity than with wild type, while maintaining the diminished ATPase activation at pCa4.5 observed with 100% mutant. In contrast, R92Q troponin gave reduced inhibition at pCa8.5 but greater activation than wild type at pCa4.5; Ca(2+) sensitivity was increased but there was no change in cooperativity. In vitro motility assay of reconstituted thin filaments confirmed the ATPase results and moreover indicated that the predominant effect of the DeltaLys-210 mutation was a reduced sliding speed. The functional consequences of this DCM mutation are qualitatively different from the R92Q or any other studied HCM troponin T mutation, suggesting that DCM and HCM may be triggered by distinct primary stimuli.