RESUMEN
The promise of using induced pluripotent stem cells (iPSCs) for cellular therapies has been hampered by the lack of easily isolatable and well characterized source cells whose genomes have undergone minimal changes during their processing. Blood-derived late-outgrowth endothelial progenitor cells (EPCs) are used for disease modeling and have potential therapeutic uses including cell transplantation and the translation of induced pluripotent stem cell (iPSC) derivatives. However, the current isolation of EPCs has been inconsistent and requires at least 40-80 mL of blood, limiting their wider use. In addition, previous EPC reprogramming methods precluded the translation of EPC-derived iPSCs to the clinic. Here a series of clinically-compatible advances in the isolation and reprogramming of EPCs is presented, including a reduction of blood sampling volumes to 10 mL and use of highly efficient RNA-based reprogramming methods together with autologous human serum, resulting in clinically relevant iPSCs carrying minimal copy number variations (CNVs) compared to their parent line.
Asunto(s)
Células Progenitoras Endoteliales/citología , Trasplante de Células Madre , Reprogramación Celular , HumanosRESUMEN
Expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc in mesodermal and endodermal derivatives, including fibroblasts, lymphocytes, liver, stomach, and beta cells, generates induced pluripotent stem (iPS) cells. It remains unknown, however, whether cell types of the ectodermal lineage are equally amenable to reprogramming into iPS cells by the same combination of factors. To test this, we have isolated genetically marked neural progenitor cells (NPCs) from neonatal mouse brains and infected them with viral vectors expressing Oct4, Sox2, Klf4, and c-Myc. Infected NPCs gave rise to iPS cells that expressed markers of embryonic stem cells, showed demethylation of pluripotency genes, formed teratomas, and contributed to viable chimeras. In contrast to other somatic cell types, NPCs expressed high levels of endogenous Sox2 and thus did not require viral Sox2 expression for reprogramming into iPS cells. Our data show that in addition to mesoderm- and endoderm-derived cell types, neural progenitor cells of the ectodermal lineage can be reprogrammed into iPS cells, suggesting that in vitro reprogramming is a universal process. These results also imply that the combination of factors necessary for reprogramming is dependent on cellular context. Disclosure of potential conflicts of interest is found at the end of this article.
Asunto(s)
Reprogramación Celular , Neuronas/citología , Neuronas/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Factores de Transcripción SOXB1/metabolismo , Animales , Animales Recién Nacidos , Diferenciación Celular , Línea Celular , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Factor 4 Similar a Kruppel , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Pluripotent embryonic stem (ES) cell lines were first isolated over 25 years ago and remain an essential tool in molecular and developmental biology to this day. In particular, the use of homologous recombination and subsequent generation of ES-derived mice has greatly facilitated research across all fields. Moreover, ES cells represent an extremely attractive model to study events in early development. In this chapter, we will describe the derivation and propagation of murine ES cells. This is followed by a description of targeting ES cells and a protocol for the generation of mice by diploid and tetraploid blastocyst injections.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Embrionarias/citología , Animales , Blastocisto/citología , Proliferación Celular , Separación Celular , Cesárea , Células Clonales , Medios de Cultivo , ADN/metabolismo , Diploidia , Embrión de Mamíferos/citología , Femenino , Fibroblastos/citología , Congelación , Inyecciones , Ratones , Poliploidía , EmbarazoRESUMEN
mRNA reprogramming results in the generation of genetically stable induced pluripotent stem (iPS) cells while avoiding the risks of genomic integration. Previously published mRNA reprogramming protocols have proven to be inconsistent and time-consuming and mainly restricted to fibroblasts, thereby demonstrating the need for a simple but reproducible protocol applicable to various cell types. So far there have been no published reports using mRNA to reprogram any cell type derived from human blood. Nonmodified synthetic mRNAs are immunogenic and activate cellular defense mechanisms, which can lead to cell death and inhibit mRNA translation upon repetitive transfection. Hence, to overcome RNA-related toxicity we combined nonmodified reprogramming mRNAs (OCT4, SOX2, KLF4, cMYC, NANOG, and LIN28 [OSKMNL]) with immune evasion mRNAs (E3, K3, and B18R [EKB]) from vaccinia virus. Additionally, we included mature, double-stranded microRNAs (miRNAs) from the 302/367 cluster, which are known to enhance the reprogramming process, to develop a robust reprogramming protocol for the generation of stable iPS cell lines from both human fibroblasts and human blood-outgrowth endothelial progenitor cells (EPCs). Our novel combination of RNAs enables the cell to tolerate repetitive transfections for the generation of stable iPS cell colonies from human fibroblasts within 11 days while requiring only four transfections. Moreover, our method resulted in the first known mRNA-vectored reprogramming of human blood-derived EPCs within 10 days while requiring only eight daily transfections.
Asunto(s)
Técnicas de Reprogramación Celular , Células Madre Pluripotentes Inducidas/metabolismo , ARN Mensajero/genética , Transfección , Células Progenitoras Endoteliales/citología , Células Progenitoras Endoteliales/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Evasión Inmune , Factor 4 Similar a Kruppel , MicroARNs/genética , ARN Mensajero/inmunología , Virus Vaccinia/genéticaRESUMEN
The method of isolation of bone marrow (BM) mesenchymal stem/stromal cells (MSCs) is a limiting factor in their study and therapeutic use. MSCs are typically expanded from BM cells selected on the basis of their adherence to plastic, which results in a heterogeneous population of cells. Prospective identification of the antigenic profile of the MSC population(s) in BM that gives rise to cells with MSC activity in vitro would allow the preparation of very pure populations of MSCs for research or clinical use. To address this issue, we used polychromatic flow cytometry and counterflow centrifugal elutriation to identify a phenotypically distinct population of mesenchymal stem/progenitor cells (MSPCs) within human BM. The MSPC activity resided within a population of rare, small CD45â»CD73âºCD90âºCD105⺠cells that lack CD44, an antigen that is highly expressed on culture-expanded MSCs. In culture, these MSPCs adhere to plastic, rapidly proliferate, and acquire CD44 expression. They form colony forming units-fibroblast and are able to differentiate into osteoblasts, chondrocytes, and adipocytes under defined in vitro conditions. Their acquired expression of CD44 can be partially downregulated by treatment with recombinant human granulocyte-colony stimulating factor, a response not found in BM-MSCs derived from conventional plastic adherence methods. These observations indicate that MSPCs within human BM are rare, small CD45â»CD73âºCD90âºCD105⺠cells that lack expression of CD44. These MSPCs give rise to MSCs that have phenotypic and functional properties that are distinct from those of BM-MSCs purified by plastic adherence.
Asunto(s)
Células de la Médula Ósea/citología , Separación Celular/métodos , Tamaño de la Célula , Citometría de Flujo/métodos , Receptores de Hialuranos/metabolismo , Células Madre Mesenquimatosas/citología , Biomarcadores/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Fraccionamiento Celular , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Centrifugación , Factor Estimulante de Colonias de Granulocitos/farmacología , Humanos , Inmunofenotipificación , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Fenotipo , Reproducibilidad de los ResultadosRESUMEN
Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS) cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts). However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that "epigenetic memory" significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle.
Asunto(s)
Reprogramación Celular , Células Madre Pluripotentes Inducidas/citología , Músculo Esquelético/citología , Adipogénesis , Animales , Antígenos Ly/metabolismo , Diferenciación Celular , Células Clonales/citología , Células Clonales/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismoRESUMEN
Induced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here we show that iPSCs obtained from mouse fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Notably, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and altered differentiation capacity. These observations may influence ongoing attempts to use iPSCs for disease modeling and could also be exploited in potential therapeutic applications to enhance differentiation into desired cell lineages.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Animales , Linfocitos B/citología , Células Cultivadas , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Epigenómica , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Ratones , Músculo Esquelético/citología , Células Madre/citología , Transcripción GenéticaRESUMEN
The first cell fate choice in the mammalian embryo, the segregation of the inner cell mass (ICM) and trophectoderm (TE), is regulated by the mutually antagonistic effects of the transcription factors, Oct4 and Cdx2, while the pluripotency factor, Nanog, is essential to specify the epiblast. We have analyzed the promoters of Nanog and Cdx2, and have found that these two transcription factors are likewise regulated reciprocally. Using an embryonic stem cell line with conditional TE differentiation, we show that Nanog overexpression suppresses the upregulation of TE markers, while Nanog knockdown upregulates the expression of TE markers. We further show that Nanog and Cdx2 bind to and repress each other's promoters. However, whereas Nanog knockout results in detectable Cdx2 expression in the ICM, we observe no overt disruption of blastocyst development, indicating that Nanog plays a subservient role to Oct4 in segregation of the ICM and TE.
Asunto(s)
Blastocisto/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Regiones Promotoras Genéticas/fisiología , Factores de Transcripción/genética , Animales , Biomarcadores , Blastocisto/citología , Factor de Transcripción CDX2 , Diferenciación Celular/fisiología , Técnicas de Cultivo de Embriones , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Femenino , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Embarazo , Factores de Transcripción/metabolismo , Trofoblastos/citología , Trofoblastos/fisiologíaRESUMEN
The reprogramming of somatic cells into induced pluripotent stem (iPS) cells upon overexpression of the transcription factors Oct4, Sox2, Klf4 and cMyc is inefficient. It has been assumed that the somatic differentiation state provides a barrier for efficient reprogramming; however, direct evidence for this notion is lacking. Here, we tested the potential of mouse hematopoietic cells at different stages of differentiation to be reprogrammed into iPS cells. We show that hematopoietic stem and progenitor cells give rise to iPS cells up to 300 times more efficiently than terminally differentiated B and T cells do, yielding reprogramming efficiencies of up to 28%. Our data provide evidence that the differentiation stage of the starting cell has a critical influence on the efficiency of reprogramming into iPS cells. Moreover, we identify hematopoietic progenitors as an attractive cell type for applications of iPS cell technology in research and therapy.
Asunto(s)
Diferenciación Celular/fisiología , Reprogramación Celular , Células Madre Hematopoyéticas/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Animales , Animales Recién Nacidos , Línea Celular , Células Cultivadas , Quimera , Fibroblastos/citología , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes/metabolismo , Factor 4 Similar a Kruppel , Lentivirus/genética , Ratones , Datos de Secuencia Molecular , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transducción GenéticaRESUMEN
Ectopic expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the fibroblast genome, generating induced pluripotent stem (iPS) cells. It remains unknown if nuclear reprogramming induced by these four factors globally resets epigenetic differences between differentiated and pluripotent cells. Here, using novel selection approaches, we have generated iPS cells from fibroblasts to characterize their epigenetic state. Female iPS cells showed reactivation of a somatically silenced X chromosome and underwent random X inactivation upon differentiation. Genome-wide analysis of two key histone modifications indicated that iPS cells are highly similar to ES cells. Consistent with these observations, iPS cells gave rise to viable high-degree chimeras with contribution to the germline. These data show that transcription factor-induced reprogramming leads to the global reversion of the somatic epigenome into an ES-like state. Our results provide a paradigm for studying the epigenetic modifications that accompany nuclear reprogramming and suggest that abnormal epigenetic reprogramming does not pose a problem for the potential therapeutic applications of iPS cells.