RESUMO
This corrects the article DOI: 10.1038/ncomms11208.
RESUMO
The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Reprogramação Celular , Fator de Transcrição GATA1/genética , Megacariócitos/citologia , Células-Tronco Pluripotentes/citologia , Proteína Proto-Oncogênica c-fli-1/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Plaquetas/citologia , Plaquetas/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Criopreservação/métodos , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lentivirus/genética , Megacariócitos/metabolismo , Análise em Microsséries , Células-Tronco Pluripotentes/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Transdução Genética , TransgenesRESUMO
Small RNA molecules, including microRNAs (miRNAs), play critical roles in regulating pluripotency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs) are similar in length to miRNAs, align to miRNA precursor (pre-miRNA) loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence. Recent next generation sequencing (NGS) studies have reported the presence of moRNAs in human neurons and cancer cells and in several tissues in mouse, including pluripotent stem cells. In order to gain additional knowledge about human moRNAs and their putative development-related expression, we applied NGS of small RNAs in human embryonic stem cells (hESCs) and fibroblasts. We found that certain moRNA isoforms are notably expressed in hESCs from loci coding for stem cell-selective or cancer-related miRNA clusters. In contrast, we observed only sparse moRNAs in fibroblasts. Consistent with earlier findings, most of the observed moRNAs derived from conserved loci and their expression did not appear to correlate with the expression of the adjacent miRNAs. We provide here the first report of moRNAs in hESCs, and their expression profile in comparison to fibroblasts. Moreover, we expand the repertoire of hESC miRNAs. These findings provide an expansion on the known repertoire of small non-coding RNA contents in hESCs.
Assuntos
Expressão Gênica , Células-Tronco Embrionárias Humanas/metabolismo , MicroRNAs/genética , Pequeno RNA não Traduzido/genética , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Biologia Computacional , Perfilação da Expressão Gênica , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/química , Anotação de Sequência Molecular , Dados de Sequência Molecular , Pequeno RNA não Traduzido/química , Alinhamento de SequênciaRESUMO
Transplantation of human embryonic stem cells (hESCs), like other allogeneic cellular transplants, require immunomodulation or immunosuppression in order to be maintained in the recipient. Costimulation blockade applied at the time of transplantation inhibits costimulatory signals in the immunological synapse leading to a state of anergy in the donor reactive T-cell population and a state of immunological tolerance in the host. In models of solid organ transplantation, tolerance is maintained by the infiltration of Foxp3(+) regulatory T cells into the graft. In order to study if regulatory T cells could be generated to hESC transplants, costimulation blockade (CTLA4Ig, anti-CD40L, anti-LFA-1) was administered for the first week after transplantation of two different hESC lines implanted under the kidney capsule of wild-type mice. hESC transplants were maintained indefinitely, and when harvested at long-term follow-up, Foxp3(+) T-cells were found surrounding the graft, implying the maintenance of tolerance through the induction of regulatory T cells. These results imply that costimulation blockade could be a useful treatment strategy for the induction of tolerance to hESC transplants and may down-modulate immune responses locally around the graft.