RESUMEN
The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations1. Here we reconstruct a phylogenetic tree of blood development using whole-genome sequencing of 511 single-cell-derived haematopoietic colonies from healthy human fetuses at 8 and 18 weeks after conception, coupled with deep targeted sequencing of tissues of known embryonic origin. We found that, in healthy fetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 weeks after conception. We used these mutations as barcodes and timed the divergence of embryonic and extra-embryonic tissues during development, and estimated the number of blood antecedents at different stages of embryonic development. Our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.
Asunto(s)
Linaje de la Célula/genética , Desarrollo Embrionario/genética , Sistema Hematopoyético/embriología , Sistema Hematopoyético/metabolismo , Mutación , Células Sanguíneas/citología , Células Sanguíneas/metabolismo , Células Clonales/citología , Células Clonales/metabolismo , Análisis Mutacional de ADN , Feto/citología , Feto/embriología , Feto/metabolismo , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Salud , Sistema Hematopoyético/citología , Humanos , Cariotipificación , Masculino , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Tasa de Mutación , Especificidad de Órganos/genética , Factores de Tiempo , Secuenciación Completa del Genoma , Flujo de TrabajoRESUMEN
Restoring pluripotency using chemical compounds alone would be a major step forward in developing clinical-grade pluripotent stem cells, but this has not yet been reported in human cells. We previously demonstrated that VPA_AFS cells, human amniocytes cultivated with valproic acid (VPA) acquired functional pluripotency while remaining distinct from human embryonic stem cells (hESCs), questioning the relationship between the modulation of cell fate and molecular regulation of the pluripotency network. Here, we used single-cell analysis and functional assays to reveal that VPA treatment resulted in a homogeneous population of self-renewing non-transformed cells that fulfill the hallmarks of pluripotency, i.e., a short G1 phase, a dependence on glycolytic metabolism, expression of epigenetic modifications on histones 3 and 4, and reactivation of endogenous OCT4 and downstream targets at a lower level than that observed in hESCs. Mechanistic insights into the process of VPA-induced reprogramming revealed that it was dependent on OCT4 promoter activation, which was achieved independently of the PI3K (phosphatidylinositol 3-kinase)/AKT/mTOR (mammalian target of rapamycin) pathway or GSK3ß inhibition but was concomitant with the presence of acetylated histones H3K9 and H3K56, which promote pluripotency. Our data identify, for the first time, the pluripotent transcriptional and molecular signature and metabolic status of human chemically induced pluripotent stem cells.
Asunto(s)
Amnios/citología , Transdiferenciación Celular/efectos de los fármacos , Reprogramación Celular/efectos de los fármacos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Biomarcadores , Ciclo Celular/genética , Transdiferenciación Celular/genética , Reprogramación Celular/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Metabolismo Energético , Epigénesis Genética , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Genes Reporteros , Glucólisis , Histonas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteína Homeótica Nanog/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Recombinantes de Fusión , Serina-Treonina Quinasas TOR/metabolismo , Activación TranscripcionalRESUMEN
Regulation of hematopoiesis during human development remains poorly defined. Here we applied single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to over 8,000 human immunophenotypic blood cells from fetal liver and bone marrow. We inferred their differentiation trajectory and identified three highly proliferative oligopotent progenitor populations downstream of hematopoietic stem cells (HSCs)/multipotent progenitors (MPPs). Along this trajectory, we observed opposing patterns of chromatin accessibility and differentiation that coincided with dynamic changes in the activity of distinct lineage-specific transcription factors. Integrative analysis of chromatin accessibility and gene expression revealed extensive epigenetic but not transcriptional priming of HSCs/MPPs prior to their lineage commitment. Finally, we refined and functionally validated the sorting strategy for the HSCs/MPPs and achieved around 90% enrichment. Our study provides a useful framework for future investigation of human developmental hematopoiesis in the context of blood pathologies and regenerative medicine.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Hematopoyesis , Linaje de la Célula/genética , Hematopoyesis/genética , Células Madre Hematopoyéticas , Humanos , RNA-Seq , Análisis de la Célula IndividualRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Expression of OCT4A is one of the hallmarks of pluripotency, defined as a stem cell's ability to differentiate into all the lineages of the three germ layers. Despite being defined as non-tumorigenic cells with high translational potential, human mid-trimester amniotic fluid stem cells (hAFSCs) are often described as sharing features with embryonic stem cells, including the expression of OCT4A, which could hinder their clinical potential. To clarify the OCT4A status of hAFSCs, we first undertook a systematic review of the literature. We then performed extensive gene and protein expression analyses to discover that neither frozen, nor fresh hAFSCs cultivated in multipotent stem cell culture conditions expressed OCT4A, and that the OCT4A positive results from the literature are likely to be attributed to the expression of pseudogenes or other OCT4 variants. To address this issue, we provide a robust protocol for the assessment of OCT4A in other stem cells.
Asunto(s)
Líquido Amniótico/citología , Regulación del Desarrollo de la Expresión Génica , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre/citología , Linaje de la Célula , Exones , Femenino , Perfilación de la Expresión Génica , Variación Genética , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Microscopía Fluorescente , Células Madre Multipotentes/citología , Embarazo , Segundo Trimestre del Embarazo , Isoformas de ProteínasRESUMEN
The human amniotic fluid stem cell (hAFSC) population consists of two morphologically distinct subtypes, spindle-shaped and round-shaped cells (SS-hAFSCs and RS-hAFSCs). Whilst SS-hAFSCs are routinely expanded in mesenchymal-type (MT) conditions, we previously showed that they acquire broader differentiation potential when cultured under embryonic-type (ET) conditions. However, the effects of culture conditions on RS-hAFSCs have not been determined. Here, we show that culturing RS-hAFSCs under ET conditions confers faster proliferation and enhances the efficiency of osteogenic differentiation of the cells. We show that this occurs via TGFß-induced activation of CD73 and the associated increase in the generation of extracellular adenosine. Our data demonstrate that culture conditions are decisive for the expansion of hAFSCs and that TGFß present in ET conditions causes the phenotype of RS-hAFSCs to revert to an earlier state of stemness. Cultivating RS-hAFSCs in ET conditions with TGFß may therefore increase their therapeutic potential for clinical applications.