RESUMEN
Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence, comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end, here, we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%-99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively, this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. VIDEO ABSTRACT.
Asunto(s)
Mesodermo/citología , Transducción de Señal , Proteínas Morfogenéticas Óseas/metabolismo , Huesos/citología , Huesos/metabolismo , Corazón/crecimiento & desarrollo , Proteínas de Homeodominio/metabolismo , Humanos , Mesodermo/metabolismo , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/metabolismo , Línea Primitiva/citología , Línea Primitiva/metabolismo , Análisis de la Célula Individual , Somitos/metabolismo , Células Madre , Proteínas Supresoras de Tumor/metabolismo , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/metabolismoRESUMEN
Understanding the relative contributions of genetic and epigenetic abnormalities to acute myeloid leukemia (AML) should assist integrated design of targeted therapies. In this study, we generated induced pluripotent stem cells (iPSCs) from AML patient samples harboring MLL rearrangements and found that they retained leukemic mutations but reset leukemic DNA methylation/gene expression patterns. AML-iPSCs lacked leukemic potential, but when differentiated into hematopoietic cells, they reacquired the ability to give rise to leukemia in vivo and reestablished leukemic DNA methylation/gene expression patterns, including an aberrant MLL signature. Epigenetic reprogramming was therefore not sufficient to eliminate leukemic behavior. This approach also allowed us to study the properties of distinct AML subclones, including differential drug susceptibilities of KRAS mutant and wild-type cells, and predict relapse based on increased cytarabine resistance of a KRAS wild-type subclone. Overall, our findings illustrate the value of AML-iPSCs for investigating the mechanistic basis and clonal properties of human AML.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Leucemia Mieloide Aguda/patología , Modelos Biológicos , Crisis Blástica/patología , Línea Celular Tumoral , Linaje de la Célula , Forma de la Célula , Reprogramación Celular , Aberraciones Cromosómicas , Células Clonales , Metilación de ADN/genética , Epigénesis Genética , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Células HEK293 , Hematopoyesis/genética , Humanos , Leucemia Mieloide Aguda/genética , Terapia Molecular Dirigida , Mutación/genética , Invasividad Neoplásica , FenotipoRESUMEN
Mesoderm is the developmental precursor to myriad human tissues including bone, heart, and skeletal muscle. Unravelling the molecular events through which these lineages become diversified from one another is integral to developmental biology and understanding changes in cellular fate. To this end, we developed an in vitro system to differentiate human pluripotent stem cells through primitive streak intermediates into paraxial mesoderm and its derivatives (somites, sclerotome, dermomyotome) and separately, into lateral mesoderm and its derivatives (cardiac mesoderm). Whole-population and single-cell analyses of these purified populations of human mesoderm lineages through RNA-seq, ATAC-seq, and high-throughput surface marker screens illustrated how transcriptional changes co-occur with changes in open chromatin and surface marker landscapes throughout human mesoderm development. This molecular atlas will facilitate study of human mesoderm development (which cannot be interrogated in vivo due to restrictions on human embryo studies) and provides a broad resource for the study of gene regulation in development at the single-cell level, knowledge that might one day be exploited for regenerative medicine.