RESUMEN
The sea urchin larval skeleton offers a simple model for formation of developmental patterns. The calcium carbonate skeleton is secreted by primary mesenchyme cells (PMCs) in response to largely unknown patterning cues expressed by the ectoderm. To discover novel ectodermal cues, we performed an unbiased RNA-Seq-based screen and functionally tested candidates; we thereby identified several novel skeletal patterning cues. Among these, we show that SLC26a2/7 is a ventrally expressed sulfate transporter that promotes a ventral accumulation of sulfated proteoglycans, which is required for ventral PMC positioning and skeletal patterning. We show that the effects of SLC perturbation are mimicked by manipulation of either external sulfate levels or proteoglycan sulfation. These results identify novel skeletal patterning genes and demonstrate that ventral proteoglycan sulfation serves as a positional cue for sea urchin skeletal patterning.
Asunto(s)
Tipificación del Cuerpo/genética , Proteoglicanos/metabolismo , Erizos de Mar/embriología , Erizos de Mar/genética , Análisis de Secuencia de ARN/métodos , Sulfatos/metabolismo , Animales , Tipificación del Cuerpo/efectos de los fármacos , Proteínas de Transporte de Catión/metabolismo , Diferenciación Celular/efectos de los fármacos , Ectodermo/efectos de los fármacos , Ectodermo/enzimología , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Mesodermo/citología , Modelos Biológicos , Níquel/toxicidad , Erizos de Mar/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Derivation of hematopoietic stem cells (HSCs) from human pluripotent stem cells remains a key goal for the fields of developmental biology and regenerative medicine. Here, we use a novel genetic reporter system to prospectively identify and isolate early hematopoietic cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (iPSCs). Cloning the human RUNX1c P1 promoter and +24 enhancer to drive expression of tdTomato (tdTom) in hESCs and iPSCs, we demonstrate that tdTom expression faithfully enriches for RUNX1c-expressing hematopoietic progenitor cells. Time-lapse microscopy demonstrated the tdTom(+) hematopoietic cells to emerge from adherent cells. Furthermore, inhibition of primitive hematopoiesis by blocking Activin/Nodal signaling promoted the expansion and/or survival of the tdTom(+) population. Notably, RUNX1c/tdTom(+) cells represent only a limited subpopulation of the CD34(+) CD45(+) and CD34(+) CD43(+) cells with a unique genetic signature. Using gene array analysis, we find significantly lower expression of Let-7 and mir181a microRNAs in the RUNX1c/tdTom(+) cell population. These phenotypic and genetic analyses comparing the RUNX1c/tdTom(+) population to CD34(+) CD45(+) umbilical cord blood and fetal liver demonstrate several key differences that likely impact the development of HSCs capable of long-term multilineage engraftment from hESCs and iPSCs.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/biosíntesis , Células Madre Hematopoyéticas/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Regiones Promotoras Genéticas/fisiología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Células Madre Embrionarias/fisiología , Hematopoyesis/fisiología , HumanosRESUMEN
Advancements in human pluripotent stem cell (hPSC) research have potential to revolutionize therapeutic transplantation. It has been demonstrated that transcription factors may play key roles in regulating maintenance, expansion, and differentiation of hPSCs. In addition to its regulatory functions in hematopoiesis and blood-related disorders, the transcription factor RUNX1 is also required for the formation of definitive blood stem cells. In this study, we demonstrated that expression of endogenous RUNX1a, an isoform of RUNX1, parallels with lineage commitment and hematopoietic emergence from hPSCs, including both human embryonic stem cells and inducible pluripotent stem cells. In a defined hematopoietic differentiation system, ectopic expression of RUNX1a facilitates emergence of hematopoietic progenitor cells (HPCs) and positively regulates expression of mesoderm and hematopoietic differentiation-related factors, including Brachyury, KDR, SCL, GATA2, and PU.1. HPCs derived from RUNX1a hPSCs show enhanced expansion ability, and the ex vivo-expanded cells are capable of differentiating into multiple lineages. Expression of RUNX1a in embryoid bodies (EBs) promotes definitive hematopoiesis that generates erythrocytes with ß-globin production. Moreover, HPCs generated from RUNX1a EBs possess ≥9-week repopulation ability and show multilineage hematopoietic reconstitution in vivo. Together, our results suggest that RUNX1a facilitates the process of producing therapeutic HPCs from hPSCs.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Células Madre Embrionarias/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Western Blotting , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula/genética , Proliferación Celular , Células Cultivadas , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Células Madre Embrionarias/citología , Proteínas Fetales/genética , Proteínas Fetales/metabolismo , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo , Expresión Génica , Células Madre Hematopoyéticas/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Microscopía Confocal , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
To evaluate hematopoietic niche cell populations isolated from human embryonic stem cells (hESCs), we tested the ability of hESC-derived stromal lines to support CD34(+) umbilical cord blood (UCB)- and hESC-derived CD34(+)45(+) cells in long-term culture initiating cell (LTC-IC) assays. Specifically, these hematopoietic populations were cocultured with hESC-derived mesenchymal stromal cells (hESC-MSCs) and hESC-derived endothelial cells (hESC-ECs), and then assessed for their LTC-IC potential in comparison to coculture with bone marrow (BM)-derived MSCs and the mouse stromal line M2-10B4. We found that the hESC-derived stromal lines supported LTC-ICs from UCB similar to M2-10B4 cells and better than BM-MSCs. However, none of the stromal populations supported LTC-IC from hESC-derived CD34(+)45(+) cells. Engraftment data using the output from LTC-IC assays showed long-term repopulation (12 weeks) of NSG mice to correlate with LTC-IC support on a given stromal layer. Therefore, hESC-derived stromal lines can be used to efficiently evaluate putative hematopoietic stem/progenitor cells derived from hESCs or other cell sources.