RESUMO
Throughout life, one's blood supply depends on sustained division of hematopoietic stem cells (HSCs) for self-renewal and differentiation. Within the bone marrow microenvironment, an adhesion-dependent or -independent niche system regulates HSC function. Here we show that a novel adhesion-dependent mechanism via integrin-ß3 signaling contributes to HSC maintenance. Specific ligation of ß3-integrin on HSCs using an antibody or extracellular matrix protein prevented loss of long-term repopulating (LTR) activity during ex vivo culture. The actions required activation of αvß3-integrin "inside-out" signaling, which is dependent on thrombopoietin (TPO), an essential cytokine for activation of dormant HSCs. Subsequent "outside-in" signaling via phosphorylation of Tyr747 in the ß3-subunit cytoplasmic domain was indispensable for TPO-dependent, but not stem cell factor-dependent, LTR activity in HSCs in vivo. This was accompanied with enhanced expression of Vps72, Mll1, and Runx1, 3 factors known to be critical for maintaining HSC activity. Thus, our findings demonstrate a mechanistic link between ß3-integrin and TPO in HSCs, which may contribute to maintenance of LTR activity in vivo as well as during ex vivo culture.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Integrina alfaVbeta3/fisiologia , Transdução de Sinais , Trombopoetina/farmacologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores/metabolismo , Western Blotting , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Comunicação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Human embryonic stem cells (hESCs) could potentially represent an alternative source for blood transfusion therapies and a promising tool for studying the ontogeny of hematopoiesis. When we cultured hESCs on either C3H10T1/2 or OP-9 cells to facilitate hematopoiesis, we found that exogenous administration of vascular endothelial growth factor promoted the emergence of sac-like structures, which we named embryonic stem cell-derived sacs (ES-sacs). These ES-sacs consisted of multiple cysts demarcated by cellular monolayers that retained some of the properties of endothelial cells. The spherical cells inside ES-sacs expressed primarily CD34, along with VE-cadherin, CD31, CD41a, and CD45, and were able to form hematopoietic colonies in semisolid culture and to differentiate into mature megakaryocytes by day 24 in the presence of thrombopoietin. Apparently, ES-sacs provide a suitable environment for hematopoietic progenitors. Relatively large numbers of mature megakaryocytes could be induced from the hematopoietic progenitors within ES-sacs, which were then able to release platelets that displayed integrin alpha IIb beta 3 activation and spreading in response to ADP or thrombin. This novel protocol thus provides a means of generating platelets from hESCs, which could serve as the basis for efficient production of platelets for clinical transfusion and studies of thrombopoiesis.
Assuntos
Plaquetas/citologia , Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Hematopoéticas/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Plaquetas/metabolismo , Células-Tronco Embrionárias/metabolismo , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imuno-Histoquímica , Megacariócitos/citologia , Megacariócitos/metabolismo , Microscopia Confocal , Ativação Plaquetária/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The Polycomb group (PcG) gene Bmi-1 has recently been implicated in the maintenance of hematopoietic stem cells (HSC) from loss-of-function analysis. Here, we demonstrate that increased expression of Bmi-1 promotes HSC self-renewal. Forced expression of Bmi-1 enhanced symmetrical cell division of HSCs and mediated a higher probability of inheritance of stemness through cell division. Correspondingly, forced expression of Bmi-1, but not the other PcG genes, led to a striking ex vivo expansion of multipotential progenitors and marked augmentation of HSC repopulating capacity in vivo. Loss-of-function analyses revealed that among PcG genes, absence of Bmi-1 is preferentially linked with a profound defect in HSC self-renewal. Our findings define Bmi-1 as a central player in HSC self-renewal and demonstrate that Bmi-1 is a target for therapeutic manipulation of HSCs.