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1.
Haematologica ; 100(11): 1396-406, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26294724

RESUMO

Expansion of erythroblasts from human peripheral blood mononuclear cells is 4- to 15-fold more efficient than that of CD34(+) cells purified from peripheral blood mononuclear cells. In addition, purified CD34(+) and CD34(-) populations from blood do not reconstitute this erythroid yield, suggesting a role for feeder cells present in blood mononuclear cells that increase hematopoietic output. Immunodepleting peripheral blood mononuclear cells for CD14(+) cells reduced hematopoietic stem and progenitor cell expansion. Conversely, the yield was increased upon co-culture of CD34(+) cells with CD14(+) cells (full contact or transwell assays) or CD34(+) cells re-constituted in conditioned medium from CD14(+) cells. In particular, CD14(++)CD16(+) intermediate monocytes/macrophages enhanced erythroblast outgrowth from CD34(+) cells. No effect of CD14(+) cells on erythroblasts themselves was observed. However, 2 days of co-culturing CD34(+) and CD14(+) cells increased CD34(+) cell numbers and colony-forming units 5-fold. Proliferation assays suggested that CD14(+) cells sustain CD34(+) cell survival but not proliferation. These data identify previously unrecognized erythroid and non-erythroid CD34(-) and CD34(+) populations in blood that contribute to the erythroid yield. A flow cytometry panel containing CD34/CD36 can be used to follow specific stages during CD34(+) differentiation to erythroblasts. We have shown modulation of hematopoietic stem and progenitor cell survival by CD14(+) cells present in peripheral blood mononuclear cells which can also be found near specific hematopoietic niches in the bone marrow.


Assuntos
Células Eritroides/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Sobrevivência Celular , Técnicas de Cocultura , Células Eritroides/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Macrófagos/citologia , Monócitos/citologia
2.
Am J Hematol ; 88(9): 723-9, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23720412

RESUMO

Erythropoiesis is a tightly regulated process which becomes decoupled from its normal differentiation program in patients with polycythemia vera (PV). Somatic mutations in JAK2 are commonly associated with this myeloid proliferative disorder. To gain insight into the molecular events that are required for abnormally developing erythroid cells to escape dependence on normal growth signals, we performed in vitro expansion of mature erythroblasts (ERY) from seven normal healthy donors and from seven polycythemic patients in the presence of IL3, EPO, SCF for 10, 11, or 13 days. Normal ERYs required exposure to the glucocorticoid dexamethasone (Dex) for expansion, while PV-derived ERYs expanded in the absence of dexamethasone. RNA expression profiling revealed enrichment of two known oncogenes, GPR56 and RAB4a, in PV-derived ERYs along with reduced expression levels of transcription factor TAL1 (ANOVA FDR < 0.05). While both normal and polycythemic-derived ERYs integrated signaling cascades for growth, they did so via different signaling pathways which are represented by their differential phospho-profiles. Our results show that normal ERYs displayed greater levels of phosphorylation of EGFR, PDGFRß, TGFß, and cKit, while PV-derived ERYs were characterized by increased phosphorylation of cytoplasmic kinases in the JAK/STAT, PI3K, and GATA1 pathways. Together these data suggest that PV erythroblast expansion and maturation may be maintained and enriched in the absence of dexamethasone through reduced TAL1 expression and by accessing additional signaling cascades. Members of this acquired repertoire may provide important insight into the pathogenesis of aberrant erythropoiesis in myeloproliferative neoplasms such as polycythemia vera.


Assuntos
Eritroblastos/metabolismo , Eritropoese/genética , Fosfoproteínas/genética , Policitemia Vera/genética , Adulto , Idoso , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Dexametasona/farmacologia , Eritroblastos/efeitos dos fármacos , Eritroblastos/patologia , Eritropoetina/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Interleucina-3/farmacologia , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/metabolismo , Policitemia Vera/metabolismo , Policitemia Vera/patologia , Proteômica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Fator de Células-Tronco/farmacologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/metabolismo
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