RESUMO
Embryonic stem cells (ESCs) can be propagated in vitro on feeder layers of mouse STO fibroblast cells. The STO cells secrete several cytokines that are essential for ESCs to maintain their undifferentiated state. In this study, we found significant growth inhibition of mouse ESCs (mESCs) cultured on STO cells infected with adenovirus containing a dominant-negative mutant form of IkappaB (rAd-dnIkappaB). This blockage of the NF-kappaB signal pathway in STO cells led to a significant decrease in [3H]thymidine incorporation and colony formation of mESCs. Expression profile of cytokines secreted from the STO cells revealed an increase in the bone morphogenetic protein4 (BMP4) transcript level in the STO cells infected with adenoviral vector encoding dominant negative IkappaB (rAd-dnIkappaB). These results suggested that the NF-kappaB signaling pathway represses expression of BMP4 in STO feeder cells. Conditioned medium from the rAd-dnIkappaB-infected STO cells also significantly reduced the colony size of mESCs. Addition of BMP4 prevented colony formation of mESCs cultured in the conditioned medium. Our finding suggested that an excess of BMP4 in the conditioned medium also inhibits proliferation of mESCs.
Assuntos
Animais , Camundongos , Proteína Morfogenética Óssea 4/genética , Diferenciação Celular/genética , Proliferação de Células , Meios de Cultivo Condicionados , Células-Tronco Embrionárias/citologia , Células Alimentadoras/citologia , Fibroblastos/citologia , Regulação da Expressão Gênica/genética , Proteínas I-kappa B/genética , Mutação , NF-kappa B/genética , Transdução de SinaisRESUMO
Capsaicin, the pungent component of chilli peppers, is known to induce mediators of hematopoiesis. We investigated the effect of capsaicin on hematopoiesis in mouse progenitor cells. Treatment of mouse bone marrow cells with capsaicin induced the formation of colony of burst-forming units-erythroid (BFU-E). We also found that the number of erythropoietin receptor (EpoR)-positive cells was increased by capsaicin. To clarify the effect of capsaicin on erythroid lineage, BFU-E colonies were separated from non-BFU-E colonies by colony-picking after in vitro culture of mouse bone marrow cells. Quantitative RT-PCR analysis revealed that capsaicin stimulated the expression of the erythroid-specific genes encoding EpoR, glycophorin A (GPA), beta-globin (Hbb-b1), GATA-1, PU.1, nuclear factor erythroid-derived 2 (NF-E2), and Kruppel-like factor 1 (KLF1) in the BFU-E colonies. Furthermore, capsaicin could effectively stimulate the transfected GATA-1 promoter in K562 cells. GATA-1 is known as an essential transcription factor for the development of erythroid cells. Our results show that development of the erythroid lineage from bone marrow cells can be induced by treatment with capsaicin, and that GATA-1 seems to play a role in this induced erythroid maturation.
Assuntos
Animais , Masculino , Camundongos , Células da Medula Óssea/citologia , Capsaicina/farmacologia , Linhagem da Célula , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Células Eritroides/citologia , Fator de Transcrição GATA1/genética , Hematopoese , Células-Tronco Hematopoéticas/citologia , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Receptores da Eritropoetina/metabolismoRESUMO
A C6 beta-chemokine, CKbeta8-1, suppressed the colony formation of CD34 + cells of human cord blood (CB). Molecular mechanisms involved in CKbeta8-1-medicated suppression of colony formation of CD34 + cells are not known. To address this issue, the level of various G1/S cell cycle regulating proteins in CKbeta8-1-treated CD34 + cells were compared with those in untreated CD34 + cells. CKbeta8-1 did not significantly alter the expression of the G1/S cycle regulation proteins (cyclin D1, D3, and E), CDK inhibitor (p27and Rb), and other cell proliferation regulation protein (p53) in CB CD34 + cells. Here we describe an in vitro system in which CB CD34 + cells were committed to a multipotent progenitor lineage of colony forming units-granulocyte/macrophage (CFU-GM) by a simple combination of recombinant human (rh) GM-CSF and rhIL-3. In this culture system, we found that cyclin E protein appeared later and disappeared faster in the CKbeta8-1-treated cells than in the control cells during CFU-GM lineage development. These findings suggested that cyclin E may play a role in suppressing the colony formation of CFU-GM by CKbeta8-1.
Assuntos
Humanos , Antígenos CD34/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula , Células Cultivadas , Quimiocinas CC/farmacologia , Ciclina E/metabolismo , Sangue Fetal/citologia , Fase G1/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Granulócitos/citologia , Substâncias de Crescimento/farmacologia , Macrófagos/citologia , Células-Tronco/citologiaRESUMO
PURPOSE: We investigated the expression of vasoactive intestinal peptide (VIP), VIP receptor 1 (VPAC1), VIP receptor 2 (VPAC2) genes in the human umbilical cord blood CD34 cells, and the ability of VIP to stimulate human primitive as well as monopotent hematopoietic progenitors. METHODS: We isolated RNA from umbilical cord blood CD34 cells, and then performed RT-PCR, and sequencing. The umbilical cord blood CD34 cells were cultured with the various concentrations of VIP for burst-forming unit of erythrocyte (BFU-E), colony-forming unit of granulocyte/monocyte (CFU-GM), colony-forming unit of graulocyte/erythrocyte/monocyte/megakaryocyte (CFU-GEMM), and colony-forming unit of megakaryocyte (CFU-Mk). RESULTS: The RNA coding for VPAC1 was detected in human umbilical cord blood CD34 cells. VIP significantly stimulated the growth of CFU-GEMM and CFU-Mk. CONCLUSION: The present results suggest that VIP is an important neuropeptide in the early proliferation of human primitive as well as megakaryocyte progenitors.