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1.
Blood ; 134(14): 1159-1175, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31366618

RESUMO

Hematopoietic transcription factor LIM domain only 2 (LMO2), a member of the TAL1 transcriptional complex, plays an essential role during early hematopoiesis and is frequently activated in T-cell acute lymphoblastic leukemia (T-ALL) patients. Here, we demonstrate that LMO2 is activated by deacetylation on lysine 74 and 78 via the nicotinamide phosphoribosyltransferase (NAMPT)/sirtuin 2 (SIRT2) pathway. LMO2 deacetylation enables LMO2 to interact with LIM domain binding 1 and activate the TAL1 complex. NAMPT/SIRT2-mediated activation of LMO2 by deacetylation appears to be important for hematopoietic differentiation of induced pluripotent stem cells and blood formation in zebrafish embryos. In T-ALL, deacetylated LMO2 induces expression of TAL1 complex target genes HHEX and NKX3.1 as well as LMO2 autoregulation. Consistent with this, inhibition of NAMPT or SIRT2 suppressed the in vitro growth and in vivo engraftment of T-ALL cells via diminished LMO2 deacetylation. This new molecular mechanism may provide new therapeutic possibilities in T-ALL and may contribute to the development of new methods for in vitro generation of blood cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Hematopoese , Proteínas com Domínio LIM/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Acetilação , Animais , Células Cultivadas , Células HEK293 , Humanos , Leucopoese , Camundongos , Modelos Moleculares , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Peixe-Zebra
2.
Transfusion ; 58(5): 1132-1142, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29473177

RESUMO

BACKGROUND: Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP). STUDY DESIGN AND METHODS: ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake. RESULTS: ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44 × 10-8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730 × 103 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2. CONCLUSIONS: Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP.


Assuntos
Plaquetas/química , Células Progenitoras Endoteliais/citologia , Leucaférese , Células-Tronco de Sangue Periférico/citologia , Antígenos CD/análise , Sistema Livre de Células/química , Humanos , Lipoproteínas LDL/análise , Manufaturas
3.
BMC Med ; 11: 146, 2013 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-23758701

RESUMO

BACKGROUND: Mesenchymal stromal cells (MSCs) are attractive for cell-based therapies ranging from regenerative medicine and tissue engineering to immunomodulation. However, clinical efficacy is variable and it is unclear how the phenotypes defining bone marrow (BM)-derived MSCs as well as donor characteristics affect their functional properties. METHODS: BM-MSCs were isolated from 53 (25 female, 28 male; age: 13 to 80 years) donors and analyzed by: (1) phenotype using flow cytometry and cell size measurement; (2) in vitro growth kinetics using population doubling time; (3) colony formation capacity and telomerase activity; and (4) function by in vitro differentiation capacity, suppression of T cell proliferation, cytokines and trophic factors secretion, and hormone and growth factor receptor expression. Additionally, expression of Oct4, Nanog, Prdm14 and SOX2 mRNA was compared to pluripotent stem cells. RESULTS: BM-MSCs from younger donors showed increased expression of MCAM, VCAM-1, ALCAM, PDGFRß, PDL-1, Thy1 and CD71, and led to lower IL-6 production when co-cultured with activated T cells. Female BM-MSCs showed increased expression of IFN-γR1 and IL-6ß, and were more potent in T cell proliferation suppression. High-clonogenic BM-MSCs were smaller, divided more rapidly and were more frequent in BM-MSC preparations from younger female donors. CD10, ß1integrin, HCAM, CD71, VCAM-1, IFN-γR1, MCAM, ALCAM, LNGFR and HLA ABC were correlated to BM-MSC preparations with high clonogenic potential and expression of IFN-γR1, MCAM and HLA ABC was associated with rapid growth of BM-MSCs. The mesodermal differentiation capacity of BM-MSCs was unaffected by donor age or gender but was affected by phenotype (CD10, IFN-γR1, GD2). BM-MSCs from female and male donors expressed androgen receptor and FGFR3, and secreted VEGF-A, HGF, LIF, Angiopoietin-1, basic fibroblast growth factor (bFGF) and NGFB. HGF secretion correlated negatively to the expression of CD71, CD140b and Galectin 1. The expression of Oct4, Nanog and Prdm14 mRNA in BM-MSCs was much lower compared to pluripotent stem cells and was not related to donor age or gender. Prdm14 mRNA expression correlated positively to the clonogenic potential of BM-MSCs. CONCLUSIONS: By identifying donor-related effects and assigning phenotypes of BM-MSC preparations to functional properties, we provide useful tools for assay development and production for clinical applications of BM-MSC preparations.


Assuntos
Imunofenotipagem , Doadores Vivos , Células-Tronco Mesenquimais/imunologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular/imunologia , Células Cultivadas , Feminino , Humanos , Imunofenotipagem/métodos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/patologia , Pessoa de Meia-Idade , Fatores Sexuais , Adulto Jovem
4.
Br J Haematol ; 153(4): 520-8, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21418181

RESUMO

Incompatible blood group antigens are highly immunogenic and can cause graft rejections. Focusing on distinct carbohydrate- and protein-based membrane structures, defined by blood group antigens, we investigated human bone marrow-derived mesenchymal stem cells (MSCs) cultured in human serum. The presence of H (CD173), ABO, RhD, RhCE, RhAG, Kell, urea transporter type B (SLC14A1, previously known as JK), and Duffy antigen receptor of chemokines (DARC) was evaluated at the levels of genome, transcriptome and antigen. Fucosyltransferase-1 (FUT1), RHCE, KEL, SLC14A1 (JK) and DARC mRNA were transcribed in MSCs. FUT1 mRNA transcription was lost during differentiation. The mRNA transcription of SLC14A1 (JK) decreased during chondrogenic differentiation, while that of DARC increased during adipogenic differentiation. All MSCs synthesized SLC14A1 (JK) but no DARC protein. However, none of the protein antigens tested occurred on the surface, indicating a lack of associated protein function in the membrane. As A and B antigens are neither expressed nor adsorbed, concerns of ABO compatibility with human serum supplements during culture are alleviated. The H antigen expression by GD2dim+ MSCs identified two distinct MSC subpopulations and enabled their isolation. We hypothesize that GD2(dim+) H(+) MSCs retain a better 'stemness'. Because immunogenic blood group antigens are lacking, they cannot affect MSC engraftment in vivo, which is promising for clinical applications.


Assuntos
Antígenos de Grupos Sanguíneos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Sistema ABO de Grupos Sanguíneos/metabolismo , Antígenos de Grupos Sanguíneos/genética , Diferenciação Celular/genética , Células Cultivadas , Sistema do Grupo Sanguíneo Duffy/biossíntese , Sistema do Grupo Sanguíneo Duffy/genética , Eritrócitos/metabolismo , Gangliosídeos/metabolismo , Humanos , Imunofenotipagem , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/genética , Células-Tronco Mesenquimais/citologia , RNA Mensageiro/genética , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Transcrição Gênica , Transportadores de Ureia
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