Contributions
Abstract: EP1299
Type: E-Poster Presentation
Session title: Stem cell transplantation - Experimental
Background
Umbilical cord blood is a source of hematopoietic stem cell (HSC) and is used in hematopoietic cell transplantation. The HSC engraft in the bone marrow and differentiate to progenitor and mature blood cells. The dynamic of the HSC can be evaluated by hematopoietic colony-forming unit (CFU) assays using methylcellulose-based medium. From the plasma membrane of hematopoietic cells, like other cells, extracellular vesicles of 0.1–1μm in size germinate, called microparticles (MPs). MPs can affect normal cell functions. Our previous study showed that there are MPs derived from hematopoietic stem cells in umbilical cord blood and they carry the surface antigen CD34+ (CD34+ MPs).
Aims
The objective of this study was to analyze the role of CD34+MPs in the differentiation program of hematopoietic progenitor cells.
Methods
Umbilical cord blood units (UCB) were collected after informed consent. The low volume units were used in this study, as not appropriate for transplantation. CD34+MPs were isolated from the plasma of UCBs by centrifugation and purified using magnetic bead based technology (Miltenyi Biotec). Their number was estimated by flow cytometry using CD34-PE and Annexin V-FITC Abs. Mononuclear cells (MNCs) were isolated from UCB by ficoll density centrifugation. MNCs were cultured in liquid culture without growth factors for 24 hours with or without CD34+MPs. Furthermore, MNCs cultured in liquid culture without growth factors for 1 or 24 hours with or without CD34+MPs and then were seeded in semisolid cultures in the presence of a cocktail of growth factors for colony-forming unit-granulocyte/macrophage (CFU-GM), erythroid burst-forming units (BFU-E), colony-forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) colony growth.
Results
The number of MNCs cultured in the presence of CD34+MPs was reduced by 15.1% compared to mononuclear cells cultured in the absence of CD34+MPs (Control) in liquid cultures of 24 hrs. In order to analyze the effect of CD34+MPs in the differentiation and proliferation of hematopoietic cells, CFU assays were performed. First MNCs were incubated for 1 hr or 24 hrs with 500, 1000 and 2000 CD34+MPs and then were seeded in semisolid culture. CFU colony numbers were decreased by 21,7% and 70,2% respectively when MNCs co-incubated for 1h with 1000 and 2000 CD34+ΜPs. When MNCs were incubated for 24 hrs with 500 CD34+ΜPs, the number of CFU colonies was increased by 26,6%, while co-incubation with 2000 CD34+MPs decreased the number by 44,2%. Furthermore, comparison between 1 and 24 hrs incubation of MNCs with CD34+MPs in liquid culture before plating in semisolid medium has shown, an increase by 77,87% and 32,8% in CFU-GEMM and CFU-GM respectively in the presence of 500 CD34+MPs while CFU-GEMM and CFU-GM colonies decreased by 11,88% and 27,3% with 1000 CD34+MPs and 73,8 and 53,1% with 2000 CD34+MPs respectively.
Conclusion
In this study we have evaluated the negative effect of CD34+MPs in the viability of MNC from cord blood. CD34+MPs were associated with CFU growth in a dose- and time- dependent manner. Low number of CD34+MPs acts beneficially for CFU viability whereas the high number of CD34+MPs decreases the number of CFU-GM and CFU-GEMM.
Keyword(s): CD34, Cord blood, Microparticles
Abstract: EP1299
Type: E-Poster Presentation
Session title: Stem cell transplantation - Experimental
Background
Umbilical cord blood is a source of hematopoietic stem cell (HSC) and is used in hematopoietic cell transplantation. The HSC engraft in the bone marrow and differentiate to progenitor and mature blood cells. The dynamic of the HSC can be evaluated by hematopoietic colony-forming unit (CFU) assays using methylcellulose-based medium. From the plasma membrane of hematopoietic cells, like other cells, extracellular vesicles of 0.1–1μm in size germinate, called microparticles (MPs). MPs can affect normal cell functions. Our previous study showed that there are MPs derived from hematopoietic stem cells in umbilical cord blood and they carry the surface antigen CD34+ (CD34+ MPs).
Aims
The objective of this study was to analyze the role of CD34+MPs in the differentiation program of hematopoietic progenitor cells.
Methods
Umbilical cord blood units (UCB) were collected after informed consent. The low volume units were used in this study, as not appropriate for transplantation. CD34+MPs were isolated from the plasma of UCBs by centrifugation and purified using magnetic bead based technology (Miltenyi Biotec). Their number was estimated by flow cytometry using CD34-PE and Annexin V-FITC Abs. Mononuclear cells (MNCs) were isolated from UCB by ficoll density centrifugation. MNCs were cultured in liquid culture without growth factors for 24 hours with or without CD34+MPs. Furthermore, MNCs cultured in liquid culture without growth factors for 1 or 24 hours with or without CD34+MPs and then were seeded in semisolid cultures in the presence of a cocktail of growth factors for colony-forming unit-granulocyte/macrophage (CFU-GM), erythroid burst-forming units (BFU-E), colony-forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) colony growth.
Results
The number of MNCs cultured in the presence of CD34+MPs was reduced by 15.1% compared to mononuclear cells cultured in the absence of CD34+MPs (Control) in liquid cultures of 24 hrs. In order to analyze the effect of CD34+MPs in the differentiation and proliferation of hematopoietic cells, CFU assays were performed. First MNCs were incubated for 1 hr or 24 hrs with 500, 1000 and 2000 CD34+MPs and then were seeded in semisolid culture. CFU colony numbers were decreased by 21,7% and 70,2% respectively when MNCs co-incubated for 1h with 1000 and 2000 CD34+ΜPs. When MNCs were incubated for 24 hrs with 500 CD34+ΜPs, the number of CFU colonies was increased by 26,6%, while co-incubation with 2000 CD34+MPs decreased the number by 44,2%. Furthermore, comparison between 1 and 24 hrs incubation of MNCs with CD34+MPs in liquid culture before plating in semisolid medium has shown, an increase by 77,87% and 32,8% in CFU-GEMM and CFU-GM respectively in the presence of 500 CD34+MPs while CFU-GEMM and CFU-GM colonies decreased by 11,88% and 27,3% with 1000 CD34+MPs and 73,8 and 53,1% with 2000 CD34+MPs respectively.
Conclusion
In this study we have evaluated the negative effect of CD34+MPs in the viability of MNC from cord blood. CD34+MPs were associated with CFU growth in a dose- and time- dependent manner. Low number of CD34+MPs acts beneficially for CFU viability whereas the high number of CD34+MPs decreases the number of CFU-GM and CFU-GEMM.
Keyword(s): CD34, Cord blood, Microparticles