Contributions
Abstract: EP1361
Type: e-Poster
Background
Microparticles (MPs) are small cell derived vesicles, sized 100nm-1μm and released from the plasma membrane of apoptotic or activated cells carrying cell surface markers from those cells. Apoptosis is a complex interaction network regulated either through death receptors like FAS or through the internal pathway of Bcl2, Bax genes. The two pathways activate the executors of cell death program, caspases, which can be activated by perforin pathway. MPs play role in intercellular communication that is associated with physiological and pathological conditions. Our previous study showed that MPs derived from CD34+cells of umbilical cord blood (UCB), which is an alternative source for hematopoietic stem cell transplantation carry hemopoietic specific miRNAs. However, the role of MPs has not been completely clarified.
Aims
The objective of this study was to study the role of stem cell derived-MPs from cord blood units in apoptosis of hematopoietic cells.
Methods
UCB were collected after informed consent. The units that were used in this study, were rejected as not appropriate for transplantation due to low volume. CD34+MPs were isolated from the plasma of UCBs by centrifugation and purified using magnetic bead MACS technology (Miltenyi Biotec). The number of CD34+MPs was estimated by flow cytometry using CD34-PE and Annexin V-FITC Abs. Mononuclear cells (MNC) were collected after density gradient centrifugation and were cultured for 24hrs in the presence of CD34+MPs. The lymphoblastic leukemia cell line MOLT4 was cultured in RPMI (Life Technologies) supplemented with 5% fetal bovine serum and 2% penicillin-strepromycin. Viability assays were performed by trypan blue exclusion. MOLT4 cells were incubated with or without CD34+MPs. RT-PCR of Fas, Bcl2, caspase 3, perforin and GAPDH genes were performed. The PCR products were analysed in agarose gel electrophoresis. mRNA expression of Bcl2 gene was also evaluated by RQ-PCR.
Results
CD34+MPs affect the expression of Bcl2, Fas and caspase 3. Specifically purified CD34+MPs were applied in MOLT4 for 4hrs and RT-PCR analysis showed, high expression of Fas and moderate increased expression of caspase 3 and Bcl2. In contrast the expression of Bcl2 was decreased after 24hrs incubation with CD34+MPs whereas the expression of Fas and caspase 3 was slightly decreased. The number of MOLT4 cells were decreased in the presence of CD34+MPs after 24hrs. Analysis of the apoptotic pathway of perforin has shown that perforin gene expression was increased in CD34+MPs treated cells in comparison to control after incubation of 4 and 24hrs with higher expression at 4hrs. These results are in accordance to the decrease of the cell number in MOLT4 culture in presence of CD34+MPs after 24hrs. Proteomic analysis showed that CD34+MPs increased the expression of Bcl2, Bax, Fas and caspase 3 proteins in cord blood MNC after 24hrs incubation whereas Bcl2 and Fas were not expressed in control cells.
Conclusion
In this study we have identified and monitored the time-dependent effect of CD34+MPs on the viability of lymphoblastic cell line MOLT4. The internal, external and perforin apoptotic pathways were activated by CD34+MPs with differential expression pattern of the apoptotic genes Fas and Perforin and the anti-apoptotic Bcl2 gene. In cord blood derived MNC, CD34+MPs increases the expression of Bcl2 as well as Fas, Bax and caspase 3. Therefore the CD34+MPs may regulate the apoptosis in normal and malignant hematopoietic cells
Session topic: 21. Stem cell transplantation - Experimental
Keyword(s): Apoptosis
Abstract: EP1361
Type: e-Poster
Background
Microparticles (MPs) are small cell derived vesicles, sized 100nm-1μm and released from the plasma membrane of apoptotic or activated cells carrying cell surface markers from those cells. Apoptosis is a complex interaction network regulated either through death receptors like FAS or through the internal pathway of Bcl2, Bax genes. The two pathways activate the executors of cell death program, caspases, which can be activated by perforin pathway. MPs play role in intercellular communication that is associated with physiological and pathological conditions. Our previous study showed that MPs derived from CD34+cells of umbilical cord blood (UCB), which is an alternative source for hematopoietic stem cell transplantation carry hemopoietic specific miRNAs. However, the role of MPs has not been completely clarified.
Aims
The objective of this study was to study the role of stem cell derived-MPs from cord blood units in apoptosis of hematopoietic cells.
Methods
UCB were collected after informed consent. The units that were used in this study, were rejected as not appropriate for transplantation due to low volume. CD34+MPs were isolated from the plasma of UCBs by centrifugation and purified using magnetic bead MACS technology (Miltenyi Biotec). The number of CD34+MPs was estimated by flow cytometry using CD34-PE and Annexin V-FITC Abs. Mononuclear cells (MNC) were collected after density gradient centrifugation and were cultured for 24hrs in the presence of CD34+MPs. The lymphoblastic leukemia cell line MOLT4 was cultured in RPMI (Life Technologies) supplemented with 5% fetal bovine serum and 2% penicillin-strepromycin. Viability assays were performed by trypan blue exclusion. MOLT4 cells were incubated with or without CD34+MPs. RT-PCR of Fas, Bcl2, caspase 3, perforin and GAPDH genes were performed. The PCR products were analysed in agarose gel electrophoresis. mRNA expression of Bcl2 gene was also evaluated by RQ-PCR.
Results
CD34+MPs affect the expression of Bcl2, Fas and caspase 3. Specifically purified CD34+MPs were applied in MOLT4 for 4hrs and RT-PCR analysis showed, high expression of Fas and moderate increased expression of caspase 3 and Bcl2. In contrast the expression of Bcl2 was decreased after 24hrs incubation with CD34+MPs whereas the expression of Fas and caspase 3 was slightly decreased. The number of MOLT4 cells were decreased in the presence of CD34+MPs after 24hrs. Analysis of the apoptotic pathway of perforin has shown that perforin gene expression was increased in CD34+MPs treated cells in comparison to control after incubation of 4 and 24hrs with higher expression at 4hrs. These results are in accordance to the decrease of the cell number in MOLT4 culture in presence of CD34+MPs after 24hrs. Proteomic analysis showed that CD34+MPs increased the expression of Bcl2, Bax, Fas and caspase 3 proteins in cord blood MNC after 24hrs incubation whereas Bcl2 and Fas were not expressed in control cells.
Conclusion
In this study we have identified and monitored the time-dependent effect of CD34+MPs on the viability of lymphoblastic cell line MOLT4. The internal, external and perforin apoptotic pathways were activated by CD34+MPs with differential expression pattern of the apoptotic genes Fas and Perforin and the anti-apoptotic Bcl2 gene. In cord blood derived MNC, CD34+MPs increases the expression of Bcl2 as well as Fas, Bax and caspase 3. Therefore the CD34+MPs may regulate the apoptosis in normal and malignant hematopoietic cells
Session topic: 21. Stem cell transplantation - Experimental
Keyword(s): Apoptosis