EVALUATION OF PLURIPOTENT MARKERS IN CELLS OBTAINED FROM DIFFERENT HUMAN HEMATOPOETIC SOURCES
(Abstract release date: 05/21/15)
EHA Library. Çelik D. 06/12/15; 102931; PB1770
Disclosure(s): ?stanbul University Faculte of MedicineDepartment of Physiology
Damla Çelik
Contributions
Contributions
Abstract
Abstract: PB1770
Type: Publication Only
Background
Clinical use of hematopoetic stem cells (HSC) is a developing field for treatment of most hemoglobinopaties, hereditary blood diseases and cancers. Even though use of HSC’s are practical they are not as effective as pluripotent stem cells (PSC). In last years the thought of possible presence of PSCs in other adult tissues has become an attractive research area for search and isolate of PSCs from adult tissues.
Aims
In our study we refined cell populations using different isolation methods. Cells obtained from three different human hematopoetic sources and analysed for expression of pluripotent markers like NANOG, OCT3/4 and SSEA4.
Methods
Human peripheral blood (PB), apheresis material (AM) and cord blood (CB) were used as hematopoietic sourses. Two isolation method performed to all materials. 1) Total mononuclear cells obtained by eryhtrocyte lysis method. 2) MNC layer and erythrocyte layer were isolated with ficoll density gradient method.
Results
The results obtained from western blotting and immunofluorescence methods showed expression of NANOG, OCT3/4 and SSEA4 proteins in PB, AM and CB. And also immunofluorescence images demonstrated cytoplasmic and nuclear presence of these proteins. OCT3/4 protein of immunofluorescence determinations showed cytoplasmic locations in all layers. Futher investigations need to be done for enlighten this protein function in hematopoetic cells because OCT3/4 has different isoforms. NANOG protein showed cytoplasmic location in PB and AM, both cytoplasmic and nucleic location in CB. Western blotting results indicated that NANOG protein create a ladder pattern mostly cause of post-translational modifications. NANOG ladder pattern also changed between layers. These findings showed different layers had different post-translational modifications so different type of NANOG protein.
Summary
As a result, these findings showed that it is necessary to investigate the function of PSC markers in differentiated adult cells. Another conclusion is, among the lysis and ficoll density gradient method, lysis method has the highest cell recovery amount.Consequently, this study provided us new informations and viewpoints about expression of PSC markers in adult tissues.
Keyword(s): Apheresis, Cord blood, Peripheral blood, Stem cell marker
Session topic: Publication Only
Type: Publication Only
Background
Clinical use of hematopoetic stem cells (HSC) is a developing field for treatment of most hemoglobinopaties, hereditary blood diseases and cancers. Even though use of HSC’s are practical they are not as effective as pluripotent stem cells (PSC). In last years the thought of possible presence of PSCs in other adult tissues has become an attractive research area for search and isolate of PSCs from adult tissues.
Aims
In our study we refined cell populations using different isolation methods. Cells obtained from three different human hematopoetic sources and analysed for expression of pluripotent markers like NANOG, OCT3/4 and SSEA4.
Methods
Human peripheral blood (PB), apheresis material (AM) and cord blood (CB) were used as hematopoietic sourses. Two isolation method performed to all materials. 1) Total mononuclear cells obtained by eryhtrocyte lysis method. 2) MNC layer and erythrocyte layer were isolated with ficoll density gradient method.
The presence of pluripotent markers such as NANOG, OCT3/4 and SSEA4 and their celullar location were detected with immunofluorescence staining in all three layers. Also western blotting method used for determinig NANOG and OCT3/4 protein expression.
Results
The results obtained from western blotting and immunofluorescence methods showed expression of NANOG, OCT3/4 and SSEA4 proteins in PB, AM and CB. And also immunofluorescence images demonstrated cytoplasmic and nuclear presence of these proteins. OCT3/4 protein of immunofluorescence determinations showed cytoplasmic locations in all layers. Futher investigations need to be done for enlighten this protein function in hematopoetic cells because OCT3/4 has different isoforms. NANOG protein showed cytoplasmic location in PB and AM, both cytoplasmic and nucleic location in CB. Western blotting results indicated that NANOG protein create a ladder pattern mostly cause of post-translational modifications. NANOG ladder pattern also changed between layers. These findings showed different layers had different post-translational modifications so different type of NANOG protein.
Summary
As a result, these findings showed that it is necessary to investigate the function of PSC markers in differentiated adult cells. Another conclusion is, among the lysis and ficoll density gradient method, lysis method has the highest cell recovery amount.Consequently, this study provided us new informations and viewpoints about expression of PSC markers in adult tissues.
Keyword(s): Apheresis, Cord blood, Peripheral blood, Stem cell marker
Session topic: Publication Only
Abstract: PB1770
Type: Publication Only
Background
Clinical use of hematopoetic stem cells (HSC) is a developing field for treatment of most hemoglobinopaties, hereditary blood diseases and cancers. Even though use of HSC’s are practical they are not as effective as pluripotent stem cells (PSC). In last years the thought of possible presence of PSCs in other adult tissues has become an attractive research area for search and isolate of PSCs from adult tissues.
Aims
In our study we refined cell populations using different isolation methods. Cells obtained from three different human hematopoetic sources and analysed for expression of pluripotent markers like NANOG, OCT3/4 and SSEA4.
Methods
Human peripheral blood (PB), apheresis material (AM) and cord blood (CB) were used as hematopoietic sourses. Two isolation method performed to all materials. 1) Total mononuclear cells obtained by eryhtrocyte lysis method. 2) MNC layer and erythrocyte layer were isolated with ficoll density gradient method.
Results
The results obtained from western blotting and immunofluorescence methods showed expression of NANOG, OCT3/4 and SSEA4 proteins in PB, AM and CB. And also immunofluorescence images demonstrated cytoplasmic and nuclear presence of these proteins. OCT3/4 protein of immunofluorescence determinations showed cytoplasmic locations in all layers. Futher investigations need to be done for enlighten this protein function in hematopoetic cells because OCT3/4 has different isoforms. NANOG protein showed cytoplasmic location in PB and AM, both cytoplasmic and nucleic location in CB. Western blotting results indicated that NANOG protein create a ladder pattern mostly cause of post-translational modifications. NANOG ladder pattern also changed between layers. These findings showed different layers had different post-translational modifications so different type of NANOG protein.
Summary
As a result, these findings showed that it is necessary to investigate the function of PSC markers in differentiated adult cells. Another conclusion is, among the lysis and ficoll density gradient method, lysis method has the highest cell recovery amount.Consequently, this study provided us new informations and viewpoints about expression of PSC markers in adult tissues.
Keyword(s): Apheresis, Cord blood, Peripheral blood, Stem cell marker
Session topic: Publication Only
Type: Publication Only
Background
Clinical use of hematopoetic stem cells (HSC) is a developing field for treatment of most hemoglobinopaties, hereditary blood diseases and cancers. Even though use of HSC’s are practical they are not as effective as pluripotent stem cells (PSC). In last years the thought of possible presence of PSCs in other adult tissues has become an attractive research area for search and isolate of PSCs from adult tissues.
Aims
In our study we refined cell populations using different isolation methods. Cells obtained from three different human hematopoetic sources and analysed for expression of pluripotent markers like NANOG, OCT3/4 and SSEA4.
Methods
Human peripheral blood (PB), apheresis material (AM) and cord blood (CB) were used as hematopoietic sourses. Two isolation method performed to all materials. 1) Total mononuclear cells obtained by eryhtrocyte lysis method. 2) MNC layer and erythrocyte layer were isolated with ficoll density gradient method.
The presence of pluripotent markers such as NANOG, OCT3/4 and SSEA4 and their celullar location were detected with immunofluorescence staining in all three layers. Also western blotting method used for determinig NANOG and OCT3/4 protein expression.
Results
The results obtained from western blotting and immunofluorescence methods showed expression of NANOG, OCT3/4 and SSEA4 proteins in PB, AM and CB. And also immunofluorescence images demonstrated cytoplasmic and nuclear presence of these proteins. OCT3/4 protein of immunofluorescence determinations showed cytoplasmic locations in all layers. Futher investigations need to be done for enlighten this protein function in hematopoetic cells because OCT3/4 has different isoforms. NANOG protein showed cytoplasmic location in PB and AM, both cytoplasmic and nucleic location in CB. Western blotting results indicated that NANOG protein create a ladder pattern mostly cause of post-translational modifications. NANOG ladder pattern also changed between layers. These findings showed different layers had different post-translational modifications so different type of NANOG protein.
Summary
As a result, these findings showed that it is necessary to investigate the function of PSC markers in differentiated adult cells. Another conclusion is, among the lysis and ficoll density gradient method, lysis method has the highest cell recovery amount.Consequently, this study provided us new informations and viewpoints about expression of PSC markers in adult tissues.
Keyword(s): Apheresis, Cord blood, Peripheral blood, Stem cell marker
Session topic: Publication Only
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