OBJECTIVES: This study aims to establish whether the pluripotent embryonic stem cell marker and nuclear transcription factor Oct-4A isoform is expressed in human umbilical cord blood CD133 stem cells (CD133 cells) and their differentiated progeny. MATERIALS AND METHODS: CD133 cells were examined for expression of the embryonic stem cell marker Oct-4A by reverse transcription-polymerase chain reaction using primers specific for the coding region of the Oct-4A isoform. Immunocytochemistry and flow cytometry were performed using an antibody raised to a peptide from the unique amino-terminal domain of the Oct-4A isoform, that does not exist in the Oct-4B isoform. Furthermore, specificity was confirmed by pre-adsorption of the antibody with the peptide immunogen. Differentiation was determined before and after expansion in culture, by flow cytometry for haematopoietic stem cell and differentiation markers. For many studies, after 7 days of culture CD133-positive and CD133-negative cells were separated by flow cytometry for additional analyses. Multilineage haematopoietic proliferative potential was determined using colony-forming assays. RESULTS: Freshly isolated CD133 cells expressed Oct-4A mRNA and protein. The cells proliferated rapidly in culture producing only a small proportion of CD133-positive cells and a much larger proportion of non-self-renewing CD133-negative cells. Proliferation was also associated with loss of other adult stem cell markers, gain of differentiated haematopoietic markers, and maintenance of potential to generate haematopoietic lineages. Oct-4A mRNA and protein were expressed throughout these changes. CONCLUSIONS: Oct-4A, which is associated with self-renewal in embryonic stem cells, neither defines nor confers self-renewal to CD133 stem cells.
OBJECTIVES: This study aims to establish whether the pluripotent embryonic stem cell marker and nuclear transcription factor Oct-4A isoform is expressed in human umbilical cord blood CD133 stem cells (CD133 cells) and their differentiated progeny. MATERIALS AND METHODS:CD133 cells were examined for expression of the embryonic stem cell marker Oct-4A by reverse transcription-polymerase chain reaction using primers specific for the coding region of the Oct-4A isoform. Immunocytochemistry and flow cytometry were performed using an antibody raised to a peptide from the unique amino-terminal domain of the Oct-4A isoform, that does not exist in the Oct-4B isoform. Furthermore, specificity was confirmed by pre-adsorption of the antibody with the peptide immunogen. Differentiation was determined before and after expansion in culture, by flow cytometry for haematopoietic stem cell and differentiation markers. For many studies, after 7 days of culture CD133-positive and CD133-negative cells were separated by flow cytometry for additional analyses. Multilineage haematopoietic proliferative potential was determined using colony-forming assays. RESULTS: Freshly isolated CD133 cells expressed Oct-4A mRNA and protein. The cells proliferated rapidly in culture producing only a small proportion of CD133-positive cells and a much larger proportion of non-self-renewing CD133-negative cells. Proliferation was also associated with loss of other adult stem cell markers, gain of differentiated haematopoietic markers, and maintenance of potential to generate haematopoietic lineages. Oct-4A mRNA and protein were expressed throughout these changes. CONCLUSIONS: Oct-4A, which is associated with self-renewal in embryonic stem cells, neither defines nor confers self-renewal to CD133 stem cells.
Authors: S Robinson; T Niu; M de Lima; J Ng; H Yang; J McMannis; S Karandish; T Sadeghi; P Fu; M del Angel; S O'Connor; R Champlin; E Shpall Journal: Cytotherapy Date: 2005 Impact factor: 5.414
Authors: Yuin-Han Loh; Qiang Wu; Joon-Lin Chew; Vinsensius B Vega; Weiwei Zhang; Xi Chen; Guillaume Bourque; Joshy George; Bernard Leong; Jun Liu; Kee-Yew Wong; Ken W Sung; Charlie W H Lee; Xiao-Dong Zhao; Kuo-Ping Chiu; Leonard Lipovich; Vladimir A Kuznetsov; Paul Robson; Lawrence W Stanton; Chia-Lin Wei; Yijun Ruan; Bing Lim; Huck-Hui Ng Journal: Nat Genet Date: 2006-03-05 Impact factor: 38.330