Oct-4A isoform is expressed in human cord blood-derived CD133 stem cells and differentiated progeny

M. Howe, J. Zhao, Y. Bodenburg, C. P. McGuckin, N. Forraz, Ronald Tilton, Randall Urban, Larry Denner

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)265-275
Number of pages11
JournalCell Proliferation
Volume42
Issue number3
DOIs
StatePublished - Jun 2009

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Fetal Blood
Protein Isoforms
Stem Cells
Embryonic Stem Cells
Flow Cytometry
Pluripotent Stem Cells
Messenger RNA
Peptides
Adult Stem Cells
Antibodies
Differentiation Antigens
Hematopoietic Stem Cells
Adsorption
Reverse Transcription
Cell Differentiation
Proteins
Transcription Factors
Immunohistochemistry
Maintenance
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Cell Biology

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Oct-4A isoform is expressed in human cord blood-derived CD133 stem cells and differentiated progeny. / Howe, M.; Zhao, J.; Bodenburg, Y.; McGuckin, C. P.; Forraz, N.; Tilton, Ronald; Urban, Randall; Denner, Larry.

In: Cell Proliferation, Vol. 42, No. 3, 06.2009, p. 265-275.

Research output: Contribution to journalArticle

Howe, M. ; Zhao, J. ; Bodenburg, Y. ; McGuckin, C. P. ; Forraz, N. ; Tilton, Ronald ; Urban, Randall ; Denner, Larry. / Oct-4A isoform is expressed in human cord blood-derived CD133 stem cells and differentiated progeny. In: Cell Proliferation. 2009 ; Vol. 42, No. 3. pp. 265-275.
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AU - Forraz, N.

AU - Tilton, Ronald

AU - Urban, Randall

AU - Denner, Larry

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N2 - 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.

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