Signaling pathways regulating self-renewal, differentiation, and multipotency of CD133+ umbilical cord blood stem cells

Our goal is to increase the number of immature umbilical cord blood stem cells (UCBSCs) for hematopoietic transplantation. Towards this goal, our lab adapted a culture technique to grow immature CD133+ UCBSCs (CD133+ cells). Although CD133+ cells proliferate rapidly in culture, a minority self-renew and remain CD133+, while a majority differentiate and become CD133-. Therefore, new strategies to identify and grow immature UCBSCs are important. Since little is known about signally mechanisms regulating self-renewal and differentiation of UCBSCs, we sought insight from embryonic stem cell (ESC) literature to guide our studies. \r\nTo identify a population of UCBSCs that grow without differentiating, we focused on Oct-4, a transcription factor essential for self-renewal in ESCs that we previously reported expression in UCBSCs. During our studies, new challenges in the field arose. Two isomers of Oct-4 were discovered, Oct-4A and Oct-4B 3, in which only Oct-4A conferred the ability of ESCs to self-renew 4. We redesigned our experiments to detect Oct-4A and discovered that freshly isolated CD133+ cells expressed Oct-4A mRNA and protein. Since these cells proliferated in culture, they lost expression of adult stem cell markers including CD133, and gained markers of hematopoietic differentiation. However, Oct-4A mRNA and protein were expressed regardless of the differentiation status. Therefore, Oct-4A, despite its essential roles in ESCs, neither defined nor conferred self-renewal of CD133+ cells.\r\nTo discover strategies to grow immature CD133+ cells without differentiation, we focused on the Wnt pathway which is essential for self-renewal in ESCs. Differentiation of CD133+ cells to CD133- cells corresponded to down-regulation of Wnt signaling. Pharmacological activation of the Wnt pathway by (2’Z,3’E)-6-Bromoindirubin-3’-oxime (BIO) inhibition of GSK-3beta resulted in accelerated differentiation, instead of decreased differentiation, of CD133+ cells. BIO-treated CD133- cells that were differentiated maintained multipotency while proliferating at similar rates to vehicle-treated CD133+ cells that self-renewed. Therefore, inhibition of GSK-3beta could be a strategy for differentiating CD133+ cells into hematopoietic progenitor cells while maintaining their proliferation capacity. \r\nIn conclusion, this project demonstrated that pathways regulating UCBSC properties are not similar to pathways regulating ESCs properties. Our findings are the first studies that derive UCBSCs properties of self-renewal, differentiation, and multipotency.\r\n