Session: 501. Hematopoietic Stem and Progenitor Biology: New Insights into the Molecular Regulation of Hematopoietic Stem Cells
Hematology Disease Topics & Pathways:
Technology and Procedures, cell expansion
To clarify the difference between mouse and human HSPCs during these cultures, we analyzed the phosphorylation status of major signaling pathways. The results showed significant decreases in PI3K and AKT pathways were observed in human cells as compared with the mouse cells, and we found that the expansion efficiency of human CD34+ cell was significantly improved by adding the PI3K activator 740Y-P. Furthermore, we found that human CD34+ cells could be expanded under PVA culture conditions for one week using 740Y-P and a TPO receptor agonist (TPO-RA) instead of SCF and TPO. In addition, CD34+ cells were successfully expanded ~10-15-fold over a 30-day culture by adding UM171 to the 740Y-P/TPO-RA, which we defined as three activator (3a) medium. The cultured cells maintained long-term hematopoietic reconstitution as confirmed by xenotransplantation assays.
We next aimed to improve the rate of HSPC expansion ex vivo, and screened 10 polymers in 3a medium. The results showed that a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG), as supportive of significantly higher cell expansion than PVA. 3a medium containing PCL-PVAc-PEG also supported cell proliferation longer-term, with a 75-fold expansion of total cells and 55-fold expansion of CD34+ cell observed after a 30-day culture. The cultured cells also maintained long-term hematopoietic reconstitution as confirmed by xenotransplantation assays.
Furthermore, PVA-based 3a medium not only expanded healthy CB CD34+ cells but also malignant HPSCs; BM mononuclear cells from a newly diagnosed chronic myeloid leukemia in chronic phase patient expanded ~15-fold, a proliferation that sensitive to imatinib.
In conclusion, we have developed a long-term expansion culture system for human HSCs without recombinant cytokines or albumin. the culture system described here provides a powerful platform for both basic scientists and clinicians interested in stem cell biology, leukemia therapeutics, and the next generation of HSC transplantation and gene therapies.
Disclosures: Sakurai: Otsuka Pharmaceutical: Honoraria; Eisai Co., Ltd: Honoraria; Nihon Shinyaku: Honoraria; Takeda Pharmaceutical: Honoraria; MSD K.K: Research Funding; Nippon Shinyaku: Research Funding.
See more of: Oral and Poster Abstracts