Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells Using a Natural Product, Garcinol

Abstract 1174

Ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs) have recently been explored to optimize autologous and allogeneic HSPC transplantation and shown to be effective in the field of stem cell biology. However, to our knowledge, identification of culture conditions that allow HSPCs expansion and long-term hematopoietic reconstitution have remained incomplete, and clinical methods to expand human HSPCs have yet to be realized.

In this study, we assumed that some small molecule compounds may preferentially activate signals that are required for optimal HSPC expansion and facilitate self-renewal of hematopoietic stem cells (HSCs). Thus, we evaluated the effects of several biologically active compounds on the ex vivo expansion of CD34⁺ hematopoietic stem and progenitor cells from human cord blood (hCB) and identified Garcinol, a plant-derived natural product as a novel modulator of HSPC proliferation.

We cultured hCB CD34⁺ cells in serum-free medium supplemented with human thrombopoietin, human stem cell factor and Garcinol for 7 days and analyzed the cellular phenotype of the cultured cells by flow cytometry and colony assay. Although the total number of cells cultured with Garcinol was similar to those cultured without Garcinol, the cultures with Garcinol showed >2-fold increase in the number of CD34⁺CD38^- hematopoietic stem and progenitor cells and contained 2-fold more high-proliferative-potential colony-forming cells (HPP-CFCs; >1mm in diameter) compared to control cultures. Correspondingly, SCID-repopulating cells (SRCs) were increased 2-fold during a 7-day culture with Garcinol compared to cultures without Garcinol. These findings suggest that Garcinol efficiently promotes the net expansion of HPSCs.

To investigate the structure-activity relationship of Garcinol, we synthesized the chemical derivatives of Garcinol and evaluated the effect of Garcinol and its derivatives, Isogarcinol and O, O'-dimethylisogarcinol, on the proliferation of CD34⁺CD38^- cells. Although Isogarcinol exhibited almost the same activity as Garcinol, O, O'-dimethyl isogarcinol was scarcely effective in the CD34⁺CD38^- cell proliferation. Correspondingly, O, O'-dimethylisogarcinol had no effect on numbers of HPP-CFCs. These results indicate that dihydroxybenzoyl moiety is crucial for the positive effect of Gacinol on HSPCs.Garcinol has been reported to be a potent inhibitor of histone acetyltransferases (HAT). Thus, we estimated the HAT activity in cells treated with Garcinol and its derivatives. Garcinol and Isogarcinol inhibited HAT activity while O, O'-dimethylisogarcinol showed much less HAT inhibitory activity as compared to Garcinol and Isogarcinol, which suggested that HAT inhibitory activity of Garcinol is correlate with the expansion of HPSCs. We are now investigating gene expression profiling in cells cultured with Garcinol using DNA microarray analysis and Q-PCR.

In conclusion, we have identified Garcinol, a plant-derived small-molecule compound, which exhibits inhibitory effect on HAT activity, as a novel stimulator of HSPC expansion. The results reported here indicate that Garcinol would be applied as a useful tool for the development of novel and efficient technologies for hematopoietic stem cell and gene therapies.