Small-Molecule Ice Recrystallization Inhibitors Improve the Post-Thaw Function of Hematopoietic Stem and Progenitor Cells

Abstract 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. Disclosures: No relevant conflicts of interest to declare.

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BIO5192, a small molecule inhibitor of VLA-4, mobilizes hematopoietic stem and progenitor cells

Blood ◽

10.1182/blood-2008-10-184721 ◽

2009 ◽

Vol 114 (7) ◽

pp. 1340-1343 ◽

Cited By ~ 118

Author(s):

Pablo Ramirez ◽

Michael P. Rettig ◽

Geoffrey L. Uy ◽

Elena Deych ◽

Matthew S. Holt ◽

...

Keyword(s):

Progenitor Cells ◽

Small Molecule ◽

Fold Increase ◽

Small Molecule Inhibitor ◽

Granulocyte Colony Stimulating Factor ◽

Hematopoietic Stem ◽

Molecule Inhibitor ◽

Stem And Progenitor Cells ◽

Stimulating Factor

Abstract Here we show that interruption of the VCAM-1/VLA-4 axis with a small molecule inhibitor of VLA-4, BIO5192, results in a 30-fold increase in mobilization of murine hematopoietic stem and progenitors (HSPCs) over basal levels. An additive affect on HSPC mobilization (3-fold) was observed when plerixafor (AMD3100), a small molecule inhibitor of the CXCR-4/SDF-1 axis, was combined with BIO5192. Furthermore, the combination of granulocyte colony-stimulating factor (G-CSF), BIO5192, and plerixafor enhanced mobilization by 17-fold compared with G-CSF alone. HSPCs mobilized by BIO5192 or the combination of BIO5192 and plerixafor mobilized long-term repopulating cells, which successfully engraft and expand in a multilineage fashion in secondary transplantation recipients. Splenectomy resulted in a dramatic enhancement of G-CSF–induced mobilization while decreasing both plerixafor- and BIO5192-induced mobilization of HSPCs. These data provide evidence for the utility of small molecule inhibitors of VLA-4 either alone or in combination with G-CSF or AMD3100 for mobilization of hematopoietic stem and progenitor cells.

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Expansion and Maintenance of Hematopoietic Stem and Progenitor Cells in Course of Long-Term Inhibition of CXCR4/CXCL12 Signaling

Blood ◽

10.1182/blood.v128.22.2648.2648 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2648-2648 ◽

Cited By ~ 1

Author(s):

Darja Karpova ◽

Julie Ritchey ◽

Matthew Holt ◽

Darlene Monlish ◽

Laura G. Schuettpelz ◽

...

Keyword(s):

Cell Cycle ◽

Steady State ◽

Continuous Infusion ◽

Progenitor Cells ◽

Small Molecule ◽

Critical Role ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract During the past two decades peripheral blood stem cells have become the favored graft source for HSCT with 80 % of allogeneic and almost 100 % of autologous HSCT performed with mobilized blood. The critical role of the interaction between the chemokine receptor CXCR4 and its chief ligand CXCL12 for retention and migration of hematopoietic stem and progenitor cells (HSPC) has been well established. Interference with CXCR4/CXCL12 signalling iscurrentlybeing exploited as a strategy to mobilize HSPC indirectly with the most clinically relevant mobilizing agent to date, G-CSF as well as directly with the bicyclam CXCR4 antagonist Plerixafor (AMD3100).In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of CXCR4/CXCL12 axis within the HSPC compartment were investigated in healthy C57BL/6 mice using the non-peptidic small molecule CXCR4 antagonists Plerixafor and ALT1188 along with the Protein-EpitopeMimeticsInhibitor POL5551. Up to 12-14 fold higher mobilization efficiency was achieved by applying the antagonists via two weeks of continuous infusion (up to 8-10x104 CFU-C and LSK/ml) as compared to bolus treatment (4-6x103 CFU-C and LSK/ml) or 5-day course of G-CSF (3-6x103 CFU-C/ml).Despite dramatic increase in numbers of circulating HSPC, the BM HSPC pool dis not decrease; in fact it expanded up to 2-4-fold compared to steady state reservoir (sham-operated control mice) as measured by immunophenotypical (LSK SLAM) and functional (e.g. serial competitive transplantation) properties of the cells. Thus, in contrast to genetically CXCR4 ablatedHSPC, the reversible long-term blockade of the receptor did not diminish the long-term repopulating capacity of HSPC. Cell cycle analysis showed a 2-3-fold increase in cycling activity of BM HSPC: only 10-20% of LSK and 30-40 % of LSK SLAM cells were found to be quiescent (in G0 phase of the cell cycle) after two weeks of CXCR4 antagonist infusion versus 50-60 % of LSK and 70 % of LSK SLAM found in G0 under homeostatic conditions. This increased proliferation was very similar to the one induced transiently at day 3 G-CSF treatmentand would conceivably explain the sustained mobilization without concomitant depletion of the BM HSPC pool. Profiling of differentially treated BM HSC (LSK SLAM) via microarray analysis did not reveal substantial effects of CXCR4 inhibitor infusion on the expression signature. Ofnote, major cytological changes typically associated with G-CSF induced mobilization, e.g. depletion of bone lining osteoblast lineage cells and macrophages, were not detected in continuous infusion of POL5551 exposed BM suggesting limitedeffects within the BM niche compartment. Moreover analysis of the BM HSPC after different washout periods at the end of continuous infusion treatment revealed a rapid (within 1-3 days after discontinuation of infusion) reestablishment of steady state HSPC numbers in the BM.Our data suggest that prolonged pharmacologic blockade of the CXCR4/CXCL12 axis using multiple small molecule inhibitorsrepresents an approach thatreleasesHSPCwith efficiency superiorto any other knownmobilization strategybut also may serve as an effective method induce cell cycling and thus expand BM HSPCs. Figure Competitive transplantation of POL5551 treated andcontrol BM (n=5 recipients per group, mean±SEM) Figure. Competitive transplantation of POL5551 treated andcontrol BM (n=5 recipients per group, mean±SEM) Disclosures Levesque: GlycoMimetics: Equity Ownership.

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