[Screening and Verification of Antioxidant Small Molecular Compounds for Expansion of Human Hematopoietic Stem Cells Ex Vitro]

Abstract

Objective: To screen the antioxidant small molecular compounds with optimal efficiency of expansing the human hematopoietic stem cells (hHSC) In vitro based on antioxidant small molecular compound database of LKT laboratory, and to verify the effects of these compounds on the biological functions of hHSC.

Methods: The umbilial cord blood CD34⁺ cells were enriched by using the MACS beads; the absolute number and percentage of CD34⁺ cells and CD34⁺ CD49f⁺ cells were detected by high throughput flow cytometry after culture of hHSC with compounds in vitro for 1 week, the SR1 (1 μmol/L) was used as positive control, the candidate compounds were screened out; then 4 compounds were selected for follow-up experiments by comprehensive evaluation of concentration, safety and expansion efficacy, the optimal used concentrations of selected compounds were determined through the concentration gradient analysis, and CFC short-term colony-forming cell test was performed by using the determined concentration so as to verify the effect of compounds on the self-renewal, multilineage differentiation.

Results: Out of 85 antioxidant small molecular compounds, 4 compounds (C2968, D3331, B1753 and B3358) with obvious expansion efficacy for CD34⁺ cells and CD34⁺ CD49f⁺ cells were screened out by high throughput flow cytometry; their optimal concentrations of 4 compounds were 0.5 μmol/L for C2968, 1.5 μmol/L for D3331 and 1.5 μmol/L for B1753 and 15 μmol/L for B3358. The CFC assay showed the colony formation number in compound-treated group significantly increased as compared with control group, moreover the self-renewal and multilineage differentiation were maintained.

Conclusion: The antioxidant small molecular compounds C2968 (0.5 μmol/L), D3331 (1.5 μmol/L), B1753 (1.5 μmol/L) and B3358 (1.5 μmol/L) possess good expansion efficacy for hHSC, they can maintain hHSC self-renewal, at the same time ensure the multilineage differentiation potentiality of hHSC.