Andrographolide promotes the ex vivo expansion of CD34+ hematopoietic stem cells derived from human umbilical cord blood

Abstract

Umbilical cord blood (UCB) units are an alternative source of human hematopoietic stem cells (HSCs) for allogeneic stem cell transplants. A large quantity of HSCs is needed but the low number of accessible cells from UCB has been a significant limitation. Improving the ex vivo growth of HSCs while preserving their functioning is required. Here, we report that andrographolide (AP) enhanced the expansion of human UCB-derived HSCs (HSPCs) and pro-moted primitive HSCs (CD34⁺CD38^-CD90⁺). AP also improved HSC functionality, evidenced by increased growth of colony-forming units and multilineage differentiation. AP upregulated genes involved in the Wnt/β-catenin and Notch signaling pathways. AP also modulated signaling pathways involved in HSC self-renewal, proliferation, survival, and differentiation, demonstrated by Nanostring analysis. The results of this study suggest that andrographolide enhances ex vivo UCB-HSC expansion while maintaining functionality and has potential for treatment of hematological diseases.

Keywords: Andrographolide; Ex vivo expansion; Hematologic disorders; Hematopoietic stem cell transplantation; Hematopoietic stem cells (HSCs); Umbilical cord blood (UCB).

Fig. 1

Effects of AP on cell morphology and expansion of UCB- CD34⁺ cells. (A) Chemical structure of AP, (B) Purity of isolated CD34⁺ cells, (C) Cell morphology before and after culturing with AP at two different concentrations (2.5 and 5 µM), photos were captured on day 7 (10× objective, scale bar; 100 μm). The control was composed of cytokines and methanol, (D) Viability of UCB-CD34⁺ cells after treatment with AP at different concentrations for 3, 5, and 7 days. Data are shown as mean ± SEM, *p < 0.05, **p < 0.01 compared to control.

Fig. 2

Effects of AP on the proliferation, expansion of UCB-CD34⁺ cells, cell cycle and senescence of expanded UCB-CD34⁺ cells. (A) Total viable cell number throughout 7 days of culture after treatment with AP (B) Fold change of BrdU + cells along with flow cytometric data showing the percentage of BrdU + cells (C) relative fold-expansion of CD34⁺ cells after treatment with AP for 7 days (D) Cell cycle analysis (E) The number of β-galactosidase positive cells (senescent cells). Data are shown as mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01, compared with the control.

Fig. 3

Effect of AP on ex vivo expansion of defined primitive HSCs. Representative FACs analysis plots of CD34⁺CD38^-CD90⁺CD45RA^- cells stained with CD34, CD38, CD90 and CD45RA antibodies at 7 days of culture. (A) CD34⁺CD45RA^- (B) CD34⁺CD90⁺CD45RA^- (C) CD34⁺CD38^-CD45RA^- (D) CD34⁺CD38^-CD90⁺ CD45RA^-. Data are represented as mean ± SEM from three independent experiments, * P < 0.05, * * P < 0.01 compared with the control.

Fig. 4

Effect of AP on the expression of characteristic genes related to HSC stemness. Relative gene expressions as fold change at 7 days of culture with AP; (A) HSC-specific markers and functionally important genes (CD34, CD90, CD133, CD117, ALDH1, ALDH2) and (B) HSC-relevant genes and transcriptional factors (BMI1, HOXB4, GATA-2, RUNX1, and CXCR4). Data is represented as mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01 compared with the control.

Fig. 5

The colony-forming potential of AP-expanded CD34⁺ cells. (A) Representative images of colony formation (BFU-E, CFU-GM and CFU-GEMM) in Methocult^® media following 14 days of culture. (B) The colony-forming potential of expanded CD34⁺ cells after culture with cytokines alone or 1, 2.5 or 5 µM AP. Colonies were counted according to colony size and cellular composition. Abbreviations: BFU-E, erythroid burst forming units; CFU-GM, granulocyte/macrophage common precursors. Comparing each colony between group: * represents CFU-E colony, # represents BFU-E colony, $ represents CFU-GM colony and γ represents CFU-GEMM colony). Data is represented as mean ± SEM from three independent experiments. Data is represented as mean ± SEM from three independent experiments. *P < 0.05, ^#P < 0.05, ^$P < 0.05 and ^γP < 0.05, compared with control group for each day.

Fig. 6

Effect of AP on gene expression of UCB-CD34⁺ cells determined by RT-qPCR. (A) key genes and target genes in Wnt/β-catenin pathway (B) key gene and target genes in Notch pathway. Bars represent the mean fold-changes of gene expression in the AP-treated cells relative to the control. *P < 0.05, **P < 0.01 compared to the control.

Fig. 7

Stem cell pathway analysis in expanded CD34 + cells treated with AP (5µM) determined by Nanostring analysis system. P < 0.05, three independent experiments.