Inhibition of p38 MAPK activity promotes ex vivo expansion of human cord blood hematopoietic stem cells

Abstract

Ex vivo expansion of hematopoietic stem cells (HSCs) depends on HSC self-renewing proliferation and functional maintenance, which can be negatively affected by HSC differentiation, apoptosis, and senescence. Therefore, inhibition of HSC senescence may promote HSC expansion. To test this hypothesis, we examined the effect of inhibition of p38 mitogen-activated protein kinase (p38) on the expansion of human umbilical cord blood (hUCB) CD133(+) cells because activation of p38 has been implicated in the induction of HSC senescence under various physiological and pathological conditions. Our results showed that ex vivo expansion of hUCB CD133(+) cells activated p38, which was abrogated by the p38 specific inhibitor SB203580 (SB). Inhibition of p38 activity with SB promoted the expansion of CD133(+) cells and CD133(+)CD38(-) cells. In addition, hUCB CD133(+) cells expanded in the presence of SB for 7 days showed about threefold increase in the clonogenic function of HSCs and engraftment in non-obese diabetic/severe combined immunodeficient mice after transplantation compared to the input cells. In contrast, the cells expanded without SB exhibited a significant reduction in these HSC functions. The enhancement of ex vivo expansion of hUCB HSCs is primarily attributable to SB-mediated inhibition of HSC senescence. In addition, inhibition of HSC apoptosis and upregulation of CXCR4 may also contribute to the enhancement. However, p38 inhibition had no significant effect on HSC differentiation and proliferation. These findings suggest that inhibition of p38 activation may represent a novel strategy to promote ex vivo expansion of hUCB HSCs.

Fig. 1

Ex vivo expansion of hUCB CD133⁺ cells activates p38. (a), the activation was abrogated when the cells were cultured with SB (b). hUCB CD133⁺ cells were cultured in STF–HSC expansion medium with DMSO (0.1%) or SB (10 μM). Activation of p38 was determined by immunofluorescence staining with anti-phosphorylated p38 (p-p38) primary antibody and Alexa fluor-555-conjugated secondary antibody (red). The nuclei of the cells were stained with Hoechst33342 (blue). Representative photomicrographs of the immunofluorescence staining are shown

Fig. 2

Inhibition of p38 promotes ex vivo expansion of hUCB CD133⁺ and CD133⁺CD38⁻ cells. hUCB CD133⁺ cells were cultured in STF–HSC expansion medium with DMSO (0.1%) or SB (10 μM) for 7 days. The cells harvested from the cultures were numerated and phenotypically analyzed by flow cytometry. Production (a) and fold expansion (b) of MNCs. Representative flow cytometric analyses (c) and percentage of CD133⁺ cells (d) and CD133⁺CD38⁻ cells (g) in freshly isolated hUCB CD133⁺ cells (day 0) and day-7 expanded cells with DMSO or SB. Production and fold expansion of CD133⁺ cells (e, f) and CD133⁺CD38⁻ cells (h, i) after 7 days of expansion. The data are presented as mean±SE (n = 5 independent cultures). All the numbers were calculated based on the cultures with 3×10⁴ input hUCB CD133⁺ cells. *p<0.01 vs. day 0 cells and #p<0.01 vs. DMSO

Fig. 3

Inhibition of p38 promotes ex vivo expansion of HSCs but not that of HPCs. hUCB CD133⁺ cells were cultured in STF–HSC expansion medium with DMSO (0.1%) or SB (10 μM) for 7 days. The number of CFUs and day-28 CAFCs was quantified in freshly isolated hUCB CD133⁺ cells (day 0) and day-7 expanded cells with DMSO or SB by CFC and day-28 CAFCs assays to measure the expansion of HPCs and HSCs, respectively. Number of total CFUs (a), different types of CFUs (b), and day-28 CAFCs (d) and fold of expansion of various types of CFUs (c) and day-28 CAFCs (e) are presented as mean±SE (n = 5 independent cultures). The numbers of CFUs and CAFCs were calculated based on the culture with 200 and 1,000 input hUCB CD133⁺ cells, respectively. *p<0.01 vs. day 0 cells and #p<0.01 vs. DMSO

Fig. 4

Inhibition of p38 increases the engraftment of ex vivo expanded hUCB CD133⁺ cells in NOD/SCID mice. Nonlethally irradiated NOD/ SCID mice were transplanted with 6×10⁴ freshly isolated hUCB CD133⁺ cells (day 0) or the cells expanded from the same number of hUCB CD133⁺ cells for 7 days with DMSO (0.1%) or SB (10 μM). Human hematopoietic cell engraftment in the mouse BM was determined 10 weeks after transplantation by flow cytometry. Representative flow cytometric analyses of human CD45⁺ hematopoietic cells (a) and CD45⁺/ CD33⁺ myeloid cells and CD45⁺/CD19⁺ B cells (c) and percentage of CD45⁺ cells (b) and CD45⁺/CD33⁺ myeloid cells and CD45⁺/CD19⁺ B cells (d) in mouse BM cells are shown. In b, each data point represents an individual mouse, the short line for each data set is the mean engraftment and the solid line shows the positive engraftment threshold (set at 0.1%). The data presented in d are mean (± SE) engraftment of human myeloid and B cells (n = 12, 16, and 17 for day 0, DMSO-expanded, and SB-expanded cells, respectively). *p<0.01 vs. day 0 cells and #p<0.01 vs. DMSO

Fig. 5

Effects of p38 inhibition on hUCB CD133⁺ cell proliferation, apoptosis, senescence, expression of CXCR4, and migration. hUCB CD133⁺ cells were cultured in STF–HSC expansion medium with DMSO (0.1%) or SB (10 μM) for 7 days. They were harvested from the culture and analyzed by various assays as described below: a representative analysis of cell divisions by CFSE staining and flow cytometry. The blue line, red line and black line represent the cells cultured with SB, DMSO, or without culture after CFSE labeling, respectively. Numbers in the histogram indicate the number of divisions (divisions 0–9 from right to left) underwent by the cells after 7 days of culture. b Representative analyses of apoptosis in expanded MNCs and CD133⁺ cells by Annexin V-FITC staining and flow cytometry. c Percentage of apoptotic cells in expanded MNCs and CD133⁺ cells. d Percentage of SA-β-gal-positive senescent cells in hUCB CD133⁺ cells after ex vivo expansion. e The ratio of p16 and p21 mRNA expression in expanded hUCB CD133⁺ cells with and without SB. f Representative analyses of CXCR4 expression in expanded CD133⁺ cells by flow cytometry. g Percentage of CXCR4-positive cells in hUCB CD133⁺ cell after ex vivo expansion. h Percentage of expanded hUCB CD133⁺ cells migrated through the transwell in response to SDF-1 attraction. The data presented in the bar graphs are mean±SE from five independent cultures. #p<0.05 vs. DMSO