Wnt3a protein reduces growth factor-driven expansion of human hematopoietic stem and progenitor cells in serum-free cultures

Abstract

Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in robust proliferation, it is accompanied with extensive differentiation and loss of self-renewal capacity. Wnt signaling has been implicated in regulating HSPC fate decisions in vivo and in promoting HSPC self-renewal by inhibition of differentiation, but the effects of Wnt on the ex vivo expansion of HSPC are controversial. Here, we demonstrate that exogenous Wnt3a protein suppresses rather than promotes the expansion of UCB-derived CD34+ cells in serum free expansion cultures. The reduced expansion was also observed in cultures initiated with Lin-CD34+CD38lowCD45RA-CD90+ cells which are highly enriched in HSC and was also observed in response to activation of beta-catenin signaling by GSK3 inhibition. The presence of Wnt3a protein during the culture reduced the frequency of multilineage CFU-GEMM and the long-term repopulation ability of the expanded HSPC. These data suggest that Wnt signaling reduces expansion of human HSPC in growth factor-driven expansion cultures by promoting differentiation of HSPC.

Fig 1. Exogenous Wnt3a reduces growth factor-driven expansion of CD34⁺ cells.

UCB-derived CD34⁺ cells were cultured in serum free SFT medium with or without the addition of Wnt3a. Cells were analyzed using flow cytometry at 7 and 14 days of culture. Shown are (A) the total nucleated cell expansion compared to input (n = 8), (B) the frequency of CD34⁺ cells within the TNC population during culture (n = 8), (C) expression of CD45 and CD34 after 14 days of culture in SFT with (lower panel) or without (upper panel) Wnt3a (representative experiment out of 8), (D) the expansion of CD34⁺ cells compared to input (n = 8) and (E) the frequency of cells expressing lineage markers after 7 and 14 days of culture (n = 6). (F) Frequency of CFU-GEMM, BFU-E and CFU-GM in 250 CD34⁺ cells cultured for 2 weeks in SFT or SFT+Wnt3a (n = 2, 3 dishes per experiment). (G) CD34⁺ cell expansion compared to SFT medium after 14 days of culture with different dosages of Wnt3a (n = 2). (H) Levels of human chimerism at several time points after transplantation with the progeny of 10⁵ CD34⁺ cells cultured for 7 days in SFT or SFT+Wnt3a medium (n = 5 mice per group). (I) Levels of human chimerism in bone marrow 17 weeks after transplantation with the progeny of 10⁵ CD34⁺ cells cultured for 7 days in SFT or SFT+Wnt3a medium (n = 5 mice per group). * p<0.05, ** p<0.01, *** p<0.001

Fig 2. Wnt3a reduces growth factor-driven expansion of HSC.

UCB-derived DAPI^-Lin^-CD34⁺CD38^lowCD45RA^lowCD90⁺-cells were sorted out of CD34-selected cells and were cultured in SFT medium with or without Wnt3a. Flowcytometric analysis was performed at day 7 and 14. Depicted are (A) the total nucleated cell expansion in SFT and SFT+Wnt3a medium at 7 and 14 days of culture (n = 4), (B) the CD34⁺ cell frequency during culture (n = 4), (C) the fold expansion of CD34⁺ cells at day 7 and 14 of culture (n = 4) and (D) the total number of DAPI^-Lin^-CD34⁺CD38^lowCD45RA^lowCD90⁺-cellsat input and after 14 days of culture in SFT or SFT+Wnt3a medium (n = 4). * p<0.05, ** p<0.01

Fig 3. Liposomal Wnt3a reduces expansion of CD34⁺ cells.

(A) Purified Wnt3a and liposomal Wnt3a were incubated for 0, 8, and 24 hours at 37°C in cell culture media, and transferred to LSL cells. Remaining Wnt activity was assayed by luminescence measurements. Activity plot displays average increase of luminescence over incubation time relative to background (n = 10). (B) CD34⁺ cell expansion in SFT medium with or without liposomal Wnt3a after 7 days of culture (n = 1).

Fig 4. Wnt3a-mediated inhibition of HSPC expansion is due to activation of the canonical Wnt3a pathway.

(A) Expansion of CD34⁺ cells after 7 and 14 days of culture in SFT, SFT+Wnt3a, SFT+Fr8CRD and SFT+Wnt3a+Fr8CRD (n = 2). (B) CD34⁺ cell frequency within the TNC population during these cultures (n = 2). (C) CD34⁺ cell expansion in SFT and SFT+CHIR99021 after 7 and 14 days of culture (n = 2).

Fig 5. Wnt3a inhibits SR1-enhanced CD34⁺ cell expansion.

UCB-derived CD34⁺ HSPC were cultured for 14 days in SFT or SFTSR1 medium with or without the addition of Wnt3a. Flowcytometric analysis was performed at day 14. Depicted is the CD34⁺ cell expansion after 14 days of culture (n = 5). * p<0.05, ** p<0.01