Differential Effects of Wnt Signaling on Proliferation and Hematopoietic Support of Adult and Fetal Bone Marrow-Derived MSCs

Human adult bone marrow-derived mesenchymal stromal cells MSC (ABMSC) are increasingly applied in the clinic to decrease graft versus host disease and to enhance hematopoietic recovery. Fetal bone marrow-derived MSC (FBMSC) display similar immune suppressive and regenerative capacities as adult MSC, and have been transplanted into patients. The proliferation capacity of FBMSC, however, is much larger than that of ABMSC. The aim of our studies is to understand the molecular mechanism of proliferation and hematopoietic support by MSC to optimize the expansion of functional MSC for clinical use.

Comparison of gene expression between ABMSCs and FBMSCs identified 687 differentially expressed genes. Of these, 16 were Wnt-related, mainly Wnt-inhibitors and Frizzled receptors. Expression of SFRP4, WISP1, WISP2, WISP3, FZD1, FZD5, FZD8 and MYCBP2 was upregulated in ABMSC, whereas DKK1, DKK2, CCND2, WNT5a, MYC, FZD2, FZD6 and FZD7 are expressed at a higher level in FBMSC. Although the expression of few genes (e.g. DKK1) was culture density dependent, other genes such as Wnt5a, DKK2 and SFRP4 were consistently differentially expressed independent of culture conditions. Therefore we investigated the role of Wnt signaling in adult and fetal bone marrow-derived MSC. Wnt3a induced a concentration dependent increase of the canonical Wnt-target genes TCF and LEF in both ABMSC and FBMSC. However, ABMSC responded faster, and at a lower concentration of Wnt3a compared to FBMSC. In addition, Wnt3a increased the proliferation of ABMSC, but not of FBMSC. Interestingly, a complete medium change was already sufficient to increase TCF/LEF expression in ABMSC, but not in FBMSC, suggesting that ABMSC produced a soluble Wnt-inhibitor. Moreover, switching MSC conditioned medium between FBMSC and ABMSC indicated that FBMSC conditioned medium significantly stimulated the expansion of ABMSC while the reverse experiments did not show an inhibiting effect of ABMSC conditioned medium on the expansion of FBMSC. Thus, ABMSC produce a factor that only affects ABMSC, but not (the factors produced by) FBMSC.

To block autocrine Wnt production, MSC were exposed for 48 h to the Inhibitor of Wnt Production 2 (IWP2). Abrogation of Wnt-production in FBMSC modestly decreased beta-catenin expression, and strongly decreased TCF/LEF expression, but did not affect ABMSC. Addition of IWP-2 to long-term cultures strongly inhibited proliferation of FBMSCs compared to ABMSCs. To unravel the role of MSC-produced Wnt factors in hematopoiesis, we co-cultured adult or fetal MSCs together with cord blood derived CD34+ cells in the presence or absence of IWP2 inhibitor. Addition of IWP2 to ABMSC decreased the short term support of hematopoietic stem and progenitors (HSPC), while IWP2 did not affect the support of HSPCs by FBMSC. Overall, ABMSCs provided a significant better short term hematopoietic support than FBMSCs. In conclusion, our data demonstrate that ABMSC produce both Wnt factors and inhibitors. FBMSC, in contrast, produce Wnt-related factors that seem to contribute more to the expansion capacity of FBMSC than to their hematopoietic support. To identify factors we current use mass spectroscopy of supernatant to determine the secretome. Modulation of (parts) of the Wnt pathway may improve clinical expansion protocols of ABMSC.