Figure 4.

The hematopoietic support by erythropoietin-treated mesenchymal stromal cells is inhibited but can be restored by Wnt pathway activation. Human mesenchymal stromal cells (MSC) were pretreated with erythropoietin (Epo) 50 IU/mL and/or lithium chloride (LiCl) 25 mM for 7 days and co-cultured with freshly isolated CD34⁺ HSPC. After 7 days, flow cytometric analysis was performed for the non-adherent cells. (A) Number of CD34⁺ cells and (B) number of CD38⁺ cells. Each dot represents the percentage of positive-stained cells; each graph represents one donor/patient. (C) After 4 weeks of co-culture, the number of cobblestone area-forming cells (CAF-C) was determined in each well. A colony-forming unit (CFU) assay was performed in methylcellulose medium for 2 additional weeks and the colonies were classified under a microscope for HSPC co-cultured with (D) young healthy MSC, (E) old healthy MSC and (F) MDS MSC. N=3–5. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001