Fig. 5.

Epigenetic targeting of MDS-MSCs reverses long-term deleterious effects on healthy CD34⁺ HSPCs. a Schematic of transplantation experiments with FACS sorted human BM CD34⁺ HSPCs from different MSC co-cultures. b Percentage chimerism of human CD45⁺ cells in the BM of irradiated (240 cGy) NSG mice 8 weeks after transplantation. Human CD34⁺ HSPC dose was fixed among all groups tested at ~1 × 10⁷ cells/kg and only HSPCs were injected. At least 8 mice were used per experimental group and each data point represents chimerism in one mouse. Values for untreated and AZA experimental groups are – healthy: 2.79 ± 0.53 and 3.07 ± 0.36, 261HR: 0.91 ± 0.55 and 0.96 ± 0.63, 325HR: 0.37 ± 0.22 and 0.45 ± 0.31; 400 h: 0.81 ± 0.43 and 2.12 ± 0.56, 455HR: 0.45 ± 0.23 and 1.99 ± 0.56, 627 h: 0.34 ± 0.19 and 2.90 ± 0.91, 768HR: 0.46 ± 0.23 and 1.71 ± 0.58, 775HR: 0.49 ± 0.10 and 2.15 ± 0.60, respectively. Overall, chimerism of human cells was significantly lower after in co-culture groups with HR-MDS-MSCs (p < 0.01 for each MDS group vs healthy MSCs). No differences were observed in the general marrow architecture of the two experimental groups (Supplementary Figure 7). Pre-treatment of MSCs with AZA led to significant improvements in BM chimerism for 5/7HR-MDS-MSC groups (400HR (2.6 × ), 455 h(4.5 × ), 627HR (76 × ), 768HR(3.7 × ) and 775HR (4.3 × )). No appreciable improvements to BM chimerism was observed in the AZA-treated healthy MSC, 261HR and 325HR groups. c In secondary recipient mice (n = 5), we found negligible chimerism of human CD45⁺ cells in the BM of HR627 and HR775 groups compared a secondarily transplanted healthy HSPC groups. However, higher levels of chimerism were found in AZA-treated HR627 and HR775 groups (p < 0.05). d In the primary transplant the engraftment frequencies (threshold of 0.5% human CD45⁺ cells in the BM) for untreated vs AZA experimental groups are – healthy: 1 and 1, 261HR: 0 and 0.13, 325HR: 0 and 0.15, 400 h: 0.13 and 0.5, 455HR: 0 and 0.63, 627HR: 0 and 0.75, 768HR: 0 and 0.5, 775HR: 0 and 0.75, respectively. e In secondary transplanted mice, the engraftment frequencies (threshold of 0.5% human CD45⁺ cells in the BM (see Supplementary Figure 8 for representative plots used to determine minimum threshold of 0.5%), for untreated vs. AZA experimental groups are—healthy: 1 and 1, 627HR: 0 and 0.20, 775HR: 0 and 0.40, respectively. Note that engraftment efficiencies were trending higher although not quite mathematically significant (p = 0.1). Together, these results demonstrate significant effects on both progenitors and HSCs after exposure to MDS stroma. The treatment of MDS stromal cells with hypomethylating agents such as AZA has the potential to correct this disorder. *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant. Non-paired student’s t test was performed. All data represented as mean ± SEM