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Ex vivo expansion of hematopoietic stem cells (HSCs) is an approach for overcoming cell insufficiency for umbilical cord blood transplantation. It was suggested that in common ex vivo cultures, the stemness specificity of HSCs is rapidly reducing due to DNA hypermethylation. Here, Nicotinamide (NAM), a DNA methyltransferase and histone deacetylase inhibitor, is used with a bioengineered Bone Marrow-like niche (BLN) for HSC ex vivo expansion. The CFSE cell proliferation assay was used for tracking HSCs division. qRT-PCR was conducted to assay the HOXB4 mRNA expression levels. The morphology of BLN-cultured cells was analyzed using scanning electron microscopy (SEM). NAM boosted the induction of HSC proliferation in the BLN group compared to the control group. In addition, the ability of HSCs to colonize was more significant in the BLN group than in the control group. Our data suggest that the presence of NAM in bioengineered niches promotes HSC proliferation. The presented approach showed that small molecules could be used in the clinical setting to overcome the limited number of CD34
+
cells in cord blood units.