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Objective-Nanofiber scaffolds with amino groups conjugated to fiber surface through different spacers (ethylene, butylenes, and hexylene groups, respectively) were prepared and the effect of spacer length on adhesion and expansion of umbilical cord blood hematopoietic stem/progenitor cells (HSPCs) was investigated.Materials and Methods-Electrospun polymer nanofiber scaffolds were functionalized with poly (acrylic acid) grafting, followed by conjugation of amino groups with different spacers. HSPCs were expanded on aminated scaffolds for 10 days. Cell proliferation, surface marker expression, clonogenic potential, and nonobese diabetic (NOD)/severe combined immunodeficient (SCID) repopulation potential of the expanded cells were evaluated following expansion culture.Results-Aminated nanofiber scaffolds with ethylene and butylene spacers showed high-expansion efficiencies (773-and 805-fold expansion of total cells, 200-and 235-fold expansion of CD34 + CD45 + cells, respectively). HSPC proliferation on aminated scaffold with hexylene spacer was significantly lower (210-fold expansion of total cells and 86-fold expansion of CD34 + CD45 + cells), but maintained the highest CD34 + CD45 + cell fraction (41.1%). Colony-forming unit granulocyte-erythrocyte-monocyte-megakaryocyte and long-term culture-initiating cell maintenance was similar for HSPCs expanded on all three aminated nanofiber scaffolds; nevertheless, the NOD/SCID mice engraftment potential of HSPCs expanded on aminoethyl and aminobutyl conjugated nanofibers was significantly higher than that on aminohexyl conjugated nanofibers.Conclusion-This study demonstrated that aminated nanofibers are superior substrates for ex vivo HSPC expansion, which was correlated with the enhanced HSPC adhesion to these aminated nanofibers. The spacer, through which amino groups were conjugated to nanofiber surface, affected the expansion outcome. Our results highlighted the importance of scaffold topography and cellsubstrate interaction to regulating HSPC proliferation and self-renewal in cytokine-supplemented expansion.Offprint requests to: Hai-Quan Mao, Ph.D., Department of Materials Science and Engineering, Johns Hopkins University, 102 Maryland Hall, 3400 N. Charles Street, Baltimore, MD 21218; E-mail: hmao@jhu.edu. In conventional ex vivo expansion culture, HSPCs are generally regarded as suspension cells and numerous protocols implement HSPC suspension cultures in flasks or bags in the presence of various combinations of early acting cytokines [3][4][5][6][7]. The importance of topographical and substrate-immobilized biochemical cues has not been taken into consideration in these culture systems. Suspension culture conditions differ drastically from conditions provided by the natural bone marrow microenvironment, where a complex network of stromal cells and extracellular matrix (ECM) serves as a stem cell niche that regulates HSPC functions such as homing, self-renewal, proliferation, and fate choice [2,8]. A growing body of evidence has suggested the importance of...