Notch signaling has been recognized as a key pathway to regulate the proliferation and differentiation of hematopoietic stem cells (HSC). In this study, the orientation-regulated immobilization of a Notch ligand was designed to achieve the efficient Notch ligand-receptor recognition for the ex vivo proliferation of a bone marrow cell population containing HSC. Protein A was chemically conjugated onto aminated glass substrates, followed by immobilizing a recombinant chimeric protein of Jagged1 and Fc domain (Jagged1-Fc) through the biospecific binding between protein A and Fc domain. Protein A adsorption was suppressed for the Jagged1-Fc-immobilized substrates, in contrast to the Jagged1-Fc-coated ones, indicating the orientation-regulated immobilization of Jagged1-Fc for the substrates. Mouse lineage negative cells (Lin(-)) were cultured on the Jagged1-Fc-immobilized substrates. Flow cytometric analyses demonstrated that c-Kit(+), Sca-1(+), Lin(-), and CD34(-) cells of an HSC population was significantly proliferated on the Jagged1-Fc-immobilized substrates 6 days after culture, whereas no proliferation was observed for the Jagged1-Fc-coated substrates in a random manner or Jagged1-Fc-immobilized ones with a Notch signaling inhibitor. It is concluded that the orientation-regulated immobilization of Jagged1-Fc increased the efficiency of Jagged1 to recognize the Notch receptors, resulting in the promoted ex vivo proliferation of the HSC population.
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