Novel Culture Medium using a Small-molecule Agonist of Thrombopoetin Receptor
Poster Mar 20, 2015
Hondo M1; Nishino T2; Inamura M1
Hematopoietic stem cells (HSCs), defined by their capacity to self-renew and differentiate into all blood cell lineages, can be applied for transplantation therapy. Since a large number of HSCs are required for clinical use, improvement of techniques for expansion of HSCs ex vivo is a critical issue. Several cytokines have been used for this purpose. Thrombopoietin (TPO) is an essential cytokine that regulates megakaryocyte production and HSC proliferation via activating signaling through its receptor c- MPL. We have developed a small-molecule agonist (NR-101) of c-MPL and report that human HSCs are expanded efficiently ex vivo with NR-101. Using a new small-molecule agonist NR- 102 which is related to NR-101, we produced a novel culture medium, ReproHSCTM. The cost for culture of human HSC can be reduced by using this small-molecule.
Here we demonstrated that ReproHSCTM efficiently expands human CD34+CD38- primitive hematopoietic cells in culture and thereby enhances repopulating capacity of HSCs in NOD/SCID mice. Human blood cord CD34+ cells were cultured with ReproHSCTM supplemented with only Stem Cell Factor (SCF) for 7 days. The total cell number was increased about 40-fold during culture. CD34+ cells and CD34+CD38- cells were expanded 12- fold and 8.5-fold, respectively. We then transplanted expanded cells with ReproHSCTM supplemented with SCF and flt3 ligand for 14 days into NOD/SCID mice and analyzed the SCID-repopulating CD45+ cells with flow cytometry. The expanded cells established engraftment better than the fresh CD34+ cells did. These results indicate that ReproHSCTM is a novel medium suitable for the expansion of HSCs ex vivo.
Multiplexing cell-based assays is possible using 3D culture models that are larger and more complex than monolayers
Real-time detection methods to measure live or dead cells provide much flexibility for multiplexing
All multiplexed assay combinations should be verified using appropriate controls for each 3D cell culture model.
Basic fibroblast growth factor (bFGF) is widely used in vitro for the maintenance and stimulation of a variety of cells. However, use of native bFGF in cell biology is limited by the fact that bFGF rapidly degrades at physiological temperatures. We have addressed this problem with an engineered form of bFGF, named Heat Stable bFGF (HS bFGF), which is stable at 37 degrees Celsius.READ MORE