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Proliferation of Hematopoietic Stem Cells Is Stimulated in 3D-clinostat Culture.   R. Yoshimoto¹, T. Kajiume², Y. Kawahara^1,3, C. Umeda¹, A. Sasaki¹, S.L. Wu¹, K. Naminohira¹, K. Kataoka², and L. Yuge^{1,3  1}Graduate School of Health Sciences, ²Graduate School of Biomedical Sciences, ³Space Bio-Laboratories Y. K., Hiroshima University, Japan. 

Hematopoietic stem cells (HSCs) are multipotent stem cells, which have abilities for self-renewal and differentiation.  Different cytokines and growth factors have been used to expand stem cells.  The well-established strategies for ex vivo expansion of HSCs include culture with cytokine combination and co-culture with bone marrow stromal cells.  A 3D-clinostat is a multi-directional gravity device for simulating microgravity.  By controlled rotation of two axes, a 3D-clinostat minimizes the cumulative gravity vector in cells cultured at the center of the device and makes 10^-3 G average over time.  In this study, we investigated the application of microgravity to HSC culture using a 3D-clinostat.

Mouse bone marrow cells were cultured for 3 to 7 days in a 3D-clinostat (group CL), or in control cultures at 1G (group C) in the presence of optimal cytokine combination.  Then the cultured cells were transferred into semisolid culture for colony-forming cell assay (CFC assay).  The number of cells was assessed by surface antigen analysis on day 3 and day 7.  After 1-wk culture in, CFCs were collected, and then 2nd CFC assay was performed.  Highly purified HSCs were defined as lineage marker depletion and expression of the cell surface markers, Sca1 and c-Kit cells (Lin(-) Sca1(+) c-Kit(+) [LSK]), such cells were established by 2nd CFC.  The fold increase of cells expanded from day 3 to day 7 was significantly greater in group CL than in group C. Furthermore, group CL included larger proportion of LSK than group C at day 7.  The number of CFCs in 2nd CFC assay was also more abundant in group CL.  In conclusion, we demonstrated that HSCs were well proliferated and maintained their multipotential in culture in simulated microgravity by 3D-clinostat.