Cytokine combinations on the potential for ex vivo expansion of murine hematopoietic stem cells

• Murine HSCs were ex vivo cultured in six combinations of cytokines.
• Serum-free medium with SCF/Flt3-L/IL-11 stimulates expansion of HSC but not mast cells.
• SCF/Flt3-L/IL-11 expanded HSCs maintain multipotency and a HSC transcription profile.
• SCF/Flt3-L/IL-11 is the optimal cytokine combination for murine HSC.

Hematopoietic stem cell (HSC) is a rare cell population, which is capable of self-renewal and differentiation to all blood lineages. The clinical potential of HSCs for treating hematological disorders has led to the use of cytokine stimulation for ex vivo expansion. However, little is known about the molecular features of the HSC populations expanded under different cytokine combinations. We studied the expansion of murine HSCs cultured with six different cytokine combinations under serum-containing or serum-free conditions for 14 days. We found that all the cytokine combinations promoted expansion of murine HSCs. Although SCF/IL-3/IL-6 induced the highest expansion of the immunophenotypic Lineage−Sca-1+c-Kit+ (LSK) cells at day 14, over 90% of them were FcεRIα+ mast cells. In contrast, the serum-free medium with SCF/Flt3-L/IL-11 effectively promoted the expansion of LSK/FcεRIα− HSCs by over 50-fold. HSCs expanded by SCF/Flt3-L/IL-11 combination formed compact hematopoietic colonies and demonstrated a higher degree of multipotency compared to the HSCs cultured with other cytokine combinations. Surprisingly, despite the same LSK/FcεRIα− immunophenotype, HSCs cultured with different cytokine combinations demonstrated differential patterns of hematopoietic gene expression. HSCs cultured with SCF/Flt3-L/IL-11 maintained a transcription profile resembling that of freshly isolated HSCs. We propose that serum-free medium supplemented with SCF/Flt3-L/IL-11 is the optimal culture condition to maintain the stemness of ex vivo expanded HSCs. This study used molecular characterization of cytokine-expanded murine HSCs to facilitate the selection of cytokine combinations that could induce fully competent HSC for clinical applications.