Chimeric feeders of mesenchymal stromal cells and stromal cells modified with constitutively active AKT expand hematopoietic stem cells

Abstract

Aim: To examine whether AKT-modified stromal cells expand human CD34⁺ hematopoietic stem cells (HSCs). Methods: Coculture, in vitro functional assays, immuno-fluorescence microscopy, flow cytometry. Results: M2-10B4 stromal cells (M2) modified with AKT1 (M2-AKT) expanded primitive CD34⁺38^- HSCs, but affected their functionality. A chimeric feeder layer comprising naive human bone marrow-derived mesenchymal stromal cells and M2-AKT not only overcame the negative effects of M2-AKT, but, unexpectedly, also gave a synergistic effect on the growth and functionality of the HSCs. Conditioned medium of bone marrow stromal cells worked as effectively, but cell-cell contact between HSCs and M2-AKT cells was necessary for the synergistic effect of M2-AKT and bone marrow-derived mesenchymal stromal cells or their CM. Conclusion: Chimeric feeders expand HSCs.

Keywords: AKT signaling; CD34; M2-10B4; bone marrow stromal cells (BMSCs); cobblestone-area-forming-cells (CAFC) assay; cocultures; conditioned medium (CM); hematopoietic stem cells (HSCs); long-term culture-initiating cell (LTC-IC) assay; mesenchymal stem/stromal cells (MSCs).