Study of the Mechanisms by Which Angptl5 and IGFBP2 Support Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells.

Abstract We previously showed that angiopoietin-like protein 5 (Angptl5) and IGF Binding Protein 2 (IGFBP2) support dramatic ex vivo expansion of human hematopoietic stem cells (HSCs). To understand the mechanisms of their action, here we studied the effects of Angptl5 and IGFBP2 on the surface phenotype, signaling activation, self-renewal, apoptosis, differentiation, and homing of human cord blood CD34+ cells. Using immunofluorescence staining, we showed that Angptl5 and IGFBP2 activate certain signaling pathways such as MAPK and Stat5 in human cord blood CD34+ cells. IGFBP2 and Angptl5 increased the expression of transcription factors HoxB4, Bmi-1, EZH2, and survivin, measured by intracellular staining flow cytometry analysis and real-time RT-PCR. IGFBP2 and Angptl5 also inhibit expression of certain transcription factors important for differentiation of myeloid, erythroid, and lymphoid lineages. To test whether IGFBP2 and Angptl5 affect the homing of HSCs, we cultured human cord blood CD34+ cells in serum-free medium supplemented with SCF, TPO, Flt3-L, IGFBP2 or Angptl5, and transplanted them into sublethally irradiated NOD/SCID mice intraveneously or intrafemorally. Both IGFBP2 and Angptl5 support ex vivo expansion of SRCs in intrafemorally injected mice, suggesting the expansion-stimulating effects elicited by both factors are not caused by modulation of HSC homing. Interestingly, when we used intrafemoral injection, we found that Angptl5 treated HSCs have enhanced engraftment in non-injected bone marrow. This suggests Angptl5 treated HSCs further facilitate the mobilization of HSCs in vivo. We conclude that IGFBP2 and Angptl5 support self-renewal and inhibit differentiation of human cord blood HSCs. Our data also suggest that a combination of expression of transcription factors important for self-renewal, survival, and differentiation of HSCs can be used as a “stemness index” that predicts the activity of cultured human HSCs.