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Insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs) and angiopoietin-like proteins (ANGPTLs) can enhance the  ex vivo  expansion of hematopoietic stem cells (HSCs) when used with a standard cytokine cocktail of stem cell factor (SCF), thrombopoietin (TPO) and FLT3 ligand (FL). In order to determine the optimal dose and combination of IGFs, IGFBPs and ANGPTLs, serial dilution and full permutation of IGFBP1, IGFBP2, IGF2 and ANGPTL3 were applied on a cryopreserved umbilical cord blood mononuclear cell (UCB-MNC)  ex vivo  expansion system.    Methods  In this system, 4 × 10 5  cells/ml of UCB-MNCs were inoculated in serum-free Stemspan® medium (Stemcell technologies, vancouver, BC, Canada) supplied with standard basal cytokine combination of 100 ng/ml SCF, 50 ng/ml FL and 100 ng/ml TPO and supported by a bone marrow mesenchymal stromal cell layer.    Results  Paradoxically, experiment results showed that the highest expansion of CD34 + CD38 − CD90 +  primitive progenitor was stimulated by cytokine combination of SCF + TPO + FL + IGFBP1 + IGFBP2 + ANGPTL3 at a low dose of 15 ng/ml IGFBP1 and 20 ng/ml IGFBP2 and ANGPTL3. This  ex vivo  expansion was further validated in 8-week-old to 10-week-old nonobese diabetic/severe combined immunodeficiency interleukin 2 gamma chain null (NOD/SCID-IL2Rγ −/− ) mice. Limiting dilution assay showed excellent correlation between the HSC  ex vivo  surface marker of CD34 + CD38 − CD90 +  and the  in vivo  competitive repopulating unit (CRU) functional assay.    Conclusion  IGFBP1, IGFBP2, IGF2 and ANGPTL3 can stimulate the expansion of CD34 + CD38 − CD90 +  primitive progenitor at low dose. The optimal combination comprises IGFBP1, IGFBP2 and ANGPTL3 together with the standard cytokine cocktail of SCF, FL and TPO. The CD34 + CD38 − CD90 +  phenotype can serve as a surrogate  ex vivo  surface marker for HSCs due to consistency with the  in vivo  CRU functional assay.

stem cell research and therapy

Low-dose insulin-like growth factor binding proteins 1 and 2 and angiopoietin-like protein 3 coordinately stimulate ex vivo expansion of human umbilical cord blood hematopoietic stem cells as assayed in NOD/SCID gamma null mice