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.

[severe combined immunodeficiency]

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 FLT 3 ligand (FL ). In order to determine the optimal dose and combination of IGFs , IGFBPs and ANGPTLs , serial dilution and full permutation of IGFBP 1 , IGFBP 2 , IGF 2 and ANGPTL 3 were applied on a cryopreserved umbilical cord blood mononuclear cell (UCB-MNC ) ex vivo expansion system .In this system , 4 × 105 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 .Paradoxically , experiment results showed that the highest expansion of CD 34 +CD 38 -CD 9 0 + primitive progenitor was stimulated by cytokine combination of SCF + TPO + FL + IGFBP 1 + IGFBP 2 + ANGPTL 3 at a low dose of 15 ng /ml IGFBP 1 and 20 ng /ml IGFBP 2 and ANGPTL 3 . 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 -IL 2 Rγ-/-) mice . Limiting dilution assay showed excellent correlation between the HSC ex vivo surface marker of CD 34 +CD 38 -CD 9 0 + and the in vivo competitive repopulating unit (CRU ) functional assay .I GFBP 1 , IGFBP 2 , IGF 2 and ANGPTL 3 can stimulate the expansion of CD 34 +CD 38 -CD 9 0 + primitive progenitor at low dose . The optimal combination comprises IGFBP 1 , IGFBP 2 and ANGPTL 3 together with the standard cytokine cocktail of SCF , FL and TPO . The CD 34 +CD 38 -CD 9 0 + phenotype can serve as a surrogate ex vivo surface marker for HSCs due to consistency with the in vivo CRU functional assay .