Ex-Vivo Expansion of Multipotent CD133+ Stem Cells with VEGF, FLT3l and SCGF.

Abstract

The rare CD133+ stem cell population contains both hematopoietic and endothelial progenitors. Successful ex-vivo expansion of this multipotent population would therefore be of great benefit in many clinical settings including stem cell transplantation and gene therapy. We developed a cell culture system containing the recombinant human cytokines vascular endothelial growth factor (VEGF), FLT3 ligand (FLT3L) and stem cell growth factor (SCGF) for ex-vivo expansion of purified human CD133+ stem cells obtained from leukapheresis products from patients pre-treated with G-CSF.FACS analysis, colony assays and NOD-SCID transplantation studies were performed to monitor stem cell and endothelial phenotype in-vitro and in-vivo. Cultivation with VEGF, FLT3L and SCGF induced a mean 2200-fold increase of total cell counts in 5 weeks. FACS analysis revealed persistence of 6–15% CD133+ stem cells indicating proliferation and survival of primitive hematopoietic stem cells. 5–6% of the proliferating cells expressed the endothelial markers CD144 (VE-Cadherin) and von-Willebrand factor (vWF). Ex-vivo expanded stem cells could be differentiated into adherent endothelial cells after withdrawal of SCGF and FLT3L allowing generation of large numbers of endothelial cells. Colony-assays showed an increase of hematopoietic and endothelial colonies after 5 weeks of ex-vivo expansion indicating simultaneous proliferation of hematopoietic and endothelial precursors under the established culture conditions (CFU-E 60-fold, CFU-GEMM 48-fold, CFU-GM 59-fold, CFU-G 99-fold, CFU-M 1356-fold and CFU-EC 1843-fold).To assess in-vivo functionality, hematopoietic stem cells expanded ex-vivo for 7, 14, 21 and 32 days were transplanted into sublethally irradiated NOD-SCID mice. For each expansion period, the mean percentage of anti-human CD45 positive bone marrow cells 3 months post-transplantation was 11, 3, 3 and 1%, respectively. Human CD45+ cells for each set of experiments contained a mean of 15, 26, 8 and 32% T-cells (CD3+), 9, 0, 7 and 21% B-cells (CD19+), 24, 2, 2 and 11% monocytes (CD14+), 21, 3, 1 and 12% granulocytes (CD33+) and 19, 37, 44 and 24% stem cells (CD34+) (d7 (n=5), d14 (n=4), d21 (n=7) and d32 (n=6) respectively).Our experiments showed multilineage engraftment of human stem cells expanded for more than 4 weeks ex-vivo. Therefore our culture system provides a tool to generate large numbers of human stem and endothelial cells for clinical purposes.