Platelet‐derived growth factor promotes ex vivo expansion of CD34⁺ cells from human cord blood and enhances long‐term culture‐initiating cells, non‐obese diabetic/severe combined immunodeficient repopulating cells and formation of adherent cells

Abstract

Summary. Platelet-derived growth factor (PDGF) is a major mitogen for connective tissue cells. In this study, we investigated the effects and mechanism of PDGF on the ex vivo expansion of cord blood CD34 + cells. Our data demonstrated that among various cytokine combinations of thrombopoietin (TPO), interleukin 1 beta (IL-1b), IL-3, IL-6 and Flt-3 ligand (Flt-3L), TPO + IL-6 + Flt-3L was most efficient in promoting the expansion of CD34 + cells, CD34 + CD38 -cells, mixed-lineage colony-forming units (CFU-GEMM) and long-term culture-initiating cells (LTC-IC) by 21AE7 ± 5AE00-, 103 ± 27AE9-, 10AE7 ± 7AE94-and 6AE52 ± 1AE51-fold, respectively, after 12-14 d of culture. The addition of PDGF increased the yield of these early progenitors by 45AE0%, 66AE5%, 45AE1% and 79AE8% respectively. More significantly, PDGF enhanced the engraftment of human CD45 + cells and their myeloid subsets (CD33 + , CD14 + cells) in non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) mice. The expression of PDGF receptor (PDGFR)-b was not detectable in fresh CD34 + cells but was upregulated after culture for 3 d. PDGF also enhanced the development of adherent cells/clusters that expressed the endothelial markers VE-cadherin and CD31. These findings suggest that PDGF is an effective cytokine for the ex vivo expansion of early stem and progenitor cells. The mechanism could be mediated by PDGFR-b on committed CD34 + progenitor cells and/or secondary to the stimulation of autologous, stromal feeder cells.

Multipotent neural precursors express neural and hematopoietic factors, and enhance ex vivo expansion of cord blood CD34+ cells, colony forming units and NOD/SCID-repopulating cells in contact and noncontact cultures

“…They were irradiated (30 Gy) in a Gammacell-1000 Elite Irradiator (MDS Nordion, Kanata, Ontario, Canada) and cultured in fibronectincoated (20 mg/ml; Gibco) 24-well culture plates (BDA-3047; BD, Franklin Lakes, NJ, USA) at a density of 4 Â 10 4 /well in IMDM containing 10% FCS for 24 h. Enriched CD34 þ cells at 2 Â 10 4 / well were added to the pre-established stromal cells, supplemented with thrombopoietin (TPO, 50 ng/ml), Flt-3 ligand (FL, 20 ng/ml) and interleukin (IL)-6 (20 ng/ml) and cultured at 371C in a humidified atmosphere of 5% CO 2 . 15 On days 7 and 10, four-and two-fold volumes of fresh medium and cytokines were added to the cultures, respectively. Expanded cells were harvested at days 7 and 14.…”

“…We assayed functional progenitor cells as colony forming unitgranulocyte macrophage (CFU-GM), burst forming unit/colony forming unit-erythroid (BFU/CFU-E) and CFU-mixed (CFU-GEMM) in 1% methylcellulose (Sigma) cultures in IMDM supplemented with 30% FCS, 1% BSA and 0.1 mM b-mercaptoethanol (b-ME) in the presence of 3 IU/ml erythropoietin (EPO; Eprex, Cilag, Zug, Switzerland), 10 ng/ml granulocyte macrophage-colony stimulating factor (GM-CSF; Leucomax, Basle, Switzerland), 10 ng/ml IL-3 and 50 ng/ml stem cell factor (SCF) as described previously. 15,16 Enriched or expanded CD34 þ cells at 3 Â 10 3 /ml were seeded in triplicate and incubated for 14 days. Colony forming unit-megakaryocytes (CFU-MK) were cultured in a plasma clot system.…”

Cobblestone area-forming cells, long-term culture-initiating cells and NOD/SCID repopulating cells in human neonatal blood: a comparison with umbilical cord blood

“…16,17 NOD/LtSZ-scid/scid mice were purchased from The Walter and Eliza Hall Institute of Medical Research (Victoria, Australia), bred and maintained in the Laboratory Animal Services Centre at the Chinese University of Hong Kong. Animals were handled under sterile conditions and maintained in microisolator cages.…”

“…All antibodies were previously selected to show no cross-reaction with the NOD/SCID mouse surface antigens. 17 After washing with PBS, 30 000 to 70 000 events were acquired for each sample and non-viable cells (propidium iodide positive) were gated out during data analysis.…”

“…17,18 Before plating, the mouse BM cells were incubated for 4 to 8 h to remove adherent, murine stromal cells that might secrete murine cytokines. 1 ϫ 10 5 BM cells from each recipient mouse were cultured in triplicate in methylcellulose containing 30% FCS, SCF (20 ng/ml), IL-3 (10 ng/ml), GM-CSF (100 ng/ml), and erythropoietin (2 U/ml).…”