Proliferation and Differentiation of Erythroid Progenitors from Cord Blood CD34 (+) Cells Using Extracellular Matrix and Stromal Cells

Choi, Hyoung Soo; Koh, Sang Hyeok; Park, Eun Sil; Kang, Hyoung Jin; Shin, Hee Young; Ahn, Hyo Seop

Abstract

PURPOSE
This study was performed to induce proliferation and differentiation of erythroid progenitors from cord blood CD34 cells using extracellular matrix (ECM) and stromal cells. METHODS: Cord blood mononuclear cells were separated using Ficoll-Hypaque and CD34 cells were purified by Mini-MACS column. Cells were cultured in IMDM medium including 10% FBS and several cytokines (EPO, flt3-ligand, SCF and TPO) upto 14 days. ECM like laminin, collagen IV, fibrinogen and fibronectin were used alone or in combination. Stromal cells were derived from BM mononuclear cells for 4 weeks of culture and irradiated before use. On day 14 of stromal coculture of cord blood CD34 cells, flow cytometric analysis were done with fluorescence isothiocyanate-conjugated (FITC) antihuman glycophorin A antibody for erythroid cells. RESULTS: ECM-treated group showed 16.3~31.3 fold increase of the cell numbers on day 14 and there were no difference from cytokines-treated group. Stromal cells induced great amount of fold increase compared with ECM-treated group on day 7 (25.4 fold vs. 2.2~5.4 fold). The cell numbers increased upto 16.4 fold on day 7, 92.8 fold on day 10, and 198.4 fold on day 14 with stromal cells. Erythroid progenitors expressing glycophorin A increased from 2.78% on day 0 to 21.57% on day 7 and 43.87% on day 14. CONCLUSION: Stromal coculture of cord blood CD34 cells induced marked proliferation and differentiation of erythroid cells compared with cytokines or ECM-treated group. Efficient in vitro erythroid culture might have implications for gene therapy in RBC defects or developing blood substitutes for transfusions.