Ex vivo CD34⁺ and CD34⁻ cell expansion in porcine microvascular endothelial cell-based coculture system and stroma-free culture system.

CB CD34⁺ cells were isolated using Miltenyi CD34⁺ selection kits and expanded in PMVEC cocultures (▪) or stroma-free cultures (■) supplemented with recombinant human IL3, IL6, GM-CSF, SCF, and FLT-3L. At days 7, 14, and 21, CD34⁺ (A) and CD34⁺CD38⁻ (B) cell content of resulting cultures was assessed flow cytometrically with the aid of appropriate monoclonal antibodies. Very similar total cellular expansion was obtained in both culture systems. (A) A significantly greater expansion of CD34⁺ cells was achieved in PMVEC cocultures—at day 21 PMVEC cocultures contain 5 times more CD34⁺ cells than stroma-free cultures. (B) No expansion of CD34⁺CD38⁻ cells was observed in stroma-free expansion cultures, whereas PMVEC cocultures at day 14 contained 346 times the input number of this primitive phenotype.