Selection of CD271+ cells and human AB serum allows a Large expansion of mesenchymal stromal cells from human bone marrow

Abstract

Mesenchymal stromal cells (MSC) are promising candidates for cell therapy and tissue engineering and may be used to treat acute graft-versus-host disease (GvHD). However, major obstacles for their clinical use are the required cell dose and the biosafety and potential immunogenicity of fetal bovine serum (FBS), which is a crucial supplement of all media currently used for the culture of MSC. In this study MSC were successfully expanded after selection of CD271 cells from human bone marrow (BM) mononuclear cells in medium supplemented with 10% pooled allogeneic human serum. We isolated MSC from 10 healthy donor BM by plastic adherence and immunomagnetic selection of the CD271(+) fraction and expanded MSC in medium supplemented with pooled human allogeneic serum and animal serum. We isolated a homogeneous multipotent population by CD271(+) selection with a proliferation rate that was higher than MSC isolated by plastic adherence, 6.8+/-1.57 compared with 2.07+/-1.40 logs. Similar to cells generated in animal serum medium, MSC from allogeneic human serum were positive for mesenchymal markers and negative for hematopoietic markers; moreover they expressed embryonic stem cell genes. A normal karyotype and differentiation capacity into adipogenic, osteogenic and chondrogenic lineages and neurosphere-like structures were preserved throughout long-term culture. Expansion of MSC is both feasible and large with a CD271-selected population in medium supplemented with 10% pooled allogeneic human serum, without loss of multipotent differentiation capacity or karyotype alterations.