Selection of CD271(+) cells and human AB serum allows a large expansion of mesenchymal stromal cells from human bone marrow.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. DISCUSSION: 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.

Characterization and expansion of mesenchymal progenitor cells from first-trimester chorionic villi of human placenta.

BACKGROUND: Mesenchymal stromal cells (MSC) have been identified in a variety of fetal and adult tissues, including bone marrow (BM), fetal blood and liver. We report on the isolation, expansion and differentiation in vitro of MSC-like cells from chorionic villi (CV). METHODS: We evaluated 10 samples of CV collected at the first trimester (gestational age 11-13 weeks). We only used cells taken from back-up culture after a successful karyotype analysis. CV cells were characterized by morphologic, immunophenotypic and molecular analysis. The differentiation ability of mesenchymal and neural lineages was detected using specific culture conditions. Cell expansion was assessed after plating cells at different densities in different media, supplemented with animal and human serum. RESULTS: CV cells showed a homogeneous population of spindle-shaped cells after the first passage. Cells expressed CD90, CD105, CD73, CD44, CD29 and CD13 but not CD45, CD14, CD34 and CD117. They expressed Oct-4, Rex-1, GATA-4 and nestin, which characterize the undifferentiated stem cell state. They differentiated into osteocytes, adipocytes, chondrocytes and neuronal cells. Cell expansion was greater than that of adult BM-derived MSC, 9 logs with fetal bovine serum and 6 logs with human serum. Despite their high proliferative capacity, we did not observe any karyotypic abnormalities after culture. DISCUSSION: Our study shows that CV cells have better potential for expansion than adult stem cells. They can proliferate in a medium with human allogeneic serum and can differentiate into mesenchymal and neural lineages. CV cells may be an excellent cell source for therapeutic applications.