Self-Renewing Human Bone Marrow Mesenspheres Promote Hematopoietic Stem Cell Expansion

• Human MSCs can be simply isolated, preserved, and expanded as nonadherent mesenspheres
• Human bone marrow mesenspheres derive from CD45−CD31−CD71−CD146+CD105+ nestin+ cells
• Human mesenspheres expand hematopoietic stem cells (HSCs) through secreted factors
• Mesensphere attachment to plastic changes their phenotype and decreases HSC support

SummaryStrategies for expanding hematopoietic stem cells (HSCs) include coculture with cells that recapitulate their natural microenvironment, such as bone marrow stromal stem/progenitor cells (BMSCs). Plastic-adherent BMSCs may be insufficient to preserve primitive HSCs. Here, we describe a method of isolating and culturing human BMSCs as nonadherent mesenchymal spheres. Human mesenspheres were derived from CD45− CD31− CD71− CD146+ CD105+ nestin+ cells but could also be simply grown from fetal and adult BM CD45−-enriched cells. Human mesenspheres robustly differentiated into mesenchymal lineages. In culture conditions where they displayed a relatively undifferentiated phenotype, with decreased adherence to plastic and increased self-renewal, they promoted enhanced expansion of cord blood CD34+ cells through secreted soluble factors. Expanded HSCs were serially transplantable in immunodeficient mice and significantly increased long-term human hematopoietic engraftment. These results pave the way for culture techniques that preserve the self-renewal of human BMSCs and their ability to support functional HSCs.

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