Abstract

Research studies of adherent stem cell populations, primarily isolated from bone marrow but also cord blood, adipose and other tissues, have shown cultures capable of regenerating more than one primitive germ layer in vitro or in vivo. This raises exciting possibilities for tissue repair in acute injury or degenerative disease, provided scalable processes for cell expansion can be developed. We have optimized an expansion process based on the Multi-potent Adult Progenitor Cell (MAPC) platform first described by Dr. C. Verfaillie. Termed Multistem, these adherent stem cell cultures have the capacity to differentiate into many different primary tissue types including but not limited to endothelial, hepatic, neuronal, adipogenic, and osteogenic lineages cells. Three fresh marrow aspirates were obtained from healthy donors and the mononuclear cells isolated using the COBE 2991 Cell Processor. Once the mononuclear cells were isolated, they were subsequently placed into Nunc cell factories that had been coated with 10 ng/ml Fibronectin A (Day 0). Serum containing media supplemented with PDGF and EGF was used, with cells incubated under low oxygen tension (5%). Cells were expanded to Day six, and then every two to three days until greater than 100 million cells were obtained (routinely Day 15–17). The growth rate was linear through approximately 24 population doublings, (yielding a theoretical 1011 cells) with a doubling rate of 24–30 hours. Release criteria qualifying successfully expansion to clinical scale include sterility, identity/purity, and tissue lineage potency. In addition, the cells were analyzed at approximately population doubling 20 for cytogenetic abnormalities and out of 20 spreads, 0 abnormal cells were detected for each of the three samples. All cultures tested positive by qPCR for expression of Oct4, a marker of pluripotency in ES cell systems. Telomerase levels were analyzed using a TRAP QPCR assay and the levels ranged from between 15 and 22 times higher than that measured in commercially purchased mesenchymal stem cells (MSC) at about population doubling 20 FACS analysis was performed on cells expanded using these conditions, with all cultures being positive for CD49c, CD90 and negative for CD45 and HLA class II surface markers. Current process improvements have allowed for the growth of human cells out to greater than 60 population doublings. These results demonstrate that large numbers of human stem and progenitor cells can be obtained using these culturing techniques, and that the cell products are consistent between donors. Donor ages ranged between 19 and 57. This is the first report of a scalable and consistent process for expansion of human stem cell cultures with pluripotent lineage capacity.

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