AbstractA continuous normal function of olfactory ensheathing glia (OEG) is to promote axonal regeneration from the olfactory neuroepithelium to the brain, and their neuroregenerative potential in other CNS sites such as the injured spinal cord has been studied for over a decade. However, human OEG are difficult to obtain in large amounts directly from tissues, and the derived primary cultures have a limited duplication capacity. Thus, although auto‐transplantation may be an obvious option for initial proof‐of‐concept trials, alternatives must be explored to obtain large quantities of homogeneous, pre‐characterized OEG for wide‐scale therapeutic use. We have cultured primary human OEG derived from olfactory bulbs (OB) obtained by necropsy and successfully extended the replicative lifespan of these cells using lentivectors encoding Bmi‐1 and TERT transgenes flanked by loxP sites. In contrast to the primary cells which could only be expanded for a limited number of passages (∼12), adult human OEG immortalized Bmi‐1/TERT divided indefinitely in culture. Clonal lines were isolated and the floxed transgenes could be excised by lentivector‐mediated Cre recombinase delivery. Primary, immortalized, and deimmortalized human OEG all expressed typical markers of this cell type and importantly, were all able to promote axonal regeneration of adult rat retinal ganglion neurons (RGN) in co‐culture assays. © 2009 Wiley‐Liss, Inc.