In this study, we isolated and characterized a population of non-human primate adipose tissue stromal cells (pATSCs) containing multipotent progenitor cells. We show that these pATSCs can differentiate into several mesodermal lineages, as well as neural lineage cells. For neural induction of pATSCs and non-human primate bone marrow stromal cells (pBMSCs), the cells were cultured in Neurobasal (NB) media supplemented with B27, basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF) and epidermal growth factor (EGF). After 4 days in culture, the pATSCs form compact, spheroid bodies that ultimately become neurospheres (NS). Free-floating neurospheres undergo extensive differentiation when cultured on PDL-laminin. Our data suggest that the neurogenic potential of pATSCs is markedly higher than that of pBMSCs. We have also performed microarray analysis and characterized the gene expression patterns in undifferentiated pATSCs. The direct comparison of gene expression profiles in undifferentiated pATSCs and pATSC-NS, and delineated specific members of important growth factor, signaling, cell adhesion and transcription factors families. Our data indicate that adipose tissue may be an alternative source of stem cells for therapy of central nervous system (CNS) defects.

Expression of TERT, the catalytic protein subunit of the telomerase complex, can be used to generate cell lines that expand indefinitely and retain multilineage potential. We have created immortal adipose stromal cell lines (ATSCs) by stably transducing nonhuman primate-derived ATSCs with a retroviral vector expressing TERT. Transduced cells (ATSC-TERT) had an increased level of telomerase activity and increased mean telomere length in the absence of malignant cellular transformation. Long-term culture of the ATSC-TERT cells demonstrated that the cells retain the ability to undergo differentiation along multiple lineages such as adipogenic, chondrogenic, and neurogenic. Untransduced cells demonstrated markedly reduced multilineage and selfrenewal potentials after 12 passages in vitro. To determine the functional role of telomerase during osteogenesis, we examined osteogenic differentiation potential of ATSC-TERT cells in vitro. Compared with naive ATSCs, which typically begin to accumulate calcium after 3-4 weeks of induction by osteogenic differentiation medium, ATSC-TERT cells were found to accumulate significant amounts of calcium after only 1 week of culture in osteogenic induction medium. The cells have increased production of osteoblastic markers, such as AP2, osteoblast-specific factor 2, chondroitin sulfate proteoglycan 4, and the tumor necrosis factor receptor superfamily, compared with control ATSCs, indicating that telomerase expression may aid in maintaining the osteogenic stem cell pool during in vitro expansion. These results show that ectopic expression of the telomerase gene in nonhuman primate ATSCs prevents senescenceassociated impairment of osteoblast functions and that telomerase therapy may be a useful strategy for bone regeneration and repair.

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