Characterization of neuronal/glial differentiation of murine adipose-derived adult stromal cells
Introduction
Cells capable of neuronal differentiation have broad potential for cellular therapies. Neural tissue has a limited capacity for repair after injury, and adult neurogenesis is limited to select regions of the brain Gage, 2000, Magavi et al., 2000, Rakic, 2002, Temple and Alvarez-Buylla, 1999. Commonly proposed cell sources for neuronal cellular therapies include embryonic stem cells (ESCs), and neural stem cells (NSCs) from embryonic or adult brain tissue Bain et al., 1995, Brustle et al., 1999, Freed et al., 2001, Lindvall et al., 1990, McKay, 1997. However, the use of ESCs and NSCs is limited by various ethical and logistical constraints, and adult peripheral tissues may be an alternative source of stem and progenitor cells. For example, recent studies have shown that adult bone marrow contains a population of mesenchymal stem cells capable of differentiating into several lineages, including neuronal and glial tissues Brazelton et al., 2000, Jiang et al., 2002, Kopen et al., 1999, Mezey et al., 2000, Sanchez-Ramos, 2000, Woodbury et al., 2000.
Adipose tissue has been identified as an alternative source of stromal cells capable of differentiating into mesodermal lineages. Previous studies have demonstrated the osteogenic, chondrogenic, adipogenic, myogenic, cardiomyogenic, and hematopoietic potential of stromal cells isolated from adipose tissue Charriere et al., 2003, Erickson et al., 2002, Halvorsen et al., 2001, Rangappa et al., 2003, Sen et al., 2001, Winter et al., 2003, Zuk et al., 2001. Recently, we and others have shown in preliminary studies that adipose-derived adult stromal (ADAS) cells can express select properties of neuronal and glial cells Ashjian et al., 2003, Safford et al., 2002, Zuk et al., 2002. In the current study, we have extended these observations to test the hypothesis that adipose tissue contains a population of cells that are capable of exhibiting phenotypic, morphologic, and excitotoxic characteristics of neuronal and glial tissues.
Section snippets
Cell harvest and culture
For isolation of murine ADAS (muADAS) cells, we used Balb/C mice (Charles River Laboratories, Wilmington, MA). Animals were housed under standard conditions, and all animal procedures were approved by the Duke University Institutional Animal Care and Use Committee. After sacrifice of the mice, we harvested subcutaneous adipose tissue, and isolated ADAS cells using a modification of published methods (Safford et al., 2002). Briefly, we mechanically dissociated the adipose tissue, performed a
In vitro viability of muADAS cells
Undifferentiated muADAS cells maintained 90–95% viability through 5 days of culture. In contrast, after exposure to neuronal induction media, muADAS cells show decreased viability of 70% at day 1, followed by a 10–25% decrease each additional day that the cells remained in neuronal induction media. By day 5, only 35% of the muADAS cells are still viable (P < 0.01), and all die within 14 days of culture. Additional experiments will clarify whether surviving induced ADAS cells retain
Discussion
Our findings suggest that murine ADAS cells can be induced to differentiate into cells with select characteristics of neuronal and glial tissues. Using a series of immunohistochemistry and immunoblotting experiments, we have found that muADAS cells can be induced to undergo morphologic and phenotypic changes consistent with developing neuronal and glial cells. Murine ADAS cells appear to exhibit excitotoxic characteristics in response to NMDA. These cells express several phenotypic properties
Acknowledgements
This work was supported by funds provided by the Owen H. Wagensteen, MD Faculty Research Fellowship of the American College of Surgeons. The authors thank Alex Freemerman, PhD and Dennis Rickman, PhD for expert advice during the course of this work.
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