Neurogenic differentiation of murine and human adipose-derived stromal cells

https://doi.org/10.1016/S0006-291X(02)00469-2Get rights and content

Abstract

The identification of cells capable of neuronal differentiation has great potential for cellular therapies. We examined whether murine and human adipose-derived adult stem (ADAS) cells can be induced to undergo neuronal differentiation. We isolated ADAS cells from the adipose tissue of adult BalbC mice or from human liposuction tissue and induced neuronal differentiation with valproic acid, butylated hydroxyanisole, insulin, and hydrocortisone. As early as 1–3 h after neuronal induction, the phenotype of ADAS cells changed towards neuronal morphology. Following neuronal induction, muADAS cells displayed immunocytochemical staining for GFAP, nestin and NeuN and huADAS cells displayed staining for intermediate filament M, nestin, and NeuN. Following neuronal induction of murine and human ADAS cells, Western blot analysis confirmed GFAP, nestin, and NeuN protein expression. Pretreatment with EGF and basic FGF augmented the neuronal differentiation of huADAS cells. The neuronal differentiation of stromal cells from adipose tissue has broad biological and clinical implications.

Section snippets

Materials and methods

Cell harvest and culture. For isolation of murine ADAS (muADAS) cells, we used BalbC mice (Charles River Laboratories). Animals were housed under standard conditions and the IACUC of Duke University approved all animal procedures. After sacrifice of the mice, we harvested subcutaneous adipose tissue and isolated ADAS cells using a modification of published methods [25]. Briefly, we mechanically dissociated the adipose tissue, performed a digestion with collagenase type I (Sigma), and

ADAS cell characterization

Within 2–3 passages after the initial plating of the primary culture, muADSCs cells appeared as a monolayer of large, flat cells (25–30μm in diameter). As the cells approached confluence, they assumed a more spindle-shaped, fibroblastic morphology. Flow cytometry analysis of muADAS cells at passages 3–5 demonstrated that the cells were negative for CD11b and CD45 (Fig. 1A), cell surface markers associated with hematopoietic cells. In contrast, the majority of muADAS cells expressed CD29 (Fig. 1B

Discussion

Neural tissue has limited potential for intrinsic repair following injury and the identification of cells capable of neuronal differentiation has generated intense interest [1], [2]. Our results demonstrate that murine and human adipose-derived stem cells cultured in the presence of specific soluble mediators possess the ability to undergo morphologic and phenotypic changes consistent with neuronal differentiation. These findings, together with previous studies, indicate that stem and

Acknowledgements

This work was supported by funds provided by the Owen H. Wagensteen, MD Faculty Research Fellowship of the American College of Surgeons, as well as by funds provided by Artecel Sciences, Inc. Dr. Rice serves as a consultant to Artecel Sciences, Inc. Note that portions of this research were presented at the 33rd Annual Meeting of the American Pediatric Surgical Association, May, 2002. The authors thank Alex Freemerman and Ashok Shetty for expert advice during the course of this work.

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