Use of Adipose-derived Stem Cells to Fabricate Scaffoldless Tissue-engineered Neural Conduits in Vitro

Peripheral nerve injuries resulting from trauma or disease often necessitate surgical intervention. Although the gold standard for such repairs uses nerve autografts, alternatives that do not require invasive harvesting of autologous nerve tissues are currently being designed and evaluated. We previously established the use of scaffoldless engineered neural conduits (ENCs) fabricated from primary cells as one such alternative in sciatic nerve repair in rats [Baltich et al. (2010) In Vitro Cell Dev Biol Anim 46(5):438–444]. The present study establishes protocols for fabricating neural conduits from adipose-derived stem cells (ASCs) differentiated to either a fibroblast or neural lineage and co-cultured into a three-dimensional (3-D) scaffoldless tissue-ENC. Addition of ascorbic acid-2-phosphate and fibroblast growth factor (FGF)-2 to the medium induced and differentiated ASCs to a fibroblast lineage in more than 90% of the cell population, as confirmed by collagen I expression. ASC-differentiated fibroblasts formed monolayers, delaminated, and formed 3-D conduits. Neurospheres were formed by culturing ASCs on nonadherent surfaces in serum-free neurobasal medium with the addition of epidermal growth factor (EGF) and FGF-2. The addition of 10 ng EGF and 10 ng FGF-2 produced larger and more numerous neurospheres than treatments of lower EGF and FGF-2 concentrations. Subsequent differentiation to glial-like cells was confirmed by the expression of S100. ASCderived fibroblast monolayers and neurospheres were cocultured to fabricate a 3-D scaffoldless tissue-ENC. Their nerve-like structure and incorporation of glial-like cells, which would associate with regenerating axons, may make these novel, stem cell-derived neural conduits an efficacious technology for repairing critical gaps following peripheral *Correspondence to: L. M. Larkin, Department of Molecular and Integrative Physiology and Biomedical Engineering, University of Michigan, Biomedical Science Research Building (BSRB), 109 Zina Pitcher Place, Room 2025, Ann Arbor, MI 48109–2200. Tel: 1-734-9368181; fax: 1-734-615-3292. E-mail address: [email protected] (L. M. Larkin). Abbreviations: AA2P, ascorbic acid-2-phosphate; ABAM, antibiotic/ antimycotic; aENC, adipose-derived stem cell engineered fibroblast conduit; ASC, adipose-derived stem cells; Col1, collagen 1; CSA, cross-sectional area; DM, differentiation medium; DPBS, Dulbecco’s phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; EGF, epidermal growth factor; ENC, engineered neural conduits; FBS, fetal bovine serum; FGF, fibroblast growth factor; GM, growth medium; MSC, mesenchymal stem cell; NBM, neural basal medium; PFA, t paraformaldehyde; Td, doubling time; TGF, transforming growth factor; 3-D, three-dimensional. 0306-4522/12 $36.00 © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.11.004 349 nerve injury. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.