Abstract
Calcitonin gene-related peptide (CGRP) promotes neuron recruitment and neurogenic activity. However, no evidence suggests that CGRP affects the ability of stem cells to differentiate toward neurogenesis. In this study, we genetically modified rat adipose-derived stem cells (ADSCs) with the CGRP gene (CGRP-ADSCs) and subsequently cultured in complete neural-induced medium. The formation of neurospheres, cellular morphology, and proliferative capacity of ADSCs were observed. In addition, the expression of the anti-apoptotic protein Bcl-2 and special markers of neural cells, such as Nestin, MAP2, RIP and GFAP, were evaluated using Western blot and immunocytochemistry analysis. The CGRP-ADSCs displayed a greater proliferation than un-transduced (ADSCs) and Vector-transduced (Vector-ADSCs) ADSCs (p<0.05), and lower rates of apoptosis, associated with the incremental expression of Bcl-2, were also observed for CGRP-ADSCs. Moreover, upon neural induction, CGRP-ADSCs formed markedly more and larger neurospheres and showed round cell bodies with more branching extensions contacted with neighboring cells widely. Furthermore, the expression levels of Nestin, MAP2, and RIP in CGRP-ADSCs were markedly increased, resulting in higher levels than the other groups (p<0.05); however, GFAP was distinctly undetectable until day 7, when slight GFAP expression was detected among all groups. Wnt signals, primarily Wnt 3a, Wnt 5a and β-catenin, regulate the neural differentiation of ADSCs, and CGRP gene expression apparently depends on canonical Wnt signals to promote the neurogenesis of ADSCs. Consequently, ADSCs genetically modified with CGRP exhibit stronger potential for differentiation and neurogenesis in vitro, potentially reflecting the usefulness of ADSCs as seed cells in therapeutic strategies for spinal cord injury.
Highlights
Spinal cord injury (SCI) is a devastating neurological injury that often results in profound functional deficits and a frequent cause of mortality worldwide [1,2,3]
Over-expression of Calcitonin gene-related peptide (CGRP) in genetically modified adipose-derived stem cells (ADSCs) The transduction efficiency for genetically modified ADSCs was evaluated according to the expression of EGFP gene using flow cytometry
Morphology and cell growth characterization of ADSCs after transduced CGRP gene The ADSCs genetically modified with CGRP exhibited bright green EGFP fluorescence
Summary
Spinal cord injury (SCI) is a devastating neurological injury that often results in profound functional deficits and a frequent cause of mortality worldwide [1,2,3]. Embryonic stem cells (ESCs) can be induced to typical ectodermal cells in phenotype, but problems of histocompatibility, inadequate tissue supply, and ethical concerns exist [11,12]. Neural stem cells (NSCs) were successfully used in neurogenesis in vitro and vivo [13,14]; this process was obviously limited for clinical use reflecting an insufficient cell population harvested from neural tissue isolated from the brain of postmortem human cortices [15]. Bone marrow stromal cells (BMSCs) can be effectively differentiated into neurons and glial cells [16,17], but bone narrow aspiration can harm patients, and problems of inadequate tissue supply are observed
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