Research articleThe neurotrophic potential of human platelet lysate substitution for fetal bovine serum in glial induction culture medium
Introduction
The primary role of the nervous system glial cells is to modulate neuronal cell functions. Glial cells are a heterogeneous population, including Schwann cells, that secrete neuronal growth (neurotrophic) factors, such as nerve growth factor (βNGF), glial cell-derived neurotrophic factor (GDNF), brain-derived growth factor (BDNF), and others, which can promote the survival and regeneration of neurons [1]. Another cell type, adipose derived stem cells (ASC), has also been shown to secrete neurotrophic factors including βNGF and BDNF [2]. Priming ASC by an in vitro exposure to a medium supplemented with fetal bovine serum (FBS) and a mixture of glial growth factors can promote a Schwann cell-like phenotype. This transition from ASC to a Schwann cell-like phenotype enhances the secretion of neurotrophic factors and, thereby, their neuroregenerative activity [3,4]. This neurotrophic activity of ASC suggests a therapeutic potential for neuronal regeneration.
However, these studies cannot be translated to clinical use because stimulating the secretion of neurotrophic factors of ASC relies on the addition of FBS. Developing a xeno-free culture system that stimulates the neurotrophic activity of human ASC will be beneficial. Human platelet lysate has been widely used by our institution and our group as a suitable substitute for FBS for the expansion of stromovascular (SV, the non-expanded cells derived from adipose tissue) and ASC [5,6]. The objective of this study was to modify an existing protocol for induction of Schwann cell-like differentiation of ASC [3] where FBS was replaced with human platelet lysate to assess the effect on the neurotrophic potential of ASC.
We compared the effects of FBS-containing (M-FBS) and xeno-free, platelet lysate enriched, induction media (M-PLT) on the expression of glial markers and the secretion of neurotrophic factors GDNF, BDNF, and βNGF. Cells in FBS-containing growth medium with no additional glial growth factors were used as controls (M-Ctrl). Adherent SV cells were also differentiated under the same three conditions, as SV cells have also been shown to accelerate peripheral nerve regeneration in animal models [7,8].
Section snippets
Reagents
Reagents were purchased from ThermoFisher Scientific, Inc., Waltham, MA unless otherwise noted.
Cell culture
The study utilized subcutaneous lipoaspirates obtained from three women (aged 37–75 years, body mass index 25–31.4) undergoing elective reconstructive surgeries and was approved by the Mayo Clinic Institutional Review Board. The tissue was enzymatically digested as previously described [6]. The study design of the subsequent cell cultures is shown in Fig. 1. The cells were plated overnight in
Flow cytometry
The flow cytometry for ASC using antibodies against surface markers showed that the cells passed previously published criteria for stemness (Fig. 2) [11,12].
ELISA
The secretion of GDNF was approximately 2.4-fold higher (p = 0.04) in ASC than in SV cells in all groups (Fig. 3A). Both cell types cultured in M-FBS and M-PLT had approximately 3.5-fold and 5-fold higher level of secretion than those grown in control medium, M-Ctrl, respectively. This increase was significant only for M-PLT (p = 0.02) but
Discussion
Results from this study identified an alternative to FBS for Schwann cell-like induction of ASCs for clinical translation. We demonstrated that human platelet lysate was a viable replacement for FBS, with comparable effects on the expression of glial markers and secretion of neurotrophic factors GDNF, BDNF, and βNGF in cultured SV cells and ASC. Interestingly, each factor was distinctive regarding interpersonal variability and the response to cell type and media.
GDNF is an established paracrine
Conclusion
Taken together, the results from this study suggest that adding human platelet lysate to glial induction medium potentiates the neurotrophic capability of ASC directly by the high content of BDNF in platelet lysate and indirectly by priming ASC to secrete more GDNF. Considering the secretome of ASC and of the proteins in the platelet lysate milieu offers other mechanisms of this beneficial combination. Many researchers have observed the protective effects of ASC in wound healing and tissue
CRediT authorship contribution statement
Maile Ravenkamp: Methodology, Investigation, Writing - original draft. Yourka D. Tchoukalova: Conceptualization, Methodology, Formal analysis, Writing - original draft. Cheryl E. Myers: Investigation, Formal analysis, Writing - review & editing. Cathy S. Madsen: Investigation, Writing - review & editing. Manisha K. Shah: Investigation, Visualization, Writing - review & editing. Nan Zhang: Formal analysis, Writing - review & editing. Devyani Lal: Funding acquisition, Writing - review & editing.
Declaration of Competing Interest
None.
Acknowledgments
This work was supported by the Mayo Clinic Investment for Extramural Grants Award (MEGA) Program.
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