Human Platelet Lysate Acts Synergistically With Laminin to Improve the Neurotrophic Effect of Human Adipose-Derived Stem Cells on Primary Neurons in vitro

Abstract

BackgroundDespite the advancements in microsurgical techniques and noteworthy research in the last decade, peripheral nerve lesions have still weak functional outcomes in current clinical practice. However, cell transplantation of human adipose-derived stem cells (hADSC) in a bioengineered conduit has shown promising results in animal studies. Human platelet lysate (hPL) has been adopted to avoid fetal bovine serum (FBS) in consideration of the biosafety concerns inherent with the use of animal-derived products in tissue processing and cell culture steps for translational purposes. In this work, we investigate how the interplay between hPL-expanded hADSC (hADSChPL) and extracellular matrix (ECM) proteins influences key elements of nerve regeneration.MethodshADSC were seeded on different ECM coatings (laminin, LN; fibronectin, FN) in hPL (or FBS)-supplemented medium and co-cultured with primary dorsal root ganglion (DRG) to establish the intrinsic effects of cell–ECM contact on neural outgrowth. Co-cultures were performed “direct,” where neural cells were seeded in contact with hADSC expanded on ECM-coated substrates (contact effect), or “indirect,” where DRG was treated with their conditioned medium (secretome effect). Brain-derived nerve factor (BDNF) levels were quantified. Tissue culture plastic (TCPS) was used as the control substrate in all the experiments.ResultshPL as supplement alone did not promote higher neurite elongation than FBS when combined with DRG on ECM substrates. However, in the presence of hADSC, hPL could dramatically enhance the stem cell effect with increased DRG neurite outgrowth when compared with FBS conditions, regardless of the ECM coating (in both indirect and direct co-cultures). The role of ECM substrates in influencing neurite outgrowth was less evident in the FBS conditions, while it was significantly amplified in the presence of hPL, showing better neural elongation in LN conditions when compared with FN and TCPS. Concerning hADSC growth factor secretion, ELISA showed significantly higher concentrations of BDNF when cells were expanded in hPL compared with FBS-added medium, without significant differences between cells cultured on the different ECM substrates.ConclusionThe data suggest how hADSC grown on LN and supplemented with hPL could be active and prone to support neuron–matrix interactions. hPL enhanced hADSC effects by increasing both proliferation and neurotrophic properties, including BDNF release.

Human platelet lysate stimulates neurotrophic properties of human adipose-derived stem cells better than Schwann cell-like cells

“…Indeed, we detected a higher number of DRG survived until the end of the assay and a longer total neurite length in the presence of HPL-ASCs compared to SC-ASCs, suggesting that culturing ASCs with HPL might be synergistic and more beneficial rather than differentiate them in SC-ASCs. Although HPL presents a high concentration of trophic molecules, it did not sustain nerve regeneration on its own, as demonstrated also by a previous published paper [ 5 ]. With the support also of our results, we can assert that HPL exerts an indirect effect on DRG enhancing ASCs neuroregenerative properties.…”

“…Therefore, an innovative therapy respecting the human-translatable regulations is needed and, in this context, ASCs are emerging as a promising strategy to promote tissue regeneration [ 12 – 15 ]. According to the GMP guidelines, the use of FBS for ASCs culture should be abandoned in favor of xeno-free alternatives, such as HPL that presents less safety and ethical limitations [ 5 , 29 ].…”

“…The neuronal progenitors undergo a series of fine cytoskeletal-driven morphological changes during differentiation processes (i.e., neuritogenesis, axonogenesis and dendritogenesis) and the cytoskeletal reorganization itself is controlled by the interaction with the ECM, which in turn modulates proliferation, self-renewal and differentiation of stem cells, regulating the shape of axons and dendrites and neurite extension [ 52 – 55 ]. Among ECM proteins increased in HPL-ASCs, fibronectin is particularly interesting since it promotes neurite outgrowth and axonal regeneration, improving neurotrophic capabilities of ASCs cultured in the presence of HPL [ 5 , 55 – 57 ]. Moreover, ALDH1L2 and ALDH have a role as ROS scavenger and are considered as markers for stem cells.…”

“…The current gold standard therapy in case of short gaps (< 2–3 mm) is an end-to-end anastomosis or an autologous nerve graft for longer gaps. However, these treatments often cause unsatisfactory clinical outcomes and poor recovery, mainly due to distal chronic denervation and fibrosis at the distal stump [ 5 , 6 ].…”

Tissue Engineering Strategies for Peripheral Nerve Regeneration

“…Recent studies have shown that human platelet lysates have neurotrophic properties (such as the release of BDNF). The synergistic effect with laminin can enhance the neurotrophic effect of ASCs on the primary neurons in vitro ( 75 ). The acquisition site of ASCs (the best subcutaneous and perennial) will also affect the differentiation results ( 76 ).…”