The neuroprotective activity of heat-treated human platelet lysate biomaterials manufactured from outdated pathogen-reduced (amotosalen/UVA) platelet concentrates

Abstract

BackgroundEffective neurorestorative therapies of neurodegenerative diseases must be developed. There is increasing interest in using human platelet lysates, rich in neurotrophic factors, as novel disease-modifying strategy of neurodegeneration. To ensure virus safety, pathogen reduction treatments should be incorporated in the preparation process of the platelet concentrates used as source material. We therefore investigated whether platelet concentrates (PC) pathogen-inactivated using a licensed photo-inactivation treatment combining photosensitive psoralen (amotosalen) and UVA irradiation (Intercept) can serve as source material to prepare platelet lysates with preserved neuroprotective activity in Parkinson’s disease models.MethodsIntercept treated-PCs were centrifuged, when reaching expiry day (7 days after collection), to remove plasma and platelet additive solution. The platelet pellet was re-suspended and concentrated in phosphate buffer saline, subjected to 3 freeze-thaw cycles (− 80 °C/37 °C) then centrifuged to remove cell debris. The supernatant was recovered and further purified, or not, by heat-treatment as in our previous investigations. The content in proteins and neurotrophic factors was determined and the toxicity and neuroprotective activity of the platelet lysates towards LUHMES cells or primary cortical/hippocampal neurons were assessed using ELISA, flow cytometry, cell viability and cytotoxicity assays and proteins analysis by Western blot.ResultsPlatelet lysates contained the expected level of total proteins (ca. 7–14 mg/mL) and neurotrophic factors. Virally inactivated and heat-treated platelet lysates did not exert detectable toxic effects on neither Lund human mesencephalic dopaminergic LUHMES cell line nor primary neurons. When used at doses of 5 and 0.5%, they enhanced the expression of tyrosine hydroxylase and neuron-specific enolase in LUHMES cells and did not significantly impact synaptic protein expression in primary neurons, respectively. Furthermore, virally-inactivated platelet lysates tested were found to exert very strong neuroprotection effects on both LUHMES and primary neurons exposed to erastin, an inducer of ferroptosis cell death.ConclusionOutdated Intercept pathogen-reduced platelet concentrates can be used to prepare safe and highly neuroprotective human heat-treated platelet pellet lysates. These data open reassuring perspectives in the possibility to develop an effective biotherapy using virally-inactivated platelet lysates rich in functional neurotrophins for neuroregenerative medicine, and for further bio-industrial development. However, the data should be confirmed in animal models.Graphical abstract

Human platelet lysate biotherapy for traumatic brain injury: preclinical assessment

“…There was no detectable sign of toxicity due to HPPL treatment following HPPL stimulation, as we also demonstrated previously. 15 , 23 As shown qualitatively in Supplementary Fig. 2B , neurites in the scratched area were significantly longer in the HPPL treated condition compared to untreated cells.…”

“…This is in line with previous characterization of the HPPL by ELISA that established its high contents of neurotrophic, angiogenic, and anti-inflammatory growth factors such as PDGF, BDNF, β-FGF, VEGF, and TGF-β1. 14 , 15 , 22 , 23 Here, we also provide a more extensive characterization of HPPL using proteomics. As revealed by the GO term analysis, bioactive proteins in the HPPL are involved in several biological processes including vascular regeneration, wound healing, metabolic processes, immune response, vesicle-mediated transport, protein transport, etc.…”

“… 53 The human platelet proteome, which is rich in a range of neurotrophic factors, may also potentially promote neurogenesis by differentiation of the neurogenic niche, thereby stimulating neurogenesis in the dentate gyrus of the hippocampus. 52 The human platelet proteome in cellular models of amyotrophic lateral sclerosis and Parkinson’s disease protected against apoptosis and ferroptosis cell death through Akt and mitogen-activated protein kinase (MEK) signalling, 14 , 22 , 23 enhanced expressions of tyrosine hydroxylase (TH) and neuron-specific enolase (NSE) in LUHMES cells, 23 was strongly neuroprotective of primary neurons exposed to elastin, 23 and decreased microglial inflammation after lipopolysaccharide stimulation and induction of neuronal cell differentiation. 15 The observation that HPPL administration resulted in biochemical and behavioural improvements in two distinct animal models suggests a broad therapeutic utility for both mild and intermediate TBIs.…”

Ferroptosis: A potential therapeutic target for neurodegenerative diseases

“…[ 164 ] In LUHMES cells and primary neurons, the virally inactivated platelet lysates strongly prevented erastin‐induced ferroptosis (Figure 3). [ 165 ]…”

Ferroptosis in Neurological Diseases

“…The FSP1-CoQ 10 -NAD(P)H pathway exists as an independent parallel system and can be used in conjunction with GPX4 and glutathione to inhibit phospholipid peroxidation and ferroptosis ( Bersuker et al, 2019 ; Doll et al, 2019 ). Virus-inactivated and heat-treated human platelet lysate has a strong neuroprotective effect against erastin-induced ferroptosis ( Nebie et al, 2019 ) and has been developed as a novel and effective neurodegenerative therapy. Human platelet lysates can inhibit ferroptosis and reduce neuronal loss in cellular models of PD and amyotrophic lateral sclerosis (ALS) ( Gouel et al, 2017 ).…”