Neuroprotective activity of a virus-safe nanofiltered human platelet lysate depleted of extracellular vesicles in Parkinson's disease and traumatic brain injury models

Abstract

Brain administration of human platelet lysates (HPL) is a potential emerging biotherapy of neurodegenerative and traumatic diseases of the central nervous system. HPLs being prepared from pooled platelet concentrates, thereby increasing viral risks, manufacturing processes should incorporate robust virus-reduction treatments. We evaluated a 19 ± 2-nm virus removal nanofiltration process using hydrophilic regenerated cellulose hollow fibers on the properties of a neuroprotective heat-treated HPL (HPPL). Spiking experiments demonstrated >5.30 log removal of 20-22-nm non-enveloped minute virus of mice-mock particles using an immuno-quantitative polymerase chain reaction assay. The nanofiltered HPPL (NHPPL) contained a range of neurotrophic factors like HPPL. There was >2 log removal of extracellular vesicles (EVs), associated with decreased expression of pro-thrombogenic phosphatidylserine and procoagulant activity. LC-MS/MS proteomics showed that ca. 80% of HPPL proteins, including neurotrophins, cytokines, and antioxidants, were still found in NHPPL, whereas proteins associated with some infections and cancer-associated pathways, pro-coagulation and EVs, were removed. NHPPL maintained intact neuroprotective activity in Lund human mesencephalic dopaminergic neuron model of Parkinson's disease (PD), stimulated the differentiation of SH-SY5Y neuronal cells and showed preserved anti-inflammatory function upon intranasal administration in a mouse model of traumatic brain injury (TBI). Therefore, nanofiltration of HPL is feasible, lowers the viral, prothrombotic and procoagulant risks, and preserves the neuroprotective and anti-inflammatory properties in neuronal pre-clinical models of PD and TBI.

Keywords: human platelet lysate; nanofiltration; neuroprotection; prion; virus.

FIGURE 1

Overall experimental design.

FIGURE 2

Platelet extracellular vesicle (PEV) content of heat‐treated platelet pellet lysate (HPPL), after 0.2–0.1‐μm filtration (HPPL‐0.1 μm), and of nanofiltered HPPL (NHPPL). (a) PEV size distribution analysis by dynamic light scattering. The red, green, and blue curves represent the first, second, and third analyses, respectively. (b) Phosphatidylserine‐expressing PEVs. Results are expressed as the means ± SD (n = 3). (**p < 0.01, ****p < 0.0001), compared to HPPL. Statistical evaluation was performed by a one‐way ANOVA followed by Fisher's least significant difference test.

FIGURE 3

(a) Venn diagram of the proteins identified in heat‐treated platelet pellet lysate (HPPL), 0.2–0.1‐μm‐filtered HPPL (HPPL 0.2–0.1 μm), and nanofiltered HPPL (NHPPL) (HPPL‐P20). (b) Gene ontology enrichment analysis of 813 proteins in common among HPPL, HPPL 0.2–0.1 μm, and HPPL P‐20: biological processes (BPs), molecular functions (MFs), and cellular components (CCs). (c) Gene ontology enrichment analysis of proteins that were removed from HPPL by 0.2–0.1‐μm filtration and (d) removed from HPPL 0.2–0.1‐μm filtration by Planova 20N: BPs, MFs, and CCs. The significance cutoff was p < 0.1.

FIGURE 4

Kyoto encyclopedia of genes and genomes pathways of 158 proteins that were removed by 0.2–0.1‐μm filters and pathways of 152 removed by Planova‐20N filtration. The significance cutoff was p < 0.1.

FIGURE 5

Assessment of the neuroprotective activity. LUHMES cells were pretreated with 5% (v/v) heat‐treated platelet pellet lysate (HPPL), 0.2–0.1‐μm‐filtered HPPL (HPPL‐0.1 μm), or nanofiltered HPPL (NHPPL) and after 1 h were exposed to the erastin neurotoxin. (a) Cell morphology after 24 h. The scale bar is 100 μm. (b) LUHMES cell viability after 24 h was quantified by a CCK‐8 assay. n = 3, ****p < 0.0001, compared to cells treated with erastin only. Statistical evaluation was performed by a one‐way ANOVA followed by Fisher's least significant difference test.

FIGURE 6

Capacity to stimulate SH‐SY5Y cell neuronal maturation. Cells were immuno‐stained with β‐III tubulin and counterstained with DAPI. Images (a) showing extension of SH‐SY5Y neurites under treatment with nanofiltered heat‐treated platelet pellet lysate (HPPL; NHPPL). HPPL, and retinoic acid which was used as a positive control to stimulate cell differentiation. The scale bar is 100 μm. (b) Quantitative measurement of the fluorescence intensity using ImageJ software. *p < 0.05; **p < 0.01. Statistical evaluation was performed by a one‐way ANOVA followed by Fisher's least significant difference test.

FIGURE 7

Modulation of neuroinflammatory markers in a mouse model of traumatic brain injury. A controlled cortical impact (CCI) was applied, and mice received either 60 μl heated platelet pellet lysate (HPPL), nanofiltered HPPL (NHPPL), or PBS, on three consecutive days by intranasal administration. Mice were sacrificed on day 7 post‐injury, the ipsilateral cortex was dissected out, and cytokine and glial marker mRNA levels were quantified by an RT‐qPCR. (n = 5–7 mice per group). Data are reported as the mean ± SEM.; *p < 0.05; **p < 0.01; ***p < 0.001 for CCI vs. Sham; ^# p < 0.05; ^## p < 0.01 for CCI‐PBS vs. CCI‐HPPL or CCI‐NHPPL by a one‐way ANOVA followed by Fisher's least significant difference test.