EFFECT OF HUMAN PLATELET LYSATE ON HUMAN MESENCHYMAIS STEM CELL SUPPLEMENTATION AND APPLICATION IN ACUTE NEURAL INJURY MODEL: STAB WOUND

Abstract

Traumatic brain injury(TBI) affects millions of people around the world resulting in acute or chronic motor deficiencies.At long-term, TBI causes cognitive/behavioral commitment impacting severely in the life quality. Currently, treatments for TBI are still limited, but mesenchymal stem cells(hMSCs) therapy, due to their plastic, neuroregenerative and immunoregulatory abilities are considered a promisor strategy. Objective: This study we intend to evaluate the impact of supplementation with Human Platelet Lysate(hPL) on the cultivation of hMSCs and its application in an acute neuronal traumatic experimental model Stab Wound(SW). Methodology: Bone marrow hMSCs were cultivated under two conditions: i)with 10% of Bovine Fetal Serum (FBS_hMSCs) or ii)with 10% of highly concentrated hPL(hPL_MSCs).Subsequently both cells population were characterized "in vitro" according to immunophenotype panel, markers of proliferation and oxidative stress(Ki-67 and JC-1).The neurodifferentiation of hMSCs was evaluated by immunofluorescence(β Tubulin-3) and RT-PCR(NeuN,Nestin,GFAP,Iba,NeuroG2 and NeuroD1 genes).The therapeutic potential was investigated "in vivo" using the SW model and histopathological analysis were performed(Iba and DCX).Results: Both hMSCs subsets were fibroblastoid and presented the characteristic MSCs phenotype profile(negative for CD14,CD31,CD45, and positive for CD73,CD90,CD105,CD117,CD133/2,HLA-DR),however hPL_MSCs showed a reduction in the expression of PDL-1 and PDL-2.Along the passages, the hPL_MSCs cultures showed progressively unexpected morphological changes (reduction in the number of nucleolus and cell augmentation/elongation),suggesting a pseudo-neurodifferentiation "in vitro", which was further confirmed by neuronal markers up-regulation.The FBS_hMSCs cultures remained morphologically unaltered. When compared to the FBS_hMSCs cultures, the hPL_MSCs also presented lower cellular stress with a reduced proliferative potential. We analyzed the brain damage tissues infused with both cells, the neuroprotective potential (DCX+ cells) was quite similar, however the neuronal sections treated with hPL_MSCs showed low presence of Iba+ inflammatory cells at the injury site. Conclusion: Preliminary, our results suggest that hPL_MSCs present classic neuronal characteristics “in vitro” with a neuroregenerative potential “in vivo”, and suggesting that hPL is a promisor inductor of neural-like cells being a potential tool for cellular therapy in TBI context. Traumatic brain injury(TBI) affects millions of people around the world resulting in acute or chronic motor deficiencies.At long-term, TBI causes cognitive/behavioral commitment impacting severely in the life quality. Currently, treatments for TBI are still limited, but mesenchymal stem cells(hMSCs) therapy, due to their plastic, neuroregenerative and immunoregulatory abilities are considered a promisor strategy. Objective: This study we intend to evaluate the impact of supplementation with Human Platelet Lysate(hPL) on the cultivation of hMSCs and its application in an acute neuronal traumatic experimental model Stab Wound(SW). Methodology: Bone marrow hMSCs were cultivated under two conditions: i)with 10% of Bovine Fetal Serum (FBS_hMSCs) or ii)with 10% of highly concentrated hPL(hPL_MSCs).Subsequently both cells population were characterized "in vitro" according to immunophenotype panel, markers of proliferation and oxidative stress(Ki-67 and JC-1).The neurodifferentiation of hMSCs was evaluated by immunofluorescence(β Tubulin-3) and RT-PCR(NeuN,Nestin,GFAP,Iba,NeuroG2 and NeuroD1 genes).The therapeutic potential was investigated "in vivo" using the SW model and histopathological analysis were performed(Iba and DCX).Results: Both hMSCs subsets were fibroblastoid and presented the characteristic MSCs phenotype profile(negative for CD14,CD31,CD45, and positive for CD73,CD90,CD105,CD117,CD133/2,HLA-DR),however hPL_MSCs showed a reduction in the expression of PDL-1 and PDL-2.Along the passages, the hPL_MSCs cultures showed progressively unexpected morphological changes (reduction in the number of nucleolus and cell augmentation/elongation),suggesting a pseudo-neurodifferentiation "in vitro", which was further confirmed by neuronal markers up-regulation.The FBS_hMSCs cultures remained morphologically unaltered. When compared to the FBS_hMSCs cultures, the hPL_MSCs also presented lower cellular stress with a reduced proliferative potential. We analyzed the brain damage tissues infused with both cells, the neuroprotective potential (DCX+ cells) was quite similar, however the neuronal sections treated with hPL_MSCs showed low presence of Iba+ inflammatory cells at the injury site. Conclusion: Preliminary, our results suggest that hPL_MSCs present classic neuronal characteristics “in vitro” with a neuroregenerative potential “in vivo”, and suggesting that hPL is a promisor inductor of neural-like cells being a potential tool for cellular therapy in TBI context.