Efeito do lisado de plaquetas humanas na suplementação de células mesenquimais estromais multipotentes humanas e aplicação em modelo de lesão aguda: stab wound: STAB WOUND
Data
2023-03-27
Autores
Silva, Karina Ribeiro da 
Orientadores
Almeida, Danilo Cândido de
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O traumatismo cranioencefálico (TCE) afeta milhões de pessoas em todo o
mundo, resultando em deficiências motoras agudas/crônicas, de caráter cognitivo
e comportamental, impactando severamente a qualidade de vida. Atualmente, os
tratamentos para o TCE ainda são bastante limitados, restringindo-se ao controle
da pressão intracraniana e a otimização da perfusão cerebral, o que previne o
edema, a inflamação e a morte celular. A terapia com células-tronco estromais
mesenquimais multipotentes (hMSCs), devido a suas habilidades neuroplásticas,
neuroregenerativas e imunoregulatórias, é considerada uma estratégia alternativa,
inovadora e promissora quando associada á outras tecnologias. Desse modo,
este presente estudo avaliou o impacto da suplementação com Lisado de
Plaquetas Humanas (hPL) no cultivo da hMSCs e sua aplicação em um modelo
experimental de traumatismo craniano agudo denominado de Stab Wound (SW).
As hMSCs de medula óssea foram cultivadas em duas condições: i) com 10% de
soro fetal bovino (FBS_hMSCs) ou ii) com 10% de hPL altamente concentrado
(hPL_MSCs). Posteriormente, ambas as populações celulares foram
caracterizadas "in vitro" de acordo com o seu painel imunofenotipico; capacidade
de diferenciação em linhagens mesenquimais e por marcadores de proliferação e
de estresse oxidativo (Ki-67 e JC-1). A neurodiferenciação das hMSCs foi
avaliada por imunofluorescência (β Tubulina-III) e por qPCR (NeuN, Nestina,
GFAP, Iba, Neuro G2 e Neuro D1). O efeito do hPL no cultivo de precursores
neuronais também foi analisado. O potencial terapêutico "in vivo" foi investigado
utilizando o modelo SW e foram realizadas análises histopatológicas (Iba, GFAP e
DCX) e de expressão genica para vias de inflamação, stresse oxidativo e função
neuronal. Ambos os subconjuntos de hMSCs apresentaram uma morfologia
fibroblastóide com um perfil fenotípico característico de hMSCs, ou seja, negativo
para CD14, CD31, CD45 e positivo para CD73, CD90, CD105, CD117, CD133, e
HLA-DR. Ambas as hMSCs apresentaram o potencial multipotente de
diferenciação, porém, esse foi menor nas células cultivadas com hPL. Ao longo
das passagens, nas culturas de hPL_MSCs foram identificadas alterações
morfológicas expressivas e progressivas, sugerindo uma neurodiferenciação
parcial "in vitro", que foi posteriormente confirmada pela regulação positiva de
marcadores neuronais. As culturas de FBS_hMSCs permaneceram
morfologicamente inalteradas durante o cultivo. Quando comparadas às culturas
de FBS_hMSCs, as hPL_MSCs apresentaram uma tendencia de apresentar
menor estresse mitocondrial celular com um potencial proliferativo reduzido,
caracterizando um perfil mais progenitor. Funcionalmente, o hPL promoveu “in
vitro” de neuroesferas fenômenos de aderência, migração e possível
diferenciação celular somente na presença de fatores de crescimento, o que não
foi observado em culturas controles. Adicionalmente, analisamos os tecidos com
lesão cerebral infundidos com ambas as hMSCs, e verificamos que o potencial
neuroreparador (células DCX+) foi maior nos cortes neuronais tratados com
hPL_MSCs, os quais também apresentaram baixa presença de células
inflamatórias (Iba+ e GFAP+) no local da lesão. Observamos nos córtex dos
animais infundidos com hPL_MSCs menor expressão de citocromo C e GFAP em
comparação aos animais que receberam FBS_hMSCs. Por outro lado, no
hipocampo dos animais que receberam hPL_MSCs, foi relatada baixa expressão
de IL-4 e de marcadores de função/plasticidade neural (AMPA-1, NMDA-1, Cap-
23, Gap-43, PSD-95) em comparação aos animais inoculados com FBS_hMSCs.
Por fim, quantificamos o fator de crescimento neuronal BDNF e verificamos sua
presença elevada nos sobrenadantes de culturas de hPL_MSCs e no soro de
animais transplantados com hPL_MSCs. Como conclusão os nossos resultados
sugerem que hPL_MSCs apresentam características neuronais clássicas "in vitro"
com potencial neuroprotetor "in vivo", sugerindo que hPL é um indutor promissor
de células neuronais “like” com plasticidade limitada, sendo uma ferramenta
alternativa e potencial para terapia celular explorativa no contexto do TCE.
Acreditamos que este estudo demonstrou o potencial papel “neuroregulador” do
hPL, e também gerou resultados fundamentais para o entendimento da
neuroplasticidade das hMSCs e sua aplicabilidade na medicina regenerativa.
Traumatic brain injury (TBI) affects millions of people worldwide, resulting in acute/chronic motor, cognitive, and behavioral impairments with severe impact on life-quality. Currently, treatments for TBI are still quite limited, restricting to intracranial pressure control and optimization of cerebral perfusion, which prevents edema, inflammation, and cell death. Multipotent mesenchymal stromal cell (hMSCs) therapy, due to its neuroplastic, neuroregenerative, and immunoregulatory abilities, is considered an alternative, innovative, and promising strategy when associated with other technologies. Thus, this study evaluated the impact of human Platelet Lysate (hPL) supplementation on hMSCs culture and its application in an experimental model of acute traumatic brain injury designed as Stab Wound (SW). Bone marrow hMSCs were cultured under two conditions: i) with 10% fetal bovine serum (FBS_hMSCs) or ii) with highly concentrated 10% hPL (hPL_MSCs). Subsequently, both cell populations were characterized "in vitro" according to their immunophenotypic panel; mesenchymal lineage differentiation capacity and by proliferation and oxidative stress markers (Ki-67 and JC-1). The hMSCs' neurodifferentiation was evaluated by immunofluorescence (β-Tubulin-III) and qPCR (i.e. NeuN, Nestin, GFAP, Iba, Neuro G2, and Neuro D1). The effect of hPL on neuronal precursor culture was also analyzed. The therapeutic potential "in vivo" was investigated using the SW model, and histopathological analyses (Iba and DCX) and gene expression for inflammation, oxidative stress, and neuronal function pathways. Both subsets of hMSCs presented a fibroblastoid morphology with a phenotypic profile characteristic of hMSCs, i.e., negative for CD14, CD31, CD45, and positive for CD73, CD90, CD105, CD117, CD133, and HLA-DR. Both hMSCs showed multipotent differentiation potential, but it was lower in cells cultured with hPL. Throughout the passages, significant and progressive morphological changes were identified in the hPL_MSCs cultures, suggesting partial "in vitro" neurodifferentiation, which was later confirmed by the positive regulation of neuronal markers. The FBS_hMSCs cultures remained morphologically inalterated during culture. When compared to FBS_hMSCs cultures, hPL_MSCs also showed xiv a tendency to has a lower cellular mitochondrial stress with a reduced proliferative potential, characterizing a more progenitor profile. Functionally, hPL promoted adhesion, migration, and possible cellular differentiation phenomena in neurosphere cultures in vitro only in the presence of growth factors, which were not observed in control cultures. Additionally, we analyzed brain tissues infused with both hMSCs, and we found that the neurorepair potential (DCX+ cells) was higher in the neuronal sections treated with hPL_MSCs, which also showed a low presence of inflammatory cells (Iba+ and GFAP+) at the injury site. We observed lower expression of cytochrome C and GFAP in the cortex of animals infused with hPL_MSCs compared to animals that received FBS_hMSCs. On the other hand, in the hippocampus of animals receiving hPL_MSCs, there was reported low expression of IL-4 and neural function/plasticity markers (AMPA-1, NMDA-1, Cap23, Gap-43, PSD-95) compared to animals inoculated with FBS_hMSCs. Finally, we quantified the neuronal growth factor BDNF and found its elevated presence in hPL_MSC culture supernatants and in serum from animals transplanted with hPL_MSC. In summary, our results suggest that hPL_MSCs exhibit classic neuronal characteristics "in vitro" with in vivo neuroprotective potential, indicating that hPL is a promising inducer of neuron-like cells with limited plasticity, serving as an alternative and potential tool for exploratory cell therapy in the context of traumatic brain injury. We believe that this study demonstrated the "neuroregulatory" potential of hPL, and also generated fundamental findings for understanding the neuroplasticity of hMSCs and their applicability in regenerative medicine.
Traumatic brain injury (TBI) affects millions of people worldwide, resulting in acute/chronic motor, cognitive, and behavioral impairments with severe impact on life-quality. Currently, treatments for TBI are still quite limited, restricting to intracranial pressure control and optimization of cerebral perfusion, which prevents edema, inflammation, and cell death. Multipotent mesenchymal stromal cell (hMSCs) therapy, due to its neuroplastic, neuroregenerative, and immunoregulatory abilities, is considered an alternative, innovative, and promising strategy when associated with other technologies. Thus, this study evaluated the impact of human Platelet Lysate (hPL) supplementation on hMSCs culture and its application in an experimental model of acute traumatic brain injury designed as Stab Wound (SW). Bone marrow hMSCs were cultured under two conditions: i) with 10% fetal bovine serum (FBS_hMSCs) or ii) with highly concentrated 10% hPL (hPL_MSCs). Subsequently, both cell populations were characterized "in vitro" according to their immunophenotypic panel; mesenchymal lineage differentiation capacity and by proliferation and oxidative stress markers (Ki-67 and JC-1). The hMSCs' neurodifferentiation was evaluated by immunofluorescence (β-Tubulin-III) and qPCR (i.e. NeuN, Nestin, GFAP, Iba, Neuro G2, and Neuro D1). The effect of hPL on neuronal precursor culture was also analyzed. The therapeutic potential "in vivo" was investigated using the SW model, and histopathological analyses (Iba and DCX) and gene expression for inflammation, oxidative stress, and neuronal function pathways. Both subsets of hMSCs presented a fibroblastoid morphology with a phenotypic profile characteristic of hMSCs, i.e., negative for CD14, CD31, CD45, and positive for CD73, CD90, CD105, CD117, CD133, and HLA-DR. Both hMSCs showed multipotent differentiation potential, but it was lower in cells cultured with hPL. Throughout the passages, significant and progressive morphological changes were identified in the hPL_MSCs cultures, suggesting partial "in vitro" neurodifferentiation, which was later confirmed by the positive regulation of neuronal markers. The FBS_hMSCs cultures remained morphologically inalterated during culture. When compared to FBS_hMSCs cultures, hPL_MSCs also showed xiv a tendency to has a lower cellular mitochondrial stress with a reduced proliferative potential, characterizing a more progenitor profile. Functionally, hPL promoted adhesion, migration, and possible cellular differentiation phenomena in neurosphere cultures in vitro only in the presence of growth factors, which were not observed in control cultures. Additionally, we analyzed brain tissues infused with both hMSCs, and we found that the neurorepair potential (DCX+ cells) was higher in the neuronal sections treated with hPL_MSCs, which also showed a low presence of inflammatory cells (Iba+ and GFAP+) at the injury site. We observed lower expression of cytochrome C and GFAP in the cortex of animals infused with hPL_MSCs compared to animals that received FBS_hMSCs. On the other hand, in the hippocampus of animals receiving hPL_MSCs, there was reported low expression of IL-4 and neural function/plasticity markers (AMPA-1, NMDA-1, Cap23, Gap-43, PSD-95) compared to animals inoculated with FBS_hMSCs. Finally, we quantified the neuronal growth factor BDNF and found its elevated presence in hPL_MSC culture supernatants and in serum from animals transplanted with hPL_MSC. In summary, our results suggest that hPL_MSCs exhibit classic neuronal characteristics "in vitro" with in vivo neuroprotective potential, indicating that hPL is a promising inducer of neuron-like cells with limited plasticity, serving as an alternative and potential tool for exploratory cell therapy in the context of traumatic brain injury. We believe that this study demonstrated the "neuroregulatory" potential of hPL, and also generated fundamental findings for understanding the neuroplasticity of hMSCs and their applicability in regenerative medicine.