Publicação
New Approaches on Hypoxic-Ischemic Encephalopathy: Translational research to diagnose and monitor stem cell therapy
| Resumo: | Hypoxic-ischemic encephalopathy is one of the leading causes of child death worldwide, and it is caused by an episode of perinatal asphyxia, which interrupts the blood supply to the brain. Due to its high energy demands, this interruption initiates glutamate excitotoxic pathways, leading to cell death. Most of the survivors develop various neurological diseases, such as cerebral palsy, seizures, and/or motor and behavioral problems. Presently, therapeutic hypothermia is the current standard of care for term newborns, but its efficacy is far from ideal. Umbilical cord mesenchymal stem cells (UC-MSCs) are gaining attention as a promising complement to the current clinical approach. However, obtaining minimal effective doses requires an extensive in vitro expansion, which compromises their therapeutic properties.UC-MSCs can sense and respond to biomechanical and chemical characteristics of the microenvironment. In the umbilical cord, the stiffness ranges between 2 and 5kPa, and the oxygen levels fluctuate from 2.4% to 3.8%, differing from the conventional in vitro culture conditions where MSCs are exposed to the stiffness of the Petri dish (2-3 GPa) and near atmospheric oxygen levels (18.5%O2). Therefore, it was hypothesized that expanding UC-MSCs on 3kPa platforms – mechanomodulation – or at 5%O2 levels – physioxia – could potentially impact the cellular proteome of readapted or primed UC-MSCs, for long (7-10 days) or short (48h) periods, respectively. Data analysis has unveiled that culturing MSCs on soft substrates for long periods promotes the expression of various proteins related to cell redox homeostasis. Conversely, culturing these cells during the same period but under low oxygen levels increases chaperone machinery proteins. These proteins can favor the clearance of misfolded proteins, possibly preventing MSCs from being driven to a senescent phenotype. Although mechanomodulation and physioxia are two distinct stimuli, both converge in downregulating the expression of histones and several ribosomal subunits, probably decreasing translational complexity. Interestingly, priming UC-MSCs leads to a differential expression of extracellular matrix proteins, suggesting that the secretome composition might also be altered in response to physiological cues. To explore this hypothesis, proteomic characterization of the secretome of UC-MSCs primed or readapted to soft or low oxygen levels was performed. Maintaining UC-MSCs on soft platforms for long periods increased the secretion of proteins associated with cell redox homeostasis, while physioxia enhanced the secretion of immunomodulatory proteins. The high secretion of these proteins might confer a therapeutical advantage by favoring a regenerative environment at the injury site. Interestingly, lowering the stiffness or oxygen converged on the downregulation of several extracellular matrix proteins (ECM) on primed and readapted cells. These results suggest that a massive reorganization of the extracellular space occurs upon culturing MSCs on conventional culture conditions, which may affect several signaling pathways initiated at the cell membrane, such as PDGF signaling pathways, consequently biasing stem cell fate. Since using the secretome as a cell-free therapy is raising interest and offers the advantages of being readily commercialized as an off-the-shelf product without immunogenicity compatibility issues, these data support that mimicking physiological culture conditions in vitro may empower its therapeutical properties.Therefore, to evaluate how physiological culture conditions could impact the therapeutic potential, physiologically primed UC-MSCs or their secretome were added to an in vitro HIE model using cortical neurons' primary cultures subjected to oxygen and glucose deprivation (OGD) insult. The treatment with the secretome of UC-MSC modulated under physioxic conditions sustained part of the neuronal network integrity and modulated several mitochondrial proteins. This suggests that the unique composition of the physioxia-modulated secretome may offer a therapeutical advantage in restoring essential cellular processes that help neurons maintain their function, compared to traditionally expanded UC-MSCs. Nevertheless, proteins whose levels were restored in the presence of UC-MSCs or their secretome were mainly involved in the re-establishment of the levels of proteins involved in translation mechanisms possibly stabilizing proteostasis, which is known to be essential for neuronal recovery. Understanding how transducing environmental cues might provide insights into how conventional culture conditions significantly alter fundamental cellular processes and support the development of a more efficient expansion protocol. Consequently, this might empower the therapeutic potential of UC-MSCs and their secretome and improve treatment outcomes compared to conventional culture conditions. |
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| Autores principais: | Caramelo, Inês Isabel Nunes |
| Assunto: | células estaminais mesenquimais encefalopatia hipóxico-isquémica fisióxia mecanomodelação proteómica hypoxic-ischemic encephalopathy mechanomodulation mesenchymal stem cells physioxia proteomics |
| Ano: | 2025 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Coimbra |
| Idioma: | inglês |
| Origem: | Estudo Geral - Universidade de Coimbra |
| Resumo: | Hypoxic-ischemic encephalopathy is one of the leading causes of child death worldwide, and it is caused by an episode of perinatal asphyxia, which interrupts the blood supply to the brain. Due to its high energy demands, this interruption initiates glutamate excitotoxic pathways, leading to cell death. Most of the survivors develop various neurological diseases, such as cerebral palsy, seizures, and/or motor and behavioral problems. Presently, therapeutic hypothermia is the current standard of care for term newborns, but its efficacy is far from ideal. Umbilical cord mesenchymal stem cells (UC-MSCs) are gaining attention as a promising complement to the current clinical approach. However, obtaining minimal effective doses requires an extensive in vitro expansion, which compromises their therapeutic properties.UC-MSCs can sense and respond to biomechanical and chemical characteristics of the microenvironment. In the umbilical cord, the stiffness ranges between 2 and 5kPa, and the oxygen levels fluctuate from 2.4% to 3.8%, differing from the conventional in vitro culture conditions where MSCs are exposed to the stiffness of the Petri dish (2-3 GPa) and near atmospheric oxygen levels (18.5%O2). Therefore, it was hypothesized that expanding UC-MSCs on 3kPa platforms – mechanomodulation – or at 5%O2 levels – physioxia – could potentially impact the cellular proteome of readapted or primed UC-MSCs, for long (7-10 days) or short (48h) periods, respectively. Data analysis has unveiled that culturing MSCs on soft substrates for long periods promotes the expression of various proteins related to cell redox homeostasis. Conversely, culturing these cells during the same period but under low oxygen levels increases chaperone machinery proteins. These proteins can favor the clearance of misfolded proteins, possibly preventing MSCs from being driven to a senescent phenotype. Although mechanomodulation and physioxia are two distinct stimuli, both converge in downregulating the expression of histones and several ribosomal subunits, probably decreasing translational complexity. Interestingly, priming UC-MSCs leads to a differential expression of extracellular matrix proteins, suggesting that the secretome composition might also be altered in response to physiological cues. To explore this hypothesis, proteomic characterization of the secretome of UC-MSCs primed or readapted to soft or low oxygen levels was performed. Maintaining UC-MSCs on soft platforms for long periods increased the secretion of proteins associated with cell redox homeostasis, while physioxia enhanced the secretion of immunomodulatory proteins. The high secretion of these proteins might confer a therapeutical advantage by favoring a regenerative environment at the injury site. Interestingly, lowering the stiffness or oxygen converged on the downregulation of several extracellular matrix proteins (ECM) on primed and readapted cells. These results suggest that a massive reorganization of the extracellular space occurs upon culturing MSCs on conventional culture conditions, which may affect several signaling pathways initiated at the cell membrane, such as PDGF signaling pathways, consequently biasing stem cell fate. Since using the secretome as a cell-free therapy is raising interest and offers the advantages of being readily commercialized as an off-the-shelf product without immunogenicity compatibility issues, these data support that mimicking physiological culture conditions in vitro may empower its therapeutical properties.Therefore, to evaluate how physiological culture conditions could impact the therapeutic potential, physiologically primed UC-MSCs or their secretome were added to an in vitro HIE model using cortical neurons' primary cultures subjected to oxygen and glucose deprivation (OGD) insult. The treatment with the secretome of UC-MSC modulated under physioxic conditions sustained part of the neuronal network integrity and modulated several mitochondrial proteins. This suggests that the unique composition of the physioxia-modulated secretome may offer a therapeutical advantage in restoring essential cellular processes that help neurons maintain their function, compared to traditionally expanded UC-MSCs. Nevertheless, proteins whose levels were restored in the presence of UC-MSCs or their secretome were mainly involved in the re-establishment of the levels of proteins involved in translation mechanisms possibly stabilizing proteostasis, which is known to be essential for neuronal recovery. Understanding how transducing environmental cues might provide insights into how conventional culture conditions significantly alter fundamental cellular processes and support the development of a more efficient expansion protocol. Consequently, this might empower the therapeutic potential of UC-MSCs and their secretome and improve treatment outcomes compared to conventional culture conditions. |
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