Biology subjects

Caramelo, I.

Publications and source records attributed to Caramelo, I..

5 recordsLinked to original sources

bioRxiv2025

Quieting the Storm: Hypoxia as a Strategy to Boost UC-MSC Therapies for Neonatal Hypoxic-Ischemic Encephalopathy

Integrating stem cell therapies into clinical settings faces several challenges, particularly in achieving the high cell yields necessary for attaining therapeutic doses. Preconditioning with hypoxic conditions has shown promise in enhancing the UC-MSCs reparative capabilities of the central nervous system. Recent evidence suggests that oxygen concentration and exposure duration can shape MSCs phenotypes, supporting the need for further optimization of this strategy in a way to achieve maximal repair. In this study, we assessed the effects of both prolonged mild hypoxia (MH; 5% oxygen for 48 hours) and short severe hypoxia (SSH; 0.1% oxygen for 24 hours) on UC-MSCs ability to alleviate motor and cognitive deficits in a rodent model of neonatal HIE. Our results show that short, severe hypoxia led to more improvements in functional recovery than prolonged mild hypoxia, supporting that specific preconditioning parameters are crucial in maximizing UC-MSC therapeutic potential. To investigate the molecular effects of hypoxia-preconditioned MSCs in the neonatal brain post-HIE, we employed untargeted proteomics on ipsilesional brain samples from control, HIE, HIE treated with naive UC-MSCs, and HIE treated with SSH-preconditioned UC-MSCs groups, 30 days after lesion induction. This approach identified protein signatures related to injury and therapeutic intervention. Pathway enrichment analysis further revealed that administration of UC-MSCs preconditioned with short severe hypoxia significantly impacted neural signaling, protein synthesis, and energy metabolism pathways, pointing to long-term mechanisms that may support neuronal repair. These findings enhance our understanding of hypoxia-preconditioning in MSCs therapy in driving a positive therapeutic response, supporting the development of more effective and feasible treatments for neonatal HIE.

neuroscience↗

bioRxiv2025

Nose-to-Brain Healing: Hypoxia-Preconditioned Mesenchymal Stem Cells Prompt Recovery in Hypoxic-Ischemic Encephalopathy Rats

Neonatal HIE poses a significant risk factor for neurodevelopment impairment. Therapeutic hypothermia, the current standard of care for this condition, has several constraints and reduced effectivity, especially in more severe cases. Thus, it is necessary to explore novel therapeutics, like MSCs. Although previous studies report that administration of MSCs (from different sources) prompted the recovery of HIE-lesioned animals, high doses are currently used. First, this study compared the efficacy of IN versus IV administration of 50,000 UC-MSCs in a rat model of neonatal HI brain injury. For this cell dose, only IN-UC-MSC therapy reduced infarct volume, an effect accompanied by improvements of motor skills and recognition memory. Also, IN-UC-MSC administration restored myelination in the corpus callosum and mitigated glial reactivity more effectively than IV administration. In a second part of the study, to potentiate the effect of UC-MSCs administration, postnatal rats that underwent HI injury received 25,000 hypoxia-preconditioned UC-MSCs or its secretome two days later, via IN route. The administration of a low-dose of hypoxia-preconditioned UC-MSCs was sufficient to induce neurological recovery and modulation of glial response. Moreover, the administration of the secretome of these cells was enough to induce the same extent of recovery. These findings support the higher potential of IN-UC-MSC administration, compared to IV administration, while enhancing our understanding of hypoxia-preconditioning and the role of the MSCs secretome in driving a positive therapeutic response, contributing to the development of more effective and feasible treatments for neonatal HIE.

neuroscience↗

bioRxiv2024

Physioxia-modulated mesenchymal stem cells secretome has higher capacity to preserve neuronal network and translation processes in hypoxic-ischemic encephalopathy in vitro model

Hypoxic-ischemic encephalopathy (HIE) is one of the leading causes of child death worldwide. Most of the survivors develop various neurological diseases, such as cerebral palsy, seizures, and/or motor and behavioral problems. HIE 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. Umbilical cord mesenchymal stem cells (UC-MSCs) are gaining attention as a promising complement to the current clinical approach, based on therapeutic hypothermia, which has shown limited efficacy. Previous data have shown that priming MSCs under physiological culture conditions, namely soft platforms (3kPa) - mechanomodulated - or physiological oxygen levels (5% O2) - physioxia - leads to changes in the cellular proteome and their secretome. To evaluate how exposing MSCs to these culture conditions could impact their 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. By comparing the neuronal proteome of sham, OGD insulted, and OGD-treated neurons, it was possible to identify proteins whose levels were restored in the presence of UC-MSCs or their secretome. Despite the different approaches that differentially altered UC-MSCs proteome and secretome, the effects converged on the re-establishment of the levels of proteins involved in translation mechanisms (such as the 40S and 60s ribosomal subunits), possibly stabilizing proteostasis, which is known to be essential for neuronal recovery. Interestingly, treatment with the secretome of UC-MSC modulated under physioxic conditions sustained part of the neuronal network integrity and modulated several mitochondrial proteins, including those proteins involved in ATP production. 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. These findings suggest that both the presence of UC-MSCs and their secretome alone can influence multiple targets and signaling pathways, collectively promoting neuronal survival following an OGD insult.

neuroscience↗

bioRxiv2024

Readaptation of mesenchymal stem cells to high stiffness and oxygen environments modulate the extracellular matrix

The therapeutic potential of mesenchymal stem cells (MSCs) has been explored over the past decades due to their ability to modulate the microenvironment through paracrine signaling. Consequently, the secretome of MCSs has emerged as a cell-free therapy rather than a cell therapy, offering the advantages of being readily commercialized as an off-the-shelf product without immunogenicity compatibility issues. As a result, strategies to manipulate and enhance the secretory profile of MSCs secretome are emerging. MSCs from the Whartons jelly niche are accommodated to the stiffness and oxygen level found at the umbilical cord (UC), which are 2 to 5kPa (Youngs modulus) and 2.4% to 3.8% O2, respectively. However in vitro culture conditions (2-3 GPa and 18.5% O2) are largely different from the one observed in vivo. Here, we present a proteomic characterization of the secretome of MSCs primed (48h) or readapted (7-10 days) to soft (3kPa) (mechanomodulated) or low oxygen levels (5% O2) (physioxia). Maintaining MSCs on soft platforms for long periods increased the secretion of proteins associated with cell redox homeostasis, such as protein disulfide isomerases and mitochondrial proteins, 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), particularly collagen fibrils, 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 not only matrix stiffness but also several signaling pathways initiated at the cell membrane, such as PDGF signaling pathways (e.g., PI3K-AKT), consequently biasing stem cell fate. In conclusion, mimicking physiological culture conditions in vitro modulates secretome composition, which may empower its therapeutical properties by enriching proteins that promote cell survival. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/609692v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@16350corg.highwire.dtl.DTLVardef@1f4c18borg.highwire.dtl.DTLVardef@1ce9c0aorg.highwire.dtl.DTLVardef@1666d0b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Mimicking physiological stiffness or oxygen levels in vitro reorganizes mesenchymal stem cells machinery toward a more naive phenotype

Mesenchymal stem cells (MSCs) offer a promising therapeutic potential for a wide variety of pathologies. However, obtaining minimal effective doses requires an extensive in vitro expansion, which compromises their stemness and therapeutic properties. The stiffness of the umbilical cord ranges between 2 and 5kPa, and the oxygen levels fluctuate from 2.4% to 3.8%, differing from the standard 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). Since MSCs can sense and respond to biomechanical and chemical characteristics of the microenvironment, it was hypothesized that expanding MSCs on 3kPa platforms - mechanomodulation - or at 5% O2 levels - physioxia - could potentially impact the cellular proteome of MSCs, for long (7-10 days) or short (48h) periods. Data analysis has unveiled that culturing MSCs on soft substrates for long periods promotes the expression of various proteins related to cell redox homeostasis, such as thioredoxins and peroxiredoxins. Conversely, culturing these cells during the same period but under low oxygen levels leads to an increase in chaperone machinery proteins, such as HSP90 or TRiC. These proteins can favor the clearance of misfolded proteins and telomerase maintenance processes, 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, possibly decreasing translational complexity, which could hypothetically favor a more naive phenotype for MSCs. Interestingly, priming UC-MSCs (48h) leads to a differential expression of proteins of the extracellular matrix and histone subtypes. Understanding the role of these proteins in transducing environmental cues might provide insights into how conventional culture conditions significantlyalter fundamental cellular processes and support the development of a more efficient protocol to expand and empower the therapeutic potential of MSCs. In the future, employing a combination of reduced stiffness and lower oxygen levels may present a promising strategic approach. HighlightsO_LICulturing MSCs on a soft substrate (3kPa) enhances the expression of antioxidant proteins, such as thioredoxins and peroxiredoxins C_LIO_LIProtein homeostasis is remodeled in MSCs cultured under physiological levels of oxygen (5% O2) through the differential expression of the chaperone machinery C_LIO_LILowering stiffness or oxygen levels during in vitro MSCs expansion decreases histones and ribosomal subunits expression, possibly favoring a more naive phenotype C_LI

cell biology↗