Neonatal Hypoxic-Ischemic Encephalopathy: Stem Cell Therapy and Identification of Biomarkers for Early Diagnosis, Prognosis, and Prediction of Treatment Success

Publications

From Hypoxia to Healing: Optimizing Stem Cell Therapy for Neonatal Hypoxic-Ischemic Brain Injury

Publication . Serrenho, Inês Isabel Pires; Baltazar, Graça Maria Fernandes; Manadas, Bruno José Fernandes Oliveira; Grãos, Mário Martins Rodrigues

Neonatal hypoxic-ischemic encephalopathy (HIE), caused by oxygen and blood flow deprivation to the neonatal brain, is a leading cause of mortality and long-term neurological disabilities in children under five. Its complex pathophysiology— comprising excitotoxicity, inflammation, oxidative stress, and delayed cell death—poses significant therapeutic challenges. Moreover, current management with therapeutic hypothermia (TH) shows limited efficacy, particularly in severe cases, and excludes many neonates due to strict enrollment criteria. To overcome these limitations, preclinical research has explored stem cell therapies (SCT), particularly using stem cells from umbilical cord blood (UCB) and tissue (UC). These therapies show promise in reducing brain injury and improving outcomes, but challenges remain in optimizing doses, delivery methods, and scalability. Building on previous work, this thesis evaluated several approaches to optimize SCT for neonatal HIE. First, administration of UCBCs alone and in combination with TH in a rat model of HIE, reduced brain lesion size and improved functional outcomes more effectively than TH alone, while also reducing glial reactivity—an effect not observed with TH alone. These findings establish UCBCs as a potential therapy, particularly when TH is unavailable or insufficient. Nonetheless, the use of these cells can present disadvantages: autologous use, limited culture and expansion, thus not being possible to manipulate with preconditioning strategies. Thus, we focused on the use of UC-derived MSCs that overcome some of the limitations previously mentioned. Also, since preconditioning strategies are being explored as ways to potentiate MSCs therapeutic effects, we hypothesized that hypoxic preconditioning could enhance UC-MSC efficacy and what explored the potential mechanisms behind neurological recovery. Proteomic analysis revealed that HIE rats treated with hypoxiapreconditioned MSCs had an enrichment of pathways related to synapse function, brain connectivity, and energy metabolism. Moreover, administration of UC-MSCs preconditioned with short hypoxia induced a greater functional recovery in HIE rats than administration of UC-MSCs preconditioned mild hypoxia. Stem cell therapy requires high doses of stem cells, which limits its clinical translatability. To optimize SCT and significantly reduce effective doses of MSCs, we evaluated the impact of two delivery methods and the combination of different strategies on the efficacy of UC-MSCs. Intranasal administration of UC-MSCs was more effective in reducing the infarct volume enhancing motor and cognitive recovery than intravenous administration, restoring myelination in the corpus callosum, and mitigating glial reactivity. Intranasal administration of half the dose of hypoxia-preconditioned UCMSCs reduced neurological deficits associated with neonatal HIE. Strikingly, intranasal administration of secretome from hypoxia-preconditioned MSCs demonstrated similar therapeutic efficacy, offering a promising cell-free alternative. Finally, to address the limitations associated with the expansion of UC-MSCs and donor variability on the therapeutic potency of these cells, we aimed to determine if induced pluripotent stem cell-derived MSCs (iMSCs) could hold the same potential in the neonatal HIE model. Intranasal administration of iMSCs or their secretome improved motor and cognitive recovery, reduced brain lesion size, modulated glial reactivity, and enhanced neurogenesis in the hippocampus of HIE rats. The comparable outcomes between iMSCs and their secretome underscore the critical role of secreted factors, presenting a scalable, cell-free option for clinical application. In summary, this thesis advances the field of regenerative medicine for neonatal HIE by identifying innovative strategies to optimize stem cell therapies. The findings demonstrate the potential of intranasal delivery and hypoxic preconditioning to reduce the required cell doses while maintaining efficacy, making these therapies more feasible for clinical use. Furthermore, secretome-based approaches offer a scalable alternative that addresses logistical challenges. Beyond neonatal HIE, these strategies may have broader implications for other neurodevelopmental and neurological disorders, as well as for veterinary medicine. While significant progress has been made, future research should aim to elucidate the mechanisms underlying MSC and secretome efficacy and advance these approaches toward clinical trials, paving the way for innovations in regenerative medicine.

2025-12-02Doctoral thesis

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