Differentiation Capacity Research Articles

HMGB1 Accelerates Wound Healing by Promoting the Differentiation of Epidermal Stem Cells via the "HMGB1-TLR4-Wnt/Notch" Axis.

Objective: Impairments in the differentiation and migratory capacity of epidermal stem cells (ESCs) are pivotal factors contributing to delayed wound healing. High mobility group box1 (HMGB1) has recently emerged as a potential target for tissue repair. Therefore, we aimed to investigate the role and molecular mechanisms of HMGB1 in ESCs during the wound-healing process. Approach: Initially, we examined the expression of HMGB1 and the differentiation of ESCs in normal skin, normal wounds and chronic wounds. Then, we assessed the ESC migration and differentiation, and the key markers in the Wnt/Notch signaling pathways, after treatment of HMGB1 and inhibitor, and the knockdown of toll-like receptor 4 (TLR4), using scratch assay, qPCR, western blotting, and immunofluorescence. Finally, we conducted mice models to analyze the healing rates and quality invivo. Results: HMGB1 was decreased across all epidermal layers, and the differentiation of ESCs was hindered in diabetic foot ulcer. In vitro, HMGB1 enhanced both the migration and differentiation of ESCs while stimulating the expression of the Wnt/Notch pathway within ESCs. However, the downregulation of TLR4 negated these effects. Finally, our invivo experiments provided evidence that HMGB1 facilitates wound healing and epidermis differentiation via TLR4 and Wnt/Notch signaling pathways. Innovation: This study innovatively introduces HMGB1 as a novel target for skin wound healing and elucidates its mechanisms of action. Conclusions: HMGB1 accelerated wound healing by promoting the differentiation of epidermal stem cells through the "HMGB1-TLR4-Wnt/Notch" axis, which reveals a new potential mechanism and target to expedite wound healing.

Platelet-derived extracellular vesicles induced through different activation pathways drive melanoma progression by functional and transcriptional changes

BackgroundBeyond their conventional roles in hemostasis and wound healing, platelets have been shown to facilitate hematogenous metastasis by interacting with cancer cells. Depending on the activation route, platelets also generate different platelet-derived extracellular vesicles (PEVs) that may educate cancer cells in the circulation or within the tumor microenvironment. We engaged different platelet-activating receptors, including glycoprotein VI and C-type lectin-like receptor 2, to generate a spectrum of PEV types. This allowed us to investigate the differential capacity of PEVs to alter cancer hallmark functions such as proliferation, invasion, and pro-angiogenic potential using melanoma as a model. Additionally, we analyzed changes in the cell transcriptomes and cancer EV profiles.MethodsTwo human melanoma cell lines (MV3 and A2058) with differential metastatic potential were studied in the 3D spheroid cultures. Human platelets were activated with collagen related peptide (CRP), fucoidan from Fucus vesiculosus (FFV), thrombin & collagen co-stimulus and Ca2+ ionophore, and PEVs were isolated by size-exclusion chromatography followed by ultrafiltration. Spheroids or cells were treated with PEVs and used in functional assays of proliferation, invasion, and endothelial tube formation as well as for the analysis of cancer EV production and their tetraspanin profiles. Differentially expressed genes and enriched signaling pathways in the PEV-treated spheroids were analyzed at 6 h and 24 h by RNA sequencing.ResultsAmong the studied PEVs, those generated by CRP and FFV exhibited the most pronounced effects on altering cancer hallmark functions. Specifically, CRP and FFV PEVs increased proliferation in both MV3 and A2058 spheroids. Distinct tetraspanin signatures of melanoma EVs were induced by all PEV types. While the PI3K-Akt and MAPK signaling pathways were activated by both CRP and FFV PEVs, they differently upregulated the immunomodulatory TGF-β and type-I interferon signaling pathways, respectively.ConclusionsOur study revealed both shared and distinct, cancer-promoting functions of PEVs, which contributed to the transcriptome and metastatic capabilities of the melanoma spheroids. Inhibiting the platelet receptors that modulate the PEVs’ cancer-promoting properties may open up new strategies for identifying promising treatment targets for cancer therapy.

Notch Pathway Deactivation Sensitizes Breast Cancer Stem Cells toward Chemotherapy Using NIR Light-Responsive Nanoparticles.

Chemotherapy remains a major therapeutic approach to cancer treatment. However, its effectiveness can be compromised by the heterogeneity of a solid tumor, in which different cancer cell populations display varied responses to chemotherapy. Such an intratumor heterogeneous structure is maintained by the cancer stem-like cells (CSCs) with inherent capacities for self-renewal and differentiation, giving rise to diverse cell populations. To address this, we proposed a combinational strategy in which tumor lesion-targeted Notch signaling regulation was achieved to disrupt CSC-mediated cancer heterogeneity, thereby sensitizing solid tumors toward paclitaxel (PTX). Specifically, gamma-secretase inhibitor LY-411,575 was co-delivered with PTX using a near-infrared (NIR) light-controlled drug delivery system to realize targeted ablation of both differentiated cancer cells and undifferentiated CSCs. By enabling precise regulation of the Notch pathway at the tumor site through NIR light, we observed significantly elevated efficacy of chemotherapy and notable prevention of postsurgical tumor relapse while minimizing systemic side effects. The devised strategy shows promise in addressing the nonspecific inhibition of stemness across various organs, a challenge that hampers the clinical translation of gamma-secretase inhibitors in cancer therapy.

Temporary Nanoencapsulation of Human Intestinal Organoids Using Silk Ionomers.

Human intestinal organoids (HIOs) are vital for modeling intestinal development, disease, and therapeutic tissue regeneration. However, their susceptibility to stress, immunological attack, and environmental fluctuations limits their utility in research and therapeutic applications. This study evaluated the effectiveness of temporary silk protein-based layer-by-layer (LbL) nanoencapsulation technique to enhance the viability and functions of HIOs against common biomedical stressors, without compromising their native functions. Cell viability and differentiation capacity are assessed, finding that nanoencapsulation significantly improved HIO survival under the various environmental perturbations studied without compromising cellular functionality. Post-stress exposures, the encapsulated HIOs still successfully differentiated into essential intestinal cell types such as enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Moreover, the silk nanocoatings effectively protected against environmental stressors such as ultraviolet (UV) light exposure, protease degradation, antibody binding, and cytokine-induced inflammation. This nanoencapsulation technique shows promise for advancing HIO applications in disease modeling, drug testing, and potential transplantation therapies.

Exploration of the growth pattern and stemness maintenance of bovine muscle stem cells in vitro culture

Cultured meat technology has rapidly advanced in recent years, offering promising prospects. However, a major challenge in industrializing this process is the procurement and expansion of seed cells, which frequently lose their proliferative potential during in vitro culture. In this study, high-purity bovine muscle stem cells were isolated using fluorescence-activated cell sorting, achieving a pax7 positivity rate of 90%. These cells demonstrated standard proliferation and differentiation capabilities in vitro. However, long-term culture led to decreased proliferation and differentiation capacities, as well as an accumulation of reactive oxygen species. To restore cellular function, we investigated the effects of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a powerful antioxidant. Treatment with 50 μM Trolox effectively reversed the decline in bovine muscle stem cells proliferation, decreased reactive oxygen species levels, and preserved cell stemness during extended culture. These results provide a foundation for muscle seed cells acquisition and preservation, improving their viability and functionality in vitro.

Differential coping capacities underlie the overall resistance of temperate seagrasses to herbivory

AbstractGrazing can impart long‐lasting changes in vegetated ecosystems. How ecosystems respond to herbivory depends on the ecological and evolutionary histories of their foundational species. The overall ecosystem functioning and associated biodiversity depend on these responses but there is still little understanding on how the intensity and duration of herbivory interact and impact vegetated ecosystems. We experimentally tested in the field the responses of three seagrass species with distinct life history traits to increasing intensities of herbivory over time. Specifically, we assessed structural responses (i.e., canopy height and shoot density) to reflect the ecosystem state. Additionally, we used mechanistic models to assess induced and constitutive responses in the different seagrass species. Results show that seagrasses coped with herbivory differentially in relation to their life history traits. Posidonia oceanica (persistent species) was resistant and only registered declines in canopy height, whereas both canopy heigh and shoot density rapidly decreased for Cymodocea nodosa (intermediate‐colonizing species) and Zostera noltei (colonizing species). Seagrasses also differed in the type of structural response, with the colonizing species experiencing reductions in shoot density, and the persistent P. oceanica registering declines in canopy height. After months of exposure to cumulative herbivory, all three species showed signs of stability. Interestingly, none of the species disappeared completely even when exposed to extreme herbivory. Mechanistic models indicate that herbivory‐induced responses are a potential explanation for these patterns. This study suggests that given the long evolutionary history of herbivory, some seagrasses may be remarkably well adapted to both intense and cumulative herbivory.

Gold Kiwi-Derived Nanovesicles Mitigate Ultraviolet-Induced Photoaging and Enhance Osteogenic Differentiation in Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BM-MSCs) play a crucial role in bone formation through their ability to differentiate into osteoblasts. Aging, however, detrimentally affects the differentiation and proliferation capacities of BM-MSCs, consequently impairing bone regeneration. Thus, mitigating the aging effects on BM-MSCs is vital for addressing bone-related pathologies. In this study, we demonstrate that extracellular nanovesicles isolated from gold kiwi (GK-NVs) protect human BM-MSCs from ultraviolet (UV)-induced photoaging, thereby alleviating aging-related impairments in cellular functions that are crucial for bone homeostasis. Notably, GK-NVs were efficiently taken up by BM-MSCs without causing cytotoxicity. GK-NVs reduced intracellular reactive oxygen species (ROS) levels upon UV irradiation, restoring impaired proliferation and migration capabilities. Furthermore, GK-NVs corrected the skewed differentiation capacities of UV-irradiated BM-MSCs by enhancing osteoblast differentiation, as evidenced by the increased expression in osteoblast-specific genes and the calcium deposition, and by reducing adipocyte differentiation, as indicated by the decreased lipid droplet formation. These findings position GK-NVs as a promising biomaterial for the treatment of bone-related diseases such as osteoporosis.

Molecular Mechanisms of Curdlan-Induced Suppression of NFATc1 Expression in Osteoclasts.

Osteoclasts derived from hematopoietic stem cells express immunoreceptors on their cell surface. Previously, we showed that the β-glucan curdlan suppressed osteoclastogenesis via binding to dectin-1, a pattern recognition receptor. Curdlan negatively regulates osteoclast differentiation and bone resorption capacity by suppressing the expression of nuclear factor of activated T cells 1 (NFATc1), a master factor for osteoclast differentiation, in a dectin-1-dependent manner; however, the mechanism involved in this process has not yet been fully elucidated. In this study, we aimed to elucidate the molecular mechanism involved in the suppression of RANKL-induced osteoclast differentiation by curdlan. Real-time RT-qPCR results showed that curdlan suppressed the expression of NFATc1 in cells of the osteoclast progenitor cell line RAW264.7 overexpressing dectin-1 (d-RAW cells), without altering the expression of negative regulators. Therefore, we examined the effect of curdlan on the NF-κB pathway, which is important for the induction of NFATc1 expression. Western blot analysis results showed that curdlan addition suppressed RANKL-induced NF-κB activation in the vector control line (c-RAW) cells with low expression of dectin-1, in d-RAW cells, and the parental RAW264.7 (RAW) cells. The results of tartrate-resistant alkaline phosphatase staining and real-time RT-qPCR showed that curdlan addition suppressed osteoclast differentiation in RAW cells, suggesting the presence of a dectin-1-independent modification system. Finally, we focused on the complement receptor 3 (CR3), which binds β-glucan, and revealed that blocking the binding of β-glucan to the CD11b molecule, a component of CR3, by neutralizing antibody, recovered the suppression of IκBα degradation by curdlan. These results suggest that the suppression of osteoclast differentiation by curdlan involves not only the dectin-1-dependent pathway but also the negative regulation of NFATc1 via modification of the NF-κB pathway via CR3 recognition. The results of this study may aid to establish treatment methods for metabolic bone diseases and inflammatory bone destruction and to clarify their pathogenesis.

Mesenchymal stem/stromal cells: dedicator to maintain tumor homeostasis.

Mesenchymal stem/stromal cells （MSCs） act as a factor in tumor recurrence after drug treatment with their involvement observed in various cancer types. As a constituent of the tumor microenvironment (TME), MSCs not only provide support to tumor growth but also establish connections with diverse cell populations within the TME, serving as mediators linking different tumor-associated components. MSCs play an important role in maintaining tumor progression due to their stem cell properties and remarkable differentiation capacity. Given the intensification of tumor research and the encouraging results achieved in recent years，the aim of this article is to investigate the supportive role of MSCs in tumor cells as well as in various cellular and non-cellular components of the tumor microenvironment. Furthermore, the article shows that MSCs do not have a specific anatomical ecological niche and describes the contribution of MSCs to the maintenance of tumor homeostasis on the basis of homing, plasticity and tumor-forming properties. By elucidating the critical roles of different components of TME, this study provides a comprehensive understanding of tumor therapy and may offer new insights into defeating cancer.

Establishment of immortalized rabbit bone marrow mesenchymal stem cells and a preliminary study of their osteogenic differentiation capability.

A stable and standardized source of mesenchymal stem cells is a prerequisite for bone repair tissue engineering research and application. We aimed to establish a stable cell line of bone marrow mesenchymal stem cells from New Zealand rabbits and explore their osteogenic differentiation capacity. Primary rabbit bone marrow mesenchymal stem cells (RBMSCs) were isolated and immortalized via retroviral expression of SV40 Large T antigen (LTA). To assess the osteogenic differentiation capacity of the cells invitro, we studied the alkaline phosphatase (ALP) expression level and calcium deposition in bone morphogenetic protein 9 (BMP9)-induced immortalized cells using ALP staining and quantification, as well as alizarin red staining. Ectopic bone formation by the cells was assessed using micro-computed tomography (μCT) and histological examination. The immortalized cell line we established using SV40 LTA, which we termed iRBMSCs, was non-tumorigenic and maintained long-term proliferative activity. We further discovered that BMP9 (MOI = 30) effectively induced the osteogenic differentiation capacity of iRBMSCs invitro, and there was a synergy with GelMA hydrogel in inducing osteogenic differentiation of the iRBMSCs invivo. We confirmed that iRBMSCs are promising as a stable cell line source for bone defect repair engineering.

Age-dependent regenerative mechanisms in the brain.

Repairing the adult mammalian brain represents one of the greatest clinical challenges in medicine. Injury to the adult brain often results in substantial loss of neural tissue and permanent functional impairment. In contrast with the adult, during development, the mammalian brain exhibits a remarkable capacity to replace lost cells. A plethora of cell-intrinsic and extrinsic factors regulate the age-dependent loss of regenerative potential in the brain. As the developmental window closes, neural stem cells undergo epigenetic changes, limiting their proliferation and differentiation capacities, whereas, changes in the brain microenvironment pose additional challenges opposing regeneration, including inflammation and gliosis. Therefore, studying the regenerative mechanisms during development and identifying what impairs them with age may provide key insights into how to stimulate regeneration in the brain. Here, we will discuss how the mammalian brain engages regenerative mechanisms upon injury or neuron loss. Moreover, we will describe the age-dependent changes that affect these processes. We will conclude by discussing potential therapeutic approaches to overcome the age-dependent regenerative decline and stimulate regeneration.

Comparison of dU/dQ, Voltage Decay, and Float Currents via Temperature Ramps and Steps in Li‐ion Batteries

Comparison of dU/dQ, Voltage Decay, and Float Currents via Temperature Ramps and Steps in Li‐ion Batteries

Gain of 1q confers an MDM4-driven growth advantage to undifferentiated and differentiating hESC while altering their differentiation capacity

Gain of 1q is a highly recurrent chromosomal abnormality in human pluripotent stem cells. In this work, we show that gains of 1q impact the differentiation capacity to derivates of the three germ layers, leading to mis-specification to cranial placode and non-neural ectoderm during neuroectoderm differentiation. Also, we found a weaker expression of lineage-specific markers in hepatoblasts and cardiac progenitors. Competition assays show that the cells retain their selective advantage during differentiation, which is mediated by a higher expression of MDM4, a gene located in the common region of gain. MDM4 drives the winner phenotype of the mutant cells in both the undifferentiated and differentiating state by reducing the cells’ sensitivity to DNA damage through decreased p53-mediated apoptosis. Finally, we found that cell density in culture plays a key role in promoting the competitive advantage of the cells by increasing DNA damage.

Deep-supercooling preservation of stem cell spheroids for chondral defect repairment

Versatile mesenchymal stem cells (MSCs) play an important role in tissue engineering and regenerative medicine. MSCs in 3D spheroid have shown higher secretion and differentiation functions than suspended counterparts, and, thus, invitro cryopreservation of MSC spheroids is an indispensable technology to bridge the spatiotemporal gaps between spheroid generation and application. Traditional cryopreservation methods are inapplicable for spheroid due to severe thermal stress, toxic cryoprotectants, and ice formation. Here, we constructed and preserved human MSC (hMSC) spheroids via deep supercooling (DSC). Spheroids were DSC preserved at -12°C without ice formation for 7days, with higher cell viability, energy level, and chondrogenic differentiation capacity than suspended hMSCs. hMSCs embedded in spheroids have close cell-cell interactions via N-cadherin to activate the AKT-cytochrome c-caspase anti-apoptotic cascade during DSC preservation. Finally, preserved hMSC spheroids were capable of chondrogenic differentiation and can be co-delivered with collagen to treat rat cartilage defects invivo.

Looking into failure mode identification driven by differential capacity in Ni-rich layered cathodes

Nickel-rich layered cathodes are one of the ideal electrode materials for high-energy lithium-ion batteries, yet suffer from capacity decay and structural degradation during cycling. Although the degradation mechanisms of electrode materials are flourishing, the analysis of performance decay and physicochemical properties dynamic evolution during cycling have not been well developed. Here, we propose a coupling analysis strategy based on differential capacity that distinguishes the failure behavior of electrode materials during cycling by the characteristic evolution of the dQ dV–1 curve recorded cycle-by-cycle. By coupling in-situ electrochemical tests with differential capacity characterization and comparing them with electrochemical characteristics recorded at different aging upper cut-off voltages cycles, the capacity decay mechanism and physicochemical properties evolution of electrode materials can be dynamically analyzed. The potential failure modes include loss of active Li inventory (LALI), loss of active structure integrity (LASI), and various dominant combinations of these factors. In addition, the distinction of aging behavior can also be applied to the failure level classification of spent electrode materials. Our findings demonstrate a general strategy for analyzing the dynamic failure mechanisms of electrode materials, thereby offering valuable insights for subsequent technology route selection in terms of recycling and reuse.

NSUN2 methylates IRF4 to affect the capacity of macrophages attached to titanium implant on osteogenic differentiation of PDLSCs and angiogenesis of HUVECs in vitro

BackgroundWe aimed to investigate the effect and underlying mechanism of titanium (Ti) implant on the polarization of macrophages and subsequent effects on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and angiogenesis of human umbilical vein endothelial cells (HUVECs) affected by macrophages.MethodsFirstly, the regulatory effect of Ti implant on macrophage polarization was investigated. Levels of M1 polarization markers and M2 polarization markers in macrophages were evaluated by immunofluorescence staining method and qPCR analysis. The osteogenic differentiation capacity of PDLSCs cultured with supernatants of macrophages of each group was evaluated by the Alizarin Red S (ARS) staining method and qPCR. Angiogenesis related genes were also evaluated in HUVECs cultured in supernatants of macrophages. To explore whether RNA m5C modification can modulate the effects of Ti implant on macrophage regulation of osteogenic differentiation and angiogenesis, we analyzed the main genes related to m5C in macrophages using RNA m5C dot blotting and qPCR methods. The interaction between NSUN2 and IRF4 was verified by m5C-RIP, RIP, and double-luciferase gene report experiments.ResultsMacrophages were activated as M1 macrophages under the interference of LPS, and macrophages attached to Ti implants were more easily activated as M2 macrophages under the action of LPS. Macrophages activated by Ti implant enhanced osteogenic differentiation of PDLSCs and angiogenesis of HUVECs. NSUN2 level was up-regulated in macrophages treated with LPS and was down-regulated by Ti implant. Over-expression of NSUN2 attenuated the effect of Ti implant on M1 polarization promotion of macrophages and enhanced the M2 polarization promotion of macrophages. Up-regulation of NSUN2 weakened the effects of Ti implant on promotion the capacity of macrophages on osteogenic differentiation of PDLSCs and angiogenesis of HUVECs. The KEGG analysis suggested that IRF4 was enriched in several inflammatory signaling pathways. Moreover, NSUN2 methylates IRF4 to affect the capacity of macrophages on osteogenic differentiation of PDLSCs and angiogenesis of HUVECs.ConclusionsTaken together, macrophages of M1 type can be stimulated by Ti implants in vitro, promoting osteogenic differentiation of PDLSCs and angiogenesis in HUVECs through NSUN2-mediated methylation of IRF4.

Improvement of osteogenic differentiation potential of placenta-derived mesenchymal stem cells by metformin via AMPK pathway activation

BackgroundPlacenta-derived human mesenchymal stem cells (PL-MSCs) have gained a lot of attention in the field of regenerative medicine due to their availability and bone-forming capacity. However, the osteogenic differentiation capacity of these cells remains inconsistent and could be improved to achieve greater efficiency. Although metformin, a widely used oral hypoglycemic agent, has been shown to increase bone formation in various cell types, its effect on osteogenic differentiation of PL-MSCs has not yet been investigated. Therefore, the objective of this study was to examine the effect of metformin on the osteogenic differentiation capacity of PL-MSCs and the underlying mechanisms.MethodsThe PL-MSCs were treated with 0.5 to 640 µM metformin and their osteogenic differentiation capacity was examined by an alkaline phosphatase (ALP) activity assay, Alizarin red S staining and expression levels of osteogenic genes. The role of adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling in mediating the effect of metformin on the osteogenic differentiation capacity of PL-MSCs was also investigated by determining levels of phosphorylated AMPK (pAMPK)/AMPK ratio and by using compound C, an AMPK inhibitor.ResultsThe results showed that 10–160 µM metformin significantly increased the viability of PL-MSCs in a dose- and time-dependent manner. Furthermore, 80–320 µM metformin also increased ALP activity, matrix mineralization, and expression levels of osteogenic genes, runt-related transcription factor 2 (RUNX2), osterix (OSX), osteocalcin (OCN) and collagen I (COL1), in PL-MSCs. Metformin increases osteogenic differentiation of PL-MSCs, at least in part, through the AMPK signaling pathway, since the administration of compound C inhibited its enhancing effects on ALP activity, matrix mineralization, and osteogenic gene expression of PL-MSCs.ConclusionsThis study demonstrated that metformin at concentrations of 80–320 μM significantly enhanced osteogenic differentiation of PL-MSCs in a dose- and time-dependent manner, primarily through activation of the AMPK signaling pathway. This finding suggests that metformin could be used with other conventional drugs to induce bone regeneration in various bone diseases. Additionally, this study provides valuable insights for future osteoporosis treatment by highlighting the potential of modulating the AMPK pathway to improve bone regeneration.

Antineoplastic therapy affects the in vitro phenotype and functionality of healthy human bone marrow-derived mesenchymal stromal cells.

While antineoplastic therapies aim to specifically target cancer cells, they may also exert adverse effects on healthy tissues, like healthy hematopoietic stem and progenitor cells (HSPC), leading to hematotoxicity as a common side effect. Mesenchymal stromal cells (MSC) are a major component of the bone marrow (BM) microenvironment, regulating normal hematopoiesis, while their susceptibility to anticancer therapies and contribution to therapy-related hematotoxicity remains largely unexplored. To address this, we investigated the effects of etoposide, temozolomide, 5-azacitidine, and venetoclax on healthy BM-derived MSC functionality. Doses below therapeutic effects of etoposide (0.1-0.25µM) inhibited cellular growth and induced cellular senescence in healthy MSC, accompanied by an increased mRNA expression of CDKN1A, decreased trilineage differentiation capacity, and insufficient hematopoietic support. Pharmacological doses of 5-azacitidine (2.5µM) shifted MSC differentiation capacity by inhibiting osteogenic capacity but enhancing the chondrogenic lineage, as demonstrated by histochemical staining and on mRNA level. At the highest clinically relevant dose, neither venetoclax (40nM) nor temozolomide (100µM) exerted any effects on MSC but clearly inhibited cellular growth of cancer cell lines and primary healthy HSPC, pointing to damage to hematopoietic cells as a major driver of hematotoxicity of these two compounds. Our findings show that besides HSPC, also MSC are sensitive to certain antineoplastic agents, resulting in molecular and functional alterations that may contribute to therapy-related myelosuppression. Understanding these interactions could be helpful for the development of strategies to preserve BM MSC functionality during different kinds of anticancer therapies.

EXTH-50. THYROID HORMONE-INDUCED DIFFERENTIATION: A PROMISING THERAPEUTIC STRATEGY FOR MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children, consisting of four major subgroups (WNT, SHH, Group 3, and Group 4) that differ in genetic/epigenetic profiles and prognoses. Despite current therapy, a significant proportion of patients still succumb to MB. Moreover, survivors often suffer severe side effects from aggressive treatments. Therefore, improved strategies to treat MB are urgently needed. MB tumor cells retain the capacity for differentiation and can undergo terminal differentiation, after which they lose their proliferative and tumorigenic capacity. This suggests that MB can potentially be treated by inducing tumor cell differentiation. However, the mechanisms underlying MB tumor cell differentiation remain unclear. Our recent studies reveal that thyroid hormone (TH) plays a critical role in promoting tumor cell differentiation in MB. Reduced TH levels free the TH receptor, TRα1, to bind to EZH2 and repress the expression of NeuroD1, a transcription factor that drives tumor cell differentiation. Increased TH levels reverse the EZH2-mediated repression of NeuroD1 by disrupting the binding between EZH2 and TRα1, thereby stimulating tumor cell differentiation and reducing MB growth. Importantly, triiodothyronine (T3, active form of TH) inhibits the growth of both SHH- and Group 3 tumors, suggesting that the tumor-inhibitory effect of T3 is not restricted by MB subgroups. Additionally, this effect is significantly enhanced by concurrent chemotherapy. These findings establish an unprecedented association between TH signaling and MB pathogenicity, providing solid evidence for TH as a promising therapeutic modality for MB treatment.

Identifying and optimizing critical process parameters for large-scale manufacturing of iPSC derived insulin-producing β-cells

BackgroundType 1 diabetes, an autoimmune disorder leading to the destruction of pancreatic β-cells, requires lifelong insulin therapy. Islet transplantation offers a promising solution but faces challenges such as limited availability and the need for immunosuppression. Induced pluripotent stem cells (iPSCs) provide a potential alternative source of functional β-cells and have the capability for large-scale production. However, current differentiation protocols, predominantly conducted in hybrid or 2D settings, lack scalability and optimal conditions for suspension culture.MethodsWe examined a range of bioreactor scaleup process parameters and quality target product profiles that might affect the differentiation process. This investigation was conducted using an optimized High Dimensional Design of Experiments (HD-DoE) protocol designed for scalability and implemented in 0.5L (PBS-0.5 Mini) vertical wheel bioreactors.ResultsA three stage suspension manufacturing process is developed, transitioning from adherent to suspension culture, with TB2 media supporting iPSC growth during scaling. Stage-wise optimization approaches and extended differentiation times are used to enhance marker expression and maturation of iPSC-derived islet-like clusters. Continuous bioreactor runs were used to study nutrient and growth limitations and impact on differentiation. The continuous bioreactors were compared to a Control media change bioreactor showing metabolic shifts and a more β-cell-like differentiation profile. Cryopreserved aggregates harvested from the runs were recovered and showed maintenance of viability and insulin secretion capacity post-recovery, indicating their potential for storage and future transplantation therapies.ConclusionThis study demonstrated that stage time increase and limited media replenishing with lactate accumulation can increase the differentiation capacity of insulin producing cells cultured in a large-scale suspension environment.