Murine Mesenchymal Stem Cells Research Articles

Murine bone-derived mesenchymal stem cells undergo molecular changes after a single passage in culture

The rarity of the mesenchymal stem cell (MSC) population poses a significant challenge for MSC research. Therefore, these cells are often expanded in vitro, prior to use. However, long-term culture has been shown to alter primary MSC properties. Additionally, early passage primary MSCs in culture are often assumed to represent the primary MSC population in situ, however, little research has been done to support this. Here, we compared the transcriptomic profiles of murine MSCs freshly isolated from the bone marrow to those that had been expanded in culture for 10 days. We identified that a single passage in culture extensively altered MSC molecular signatures associated with cell cycling, differentiation and immune response. These findings indicate the critical importance of the MSC source, highlighting the need for optimization of culture conditions to minimize the impact on MSC biology and a transition towards in vivo methodologies for the study of MSC function.

Cooling‐promoted myogenic differentiation of murine bone marrow mesenchymal stem cells through TRPM8 activation in vitro

AbstractTRPM8 agonist has been reported to promote osteogenic differentiation of mesenchymal stem cells (MSCs), therefore we evaluated whether cooling‐induced activation of TRPM8 promotes myogenic differentiation of MSCs. We used 5‐azacytidine as a myogenic differentiation inducer in murine bone marrow‐derived MSCs. Addition of menthol, a TRPM8 agonist, to the differentiation induction medium significantly, increased the percentage of MyoD‐positive cells, a specific marker of myogenic differentiation. We performed intracellular Ca2+ imaging experiments using fura‐2 to confirm TRPM8 activation by cooling stimulation. The results confirmed that intracellular Ca2+ concentration ([Ca2+]i) increases due to TRPM8 activation, and TRPM8 antagonist inhibits increase in [Ca2+]i at medium temperatures below 19°C. We also examined the effect of cooling exposure time on myogenic differentiation of MSCs using an external cooling stimulus set at 17°C. The results showed that 60 min of cooling had an acceleratory effect on differentiation (2.18 ± 0.27 times). We observed that the TRPM8 antagonist counteracted the differentiation‐promoting effect of the cooling. These results suggest that TRPM8 might modulate the multiple differentiation pathways of MSCs, and that cooling is an effective way of activating TRPM8, which regulates MSCs differentiation in vitro.

Hydrothermal Transformation of Eggshell Calcium Carbonate into Apatite Micro-Nanoparticles: Cytocompatibility and Osteoinductive Properties.

The eggshell is a biomineral consisting of CaCO3 in the form of calcite phase and a pervading organic matrix (1-3.5 wt.%). Transforming eggshell calcite particles into calcium phosphate (apatite) micro-nanoparticles opens the door to repurposing the eggshell waste as materials with potential biomedical applications, fulfilling the principles of the circular economy. Previous methods to obtain these particles consisted mainly of two steps, the first one involving the calcination of the eggshell. In this research, direct transformation by a one-pot hydrothermal method ranging from 100-200 °C was studied, using suspensions with a stoichiometric P/CaCO3 ratio, K2HPO4 as P reagent, and eggshells particles (Ø < 50 μm) both untreated and treated with NaClO to remove surface organic matter. In the untreated group, the complete conversion was achieved at 160 °C, and most particles displayed a hexagonal plate morphology, eventually with a central hole. In the treated group, this replacement occurred at 180 °C, yielding granular (spherulitic) apatite nanoparticles. The eggshell particles and apatite micro-nanoparticles were cytocompatible when incubated with MG-63 human osteosarcoma cells and m17.ASC murine mesenchymal stem cells and promoted the osteogenic differentiation of m17.ASC cells. The study results are useful for designing and fabricating biocompatible microstructured materials with osteoinductive properties for applications in bone tissue engineering and dentistry.

Sumecton reinforced gelatin-based scaffolds for cell-free bone regeneration

Bone tissue engineering has risen to tackle the challenges of the current clinical need concerning bone fractures that is already considered a healthcare system problem. Scaffold systems for the repair of this tissue have yielded different combinations including biomaterials with nanotechnology or biological agents. Herein, three-dimensional porous hydrogels were engineered based on gelatin as a natural biomaterial and reinforced with synthetic saponite nanoclays. Scaffolds were biocompatible and shown to enhance the inherent properties of pristine ones, in particular, proved to withstand pressures similar to load-bearing tissues. Studies with murine mesenchymal stem cells found that scaffolds had the potential to proliferate and promote cell differentiation. In vivo experiments were conducted to gain insight about the ability of these cell-free scaffolds to regenerate bone, as well as to determine the role that these nanoparticles in the scaffold could play as a drug delivery system. SDF-1 loaded scaffolds showed the highest percentage of bone formation, which was corroborated by osteogenic markers and new blood vessels. Albeit a first attempt in the field of synthetic nanosilicates, these results suggest that the designed constructs may serve as delivery platforms for biomimetic agents to mend bony defects, circumventing high doses of therapeutics and cell-loading systems.

Modulation of Cell Response through the Covalent Binding of Fibronectin to Titanium Substrates.

Titanium (Ti-6Al-4V) substrates were functionalized through the covalent binding of fibronectin, and the effect of the existence of this extracellular matrix protein on the surface of the material was assessed by employing mesenchymal stem cell (MSC) cultures. The functionalization process comprised the usage of the activation vapor silanization (AVS) technique to deposit a thin film with a high surface density of amine groups on the material, followed by the covalent binding of fibronectin to the amine groups using the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) crosslinking chemistry. The biological effect of the fibronectin on murine MSCs was assessed in vitro. It was found that functionalized samples not only showed enhanced initial cell adhesion compared with bare titanium, but also a three-fold increase in the cell area, reaching values comparable to those found on the polystyrene controls. These results provide compelling evidence of the potential to modulate the response of the organism to an implant through the covalent binding of extracellular matrix proteins on the prosthesis.

Optimal Preparation Protocol of Cell-Encapsulating Alginate Capsules for Long-Term Cell-Based Therapy

Cell-based therapy is an excellent therapeutic modality that involves cell transplantation into patients; however, given that most transplanted cells die immediately post-transplantation, the application of this strategy remains limited. Cell encapsulation is a promising technique for prolonging the survival of transplanted cells, although a definitive encapsulation protocol is yet to be established. Herein, we selected sodium alginate as a polymer for cell encapsulation and optimized the structure and function of cell-encapsulating alginate capsules. First, alginate capsules were prepared using various concentrations of sodium alginate and calcium chloride solution. The NanoLuc luciferase (Nluc)-expressing murine mesenchymal stem cell line C3H10T1/2 was used to prepare the alginate capsules, and cell survival was evaluated after transplantation into mice. The structural properties of the alginate capsules were dependent on the preparation conditions. Capsules with adequate hardness were obtained using 1% sodium alginate and 10% calcium chloride solutions. Alginate capsules encapsulating 5 × 103 C3H10T1/2/Nluc cells/10 μL maintained a constant cell number over time under in vitro culture conditions. After transplantation into mice, C3H10T1/2/Nluc cells encapsulated in alginate capsules exhibited a significantly longer survival (≥40 days) than suspended cells. Based on these findings, cell-encapsulating alginate capsules with optimal properties can be used for long-term cell-based therapies.

A Conceptual Approach for Examining Effects of the Adolescent Bone Marrow Milieu on MSC Phenotype.

Children with bone fragility often exhibit elevated bone marrow lipid levels, which may affect mesenchymal stem cell (MSC) differentiation potential and ultimately bone strength via cell-autonomous and/or non-cell-autonomous factors. Here, we use standard co-culture techniques to study biological effects of bone marrow cell-derived secretome on MSC. Bone marrow was collected during routine orthopedic surgery, and the entire marrow cell preparation, with or without red blood cell (RBC) reduction, was plated at three different densities. Conditioned medium (secretome) was collected after 1, 3, and 7 days. ST2 cells, a murine MSC line, were then cultured in the secretomes. Exposure to the secretomes was associated with reductions of up to 62% in MSC MTT outcomes that depended on the duration of secretome development, as well as marrow cell plating density. Reduced MTT values were not associated with diminished cell number and viability assessed using Trypan Blue exclusion. Expression of pyruvate dehydrogenase kinase 4 was modestly elevated, and β-actin levels were transiently reduced in ST2 cells exposed to secretome formulations that had elicited maximal reductions in MTT outcomes. The findings from this study can inform the design of future experimental studies to examine contributions of cell-autonomous and non-cell-autonomous factors in the bone marrow to MSC differentiation potential, bone formation, and skeletal growth. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Abstract 82: Role of MSC-derived CXCL1 in bone-metastatic prostate cancer

Abstract In 2022, the American Cancer Society estimates that over 34,000 men will die of PCa, largely due to metastatic PCa. Up to 90% of advanced prostate cancers metastasize to bone, and there are currently no curative therapies for bone metastatic prostate cancer (BM-PCa). BM-PCa cells interact with bone-forming osteoblasts and bone-resorbing osteoclasts to induce aberrant bone remodeling, which leads to skeletal related adverse events such as increased risk of fractures, bone pain, and poor quality of life. Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into osteoblasts. Cook lab data show BM-PCa cells interact with MSCs and promote MSC differentiation into osteoblasts. Microarray analysis of MSC gene expression changes in response to BM-PCa show significant upregulation of CXCL8. To determine the importance of MSC-derived CXCL8 to BM-PCa, CXCL1 (the murine homolog of CXCL8) was genetically deleted in murine MSCs. While CXCL1 knock out (KO) had no significant impact on MSC proliferation, loss of CXCL1 decreased MSC migration, which suggests upregulation of MSC CXCL1 by BM-PCa cells functions to increase MSC migration into the tumor microenvironment. Additionally, CXCL1 KO MSCs have altered osteoblastic differentiation, whereas adipogenic differentiation was unchanged. RNA sequencing of MSCs stably expressing CXCL1-targeted shRNAs compared to scrambled controls show extensive gene expression changes, including altered expression of multiple osteogenic genes. In vivo data show intratibial co-injection of CXCL1 KO MSCs with murine RM1 BM-PCa cells in an immunocompetent mouse model increases tumor burden compared with co-injection of wildtype MSCs and BM-PCa cells, indicating a role for MSC-derived CXCL1 in suppressing BM-PCa tumor growth in bone. This project shows for the first time the importance of CXCL1 in MSC migration, osteogenesis, gene expression, suppression of BM-PCa progression in bone and tumor-induced bone remodeling. Citation Format: Catherine S. Johnson, Diane Costanzo-Garvey, Massar Alsamraae, Jeremy S. Frieling, Leah M. Cook. Role of MSC-derived CXCL1 in bone-metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 82.

Sulfasalazine and Chromotrope 2B reduce oxidative stress in murine bone marrow-derived mesenchymal stem cells

With advancing age of stem cells, dysregulation of various processes at the cellular level occurs, thereby decreasing their regeneration potential. One of the changes that occurs during the aging process is the accumulation of reactive oxygen species (ROS), which accelerates the processes of cellular senescence and cell death. The aim of this study is to evaluate two antioxidant compounds; Chromotrope 2B and Sulfasalazine, for their antioxidant effects on young and old rat bone marrow mesenchymal stem cells (MSCs). Oxidative stress was induced in MSCs by 5 µM dexamethasone for 96h and the cells were treated with Chromotrope 2B or Sulfasalazine, 50 µM each. The effects of antioxidant treatment following oxidative stress induction was evaluated by transcriptional profiling of genes involved in the oxidative stress and telomere maintenance. Expression levels of Cat, Gpx7, Sod1, Dhcr24, Idh1, and Txnrd2 were found to be increased in young MSCs (yMSCs) as a result of oxidative stress, while Duox2, Parp1, and Tert1 expression were found to be decreased as compared to the control. In old MSCs (oMSCs), the expressions of Dhcr24, Txnrd2, and Parp1 increased, while that of Duox2, Gpx7, Idh1, and Sod1 decreased following oxidative stress. In both MSC groups, Chromotrope 2B prompted decrease in the ROS generation before and after the induction of oxidative stress. In oMSCs, ROS content was significantly reduced in the Sulfasalazine treated group. Our findings suggest that both Chromotrope 2B and Sulfasalazine possess the potential to reduce the ROS content in both age groups, though the latter was found to be more potent. These compounds can be used to precondition MSCs to enhance their regenerative potential for future cell-based therapeutics.

Effect of spheroid size on gene expression profiles of a mouse mesenchymal stem cell line in spheroid culture.

Mesenchymal stem cell (MSC)-based therapies offer potential for bone repair. MSC spheroid cultures may harbor enhanced therapeutic potential over MSC monolayers through increased secretion of trophic factors. However, the impact of spheroid size on trophic factor expression is unclear. We investigated the effect of spheroid size on trophic factor-related gene expression. KUM10, a murine MSC line was used. RNA-seq was used to screen the transcriptional profiles of MSC monolayer and spheroid cultures. Differentially expressed genes identified in RNA-seq were evaluated by q-PCR in cultures of 5 × 104 (S group), 5 × 105 (M group), 5 × 106 (L group) cells/well. Comparison of expression levels between KUM10 monolayer and spheroid cultures identified 2140 differentially expressed genes, of which 1047 were upregulated and 1093 were downregulated in KUM10 spheroids. Among these, 12 upregulated genes (Bmp2, Fgf9, Fgf18, Ngf, Pdgfa, Pdgfb, Tgfb1, Vegfa, Vegfc, Wnt4, Wnt5a, Wnt10a) were associated with secretory growth factors. Of these, expression of Fgf9, Fgf18, Vegfa and Vegfc was elevated in the L group, and Pdgfb and Tgfb1 was elevated in the S group. Spheroid size may impact trophic factor expression. Our results will be useful for future studies assessing the utility of MSC spheroids for treating bone injury.

A sustainable one-pot method to transform seashell waste calcium carbonate to osteoinductive hydroxyapatite micro-nanoparticles.

We have developed a straightforward, one-pot, low-temperature hydrothermal method to transform oyster shell waste particles (bCCP) from the species Crassostrea gigas (Mg-calcite, 5 wt% Mg) into hydroxyapatite (HA) micro/nanoparticles. The influence of the P reagents (H3PO4, KH2PO4, and K2HPO4), P/bCCP molar ratios (0.24, 0.6, and 0.96), digestion temperatures (25-200 °C), and digestion times (1 week-2 months) on the transformation process was thoroughly investigated. At 1 week, the minimum temperature to yield the full transformation significantly reduced from 160 °C to 120 °C when using K2HPO4 instead of KH2PO4 at a P/bCCP ratio of 0.6, and even to 80 °C at a P/bCCP ratio of 0.96. The transformation took place via a dissolution-reprecipitation mechanism driven by the favorable balance between HA precipitation and bCCP dissolution, due to the lower solubility product of HA than that of calcite at any of the tested temperatures. Both the bCCP and the derived HA particles were cytocompatible for MG-63 human osteosarcoma cells and m17.ASC murine mesenchymal stem cells, and additionally, they promoted the osteogenic differentiation of m17.ASC, especially the HA particles. Because of their physicochemical features and biological compatibility, both particles could be useful osteoinductive platforms for translational applications in bone tissue engineering.

Tafazzin Knockdown in Murine Mesenchymal Stem Cells Enhances the Tafazzin Knockdown Mediated Elevation in Interleukin-10 Secretion from Murine B Lymphocytes

Tafazzin Knockdown in Murine Mesenchymal Stem Cells Enhances the Tafazzin Knockdown Mediated Elevation in Interleukin-10 Secretion from Murine B Lymphocytes

SIRT1 haplo-insufficiency results in reduced cortical bone thickness, increased porosity and decreased estrogen receptor alpha in bone in adult 129/Sv female mice.

Sirtuin 1 (SIRT1) is a key player in aging and metabolism and regulates bone mass and architecture. Sexual dimorphism in skeletal effects of SIRT1 has been reported, with an unfavorable phenotype primarily in female mice. To investigate the mechanisms of gender differences in SIRT1 skeletal effect, we investigated femoral and vertebral cortical and cancellous bone in global Sirt1 haplo-insufficient 129/Sv mice aged 2,7,12 months lacking Sirt1 exons 5,6,7 (Sirt1+/Δ ) and their wild type (WT) counterparts. In females, femoral bone mineral content, peak cortical thickness, and trabecular bone volume (BV/TV%), number and thickness were significantly lower in Sirt1+/Δ compared to WT mice. Increased femoral cortical porosity was observed in 7-month-old Sirt1+/Δ compared to WT female mice, accompanied by reduced biomechanical strength. No difference in vertebral indices was detected between Sirt1+/Δ and WT female mice. SIRT1 decreased with aging in WT female mice and was lower in vertebrae and femur in 18- and 30- versus 3-month-old 129/Sv and C57BL/6J female mice, respectively. Decreased bone estrogen receptor alpha (ERα) was observed in Sirt1+/Δ compared to WT female mice and was significantly higher in Sirt1 over-expressing C3HT101/2 murine mesenchymal stem cells. In males no difference in femoral indices was detected in Sirt1+/Δ versus WT mice, however vertebral BV/TV%, trabecular number and thickness were higher in Sirt1+/Δ vs. WT mice. No difference in androgen receptor (AR) was detected in bone in Sirt1+/Δ vs. WT male mice. Bone SIRT1 was significantly lower in male compared to female WT mice, suggesting that SIRT1 maybe more significant in female than male skeleton. These findings demonstrate that 50% reduction in SIRT1 is sufficient to induce the hallmarks of skeletal aging namely, decreased cortical thickness and increased porosity in female mice, highlighting the role of SIRT1 as a regulator of cortical bone quantity and quality. The effects of SIRT1 in cortical bone are likely mediated in part by its regulation of ERα. The age-associated decline in bone SIRT1 positions SIRT1 as a potential therapeutic target to ameliorate age-related cortical bone deterioration in females. The crosstalk between ERα, AR and SIRT1 in the bone microenvironment remains to be further investigated.

MSCs-ILC2s-Tecs Axis Promotes the Recovery of Thymic Function in aGVHD

Background: Previous studies found that mesenchymal stem cells (MSCs) administrated at the acute graft versus host disease (aGVHD) phase effectively decreased the incidence and severity of chronic GVHD (cGVHD) by boosting thymic regeneration. However, the mechanism for MSCs repairing the damaged thymus caused by aGVHD is unclear. It has been reported that innate lymphoid cells (ILCs) are also important for the structural remodeling of the thymus in recipients after allogeneic bone marrow transplantation (allo-BMT). Here, we explored the mechanisms on how MSCs promote thymus recovery post allo-BMT through ILCs. Methods: We carried out studies in a aGVHD mice model of fully MHC-mismatched myeloablative bone marrow transplantation (C57BL/6 to BALB/c). MSCs or ILC2s were generated and administrated intravenously at day 7 post-BMT to the treated groups to compare therapeutic effects of GVHD with the untreated group. Clinical scores and survival were recorded once every days. Flow cytometery was employed for analyzing the number and proportion of ILCs and their subtypes (ILCs Lin-CD45+iCD3-CD127+, type 1 ILCs (ILC1s) Tbet+, type 2 ILCs (ILC2s) GATA3+, type 3 ILCs (ILC3s) RORγt+) in the thymus to assess the effect of MSCs on ILCs in the thymus at day 14 post-BMT. We analyzed the thymic T cells subsets and thymic epithelial cells (TECs) substes among these two groups to evaluate the repair effect of ILC2s for thymus. Thymic ILC2s were cultured alone or co-cultured with murine MSCs in vitro to investigate the effect and underlying mechanism of MSCs on ILC2s. Results: We found that ILC2s in the thymus are sensitive to conditioning therapy and and further impaired by aGVHD. But infusion of MSCs was effective in reducing the lethality of aGVHD and promote expansion of thymic ILC2s in aGVHD mice compared with the control group. The absolute number of ILC2s in the thymus had a significant negative correlation with GVHD scores in aGVHD mice. Then we found that ILC2s infusion significant increases in thymus size and weight, as well as the number of total thymocytes and the more organized cortical medullary structure compared to the untreated groups. The number of TECs and its proportion of thymus stroma were significantly improved, including cortical TEC and medullary TECs. As for thymocyte, ILC2s infusion significantly increased the number and proportion of CD4+CD8+T cells. In vitro study showed co-cultured ILC2s had a increased proliferation compared to ILC2s cultured alone, and anti-IL33 antibody inhibited the pro-proliferative effect of MSCs on ILC2. Then we perform in vivo validation by injecting IL33 knocked down MSCs and found that the effect of MSC in repairing the damaged thymus was reduced. And the number and proportion of thymic ILC2s were also significantly decreased in the thymus. Furthermore, treatment with rIL33 on day 7-11 also ameliorated aGVHD and promoted the proliferation of thymic ILC2s in aGVHD mice as well as MSCs infusion. Conclusion: These findings suggest that MSCs participate in thymus regeneration from aGVHD damage through ILC2s-mediated tissue recovery. The study outlined here provide a new insight into the mechanism of MSC therapy for aGVHD.

Poly(acrylamide) Spheroids with Tunable Elasticity for Scalable Cell Culture Applications

AbstractMany biological tissues resemble hydrogels and display Young's moduli below 50 kPa, corresponding to most cell types for the natural environmental conditions to grow. Contrastingly, conventional cell culture usually involves rigid substrates resulting in stiff priming effects, which are of increasing concern when it comes to scalable culturing of adhesive cells for regenerative purposes. As a solution to this problem, the employment of synthetic poly(acryl amide) (PAAm)‐based hydrogel beads with tissue‐matched mechanical properties are proposed as soft matrix for culture modes suitable for tidal bioreactor culture. Herein, technology is described to generate spherical, mm‐scaled PAAm hydrogel beads with adjustable, soft elastic properties that are produced by a continuous microfluidic approach. A simple and robust method is demonstrated to functionalize the spheroids with protein ligands, and the suitability of the matrix for cell cultivation is successfully demonstrated with three different cell types (murine mesenchymal stem cells, renal carcinoma cells, and human induced pluripotent stem cells) in model experiments and in a tidal bioreactor system. This versatile approach will pave the way toward novel cell culture systems based on bioreactors that allow scalable, soft carrier‐based expansion of cells on matrices with tissue‐matched elasticity.

Cinnamaldehyde regulates mitochondrial quality against hydrogen peroxide induced apoptosis in mouse lung mesenchymal stem cells via the PINK1/Parkin signaling pathway.

Idiopathic pulmonary fibrosis (IPF) is a fatal respiratory disease without effective treatments. Mitochondrial dysfunction weakens the ability of mesenchymal stem cells (MSCs) to repair the distal lung epithelium, which is a probable pathogenesis of IPF. In previous research, we found that cinnamaldehyde (CA) can maintain the mitochondrial morphology of MSCs. This present study evaluated the effect and mechanism of CA on murine lung MSCs using the hydrogen peroxide model. Antioxidant effects and mitochondrial function were determined using flow cytometry. The mRNA levels of mitochondrial dynamics and the expressions of autophagy-related proteins were also detected. CA can increase the levels of SOD, MMP and ATP, decrease the rate of ROS and apoptosis, and restore the mitochondrial structure. CA can also improve the mRNA expression of MFN1, MFN2, FIS1, DRP1, OPA1, and PGC-1α, increase the expression of LC3 II and p62 and promote the PINK1/Parkin signaling pathway. Our results demonstrated that CA can control mitochondrial quality and avoid apoptosis, which may be associated with the regulation of the PINK1/Parkin signaling pathway.

Hypoxia promotes osteogenesis by facilitating acetyl-CoA-mediated mitochondrial-nuclear communication.

Bone-derived mesenchymal stem cells (MSCs) reside in a hypoxic niche that maintains their differentiation potential. While hypoxia (low oxygen concentration) was reported to critically support stem cell function and osteogenesis, the molecular events triggering changes in stem cell fate decisions in response to normoxia (high oxygen concentration) remain elusive. Here, we study the impact of normoxia on mitochondrial-nuclear communication during stem cell differentiation. We show that normoxia-cultured murine MSCs undergo profound transcriptional alterations which cause irreversible osteogenesis defects. Mechanistically, high oxygen promotes chromatin compaction and histone hypo-acetylation, particularly on promoters and enhancers of osteogenic genes. Although normoxia induces metabolic rewiring resulting in elevated acetyl-CoA levels, histone hypo-acetylation occurs due to the trapping of acetyl-CoA inside mitochondria owing to decreased citrate carrier (CiC) activity. Restoring the cytosolic acetyl-CoA pool remodels the chromatin landscape and rescues the osteogenic defects. Collectively, our results demonstrate that the metabolism-chromatin-osteogenesis axis is perturbed upon exposure to high oxygen levels and identifies CiC as a novel, oxygen-sensitive regulator of the MSC function.

Abstract IA005: On the use of stem cells to generate sarcomas in vitro models

Abstract Sarcomas are rare cancers arising in soft tissue and bone. They are difficult to diagnose and treat, with 38-48% mortality for this often young age group. It is now generally accepted that molecular changes in cancer open avenues for personalized medicine. However, to achieve this for sarcoma there are several challenges, as representative model systems are scarce. Moreover, sarcomas are heterogeneous and &amp;gt;70 sarcoma types are recognized. A simple dichotomy based on molecular pathways of simple versus complex genomics is often used as a conceptual framework. My group has used mesenchymal stem cells to generate a model for complex genome sarcomas such as osteosarcoma. Murine and canine mesenchymal stem cells (MSCs) can be used to model spontaneous malignant transformation towards sarcomas with complex genomics. These MSCs have an abnormal karyotype, many structural variants and point mutations at whole genome sequencing analysis, and form sarcomas (osteosarcoma, undifferentiated pleomorphic and spindle cell sarcoma) after injection into mice. Cross-species analysis reveals that p53 loss is an early event in sarcomagenesis, and MSCs with a knockout in Trp53 transform earlier compared to wild-type MSCs. In addition, loss of p16Ink4a is shown to be a driver of osteosarcomagenesis: the majority of spontaneously transformed murine MSCs has alterations in p15Ink4b, p16Ink4a, or p19Arf and knockout of these genes in MSCs results in accelerated transformation. Of interest, osteosarcoma cells with defective p16INK4A are sensitive to the CDK4/CDK6 inhibitor Palbociclib, suggesting that a subset of osteosarcoma patients with intact Rb, but defective p16 or overexpression of CDK4 and/or CDK6 (in total 20-23%) might benefit from CDK4/CDK6 inhibition. Bone and soft tissue tumors with a simple genome often carry chromosomal translocations. We have generated a model for pseudomyogenic hemangioendothelioma (PHE), a rare locally aggressive vascular tumor. PHE has a tumor-specific t(7;19)(q22;q13) translocation resulting in a SERPINE1-FOSB fusion. We used CRISPR/Cas9 to generate this fusion in induced pluripotent stem cells (hiPSCs), which were subsequently differentiated into ECs (hiPSC-ECs) to address this. Comparison of parental with PHE hiPSC-ECs shows invasive growth in mice after transplantation, and transcriptome analysis identifies PI3K-Akt and MAPK signaling pathways as possible therapeutic targets. The modified hiPSC-ECs thus recapitulate functional features of PHE and demonstrate how these stem cell based translocation models can be used to understand tumorigenic mechanisms and identify therapeutic targets. Citation Format: Judith V.M.G. Boveé. On the use of stem cells to generate sarcomas in vitro models [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA005.

3D Bioprinted Hydroxyapatite or Graphene Oxide Containing Nanocellulose-Based Scaffolds for Bone Regeneration.

Bone tissue is usually damaged after big traumas, tumors, and increasing aging-related diseases such as osteoporosis and osteoarthritis. Current treatments are based on implanting grafts, which are shown to have several inconveniences. In this regard, tissue engineering through the 3D bioprinting technique has arisen to manufacture structures that would be a feasible therapeutic option for bone regenerative medicine. In this study, nanocellulose-alginate (NC-Alg)-based bioink is improved by adding two different inorganic components such as hydroxyapatite (HAP) and graphene oxide (GO). First, ink rheological properties and biocompatibility are evaluated as well as the influence of the sterilization process on them. Then, scaffolds are characterized. Finally, biological studies of embedded murine D1 mesenchymal stem cells engineered to secrete erythropoietin are performed. Results show that the addition of both HAP and GO prevents NC-Alg ink from viscosity lost in the sterilization process. However, GO is reduced due to short cycle autoclave sterilization, making it incompatible with this ink. In addition, HAP and GO have different influences on scaffold architecture and surface as well as in swelling capacity. Scaffolds mechanics, as well as cell viability and functionality, are promoted by both elements addition. Additionally, GO demonstrates an enhanced bone differentiation capacity.

The Effects of Hypoxic Preconditioned Murine Mesenchymal Stem Cells on Post-Infarct Arrhythmias in the Mouse Model.

Ventricular arrhythmias associated with myocardial infarction (MI) have a significant impact on mortality in patients following heart attack. Therefore, targeted reduction of arrhythmia represents a therapeutic approach for the prevention and treatment of severe events after infarction. Recent research transplanting mesenchymal stem cells (MSC) showed their potential in MI therapy. Our study aimed to investigate the effects of MSC injection on post-infarction arrhythmia. We used our murine double infarction model, which we previously established, to more closely mimic the clinical situation and intramyocardially injected hypoxic pre-conditioned murine MSC to the infarction border. Thereafter, various types of arrhythmias were recorded and analyzed. We observed a homogenous distribution of all types of arrhythmias after the first infarction, without any significant differences between the groups. Yet, MSC therapy after double infarction led to a highly significant reduction in simple and complex arrhythmias. Moreover, RNA-sequencing of samples from stem cell treated mice after re-infarction demonstrated a significant decline in most arrhythmias with reduced inflammatory pathways. Additionally, following stem-cell therapy we found numerous highly expressed genes to be either linked to lowering the risk of heart failure, cardiomyopathy or sudden cardiac death. Moreover, genes known to be associated with arrhythmogenesis and key mutations underlying arrhythmias were downregulated. In summary, our stem-cell therapy led to a reduction in cardiac arrhythmias after MI and showed a downregulation of already established inflammatory pathways. Furthermore, our study reveals gene regulation pathways that have a potentially direct influence on arrhythmogenesis after myocardial infarction.