Age-related Morphofunctional Changes in Sickle Cell Mice Bone Marrow Mesenchymal Stromal Cells.

BACKGROUND AND OBJECTIVES: Bone marrow mesenchymal stromal cells (BM-MSCs) are key elements of the hematopoietic niche and participate in the regulatory mechanisms of hematopoietic stem cells (HSCs). Hematological diseases can affect MSCs and their functions. However, the dysregulations caused by sickle cell disease (SCD) are not fully elucidated. This work explored changes in BM-MSCs and their relationship with age using sickle cell mice (Townes-SS). MATERIALS AND METHODS: BM-MSCs were isolated from Townes-SS, and control groups 30- and 60-day-old Townes-AA and C57BL/6 J. RESULTS: The BM-MSCs showed no morphological differences in culture and demonstrated a murine MSC-like immunophenotypic profile (Sca-1+, CD29+, CD44+, CD90.2+, CD31-, CD45-, and CD117-). Subsequently, all BM-MSCs were able to differentiate into adipocytes and osteocytes in vitro. Finally, 30-day-old BM-MSCs of Townes-SS showed higher expression of genes related to the maintenance of HSCs (Cxcl12, Vegfa, and Angpt1) and lower expression of pro-inflammatory genes (Tnfa and Il-6). However, 60-day-old BM-MSCs of Townes-SS started to show expression of genes related to reduced HSC maintenance and increased expression of pro-inflammatory genes. CONCLUSION: These results indicates age as a modifying factor of gene expression of BM-MSCs in the context of SCD.

Effects of bone marrow mesenchymal stem cell-conditioned medium on the proliferation and migration of liposarcoma cells.

INTRODUCTION: Liposarcoma constitutes a prevalent subtype of soft tissue sarcoma, represents approximately 20% of all sarcomas. However, conventional chemotherapeutic agents have shown restricted effectiveness in treating liposarcoma patients. Accumulating evidence indicates that mesenchymal stem cells (MSCs) have the characteristic of migration to tumor site, promote or suppress tumors. How human bone marrow mesenchymal stem cells (BMSCs) contribute to liposarcoma phenotype remains poorly understood. This study aims to investigate the effects of human bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on the proliferation and migration of liposarcoma cell lines 93T449 and SW872, as well as explore potential underlying mechanisms of BMSC-CM action on these cells. MATERIALS AND METHODS: We transfected BMSCs with lentiviral constructs to knock down the transcriptional co-activator Yes-associated protein 1 (YAP1), conditioned medium (CM) obtained from BMSCs and shYAP1-BMSC, respectively. Liposarcoma cell lines 93T449 and SW872 were co-cultured with BMSC-CM or shYAP1-BMSC-CM. Cell proliferation ability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was evaluated using flow cytometric assay. A wound healing assay was used to analyze cell migration. The expression levels of YAP1, Bcl-2, and matrix metalloproteinase-2 (MMP-2) were determined by western blot assay. RESULTS: Co-culturing liposarcoma cell lines 93T449 and SW872 with BMSC-CM promoted tumor cell proliferation, while shYAP1-BMSC-CM significantly inhibited cell viability and migration, induced apoptosis, and downregulated Bcl-2 and MMP-2 expression. CONCLUSIONS: These findings provide new insights into the impact of BMSC-CM on liposarcoma and suggest its possible involvement in liposarcoma cell growth.

Tumor microenvironment restricts IL-10 induced multipotent progenitors to myeloid-lymphatic phenotype.

Lymphangiogenesis is induced by local pro-lymphatic growth factors and bone marrow (BM)-derived myeloid-lymphatic endothelial cell progenitors (M-LECP). We previously showed that M-LECP play a significant role in lymphangiogenesis and lymph node metastasis in clinical breast cancer (BC) and experimental BC models. We also showed that differentiation of mouse and human M-LECP can be induced through sequential activation of colony stimulating factor-1 (CSF-1) and Toll-like receptor-4 (TLR4) pathways. This treatment activates the autocrine interleukin-10 (IL-10) pathway that, in turn, induces myeloid immunosuppressive M2 phenotype along with lymphatic-specific proteins. Because IL-10 is implicated in differentiation of numerous lineages, we sought to determine whether this pathway specifically promotes the lymphatic phenotype or multipotent progenitors that can give rise to M-LECP among other lineages. Analyses of BM cells activated either by CSF-1/TLR4 ligands in vitro or orthotopic breast tumors in vivo showed expansion of stem/progenitor population and coincident upregulation of markers for at least four lineages including M2-macrophage, lymphatic endothelial, erythroid, and T-cells. Induction of cell plasticity and multipotency was IL-10 dependent as indicated by significant reduction of stem cell markers and those for multiple lineages in differentiated cells treated with anti-IL-10 receptor (IL-10R) antibody or derived from IL-10R knockout mice. However, multipotent CD11b+/Lyve-1+/Ter-119+/CD3e+ progenitors detected in BM appeared to split into a predominant myeloid-lymphatic fraction and minor subsets expressing erythroid and T-cell markers upon establishing tumor residence. Each sub-population was detected at a distinct intratumoral site. This study provides direct evidence for differences in maturation status between the BM progenitors and those reaching tumor destination. The study results suggest preferential tumor bias towards expansion of myeloid-lymphatic cells while underscoring the role of IL-10 in early BM production of multipotent progenitors that give rise to both hematopoietic and endothelial lineages.

A Robust In Vitro Culture Model and Generation of Memory Monocytes.

Innate monocytes can be trained or reprogrammed to adopt distinct memory states, such as low-grade inflammation and immune exhaustion, bearing fundamental relevance to the pathogenesis of both acute diseases such as sepsis as well as chronic diseases such as atherosclerosis. Therefore, it is critically important to develop a regimen for generating memory monocytes in vitro in order to better define key monocyte memory states with diverse potentials for proliferation, differentiation, and activation, as well as underlying mechanisms. Here, we describe an efficient in vitro system to propagate a large number of highly purified murine memory monocytes through sustaining bone marrow-derived monocytes with macrophage colony-stimulating factor (M-CSF, 10 ng/mL)-containing medium, together with other polarization agents such as lipopolysaccharide (LPS) for a 5-day period. This method can yield high-purity monocytes, capable of exhibiting dynamic memory behaviors upon training with various polarizing agents.

Fluid shear force and hydrostatic pressure jointly promote osteogenic differentiation of BMSCs by activating YAP1 and NFAT2.

Natural bone tissue features a complex mechanical environment, with cells responding to diverse mechanical stimuli, including fluid shear stress (FSS) and hydrostatic pressure (HP). However, current in vitro experiments commonly employ a singular mechanical stimulus to simulate the mechanical environment in vivo. The understanding of the combined effects and mechanisms of multiple mechanical stimuli remains limited. Hence, this study constructed a mechanical stimulation device capable of simultaneously applying FSS and HP to cells. This study investigated the impact of FSS and HP on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and examined the distinctions and interactions between the two mechanisms. The results demonstrated that both FSS and HP individually enhanced the osteogenic differentiation of BMSCs, with a more pronounced effect observed through their combined application. BMSCs responded to external FSS and HP stimulation through the integrin-cytoskeleton and Piezo1 ion channel respectively. This led to the activation of downstream biochemical signals, resulting in the dephosphorylation and nuclear translocation of the intracellular transcription factors Yes Associated Protein 1 (YAP1) and nuclear factor of activated T cells 2 (NFAT2). Activated YAP1 could bind to NFAT2 to enhance transcriptional activity, thereby promoting osteogenic differentiation of BMSCs more effectively. This study highlights the significance of composite mechanical stimulation in BMSCs' osteogenic differentiation, offering guidance for establishing a complex mechanical environment for in vitro functional bone tissue construction.

Characterization of relapse-supportive monocytic cells in acute myeloid leukemia.

The precise identities of bone marrow resident cells contributing to AML relapse are not fully known. Hollands et al. report early evidence to support the existence of an aberrant monocytic cell population that appears to promote LSC expansion after cytarabine treatment.

Specific lipid magnetic sphere sorted CD146-positive bone marrow mesenchymal stem cells can better promote articular cartilage damage repair.

BACKGROUND: The characteristics and therapeutic potential of subtypes of bone marrow mesenchymal stem cells (BMSCs) are largely unknown. Also, the application of subpopulations of BMSCs in cartilage regeneration remains poorly characterized. The aim of this study was to explore the regenerative capacity of CD146-positive subpopulations of BMSCs for repairing cartilage defects. METHODS: CD146-positive BMSCs (CD146 + BMSCs) were sorted by self-developed CD146-specific lipid magnetic spheres (CD146-LMS). Cell surface markers, viability, and proliferation were evaluated in vitro. CD146 + BMSCs were subjected to in vitro chondrogenic induction and evaluated for chondrogenic properties by detecting mRNA and protein expression. The role of the CD146 subpopulation of BMSCs in cartilage damage repair was assessed by injecting CD146 + BMSCs complexed with sodium alginate gel in the joints of a mouse cartilage defect model. RESULTS: The prepared CD146-LMS had an average particle size of 193.7 ± 5.24 nm, an average potential of 41.9 ± 6.21 mv, and a saturation magnetization intensity of 27.2 Am2/kg, which showed good stability and low cytotoxicity. The sorted CD146 + BMSCs highly expressed stem cell and pericyte markers with good cellular activity and cellular value-added capacity. Cartilage markers Sox9, Collagen II, and Aggrecan were expressed at both protein and mRNA levels in CD146 + BMSCs cells after chondrogenic induction in vitro. In a mouse cartilage injury model, CD146 + BMSCs showed better function in promoting the repair of articular cartilage injury. CONCLUSION: The prepared CD146-LMS was able to sort out CD146 + BMSCs efficiently, and the sorted subpopulation of CD146 + BMSCs had good chondrogenic differentiation potential, which could efficiently promote the repair of articular cartilage injury, suggesting that the sorted CD146 + BMSCs subpopulation is a promising seed cell for cartilage tissue engineering.

Identification of HIST1H2BH as the hub gene associated with multiple myeloma using integrated bioinformatics analysis.

OBJECTIVES: Identifying the specific biomarkers and molecular signatures of MM might provide novel evidence for MM prognosis and targeted therapy. METHODS: Bioinformatic analyses were performed through GEO and TCGA datasets. The differential expression of HIST1H2BH in MM sample was validated by the qRT-PCR. And the CCK-8 assay was performed to detect the proliferation activity of HIST1H2BH on MM cell lines. RESULTS: A total of 793 DEGs were identified between bone marrow plasma cells from newly diagnosed myeloma and normal donors in GSE6477. Among them, four vital genes (HIST1H2AC, HIST1H2BH, CCND1 and TCF7L2) modeling were constructed. The increased HIST1H2BH expression was correlated with worse survival of MM based on TCGA datasets. The transcriptional expression of HIST1H2BH was significantly up-regulated in primary MM patients. And knockdown HIST1H2BH decreased the proliferation of MM cell lines. CONCLUSIONS: We have identified up-regulated HIST1H2BH in MM patients associated with poor prognosis using integrated bioinformatical methods.

Ablation of Wnt signaling in bone marrow stromal cells overcomes microenvironment-mediated drug resistance in acute myeloid leukemia.

The survival of leukemic cells is significantly influenced by the bone marrow microenvironment, where stromal cells play a crucial role. While there has been substantial progress in understanding the mechanisms and pathways involved in this crosstalk, limited data exist regarding the impact of leukemic cells on bone marrow stromal cells and their potential role in drug resistance. In this study, we identify that leukemic cells prime bone marrow stromal cells towards osteoblast lineage and promote drug resistance. This biased differentiation of stroma is accompanied by dysregulation of the canonical Wnt signaling pathway. Inhibition of Wnt signaling in stroma reversed the drug resistance in leukemic cells, which was further validated in leukemic mice models. This study evaluates the critical role of leukemic cells in establishing a drug-resistant niche by influencing the bone marrow stromal cells. Additionally, it highlights the potential of targeting Wnt signaling in the stroma by repurposing an anthelmintic drug to overcome the microenvironment-mediated drug resistance.

[Exosomes derived from bone marrow mesenchymal stem cells regulate NF-κB pathway and reduce lung ischemia-reperfusion injury in rats by miR-335].

This study aimed to investigate the effect of exosomes derived from bone marrow mesenchymal stem cells (BMSCs-EXO) on lung ischemia-reperfusion injury (IRI) in rats and to explore the role of miR-335. The model of rat lung IRI was established by clipping the hilum of left lung for 60 min and opening for 180 min. Forty Sprague-Dawley rats were randomly divided into sham group, IRI group, IRI+PBS group, IRI+EXO group, and IRI+miR-335 inhibitor EXO (IRI+inhibitor-EXO) group (n = 8). Rats in the sham group underwent thoracotomies without IRI. Rats in the IRI group were used to establish IRI model without any additional treatment. In the IRI+PBS, IRI+EXO, and IRI+inhibitor-EXO groups, the rats were used to establish IRI model and given PBS, EXO from BMSCs without any treatment, and EXO from BMSCs with miR-335 inhibitor treatment before reperfusion, respectively. Blood gases were analyzed during the experiment. Lung tissue wet/dry ratio (W/D), interleukin 1ß (IL-1ß), tumor necrosis factor α (TNF-α), myeloperoxidase (MPO), malondialdehyde (MDA), and superoxide dismutase (SOD) were measured at the end of reperfusion. Mitochondria were observed by electron microscopy and the Flameng scores were counted. Lung histopathology and apoptosis (TUNEL staining) were observed by light microscopy, and the lung injury scores (LIS) and apoptosis index (AI) were detected. The miR-335 expression was detected by RT-qPCR, and the expression of caspase-3, cleaved-caspase-3, caspase-9, cleaved-caspase-9, and NF-κB proteins were detected by Western blot at the end of reperfusion. The results showed that compared with the sham group, the oxygenation index, pH, and base excess (BE) were significantly lower in the IRI group and IRI+PBS group after reperfusion, whereas those indices were significantly higher in the IRI+EXO group than those in the IRI+PBS group (P < 0.05). Compared with the sham group, there were significant increases in W/D, IL-1ß, TNF-α, MPO, MDA, LIS, AI, Flameng score, caspase-3, cleaved-caspase-3, caspase-9, and cleaved-caspase-9, however significant decreases in the SOD, miR-335 and NF-κB in the IRI group (P < 0.05). These indices in the IRI and IRI+PBS groups showed no significant differences. Compared with the IRI+PBS group, there were significant decreases in W/D, IL-1ß, TNF-α, MPO, MDA, LIS, AI, Flameng score, caspase-3, cleaved-caspase-3, caspase-9, and cleaved-caspase-9, however significant increases in the SOD, miR-335 and NF-κB in the IRI+EXO group (P < 0.05). While, the changes of the above mentioned indices were reversed in the IRI+inhibitor-EXO group compared with IRI+EXO group, which were still better than those in the IRI+PBS group (P < 0.05). The results suggest that BMSCs-EXO could attenuate lung IRI in rats, activate NF-κB pathway, and maintain mitochondrial stability by up-regulating miR-335.

[Effects of BMAL2 on Aerobic Glycolysis and Cell Proliferation in Acute Myeloid Leukemia Cells].

OBJECTIVE: To explore the expression of basic helix-loop-helix ARNT like 2 (BMAL2) in acute myeloid leukemia (AML) patients and its correlation with prognosis, and analyze its effects on the aerobic glycolysis and proliferation of AML cells. METHODS: The expressions of BMAL2 in bone marrow mononuclear cells (BMMCs) of AML patients and normal control group were detected by RT-qPCR. The correlation of BMAL2 expression with prognosis of AML patients was analyzed using public database of National Center for Biotechnology Information (NCBI). The interfering in BMAL2 expression of HL-60 and Kasumi-1 cells was performed using lentiviral vector-mediated shRNA. Cell glucose metabolism and proliferation were detected by using glucose uptake experiment, lactate content test, CCK-8 assay and cell colony formation test. RESULTS: The expression level of BMAL2 mRNA in BMMCs of AML patients was significantly higher than normal control group (P < 0.01). The overall survival time of AML patients with high expression of BMAL2 was significantly shorter than those with low expression of BMAL2 (P < 0.05). Knockdown of BMAL2 significantly reduced glucose uptake and lactate production in AML cell line HL-60 and Kasumi-1 cells. The results of RT-PCR and Western blot showed that BMAL2 promoted aerobic glycolysis by enhancing the expression of HIF1A in AML cells, thereby promoting cell proliferation. CONCLUSION: BMAL2 is highly expressed in AML patients, and promotes aerobic glycolysis by enhancing the expression of HIF1A, thereby promoting cell proliferation.

[Chidamide Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stromal Cells from Patients with Myelodysplastic Syndromes].

OBJECTIVE: To explore the effects and mechanisms of chidamide on the osteogenic differentiation of bone marrow mesenchymal stromal cells (MSC) from myelodysplastic syndromes (MDS). METHODS: MSC were isolated and cultured from bone marrow of MDS patients and healthy donors. CCK-8 assay was used to detect the effects of chidamide on the proliferation of MSC. The effects of chidamide on the activity of histone deacetylase (HDAC) in MSC was measured by a fluorescence assay kit and Western blot. Alkaline phosphatase (ALP) activity was detected on day 3 and calcium nodule formation was observed by Alizarin Red staining on day 21 after osteogenic differentiation. The expression of early and late osteogenic genes was detected on day 7 and day 21, respectively. RT-PCR and Western blot were used to detect the effects of chidamide on mRNA and protein expression of RUNX2 which is the key transcription factor during osteogenesis. RESULTS: As the concentration of chidamide increased, the proliferation of MSC was inhibited. However, at a low concentration (1 µmol/L), chidamide had no significant inhibitory effect on MSC proliferation but significantly inhibited HDAC activity. In MSC from both MDS patients and healthy donors, chidamide (1 µmol/L) significantly increased ALP activity, calcium nodule formation, thereby mRNA expression of osteogenic genes, and restored the reduced osteogenic differentiation ability of MDS-MSC compared to normal MSC. Mechanistic studies showed that the osteogenic-promoting effect of chidamide may be related to the upregulation of RUNX2 . CONCLUSION: Chidamide can inhibit HDAC activity in MSC, upregulate the expression of the osteogenic transcription factor RUNX2, and promote the osteogenic differentiation of MDS-MSC.

[The Effect of SIRT5 Deletion on Recovery of Hematopoietic Stem Cells after Injury in Mouse].

OBJECTIVE: To investigate the effect of deacylase Sirtuin 5 in the recovery of hematopoietic stem cells (HSCs) after treated by 5-FU in mouse. METHODS: Flow cytometry was used to analyze the effect of SIRT5 deletion on the proportion of hematopoietic stem/progenitor cells (HSPCs) in bone marrow (BM), the proportion of T cells, B cells and myeloid cells (TBM) in peripheral blood (PB) and spleen, and the development of T cells in thymus. Mouse were treated with 5-FU to study the effect of SIRT5 deletion on the cell cycle, apoptosis and the proportion of HSPCs in BM. The effect of SIRT5 deletion on the proliferation of HSCs was analyzed by flow sorting in vitro. RESULTS: SIRT5 deletion did not affect the development of T cells in thymus and the proportion of TBM cells in PB and spleen compared with wild type mice. SIRT5 deletion increased proportion of HSPCs in BM. After 5-FU treatment, the proportion of HSCs in SIRT5 deletion mice was significant decreased (P < 0.05), the HSPC in SIRT5 deletion mice was activated from G0 to G1 phase (P < 0.05), and the proportion of early apoptosis increased (P < 0.05). By monoclonal culture in vitro, the ability of HSCs to form clones in SIRT5 deletion mice was decreased significantly (P < 0.05). CONCLUSION: SIRT5 deletion lead to a decreased the ability of HSCs to clone in vitro. SIRT5 deletion is not conducive to the recovery of HSPCs injury in mice under hematopoietic stress.

[Establishment and Evaluation Strategy of an in vitro Cell Model of Bone Marrow Microenvironment Injury in Mouse Acute Graft-Versus-Host Disease].

OBJECTIVE: To establish a mesenchymal stem cell（MSC）-based in vitro cell model for the evaluation of mouse bone marrow acute graft-versus-host disease (aGVHD). METHODS: Female C57BL/6N mice aged 6-8 weeks were used as bone marrow and lymphocyte donors, and female BALB/c mice aged 6-8 weeks were used as aGVHD recipients. The recipient mouse received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) in 6-8 hours post irradiation to establish a bone marrow transplantation (BMT) mouse model (n=20). In addition, the recipient mice received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) and spleen lymphocytes (2×106/mouse) in 6-8 hours post irradiation to establish a mouse aGVHD model (n=20). On the day 7 after modeling, the recipient mice were anesthetized and the blood was harvested post eyeball enucleation. The serum was collected by centrifugation. Mouse MSCs were isolated and cultured with the addition of 2%, 5%, and 10% recipient serum from BMT group or aGVHD group respectively. The colony-forming unit-fibroblast（CFU-F) experiment was performed to evaluate the potential effects of serums on the self-renewal ability of MSC. The expression of CD29 and CD105 of MSC was evaluated by immunofluorescence staining. In addition, the expression of self-renewal-related genes including Oct-4, Sox-2, and Nanog in MSC was detected by real-time fluorescence quantitative PCR（RT-qPCR). RESULTS: We successfully established an in vitro cell model that could mimic the bone marrow microenvironment damage of the mouse with aGVHD. CFU-F assay showed that, on day 7 after the culture, compared with the BMT group, MSC colony formation ability of aGVHD serum concentrations groups of 2% and 5% was significantly reduced （P < 0．05）; after the culture, at day 14, compared with the BMT group, MSC colony formation ability in different aGVHD serum concentration was significantly reduced （P < 0．05）. The immunofluorescence staining showed that, compared with the BMT group, the proportion of MSC surface molecules CD29+ and CD105+ cells was significantly dereased in the aGVHD serum concentration group (P < 0.05), the most significant difference was at a serum concentration of 10% （P < 0．001, P < 0.01）. The results of RT-qPCR detection showed that the expression of the MSC self-renewal-related genes Oct-4, Sox-2, and Nanog was decreased, the most significant difference was observed at an aGVHD serum concentration of 10% （P < 0.01,P < 0．001,P < 0．001）. CONCLUSION: By co-culturing different concentrations of mouse aGVHD serum and mouse MSC, we found that the addition of mouse aGVHD serum at different concentrations impaired the MSC self-renewal ability, which providing a new tool for the field of aGVHD bone marrow microenvironment damage.

[Screening and Identification of lncRNA Related to Adipocity of Bone Marrow Microenvironment in Aplastic Anemia].

OBJECTIVE: To systematically screen and identify long noncoding RNA (lncRNA) associated with bone marrow adiposity changes in aplastic anemia (AA). METHODS: The PPARγ and C/EBPα ChIP-Seq data in ChIPBase was analyzed by bioinformatics and the potential lncRNA co-transcriptionally regulated by PPARγ and C/EBPα was screened. The expression of candidate lncRNA was verified by qRT-PCR in the in vitro adipogenic differentiation model of BM-MSC, BM-MSC infected with lenti-shPPARγ and lenti-shC/EBPα as well as clinical BM-MSC samples derived from AA and controls. RESULTS: PPARγ and C/EBPα were significantly highly expressed in AA BM-MSC, and knock-down of PPARγ and C/EBPα impaired the adipogenic capacity of AA BM-MSC. PPARγ and C/EBPα cotranscriptionally activate LINC01230 promoter activity in binding sites dependant manner. The LINC01230 was also aberrantly highly expressed in AA BM-MSC compared with controls. CONCLUSION: PPARγ and C/EBPα are aberrantly expressed in AA BM-MSC and may promote the adipogenic differentiation of AA BM-MSC, and to a certain extent mediate the bone marrow adiposity alteration by transcriptionally activating LINC01230 expression.

Mg-ZIF nanozyme regulates the switch between osteogenic and lipogenic differentiation in BMSCs via lipid metabolism.

The accumulation of reactive oxygen species (ROS) within the bone marrow microenvironment leads to diminished osteogenic differentiation and heightened lipogenic differentiation of mesenchymal stem cells residing in the bone marrow, ultimately playing a role in the development of osteoporosis (OP). Mitigating ROS levels is a promising approach to counteracting OP. In this study, a nanozyme composed of magnesium-based zeolitic imidazolate frameworks (Mg-ZIF) was engineered to effectively scavenge ROS and alleviate OP. The results of this study indicate that Mg-ZIF exhibits significant potential in scavenging ROS and effectively promoting osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Additionally, Mg-ZIF was found to inhibit the differentiation of BMSCs into adipose cells. In vivo experiments further confirmed the ability of Mg-ZIF to mitigate OP by reducing ROS levels. Mechanistically, Mg-ZIF enhances the differentiation of BMSCs into osteoblasts by upregulating lipid metabolic pathways through ROS scavenging. The results indicate that Mg-ZIF has potential as an effective therapeutic approach for the treatment of osteoporosis.

Mechanical Stretch-Induced ATP Release from Osteocytes Promotes Osteogenesis of Bone Marrow Mesenchymal Stem Cells.

BACKGROUND: Mandibular distraction osteogenesis (MDO) is a highly effective method for bone regeneration, commonly employed in treating craniofacial defects and deformities. Osteocytes sense mechanical forces in the pericellular space, relay external stimuli to biochemical changes, and send signals to other effector cells, including bone marrow mesenchymal stem cells (BM-MSCs), to regulate bone resorption and formation. Piezo1 potentially affects the secretion signal molecules of bone cells under mechanical stretch. The primary aim of this study was to enhance our comprehension of the molecular biology underlying this therapeutic approach and to identify specific signaling molecules that facilitate bone formation in response to stretch forces. METHODS: Mechanical stretching was applied to negative controls and Piezo1 knockdown osteocyte-like MLO-Y4 cells. Alkaline phosphatase and Alizarin Red S staining were used to survey the osteogenic potential of BM-MSCs. The production and secretion content of adenosine triphosphate (ATP) was measured using ATP content determination analysis. Pathway-related and osteo-specific genes and proteins were evaluated using real-time polymerase chain reaction (RT-PCR), Western blots, and immunofluorescence. Mitochondrial organization was examined with a transmission electron microscope. RESULTS: The conditioned medium of stretch-exposed MLO-Y4s significantly upregulated osteogenesis-related indicators of BM-MSCs (p < 0.001). The upregulation of BM-MSC osteogenesis was associated with ATP release from osteocytes. Mechanically induced calcium transfer and transcriptional coactivator with PDZ-binding motif (TAZ) nuclear translocation mediated by Piezo1 could promote mitochondrial fission and ATP release. Osteocytes detected stretch forces through Piezo1, triggering calcium influx, TAZ nuclear translocation, and ATP production. CONCLUSIONS: The stretch stimulation of Piezo1 induces calcium influx, which in turn promotes calcium-related TAZ nuclear translocation, changes in mitochondrial dynamics, and the release of ATP in osteocytes. This signaling cascade leads to an up-regulation in the osteogenic capacity of BM-MSCs. Mitochondrial energy metabolism of mechanosensitive protein Piezo1-dependent and ATP release may provide a new effective intervention method for mechanically related bone remodeling.

Validation of Human Bone Marrow-derived Mesenchymal Stem Cells and MCF-7 Breast Cancer Cells Co-culture Using a 3D Perfused Biomimetic Microfluidic Platform.

BACKGROUND/AIM: Microfluidic experimental models allow to study the mutual interrelation between tumor development and the microvasculature avoiding animal use and lacking interspecies differences. This study aimed to develop and characterize a 3D tissue culture model employing a two-compartment microfluidic chip-perfused platform to visualize and quantify human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and MCF-7 breast cancer cell-cell interactions in real time. MATERIALS AND METHODS: MCF-7 cells were implanted in the tumor chamber and hBM-MSCs were injected into microvascular channels. hBM-MSCs culture media was perfused into microvascular compartments. The microfluidic device was microscopically examined weekly for four weeks. RESULTS: VE- and E-cadherin immunofluorescence validated hBM-MSCs differentiation into endothelial cells and MCF-7 cell tumor formation. hBM-MSCs differentiation was highly heterogeneous along the microvascular channels, due to different perfusion flow. hBM-MSCs lining microvascular channels acquired VE-cadherin positive endothelial phenotype and continuously covered microchannels as an endothelium like layer. MCF-7 cells were constantly grown as spheroidal aggregates and later formed a compact area of E-cadherin-positive tumor cells inside tumor compartment. CONCLUSION: Our study provides valuable knowledge on the properties of hBM-MSCs as vasculogenesis-supporting cells when co-cultured with MCF-7 cells on a 3D perfused biomimetic microfluidic device. This newly established model may serve as an experimental platform for testing anti-tumor/anti-angiogenic drugs.

Stimulation of mouse hematopoietic stem cells by angiogenin and DNA preparations.

Immature hematopoietic progenitors are a constant source for renewal of hemocyte populations and the basic component of the tissue and cell repair apparatus. A unique property of these cells of internalizing extracellular double-stranded DNA has been previously shown. The leukostimulatory effect demonstrated in our pioneering studies was considered to be due to the feature of this cell. In the present research, we have analyzed the effects of DNA genome reconstructor preparation (DNAgr), DNAmix, and human recombinant angiogenin on both hematopoietic stem cells and multipotent progenitors. Treatment with bone marrow cells of experimental mice with these preparations stimulates colony formation by hematopoietic stem cells and proliferation of multipotent descendants. The main lineage responsible for this is the granulocyte-macrophage hematopoietic lineage. Using fluorescent microscopy as well as FACS assay, co-localization of primitive c-Kit- and Sca-1-positive progenitors and the TAMRA-labeled double-stranded DNA has been shown. Human recombinant angiogenin was used as a reference agent. Cells with specific markers were quantified in intact bone marrow and colonies grown in the presence of inducers. Quantitative analysis revealed that a total of 14,000 fragment copies of 500 bp, which is 0.2% of the haploid genome, can be delivered into early progenitors. Extracellular double-stranded DNA fragments stimulated the colony formation in early hematopoietic progenitors from the bone marrow, which assumed their effect on cells in G0. The observed number of Sca1+/c-Kit+ cells in colonies testifies to the possibility of both symmetrical and asymmetrical division of the initial hematopoietic stem cell and its progeny.

Comparison between the Regenerative and Therapeutic Impacts of Bone Marrow Mesenchymal Stem Cells and Adipose Mesenchymal Stem Cells Pre-Treated with Melatonin on Liver Fibrosis.

BACKGROUND: The distinctive feature of liver fibrosis is the progressive replacement of healthy hepatic cells by the extracellular matrix protein, which is abundant in collagen I and III, with impaired matrix remodeling. The activation of myofibroblastic cells enhances the fibrogenic response of complex interactions of hepatic stellate cells, fibroblasts, and inflammatory cells to produce the excessive deposition of the extracellular protein matrix. This process is activated by multiple fibrogenic mediators and cytokines, such as TNF-α and IL-1ß, accompanied with a decrease in the anti-fibrogenic factor NF-κß. Mesenchymal stem cells (MSCs) represent a promising therapy for liver fibrosis, allowing for a more advanced regenerative influence when cultured with extrinsic or intrinsic proliferative factors, cytokines, antioxidants, growth factors, and hormones such as melatonin (MT). However, previous studies showed conflicting findings concerning the therapeutic effects of adipose (AD) and bone marrow (BM) MSCs; therefore, the present work aimed to conduct a comparative and comprehensive study investigating the impact of MT pre-treatment on the immunomodulatory, anti-inflammatory, and anti-apoptotic effects of AD- and BM-MSCs and to critically analyze whether MT-pre-treated AD-MSCs and BM-MSCs reveal equal or different therapeutic and regenerative potentials in a CCl4-injured liver experimental rat model. MATERIALS AND METHODS: Six groups of experimental rats were used, with ten rats in each group: group I (control group), group II (CCl4-treated group), group III (CCl4- and BM-MSC-treated group), group IV (CCl4 and MT-pre-treated BM-MSC group), group V (CCl4- and AD-MSC-treated group), and group VI (CCl4 and MT-pre-treated AD-MSC group). Liver function tests and the gene expression of inflammatory, fibrogenic, apoptotic, and proliferative factors were analyzed. Histological and immunohistochemical changes were assessed. RESULTS: The present study compared the ability of AD- and BM-MSCs, with and without MT pre-treatment, to reduce hepatic fibrosis. Both types of MSCs improved hepatocyte function by reducing the serum levels of ALT, aspartate aminotransferase (AST), alkaline phosphatase (AKP), and total bilirubin (TBIL). In addition, the changes in the hepatocellular architecture, including the hepatocytes, liver sinusoids, central veins, portal veins, biliary ducts, and hepatic arteries, showed a decrease in hepatocyte injury and cholestasis with a reduction in inflammation, apoptosis, and necrosis of the hepatic cells, together with an inhibition of liver tissue fibrosis. These results were augmented by an analysis of the expression of the pro-inflammatory cytokines TNFα and IL-1ß, the anti-fibrogenic factor NF-κß, the apoptotic factor caspase-3, and the proliferative indicators antigen Ki-67 and proliferating cell nuclear antigen (PCNA). These findings were found to be statistically significant, with the restoration of normal parameters in the rats that received AD-MSCs pre-treated with MT, denoting optimal regenerative and therapeutic effects. CONCLUSIONS: AD-MSCs pre-treated with MT are the preferred choice in improving hepatic fibrosis and promoting the therapeutic and regenerative ability of liver tissue. They represent a very significant tool for future stem cell use in the tissue regeneration strategy for the treatment of liver diseases.