[Zinc finger protein-36 deficiency inhibits osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells and preosteoblasts by activating the ERK/MAPK pathway].

OBJECTIVE: To explore the role of zinc finger protein 36(ZFP36) in regulating osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and preosteoblasts. METHODS: ZFP36 expression was observed in primary mouse BMSCs and mouse preosteoblasts (MC3T3-E1 cells) during induced osteogenic differentiation. Zfp36-deficient cell models were constructed in the two cells using RNA interference technique and the changes in differentiation capacities of the transfected cells into osteoblasts were observed. Transcriptome sequencing was used to investigate the potential mechanisms of ZFP36 for regulating osteoblast differentiation of the two cells. U0126, a ERK/MAPK signal suppressor, was used to verify the regulatory mechanism of Zfp36 in osteogenic differentiation of Zfp36-deficient cells. RESULTS: During the 14-day induction of osteogenic differentiation, both mouse BMSCs and MC3T3-E1 cells exhibited increased expression of ZFP36, and its mRNA expression reached the peak level on Day 7(P < 0.0001). The Zfp36-deficient cell models showed reduced intensity of alkaline phosphatase (ALP) staining and alizarin red staining with significantly lowered expressions of the osteogenic marker genes including Alpl, Sp7, Bglap and Ibsp (P < 0.01). Transcriptome sequencing verified the reduction of bone mineralization-related gene expressions in Zfp36-deficient cells and indicated the involvement of ERK signaling in the potential regulatory mechanism of Zfp36. Immunoblotting showed that pERK protein expression increased significantly in Zfp36-deficient cells compared with the control cells. In Zfp36-deficient MC3T3-E1 cells, inhibition of activated ERK/MAPK signaling with U0126 resulted in obviously enhanced ALP staining and significantly increased expressions of osteoblast differentiation markers Runx2 and Bglap (P < 0.05). CONCLUSIONS: ZFP36 is involved in the regulation of osteoblast differentiation of mouse BMSCs and preosteoblasts, and ZFP36 deficiency causes inhibition of osteoblast differentiation of the cells by activating the ERK/MAPK signaling pathway.

Aggregation of human osteoblasts unlocks self-reliant differentiation and constitutes a microenvironment for 3D-co-cultivation with other bone marrow cells.

Skeletal bone function relies on both cells and cellular niches, which, when combined, provide guiding cues for the control of differentiation and remodeling processes. Here, we propose an in vitro 3D model based on human fetal osteoblasts, which eases the study of osteocyte commitment in vitro and thus provides a means to examine the influences of biomaterials, substances or cells on the regulation of these processes. Aggregates were formed from human fetal osteoblasts (hFOB1.19) and cultivated under proliferative, adipo- and osteoinductive conditions. When cultivated under osteoinductive conditions, the vitality of the aggregates was compromised, the expression levels of the mineralization-related gene DMP1 and the amount of calcification and matrix deposition were lower, and the growth of the spheroids stalled. However, within spheres under growth conditions without specific supplements, self-organization processes occur, which promote extracellular calcium deposition, and osteocyte-like cells develop. Long-term cultivated hFOB aggregates were free of necrotic areas. Moreover, hFOB aggregates cultivated under standard proliferative conditions supported the co-cultivation of human monocytes, microvascular endothelial cells and stromal cells. Overall, the model presented here comprises a self-organizing and easily accessible 3D osteoblast model for studying bone marrow formation and in vitro remodeling and thus provides a means to test druggable molecular pathways with the potential to promote life-long bone formation and remodeling.

Recruitment of plasma cells from IL-21-dependent and IL-21-independent immune reactions to the bone marrow.

Bone marrow plasma cells (BMPC) are the correlate of humoral immunity, consistently releasing antibodies into the bloodstream. It remains unclear if BMPC reflect different activation environments or maturation of their precursors. Here we define human BMPC heterogeneity and track the recruitment of antibody-secreting cells (ASC) from SARS-CoV-2 vaccine immune reactions to the bone marrow (BM). Trajectories based on single-cell transcriptomes and repertoires of peripheral and BM ASC reveal sequential colonisation of BMPC compartments. In activated B cells, IL-21 suppresses CD19 expression, indicating that CD19low-BMPC are derived from follicular, while CD19high-BMPC originate from extrafollicular immune reactions. In primary immune reactions, both CD19low- and CD19high-BMPC compartments are populated. In secondary immune reactions, most BMPC are recruited to CD19high-BMPC compartments, reflecting their origin from extrafollicular reactivations of memory B cells. A pattern also observable in vaccinated-convalescent individuals and upon diphtheria/tetanus/pertussis recall-vaccination. Thus, BMPC diversity reflects the evolution of a given humoral immune response.

Technology Behind Cell Therapy Augmentation of Fracture Healing: Concentrated Bone Marrow Aspirate.

With an aging population, and an anticipated increase in overall fracture incidence, a sound understanding of bone healing and how technology can optimize this process is crucial. Concentrated bone marrow aspirate (cBMA) is a technology that capitalizes on skeletal stem and progenitor cells (SSPCs) to enhance the regenerative capacity of bone. This overview highlights the science behind cBMA, discusses the role of SSPCs in bone homeostasis and fracture repair, and briefly details the clinical evidence supporting the use of cBMA in fracture healing. Despite promising early clinical results, a lack of standardization in harvest and processing techniques, coupled with patient variability, presents challenges in optimizing the use of cBMA. However, cBMA remains an emerging technology that may certainly play a crucial role in the future of fracture healing augmentation.

Unique Approach for Isolating Rat Bone Marrow Neutrophils with Comparable Capacity.

The primary aim of this research was to develop a reliable and efficient approach for isolating neutrophil extracellular traps (NETs) from rat bone marrow. This effort arose due to limitations associated with the traditional method of extracting NETs from peripheral blood, mainly due to the scarcity of available neutrophils for isolation. The study revealed two distinct methodologies for obtaining rat neutrophils from bone marrow: a streamlined one-step procedure that yielded satisfactory purification levels, and a more time-intensive two-step process that exhibited enhanced purification efficiency. Importantly, both techniques yielded a substantial quantity of viable neutrophils, ranging between 50 to 100 million per rat. This efficiency mirrored the results obtained from isolating neutrophils from both human and murine sources. Significantly, neutrophils derived from rat bone marrow exhibited comparable abilities to secrete NETs when compared with neutrophils obtained from peripheral blood. However, the bone marrow-based method consistently produced notably larger quantities of both neutrophils and NETs. This approach demonstrated the potential to obtain significantly greater amounts of these cellular components for further downstream applications. Notably, these isolated NETs and neutrophils hold promise for a range of applications, spanning the realms of inflammation, infection, and autoimmune diseases.

The effects of exosomes originating from different cell sources on the differentiation of bone marrow mesenchymal stem cells into schwann cells.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can differentiate into Schwann cells (SCs) during peripheral nerve injury; in our previous research, we showed that SC-derived exosomes (SC-exos) played a direct induction role while fibroblast-derived exosomes (Fb-exos) had no obvious induction role. The induction role of neural stem cell (NSC)-derived exosomes (NSC-exos) has also been widely confirmed. However, no studies have compared the induction effects of these three types of cells at the same time. Therefore, by investigating the effect of these three cell-derived exosomes upon the induction of BMSCs to differentiate into SCs, this study explored the role of different exosomes in promoting the differentiation of stem cells into SCs cells, and conducted a comparison between the two groups by RNA sequencing to further narrow the range of target genes and related gene pathways in order to study their related mechanisms. MATERIALS AND METHODS: We extracted exosomes from SCs, fibroblasts (Fb) and neural stem cells (NSC) and then investigated the ability of these exosomes to induce differentiation into BMSCs under different culture conditions. The expression levels of key proteins and gene markers were detected in induced cells by fluorescence immunoassays, western blotting and polymerase chain reaction (PCR); then, we statistically compared the relative induction effects under different conditions. Finally, we analyzed the three types of exosomes by RNA-seq to predict target genes and related gene pathways. RESULTS: BMSCs were cultured by three media: conventional (no induction), pre-induction or pre-induction + original induction medium (ODM) with exosomes of the same cell origin under different culture conditions. When adding the three different types of exosomes separately, the overall induction of BMSCs to differentiate into SCs was significantly increased (P < 0.05). The induction ability was ranked as follows: pre-induction + ODM + exosome group > pre-induction + exosome group > non-induction + exosome group. Using exosomes from different cell sources under the same culture conditions, we observed the following trends under the three culture conditions: RSC96-exos group ≥ NSC-exos group > Fb-exos group. The overall ability to induce BMSCs into SCs was significantly greater in the RSC96-exos group and the NSC-exos group. Although there was no significant difference in induction efficiency when comparing these two groups, the overall induction ability of the RSC96-exos group was slightly higher than that of the NSC-exos group. By combining the differentiation induction results with the RNA-seq data, the three types of exosomes were divided into three comparative groups: RSC vs. NSC, RSC vs. Fb and NSC vs. Fb. We identified 203 differentially expressed mRNA target genes in these three groups. Two differentially expressed genes were upregulated simultaneously, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). We did not identify any co-upregulated target genes for the miRNAs, but did identify one target gene of the lncRNAs, namely ENSRNOG00000065005. Analysis identified 90 GO terms related to nerves and axons in the mRNAs; in addition, KEGG enrichment and GASA analysis identified 13 common differential expression pathways in the three groups. CONCLUSIONS: Our analysis found that pre-induction + ODM + RSC96/NSC-exos culture conditions were most conducive with regards to induction and differentiation. RSC96-exos and NSC-exos exhibited significantly greater differentiation efficiency of BMSCs into SCs. Although there was no statistical difference, the data indicated a trend for RSC96-exos to be advantageous We identified 203 differentially expressed mRNAs between the three groups and two differentially expressed target mRNAs were upregulated, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). 90 GO terms were related to nerves and axons. Finally, we identified 13 common differentially expressed pathways across our three types of exosomes. It is hoped that the efficiency of BMSCs induction differentiation into SCs can be improved, bringing hope to patients and more options for clinical treatment.

Bone marrow mesenchymal stem cell-derived exosomes shuttle microRNAs to endometrial stromal fibroblasts that promote tissue proliferation /regeneration/ and inhibit differentiation.

BACKGROUND: Human bone marrow-derived stem cells (hBMDSCs) are well characterized mediators of tissue repair and regeneration. An increasing body of evidence indicates that these cells exert their therapeutic effects largely through their paracrine actions rather than clonal expansion and differentiation. Here we studied the role of microRNAs (miRNAs) present in extracellular vesicles (EVs) from hBMDSCs in tissue regeneration and cell differentiation targeting endometrial stromal fibroblasts (eSF). METHODS: Extracellular vesicles (EVs) are isolated from hBMDSCs, characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) techniques. Extracted total RNA from EVs was subjected to RNA seq analysis. Transfection and decidualization studies were carried out in endometrial stromal fibroblasts (eSF). Gene expression was analyzed by qRTPCR. Unpaired t-test with Welch's correction was used for data analysis between two groups. RESULTS: We identified several microRNAs (miRNAs) that were highly expressed, including miR-21-5p, miR-100-5p, miR-143-3p and let7. MiR-21 is associated with several signaling pathways involved in tissue regeneration, quiescence, cellular senescence, and fibrosis. Both miR-100-5p and miR-143-3p promoted cell proliferation. MiR-100-5p specifically promoted regenerative processes by upregulating TGF-ß3, VEGFA, MMP7, and HGF. MiR-100-5p blocked differentiation or decidualization as evidenced by morphologic changes and downregulation of decidualization mediators including HOXA10, IGFBP1, PRL, PR-B, and PR. CONCLUSION: EVs delivered to tissues by hBMDSCs contain specific miRNAs that prevent terminal differentiation and drive repair and regeneration. Delivery of microRNAs is a novel treatment paradigm with the potential to replace BMDSCs in cell-free regenerative therapies.

Persistent Mesodermal Differentiation Capability of Bone Marrow MSCs Isolated from Aging Patients with Low-Energy Traumatic Hip Fracture and Osteoporosis: A Clinical Evidence.

A low-energy hit, such as a slight fall from a bed, results in a bone fracture, especially in the hip, which is a life-threatening risk for the older adult and a heavy burden for the social economy. Patients with low-energy traumatic bone fractures usually suffer a higher level of bony catabolism accompanied by osteoporosis. Bone marrow-derived stem cells (BMSCs) are critical in osteogenesis, leading to metabolic homeostasis in the healthy bony microenvironment. However, whether the BMSCs derived from the patients who suffered osteoporosis and low-energy traumatic hip fractures preserve a sustained mesodermal differentiation capability, especially in osteogenesis, is yet to be explored in a clinical setting. Therefore, we aimed to collect BMSCs from clinical hip fracture patients with osteoporosis, followed by osteogenic differentiation comparison with BMSCs from healthy young donors. The CD markers identification, cytokines examination, and adipogenic differentiation were also evaluated. The data reveal that BMSCs collected from elderly osteoporotic patients secreted approximately 122.8 pg/mL interleukin 6 (IL-6) and 180.6 pg/mL vascular endothelial growth factor (VEGF), but no PDGF-BB, IL-1b, TGF-b1, IGF-1, or TNF-α secretion. The CD markers and osteogenic and adipogenic differentiation capability in BMSCs from these elderly osteoporotic patients and healthy young donors are equivalent and compliant with the standards defined by the International Society of Cell Therapy (ISCT). Collectively, our data suggest that the elderly osteoporotic patients-derived BMSCs hold equivalent differentiation and proliferation capability and intact surface markers identical to BMSCs collected from healthy youth and are available for clinical cell therapy.

Exosomes derived from TNF-α-treated bone marrow mesenchymal stem cells ameliorate myocardial infarction injury in mice.

Exosomes derived from bone marrow mesenchymal stem cells (BMSCs) exhibit considerable therapeutic potential for myocardial regeneration. In our investigation, we delved into their impact on various aspects of myocardial infarction (MI), including cardiac function, tissue damage, inflammation, and macrophage polarization in a murine model. We meticulously isolated the exosomes from TNF-α-treated BMSCs and evaluated their therapeutic efficacy in a mouse MI model induced by coronary artery ligation surgery. Our comprehensive analysis, incorporating ultrasound, serum assessment, Western blot, and qRT-PCR, revealed that exosomes from TNF-α-treated BMSCs demonstrated significant therapeutic potential in reducing MI-induced injury. Treatment with these exosomes resulted in improved cardiac function, reduced infarct area, and increased left ventricular wall thickness in MI mice. On a mechanistic level, exosome treatment fostered M2 macrophage polarization while concurrently suppressing M1 polarization. Hence, exosomes derived from TNF-α-treated BMSCs emerge as a promising therapeutic strategy for alleviating MI injury in a mouse model.

Plasma-Based Scaffold Containing Bone-Marrow Mononuclear Cells Promotes Wound Healing in a Mouse Model of Pressure Injury.

Pressure injuries, or pressure ulcers, are a common problem that may lead to infections and major complications, besides being a social and economic burden due to the costs of treatment and hospitalization. While surgery is sometimes necessary, this also has complications such as recurrence or wound dehiscence. Among the newer methods of pressure injury treatment, advanced therapies are an interesting option. This study examines the healing properties of bone marrow mononuclear cells (BM-MNCs) embedded in a plasma-based scaffold in a mouse model. Pressure ulcers were created on the backs of mice (2 per mouse) using magnets and assigned to a group of ulcers that were left untreated (Control, n = 15), treated with plasma scaffold (Plasma, n = 15), or treated with plasma scaffold containing BM-MNC (Plasma + BM-MNC, n = 15). Each group was examined at three time points (3, 7, and 14 days) after the onset of treatment. At each time point, animals were subjected to biometric assessment, bioluminescence imaging, and tomography. Once treatment had finished, skin biopsies were processed for histological and wound healing reverse transcription polymerase chain reaction (RT-PCR) array studies. While wound closure percentages were higher in the Plasma and Plasma + BM-MNC groups, differences were not significant, and thus descriptive data are provided. In all individuals, the presence of donor cells was revealed by immunohistochemistry on posttreatment onset Days 3, 7, and 14. In the Plasma + BM-MNC group, less inflammation was observed by positron emission tomography-computed tomography (PET/CT) imaging of the mice at 7 days, and a complete morphometabolic response was produced at 14 days, in accordance with histological results. A much more pronounced inflammatory process was observed in controls than in the other two groups, and this persisted until Day 14 after treatment onset. RT-PCR array gene expression patterns were also found to vary significantly, with the greatest difference noted between both treatments at 14 days when 11 genes were differentially expressed.

Systematic review and meta-analysis of the effect of bone marrow-derived cell therapies on hind limb perfusion.

Preclinical and clinical studies on the administration of bone marrow-derived cells to restore perfusion show conflicting results. We conducted a systematic review and meta-analysis on preclinical studies to assess the efficacy of bone marrow-derived cells in the hind limb ischemia model and identify possible determinants of therapeutic efficacy. In vivo animal studies were identified using a systematic search in PubMed and EMBASE on 10 January 2022. 85 studies were included for systematic review and meta-analysis. Study characteristics and outcome data on relative perfusion were extracted. The pooled mean difference was estimated using a random effects model. Risk of bias was assessed for all included studies. We found a significant increase in perfusion in the affected limb after administration of bone marrow-derived cells compared to that in the control groups. However, there was a high heterogeneity between studies, which could not be explained. There was a high degree of incomplete reporting across studies. We therefore conclude that the current quality of preclinical research is insufficient (low certainty level as per GRADE assessment) to identify specific factors that might improve human clinical trials.

rhBMP-2 induces terminal differentiation of human bone marrow mesenchymal stromal cells only by synergizing with other signals.

BACKGROUND: Recombinant human bone morphogenetic protein 2 (rhBMP-2) and human bone marrow mesenchymal stromal cells (hBM-MSCs) have been thoroughly studied for research and translational bone regeneration purposes. rhBMP-2 induces bone formation in vivo, and hBM-MSCs are its target, bone-forming cells. In this article, we studied how rhBMP-2 drives the multilineage differentiation of hBM-MSCs both in vivo and in vitro. METHODS: rhBMP-2 and hBM-MSCs were tested in an in vivo subcutaneous implantation model to assess their ability to form mature bone and undergo multilineage differentiation. Then, the hBM-MSCs were treated in vitro with rhBMP-2 for short-term or long-term cell-culture periods, alone or in combination with osteogenic, adipogenic or chondrogenic media, aiming to determine the role of rhBMP-2 in these differentiation processes. RESULTS: The data indicate that hBM-MSCs respond to rhBMP-2 in the short term but fail to differentiate in long-term culture conditions; these cells overexpress the rhBMP-2 target genes DKK1, HEY-1 and SOST osteogenesis inhibitors. However, in combination with other differentiation signals, rhBMP-2 acts as a potentiator of multilineage differentiation, not only of osteogenesis but also of adipogenesis and chondrogenesis, both in vitro and in vivo. CONCLUSIONS: Altogether, our data indicate that rhBMP-2 alone is unable to induce in vitro osteogenic terminal differentiation of hBM-MSCs, but synergizes with other signals to potentiate multiple differentiation phenotypes. Therefore, rhBMP-2 triggers on hBM-MSCs different specific phenotype differentiation depending on the signalling environment.

Comparative evaluation of bone marrow and dental pulp mesenchymal stem cells for motor functional recovery in rat sciatic nerve injury.

This study delves into the impact of mesenchymal stem cells derived from bone marrow (BM-MSCs) and those sourced from dental pulp (DP-MSCs) on the recovery of motor function and morphological aspects of the rat's sciatic nerve after crush injuries. The findings highlight that the groups treated with BM-MSCs, DP-MSCs or a combination of both (BM + DP-MSCs) displayed enhanced sciatic functional index values when juxtaposed with the sham group. This points to bettered motor functionalities. A deeper morphological analysis showed that all the groups had retained perineurium structure and fascicular arrangement. Notably, the sham and BM-MSCs groups had very few inconsistencies. All groups showed standard vascular density. Remarkably, the combined treatment group (BM + DP-MSCs) presented diminished oedema and a lower count of inflammatory cells. Through immunohistochemical methods, the presence of S100 expression was noted in the groups that underwent treatment. In summation, the study suggests that both BM-MSCs and DP-MSCs, whether used singly or in combination, can significantly aid in motor function restoration and morphological enhancements. An interesting observation from our research and earlier studies is that stem cells from dental pulp, which are sourced with less discomfort from milk and wisdom teeth, show a heightened propensity to evolve into nerve cells. This is in contrast to the more uncomfortably acquired BM-MSCs.

[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.

A Senescence-Associated Secretory Phenotype of Bone Marrow Mesenchymal Stem Cells Inhibits the Viability of Breast Cancer Cells.

Breast cancer, the most prevalent malignancy in women, often progresses to bone metastases, especially in older individuals. Dormancy, a critical aspect of bone-metastasized breast cancer cells (BCCs), enables them to evade treatment and recur. This dormant state is regulated by bone marrow mesenchymal stem cells (BMMSCs) through the secretion of various factors, including those associated with senescence. However, the specific mechanisms by which BMMSCs induce dormancy in BCCs remain unclear. To address this gap, a bone-specific senescence-accelerated murine model, SAMP6, was utilized to minimize confounding systemic age-related factors. Confirming senescence-accelerated osteoporosis, distinct BMMSC phenotypes were observed in SAMP6 mice compared to SAMR1 counterparts. Notably, SAMP6-BMMSCs exhibited premature senescence primarily due to telomerase activity loss and activation of the p21 signaling pathway. Furthermore, the effects of conditioned medium (CM) derived from SAMP6-BMMSCs versus SAMR1-BMMSCs on BCC proliferation were examined. Intriguingly, only CM from SAMP6-BMMSCs inhibited BCC proliferation by upregulating p21 expression in both MCF-7 and MDA-MB-231 cells. These findings suggest that the senescence-associated secretory phenotype (SASP) of BMMSCs suppresses BCC viability by inducing p21, a pivotal cell cycle inhibitor and tumor suppressor. This highlights a heightened susceptibility of BCCs to dormancy in a senescent microenvironment, potentially contributing to the increased incidence of breast cancer bone metastasis and recurrence observed with aging.

Bone Marrow Nucleated Cells and Bone Marrow-Derived CD271+ Mesenchymal Stem Cell in Treatment of Encephalopathy and Drug-Resistant Epilepsy.

The broad spectrum of brain injuries in preterm newborns and the plasticity of the central nervous system prompts us to seek solutions for neurodegeneration to prevent the consequences of prematurity and perinatal problems. The study aimed to evaluate the safety and efficacy of the implantation of autologous bone marrow nucleated cells and bone marrow mesenchymal stem cells in different schemes in patients with hypoxic-ischemic encephalopathy and immunological encephalopathy. Fourteen patients received single implantation of bone marrow nucleated cells administered intrathecally and intravenously, followed by multiple rounds of bone marrow mesenchymal stem cells implanted intrathecally, and five patients were treated only with repeated rounds of bone marrow mesenchymal stem cells. Seizure outcomes improved in most cases, including fewer seizures and status epilepticus and reduced doses of antiepileptic drugs compared to the period before treatment. The neuropsychological improvement was more frequent in patients with hypoxic-ischemic encephalopathy than in the immunological encephalopathy group. Changes in emotional functioning occurred with similar frequency in both groups of patients. In the hypoxic-ischemic encephalopathy group, motor improvement was observed in all patients and the majority in the immunological encephalopathy group. The treatment had manageable toxicity, mainly mild to moderate early-onset adverse events. The treatment was generally safe in the 4-year follow-up period, and the effects of the therapy were maintained after its termination.

Complementary and Inducible creERT2 Mouse Models for Functional Evaluation of Endothelial Cell Subtypes in the Bone Marrow.

In the adult bone marrow (BM), endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche, which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM, distinct vascular arteriole, transitional, and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However, the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover, constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this, we describe two tamoxifen-inducible cre-expressing lines, Vegfr3-creERT2 and Cx40-creERT2, that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow, without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre-dependent recombination constitutively-activates MAPK signaling within adult endothelium, we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creERT2-expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM, providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis.

A self-amplifying loop of TP53INP1 and P53 drives oxidative stress-induced apoptosis of bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cell (BMSC) transplantation is a promising regenerative therapy; however, the survival rate of BMSCs after transplantation is low. Oxidative stress is one of the main reasons for the high apoptosis rate of BMSCs after transplantation, so there is an urgent need to explore the mechanism of oxidative stress-induced apoptosis of BMSCs. Our previous transcriptome sequencing results suggested that the expression of P53-induced nuclear protein 1 (TP53INP1) and the tumor suppressor P53 (P53) was significantly upregulated during the process of oxidative stress-induced apoptosis of BMSCs. The present study further revealed the role and mechanism of TP53INP1 and P53 in oxidative stress-induced apoptosis in BMSCs. Overexpression of TP53INP1 induced apoptosis of BMSCs, knockdown of TP53INP1 alleviated oxidative stress apoptosis of BMSCs. Under oxidative stress conditions, P53 is regulated by TP53INP1, while P53 can positively regulate the expression of TP53INP1, so the two form a positive feedback loop. To clarify the mechanism of feedback loop formation. We found that TP53INP1 inhibited the ubiquitination and degradation of P53 by increasing the phosphorylation level of P53, leading to the accumulation of P53 protein. P53 can act on the promoter of the TP53INP1 gene and increase the expression of TP53INP1 through transcriptional activation. This is the first report on a positive feedback loop formed by TP53INP1 and P53 under oxidative stress. The present study clarified the formation mechanism of the positive feedback loop. The TP53INP1-P53 positive feedback loop may serve as a potential target for inhibiting oxidative stress-induced apoptosis in BMSCs.

KDELR2 promotes bone marrow mesenchymal stem cell osteogenic differentiation via GSK3ß/ß-catenin signaling pathway.

Nonunion is a challenging complication of fractures for the surgeon. Recently the Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum protein retention receptor 2 (KDELR2) has been found that involved in osteogenesis imperfecta. However, the exact mechanism is still unclear. In this study, we used lentivirus infection and mouse fracture model to investigate the role of KDELR2 in osteogenesis. Our results showed that KDELR2 knockdown inhibited the osteogenic differentiation of mBMSCs, whereas KDELR2 overexpression had the opposite effect. Furthermore, the levels of active-ß-catenin and phospho-GSK3ß (Ser9) were upregulated by KDELR2 overexpression and downregulated by KDELR2 knockdown. In the fracture model, mBMSCs overexpressing KDELR2 promoted healing. In conclusion, KDELR2 promotes the osteogenesis of mBMSCs by regulating the GSK3ß/ß-catenin signaling pathway.

The nuclear factor ID3 endows macrophages with a potent anti-tumour activity.

Macrophage activation is controlled by a balance between activating and inhibitory receptors1-7, which protect normal tissues from excessive damage during infection8,9 but promote tumour growth and metastasis in cancer7,10. Here we report that the Kupffer cell lineage-determining factor ID3 controls this balance and selectively endows Kupffer cells with the ability to phagocytose live tumour cells and orchestrate the recruitment, proliferation and activation of natural killer and CD8 T lymphoid effector cells in the liver to restrict the growth of a variety of tumours. ID3 shifts the macrophage inhibitory/activating receptor balance to promote the phagocytic and lymphoid response, at least in part by buffering the binding of the transcription factors ELK1 and E2A at the SIRPA locus. Furthermore, loss- and gain-of-function experiments demonstrate that ID3 is sufficient to confer this potent anti-tumour activity to mouse bone-marrow-derived macrophages and human induced pluripotent stem-cell-derived macrophages. Expression of ID3 is therefore necessary and sufficient to endow macrophages with the ability to form an efficient anti-tumour niche, which could be harnessed for cell therapy in cancer.