Bone marrow mesenchymal stem cell-derived exosomal microRNA-382 promotes osteogenesis in osteoblast via regulation of SLIT2.

BACKGROUND: Osteoporosis (OP) is a systemic skeletal disorder with increased bone fragility. Human bone marrow mesenchymal stem cells (hBMSCs) have multi-lineage differentiation ability, which may play important roles in osteoporosis. In this study, we aim to investigate the role of hBMSC-derived miR-382 in osteogenic differentiation. METHODS: The miRNA and mRNA expressions in peripheral blood monocytes between persons with high or low bone mineral density (BMD) were compared. Then we collected the hBMSC-secreted sEV and examined the dominant components. The over-expression of the miR-382 in MG63 cell and its progression of osteogenic differentiation were investigated by qRT-PCR, western blot and alizarin red staining. The interaction between miR-382 and SLIT2 was confirmed by dual-luciferase assay. The role of SLIT2 was also confirmed through up-regulation in MG63 cell, and the osteogenic differentiation-associated gene and protein were tested. RESULTS: According to bioinformatic analysis, a series of differential expressed genes between persons with high or low BMD were compared. After internalization of hBMSC-sEV in MG63 cells, we observed that the ability of osteogenic differentiation was significantly enhanced. Similarly, after up-regulation of miR-382 in MG63 cells, osteogenic differentiation was also promoted. According to the dual-luciferase assay, the targeting function of miR-382 in SLIT2 was demonstrated. Moreover, the benefits of hBMSC-sEV in osteogenesis were abrogated through up-regulation of SLIT2. CONCLUSION: Our study provided evidence that miR-382-contained hBMSC-sEV held great promise in osteogenic differentiation in MG63 cells after internalization by targeting SLIT2, which can be served as molecular targets to develop effective therapy.

Are the Properties of Bone Marrow-Derived Mesenchymal Stem Cells Influenced by Overweight and Obesity?

Bone marrow-derived mesenchymal stem cells (BMSCs) are promising candidates for cell-based therapies. Growing evidence has indicated that overweight/obesity can change the bone marrow microenvironment, which affects some properties of BMSCs. As the overweight/obese population rapidly increases, they will inevitably become a potential source of BMSCs for clinical application, especially when receiving autologous BMSC transplantation. Given this situation, the quality control of these cells has become particularly important. Therefore, it is urgent to characterize BMSCs isolated from overweight/obese bone marrow environments. In this review, we summarize the evidence of the effects of overweight/obesity on the biological properties of BMSCs derived from humans and animals, including proliferation, clonogenicity, surface antigen expression, senescence, apoptosis, and trilineage differentiation, as well as the underlying mechanisms. Overall, the conclusions of existing studies are not consistent. Most studies demonstrate that overweight/obesity can influence one or more characteristics of BMSCs, while the involved mechanisms are still unclear. Moreover, insufficient evidence proves that weight loss or other interventions can rescue these qualities to baseline status. Thus, further research should address these issues and prioritize developing methods to improve functions of overweight- or obesity-derived BMSCs.

Impact of Double-Stranded RNA Internalization on Hematopoietic Progenitors and Krebs-2 Cells and Mechanism.

It is well-established that double-stranded RNA (dsRNA) exhibits noticeable radioprotective and radiotherapeutic effects. The experiments conducted in this study directly demonstrated that dsRNA was delivered into the cell in its native form and that it induced hematopoietic progenitor proliferation. The 68 bp synthetic dsRNA labeled with 6-carboxyfluorescein (FAM) was internalized into mouse hematopoietic progenitors, c-Kit+ (a marker of long-term hematopoietic stem cells) cells and CD34+ (a marker of short-term hematopoietic stem cells and multipotent progenitors) cells. Treating bone marrow cells with dsRNA stimulated the growth of colonies, mainly cells of the granulocyte-macrophage lineage. A total of 0.8% of Krebs-2 cells internalized FAM-dsRNA and were simultaneously CD34+ cells. dsRNA in its native state was delivered into the cell, where it was present without any signs of processing. dsRNA binding to a cell was independent of cell charge. dsRNA internalization was related to the receptor-mediated process that requires energy from ATP. Synthetic dsRNA did not degrade in the bloodstream for at least 2 h. Hematopoietic precursors that had captured dsRNA reinfused into the bloodstream and populated the bone marrow and spleen. This study, for the first time, directly proved that synthetic dsRNA is internalized into a eukaryotic cell via a natural mechanism.

Osteoregenerative Potential of 3D-Printed Poly ε-Caprolactone Tissue Scaffolds In Vitro Using Minimally Manipulative Expansion of Primary Human Bone Marrow Stem Cells.

The repair of orthopedic and maxillofacial defects in modern medicine currently relies heavily on the use of autograft, allograft, void fillers, or other structural material composites. This study examines the in vitro osteo regenerative potential of polycaprolactone (PCL) tissue scaffolding, fabricated via a three-dimensional (3D) additive manufacturing technology, i.e., a pneumatic micro extrusion (PME) process. The objectives of this study were: (i) To examine the innate osteoinductive and osteoconductive potential of 3D-printed PCL tissue scaffolding and (ii) To perform a direct in vitro comparison of 3D-printed PCL scaffolding with allograft Allowash® cancellous bone cubes with regards to cell-scaffold interactions and biocompatibility with three primary human bone marrow (hBM) stem cell lines. This study specifically examined cell survival, cell integration, intra-scaffold cell proliferation, and differentiation of progenitor cells to investigate the potential of 3D-printed PCL scaffolds as an alternative to allograft bone material for the repair of orthopedic injuries. We found that mechanically robust PCL bone scaffolds can be fabricated via the PME process and the resulting material did not elicit detectable cytotoxicity. When the widely used osteogenic model SAOS-2 was cultured in PCL extract medium, no detectable effect was observed on cell viability or proliferation with multiple test groups showing viability ranges of 92.2% to 100% relative to a control group with a standard deviation of ±10%. In addition, we found that the honeycomb infill pattern of the 3D-printed PCL scaffold allowed for superior mesenchymal stem-cell integration, proliferation, and biomass increase. When healthy and active primary hBM cell lines, having documented in vitro growth rates with doubling times of 23.9, 24.67, and 30.94 h, were cultured directly into 3D-printed PCL scaffolds, impressive biomass increase values were observed. It was found that the PCL scaffolding material allowed for biomass increase values of 17.17%, 17.14%, and 18.18%, compared to values of 4.29% for allograph material cultured under identical parameters. It was also found that the honeycomb scaffold infill pattern was superior to the cubic and rectangular matrix structures, and provided a superior microenvironment for osteogenic and hematopoietic progenitor cell activity and auto-differentiation of primary hBM stem cells. Histological and immunohistochemical studies performed in this work confirmed the regenerative potential of PCL matrices in the orthopedic setting by displaying the integration, self-organization, and auto-differentiation of hBM progenitor cells within the matrix. Differentiation products including mineralization, self-organizing "proto-osteon" structures, and in vitro erythropoiesis were observed in conjunction with the documented expression of expected bone marrow differentiative markers including CD-99 (>70%), CD-71 (>60%), and CD-61 (>5%). All of the studies were conducted without the addition of any exogenous chemical or hormonal stimulation and exclusively utilized the abiotic and inert material polycaprolactone; setting this work apart from the vast majority of contemporary investigations into synthetic bone scaffold fabrication In summary, this study demonstrates the unique clinical potential of 3D-printed PCL scaffolds for stem cell expansion and incorporation into advanced microstructures created via PME manufacturing to generate a physiologically inert temporary bony defect graft with significant autograft features for enhanced end-stage healing.

In Vitro Generation of Murine Bone Marrow-Derived Dendritic Cells.

Dendritic cells (DCs) are mononuclear phagocytes of hematopoietic origin residing in lymphoid and nonlymphoid tissues. DCs are often referred as the sentinels of the immune system as they can sense pathogens and danger signals. Upon activation, DCs migrate to the draining lymph nodes and present antigens to naïve T cells to trigger adaptive immunity. Hematopoietic progenitors for DCs reside in the adult bone marrow (BM). Therefore, BM cell culture systems have been developed to generate large amounts of primary DCs in vitro conveniently enabling to analyze their developmental and functional features. Here, we review various protocols enabling to generate DCs in vitro from murine BM cells and discuss the cellular heterogeneity of each culture system.

Dynamics of Changes in the Properties of Multipotent Mesenchymal Stromal Cells in Patients with Acute Leukemia.

In acute leukemia, the stromal microenvironment of the bone marrow that regulates hematopoiesis is modified under the influence of malignant cells. Chemotherapy also adversely affects stromal cells. Multipotent mesenchymal stromal cells (MSC) are involved in the formation of the stromal microenvironment and in the regulation of normal and tumor hematopoietic cells. The properties of MSC from the bone marrow of patients with acute myeloid and lymphoid leukemia were studied at the onset of the disease and after achieving remission. The immunophenotype and the level of gene expression were analyzed in MSC of 34 patients. In MSC from patients with acute leukemia, the expression of CD105 and CD274 was significantly reduced in comparison with MSC from healthy donors. At the onset of the disease, the expression of IL6, JAG1, PPARG, IGF1, and PDGFRA was enhanced, while the expression of IL1B, IL8, SOX9, ANG1, and TGFB was reduced. All these changes affect the course of the disease in patients and can be the targets of therapeutic intervention.

Laser Thermo-Photobiomodulation of Bone Marrow Mesenchymal Stem Cells.

We studied the effect of laser radiation of moderate intensity with a wavelength of 970 nm on the efficiency of colony formation of rat bone marrow mesenchymal stem cells (MSC) in vitro. In this case, photobimodulation and thermal heating of MSC occur simultaneously. This combined laser treatment allows increasing the number of colonies by 6 times in comparison with the control and by more than 3 times in comparison with thermal heating alone. The mechanism of such an increase is associated with combined thermal and light effects of laser radiation of moderate intensity, which stimulates cell proliferation. This phenomenon can be used as the basis for solving the most important task of cell transplantation, associated with the expansion of autologous stem cells and activation of their proliferative potential.

Downregulated FGFR3 Expression Inhibits In Vitro Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells of Mice with TBXT Gene Mutation.

We studied the effect of fibroblast growth factor receptor 3 (FGFR3) inhibitor BGJ-398 on the differentiation of bone marrow mesenchymal stem cells (BM MSC) into osteoblasts in wild type (wt) mice and in animals with mutation in TBXT gene (mt) and possible differences in the pluripotency of these cells. Cytology tests showed that the cultured BM MSC could differentiate into osteoblasts and adipocytes. The effect of different BGJ-398 concentrations on the expression of FGFR3, RUNX2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7, and SMAD8 were studied by quantitative reverse transcription PCR. The expression of RUNX2 protein was evaluated by Western blotting. BM MSC of mt and wt mice did not differ in pluripotency and expressed the same membrane marker antigens. BGJ-398 inhibitor reduced the expression of FGFR3 and RUNX2. In BM MSC from mt and wt mice have similar gene expression (and its changing) in FGFR3, RUNX2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7, and SMAD8 genes. Thus, our experiments confirmed the effect of decreased expression of FGFR3 on osteogenic differentiation of BM MSC from wt and mt mice. However, BM MSC from mt and wt mice did not differ in pluripotency and are an adequate model for laboratory research.

Prognostic subtypes of thyroid cancer was constructed based on single cell and bulk-RNA sequencing data and verified its authenticity.

There has been an increase in the mortality rate of thyroid cancer (THCA), which is the most common endocrine malignancy. We identified six distinct cell types in the THAC microenvironment by analyzing single-cell RNA sequencing (Sc-RNAseq) data from 23 THCA tumor samples, indicating high intratumoral heterogeneity. Through re-dimensional clustering of immune subset cells, myeloid cells, cancer-associated fibroblasts, and thyroid cell subsets, we deeply reveal differences in the tumor microenvironment of thyroid cancer. Through an in-depth analysis of thyroid cell subsets, we identified the process of thyroid cell deterioration (normal, intermediate, malignant cells). Through cell-to-cell communication analysis, we found a strong link between thyroid cells and fibroblasts and B cells in the MIF signaling pathway. In addition, we found a strong correlation between thyroid cells and B cells, TampNK cells, and bone marrow cells. Finally, we developed a prognostic model based on differentially expressed genes in thyroid cells from single-cell analysis. Both in the training set and the testing set, it can effectively predict the survival of thyroid patients. In addition, we identified significant differences in the composition of immune cell subsets between high-risk and low-risk patients, which may be responsible for their different prognosis. Through in vitro experiments, we identify that knockdown of NPC2 can significantly promote thyroid cancer cell apoptosis, and NPC2 may be a potential therapeutic target for thyroid cancer. In this study, we developed a well-performing prognostic model based on Sc-RNAseq data, revealing the cellular microenvironment and tumor heterogeneity of thyroid cancer. This will help to provide more accurate personalized treatment for patients in clinical diagnosis.

Endothelial and Leptin Receptor+ cells promote the maintenance of stem cells and hematopoiesis in early postnatal murine bone marrow.

Mammalian hematopoietic stem cells (HSCs) colonize the bone marrow during late fetal development, and this becomes the major site of hematopoiesis after birth. However, little is known about the early postnatal bone marrow niche. We performed single-cell RNA sequencing of mouse bone marrow stromal cells at 4 days, 14 days, and 8 weeks after birth. Leptin-receptor-expressing (LepR+) stromal cells and endothelial cells increased in frequency during this period and changed their properties. At all postnatal stages, LepR+ cells and endothelial cells expressed the highest stem cell factor (Scf) levels in the bone marrow. LepR+ cells expressed the highest Cxcl12 levels. In early postnatal bone marrow, SCF from LepR+/Prx1+ stromal cells promoted myeloid and erythroid progenitor maintenance, while SCF from endothelial cells promoted HSC maintenance. Membrane-bound SCF in endothelial cells contributed to HSC maintenance. LepR+ cells and endothelial cells are thus important niche components in early postnatal bone marrow.

In Vitro Generation of Murine Dendritic Cells from Hoxb8-Immortalized Hematopoietic Progenitors.

Mouse dendritic cells (DCs) are routinely generated based on cells isolated form the bone marrow (BM) and cultured in the presence of growth factors that support DC development, such as FMS-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Guo et al., J Immunol Methods 432:24-29, 2016). In response to these growth factors, DC progenitors expand and differentiate, while other cell types die during the in vitro culture period, ultimately leading to relatively homogenous DC populations. An alternative method, which is discussed in detail in this chapter, relies on conditional immortalization of progenitor cells with DC potential in vitro using an estrogen-regulated form of Hoxb8 (ERHBD-Hoxb8). Such progenitors are established by retroviral transduction of largely unseparated BM cells with a retroviral vector expressing ERHBD-Hoxb8. Treatment of ERHBD-Hoxb8-expressing progenitors with estrogen results in Hoxb8 activation, which blocks cell differentiation and allows for expansion of homogenous progenitor cell populations in the presence of FLT3L. These cells, referred to as Hoxb8-FL cells, retain lineage potential for lymphocyte and myeloid lineages, including the DC lineage. Upon removal of estrogen (inactivation of Hoxb8), Hoxb8-FL cells differentiate into highly homogenous DC populations in the presence of GM-CSF or FLT3L akin to their endogenous counterparts. Given their unlimited proliferative capacity and amenability for genetic manipulation, for example, by CRISPR/Cas9, these cells provide a large number of options to investigate DC biology. Here, I am describing the method to establish Hoxb8-FL cells from mouse BM, as well as procedures for DC generation and gene deletion using lentivirally delivered CRISPR/Cas9.

In Vitro Generation of Murine CD8α+ DEC205+ XCR1+ Cross-Presenting Dendritic Cells from Bone Marrow-Derived Hematopoietic Progenitors.

Dendritic cells (DCs) comprise a heterogeneous population of antigen (Ag)-presenting cells that play a critical role in both innate and adaptive immunity. DCs orchestrate protective responses against pathogens and tumors while mediating tolerance to host tissues. Evolutionary conservation between species has allowed the successful use of murine models to identify and characterize DC types and functions relevant to human health. Among DCs, type 1 classical DCs (cDC1) are uniquely capable of inducing antitumor responses and therefore present a promising therapeutic target. However, the rarity of DCs, particularly cDC1, limits the number of cells that can be isolated for study. Despite significant effort, progress in the field has been hampered by inadequate methods to produce large quantities of functionally mature DCs in vitro. To overcome this challenge, we developed a culture system in which mouse primary bone marrow cells are cocultured with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) to produce CD8α+ DEC205+ XCR1+ cDC1 (Notch cDC1). This novel method provides a valuable tool to facilitate the generation of unlimited cDC1 for functional studies and translational applications such as antitumor vaccination and immunotherapy.

[Adoptive bone marrow cells can reduce the liver injury of metabolic-dysfunction-associated fatty liver disease in mice by differentiating into natural killer T (NKT) cells and increasing their own lipid content].

Objective To investigate the therapeutic effect of bone marrow cell adoptive therapy on metabolic-dysfunction-associated fatty liver disease (MAFLD) in mice and its possible cell population. Methods The staining was used to detect the liver lesions of MAFLD in C57BL/6 mice induced by methionine and choline deficiency diet (MCD) and the adoptive therapeutic effect of bone marrow cells on MAFLD was evaluated by detecting the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The mRNA expressions of low density lipoprotein receptor (LDLR) and interleukin-4 (IL-4) in liver immune cells (including T, NKT, Kupffer cells and other cell populations) were detected by real-time quantitative PCR. The bone marrow cells labeled with 5, 6- carboxyfluorescein diacetate succinimidyl ester (CFSE) were injected into the tail vein of mice. The proportion of CFSE positive cells in liver tissue was observed by the frozen section, and the proportion of labeled cells in the liver and spleen was tracked by flow cytometry. The expression of CD3, CD4, CD8, NK1.1, CD11b and Gr-1 in CFSE labeled adoptive cells was detected by flow cytometry. The intracellular lipid content of NKT cells in liver tissue was evaluated by Nile Red lipid staining. Results The injury of liver tissue and the levels of serum ALT and AST in MAFLD mice were significantly reduced. At the same time, liver immune cells up-regulated the expression of IL-4 and LDLR. LDLR knockout mice induced more severe MAFLD after giving MCD diet. Bone marrow adoptive cells had a significant therapeutic effect and differentiated more NKT cells to colonize the liver. At the same time, the intracellular lipids of these NKT cells increased significantly. Conclusion Bone marrow cell adoptive therapy can reduce liver injury in MAFLD mice by differentiating more NKT cells and increasing the intracellular lipid content of these cells.

[The increased expression of stromal cell-derived factor-1 (SDF-1) alleviates airway inflammation in asthmatic rats by promoting the migration of bone marrow mesenchymal stem cells (BMSCs)].

Objective To observe the correlation of stromal cell-derived factor 1 (SDF-1) with bone marrow mesenchymal stem cell (BMSCs) migration and airway inflammation in asthmatic rats. Methods Twenty-four clean SD rats were randomly divided into normal control (NC) group, model control (MC) group, and BMSCs group. Asthma model was established by OVA. In the BMSCs group, 1×106 BMSCs (1 mL) were transplanted into the tail vein on the day the model was completed. Pathological changes in lung tissues were evaluated by HE staining. The count of inflammatory cells in bronchoalveolar lavage fluid(BALF) was evaluated by Wright-Giemsa staining. The concentrations of IL-4, IL-5, IL-13, IgE, IgG1 and IgG2a in BALF were tested by ELISA. The expression of SDF-1 and STAT6 mRNA in lung tissue was measured by real time quantitative PCR. The expression of SDF-1 protein in bronchial epithelial cells were evaluated by Immunofluorescence staining. The expression of SDF-1 and STAT6 protein in lung tissue were measured by Western blot analysis. Results Compared with the normal group, the number of relative inflammatory cell counts and the concentrations of IL-4, IL-5, IL-13, IgE, IgG1, and IgG2a in BALF of the MC group increased significantly. The mRNA and protein expression of SDF-1 and STAT6 in lung tissue increased significantly. Compared with the MC group, inflammatory cells and inflammatory cytokines of BALF of BMSCs group were decreased in numbers, as was the expression of SDF-1 and STAT6 in lung tissues. Compared with the MC group, the expression of SDF-1 gene in lung tissues was increased, as was the expression of SDF-1 protein in bronchial epithelial cells. Conclusion In the process of asthmatic inflammation, the expression of chemokine SDF-1 in the damaged site increases, and promotes the migration of exogenous BMSCs to the lung tissue of asthmatic rats. BMSCs can regulate immune imbalance of Th1/Th2 cells by homing to damaged lung tissue, thus inhibiting asthmatic airway inflammation.

Bone marrow-derived dedifferentiated fat cells exhibit similar phenotype as bone marrow mesenchymal stem cells with high osteogenic differentiation and bone regeneration ability.

BACKGROUND: Mesenchymal stem cells (MSCs) are known to have different differentiation potential depending on the tissue of origin. Dedifferentiated fat cells (DFATs) are MSC-like multipotent cells that can be prepared from mature adipocytes by ceiling culture method. It is still unknown whether DFATs derived from adipocytes in different tissue showed different phenotype and functional properties. In the present study, we prepared bone marrow (BM)-derived DFATs (BM-DFATs), BM-MSCs, subcutaneous (SC) adipose tissue-derived DFATs (SC-DFATs), and adipose tissue-derived stem cells (ASCs) from donor-matched tissue samples. Then, we compared their phenotypes and multilineage differentiation potential in vitro. We also evaluated in vivo bone regeneration ability of these cells using a mouse femoral fracture model. METHODS: BM-DFATs, SC-DFATs, BM-MSCs, and ASCs were prepared from tissue samples of knee osteoarthritis patients who received total knee arthroplasty. Cell surface antigens, gene expression profile, and in vitro differentiation capacity of these cells were determined. In vivo bone regenerative ability of these cells was evaluated by micro-computed tomography imaging at 28 days after local injection of the cells with peptide hydrogel (PHG) in the femoral fracture model in severe combined immunodeficiency mice. RESULTS: BM-DFATs were successfully generated at similar efficiency as SC-DFATs. Cell surface antigen and gene expression profiles of BM-DFATs were similar to those of BM-MSCs, whereas these profiles of SC-DFATs were similar to those of ASCs. In vitro differentiation analysis revealed that BM-DFATs and BM-MSCs had higher differentiation tendency toward osteoblasts and lower differentiation tendency toward adipocytes compared to SC-DFATs and ASCs. Transplantation of BM-DFATs and BM-MSCs with PHG enhanced bone mineral density at the injection sites compared to PHG alone in the mouse femoral fracture model. CONCLUSIONS: We showed that phenotypic characteristics of BM-DFATs were similar to those of BM-MSCs. BM-DFATs exhibited higher osteogenic differentiation potential and bone regenerative ability compared to SC-DFATs and ASCs. These results suggest that BM-DFATs may be suitable sources of cell-based therapies for patients with nonunion bone fracture.

Choosing the source of healthy controls for studies on myeloid malignancies: all bone marrow cells are created equal, but some are more equal than others.

Bone marrow samples from discarded femoral heads are often used as healthy controls in studies investigating the in vitro characteristics of cells from patients with hematologic malignancies. Since patient samples are usually derived from iliac crest aspirates, this carries the risk that the properties of the cells from both sources might be different due to the site and method of harvesting. Comparing BM cells from iliac crest aspirates and femoral heads from age-matched healthy donors, we show that, while mesenchymal stromal cells have indistinguishable properties between both sources, hematopoietic stem and progenitor cells (HSPC) from femoral heads show a considerable proliferative advantage in vitro. These data therefore suggest that experiments comparing leukemic cells from the iliac crest to healthy HSPC obtained from femoral heads should be interpreted with caution.

Fibulin2: a negative regulator of BMSC osteogenic differentiation in infected bone fracture healing.

Bone fracture remains a common occurrence, with a population-weighted incidence of approximately 3.21 per 1000. In addition, approximately 2% to 50% of patients with skeletal fractures will develop an infection, one of the causes of disordered bone healing. Dysfunction of bone marrow mesenchymal stem cells (BMSCs) plays a key role in disordered bone repair. However, the specific mechanisms underlying BMSC dysfunction caused by bone infection are largely unknown. In this study, we discovered that Fibulin2 expression was upregulated in infected bone tissues and that BMSCs were the source of infection-induced Fibulin2. Importantly, Fibulin2 knockout accelerated mineralized bone formation during skeletal development and inhibited inflammatory bone resorption. We demonstrated that Fibulin2 suppressed BMSC osteogenic differentiation by binding to Notch2 and inactivating the Notch2 signaling pathway. Moreover, Fibulin2 knockdown restored Notch2 pathway activation and promoted BMSC osteogenesis; these outcomes were abolished by DAPT, a Notch inhibitor. Furthermore, transplanted Fibulin2 knockdown BMSCs displayed better bone repair potential in vivo. Altogether, Fibulin2 is a negative regulator of BMSC osteogenic differentiation that inhibits osteogenesis by inactivating the Notch2 signaling pathway in infected bone.

Engineered Bone Marrow Stem Cell-Sheets Alleviate Renal Damage in a Rat Chronic Glomerulonephritis Model.

Although mesenchymal stem cell (MSC)-based regenerative therapy is being developed for the treatment of kidney diseases, cell delivery and engraftment still need to be improved. Cell sheet technology has been developed as a new cell delivery method, to recover cells as a sheet form retaining intrinsic cell adhesion proteins, which promotes its transplantation efficiency to the target tissue. We thus hypothesized that MSC sheets would therapeutically reduce kidney disease with high transplantation efficiency. When the chronic glomerulonephritis was induced by two injections of the anti-Thy 1.1 antibody (OX-7) in rats, the therapeutic efficacy of rat bone marrow stem cell (rBMSC) sheet transplantation was evaluated. The rBMSC-sheets were prepared using the temperature-responsive cell-culture surfaces and transplanted as patches onto the surface of two kidneys of each rat at 24 h after the first injection of OX-7. At 4 weeks, retention of the transplanted MSC-sheets was confirmed, and the animals with MSC-sheets showed significant reductions in proteinuria, glomerular staining for extracellular matrix protein, and renal production of TGFß1, PAI-1, collagen I, and fibronectin. The treatment also ameliorated podocyte and renal tubular injury, as evidenced by a reversal in the reductions of WT-1, podocin, and nephrin and by renal overexpression of KIM-1 and NGAL. Furthermore, the treatment enhanced gene expression of regenerative factors, and IL-10, Bcl-2, and HO-1 mRNA levels, but reduced TSP-1 levels, NF-kB, and NAPDH oxidase production in the kidney. These results strongly support our hypothesis that MSC-sheets facilitated MSC transplantation and function, and effectively retarded progressive renal fibrosis via paracrine actions on anti-cellular inflammation, oxidative stress, and apoptosis and promoted regeneration.