Bone Marrow Multipotent Stromal Cells Research Articles

Bone marrow mesenchymal stromal cells support regeneration of intestinal damage in a colitis mouse model, independent of their CXCR4 expression.

Inflammatory bowel disease (IBD) is characterized by a chronically dysregulated immune response in the gastrointestinal tract. Bone marrow multipotent mesenchymal stromal cells have an important immunomodulatory function and support regeneration of inflamed tissue by secretion of soluble factors as well as through direct local differentiation. CXCR4 is the receptor for CXCL12 (SDF-1, stromal-derived factor-1) and has been shown to be the main chemokine receptor, required for homing of MSCs. Increased expression of CXCL12 by inflamed intestinal tissue causes constitutive inflammation by attracting lymphocytes but can also be used to direct MSCs to sites of injury/inflammation. Trypsin is typically used to dissociate MSCs into single-cell suspensions but has also been shown to digest surface CXCR4. Here, we assessed the regenerative effects of CXCR4high and CXCR4low MSCs in an immune-deficient mouse model of DSS-induced colitis. We found that transplantation of MSCs resulted in clinical improvement and histological recovery of intestinal epithelium. In contrary to our expectations, the levels of CXCR4 on transplanted MSCs did not affect their regenerative supporting potential, indicating that paracrine effects of MSCs may be largely responsible for their regenerative/protective effects.

Acute Myeloid Leukemia Causes Serious and Partially Irreversible Changes in Secretomes of Bone Marrow Multipotent Mesenchymal Stromal Cells.

In patients with acute myeloid leukemia (AML), malignant cells modify the properties of multipotent mesenchymal stromal cells (MSCs), reducing their ability to maintain normal hematopoiesis. The aim of this work was to elucidate the role of MSCs in supporting leukemia cells and the restoration of normal hematopoiesis by analyzing ex vivo MSC secretomes at the onset of AML and in remission. The study included MSCs obtained from the bone marrow of 13 AML patients and 21 healthy donors. The analysis of proteins contained in the MSCs-conditioned medium demonstrated that secretomes of patient MSCs differed little between the onset of AML and remission; pronounced differences were observed between MSC secretomes of AML patients and healthy donors. The onset of AML was accompanied by a decrease in the secretion of proteins related to ossification, transport, and immune response. In remission, but not at the onset, secretion of proteins responsible for cell adhesion, immune response, and complement was reduced compared to donors. We conclude that AML causes crucial and, to a large extent, irreversible changes in the secretome of bone marrow MSCs ex vivo. In remission, functions of MSCs remain impaired despite the absence of tumor cells and the formation of benign hematopoietic cells.

Possible Kidney Complications after Application of Cell Technologies for the Repair of the Resected Liver.

The state of rat kidneys after injection of bone marrow multipotent stromal cells (MSC) labeled with Vybrant CM-Dil into intact or resected liver was studied by fluorescence microscopy. The main structural changes in the kidneys after MSC injection into intact and partially resected liver manifested as granular dystrophy and necrobiotic/necrotic changes in single epithelial cells of the distal tubules and collecting ducts, thrombosis of some vessels, progression of an ascending urinary tract infection (detection of dust-like fluorescent objects), which can be due to the immunomodulating or even immunosuppressive influence of MSC and their detritus. MSC injected into the intact or resected liver, as well as the products of their degradation were not detected in the kidneys at all terms of observation. After injection of MSC into partially resected liver, manifestations of bacterial contamination of the renal medulla appeared later. The injection of MSC into the liver can be complicated by thrombosis of the renal vessels, which should be taken into account when using this administration route in the cell therapy.

Peptide derived from stromal cell-derived factor 1δ enhances the in vitro expression of osteogenic proteins via bone marrow stromal cell differentiation and promotes bone formation in in vivo models.

Mesenchymal stem cells (MSCs) rely on chemokines and chemokine receptors to execute their biological and physiological functions. Stromal cell-derived factor-1 (SDF-1) is upregulated in injury sites, where it acts as a chemotactic agent, attracting CXCR4-expressing MSCs, which play a pivotal role in the healing and regeneration of tissue throughout the body. Furthermore, SDF-1 expression has been observed in regions experiencing inflammation-induced bone destruction and fracture sites. In this study, we identified a novel peptide called bone-forming peptide-5 (BFP-5), derived from SDF-1δ, which can promote the osteogenesis of MSCs as well as bone formation and healing. Multipotent bone marrow stromal cells treated with BFP-5 showed enhanced alizarin red S staining and higher alkaline phosphatase (ALP) activity. Moreover, ALP and osterix proteins were more abundantly expressed when cells were treated with BFP-5 than SDF-1α. Histology and microcomputed tomography data at 12 weeks demonstrated that both rabbit and goat models transplanted with polycaprolactone (PCL) scaffolds coated with BFP-5 showed significantly greater bone formation than animals transplanted with PCL scaffolds alone. These findings suggest that BFP-5 could be useful in the development of related therapies for conditions associated with bones.

Unraveling and Visualizing the Interactions of Acute Myeloid Leukemia with the Bone Marrow Microenvironment in Vitro and In Vivo

The bone marrow (BM) is a dynamic stem cell microenvironment, harboring hematopoietic stem cells (HSCs) and various non-blood components such as endothelium, mural cells, multipotent stromal cells (MSCs) and their derivatives including osteoblasts, adipocytes, and fibroblasts. This stromal niche supports HSC-fate decisions and has been proposed as a major determinant of chemoresistance in hematological malignancies, such as acute myeloid leukemia (AML). Following chemotherapy, AML patients often suffer from relapse, in part due to chemoresistant leukemic stem cells (LSCs). There have been various indications in literature that leukemic cells remodel their niche, creating a permissive microenvironment for LSCs. However, which cell types respond and which underlying molecular mechanisms are involved skewing the compositional changes in the BM microenvironment remain unclear. Here, we aim to investigate how high LSC frequency (LSChigh) versus low LSC frequency (LSClow) burden AML initiates BM niche remodeling in the favor of AML expansion, with a particular focus on the MSC lineages. We further aim to determine which stromal cell types interact with LSCs and how their composition is altered upon AML development. Additionally, we aim to visualize which cell types interact with and are positioned in close proximity to LSCs. We combine single-cell transcriptomics of AML xenograft models and in vitro co-cultures of MSCs with primary AML. First, we set up clinically relevant murine xenograft models in which primary AML cells with either a low vs high LSC frequency (Garg et al., 2019) were injected in immune-deficient NSGW41 mice. As control, we injected cord blood-derived HSPCs. 6-7 weeks after engraftment, we harvested and digested the bones using our established protocol (adapted from Baccin et al., 2020) to recover the stromal cells with high efficiency. Besides, we also kept a femur per mouse for follow-up (multiplexed) immunofluorescence imaging. We enriched for human and mouse HSPCs, human AML cells, and mouse niche cells using FACS-based sorting, followed by scRNA-seq. Additionally, we isolated BM-derived mononuclear cells from healthy donors and AML patients and expanded the MSCs in vitro. We set up 4 independent co-cultures with MSCs and primary AML cells with LSChigh versus LSClow frequency. After 48 hours, we performed scRNA-seq to investigate transcriptomic changes and subsequent variant biological processes in the MSCs. scRNA-seq profiling of the BM niche of our AML xenograft models demonstrates that AML presence significantly impacts the niche composition, leading to the expansion of Cxcl12-abundant-reticular adipocytes (adipo-CAR) and the decline of osteoblasts. When comparing LSChigh versus LSClow, we detect significant differences in abundance of certain stromal cells, including endothelium, adipo-CARs, and fibroblast subtypes. This suggests that the LSC-burden plays a critical role in the extent of niche remodeling. To further uncover the direct effects of LSCs on MSCs, we have established an AML-MSC co-culture system and perform scRNA-seq on the MSCs. In line with our in vivo data, we observe that cultured BM MSCs, especially upon exposure to LSChigh AML, suppress osteoblast lineage genes while overexpressing fibrosis-related genes. We are validating our observations using multiplexed immunofluorescence to further investigate the direct cell-cell communication of LSCs with their microenvironment. Taken together, our data in murine AML models and human AML-MSC co-cultures unravel new insights into the BM stem cell niche composition and highlight a specific LSC-induced skewing of the mesenchymal lineage. Our data helps to better understand to which extent the BM microenvironment impacts AML disease progression and relapse due to residual LSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dissemination of Multipotent Stromal Cells in the Organism after Their Injection into Intact and Resected Liver in the Experiment.

The possibility of dissemination of bone marrow multipotent stromal cells stained with Vybrant CM-Dil after injection into an intact and resected liver was studied using luminescence microscopy. Labeled cells were found in the kidneys, spleen, lungs, axillary, mesenteric, and inguinal lymph nodes. We observed dissemination of multipotent stromal cells and their detritus throughout the body that occurred only after filtration in the lungs, where most cells underwent destruction. Perivascularly located macrophages in various organs can phagocytize multipotent stromal cells and their detritus from blood vessels. The content of objects labeled with Vybrant CM-Dil in distant organs was significantly lower after multipotent stromal cell injection into the resected liver, which was associated with the deposition of cells in the damaged area of the organ and their partial entry into the abdominal cavity.

Nanoparticles of naturally occurring PPAR-γ inhibitor betulinic acid ameliorates bone marrow adiposity and pathological bone loss in ovariectomized rats via Wnt/β-catenin pathway

AimsPostmenopausal osteoporosis is one of the world's biggest yet unnoticed health issues. After ovariectomy, declined estrogen level significantly contributes to the elevation of bone marrow adiposity and bone loss leading to osteoporosis. Therapeutics to prevent osteoporosis addressing various aspects are now in short supply. In this study we made an approach to synthesize nanoparticles of naturally occurring PPAR-γ inhibitor, betulinic acid (BA/NPs) and tested the same in altered bone metabolisms developed after ovariectomy. Main methodsThe osteogenic efficacy of BA/NPs was established in human and rat primary osteoblast cells using qRT-PCR and immunoblot analysis. Furthermore, lineage allocations of multipotent bone marrow stromal cells were evaluated. Various aspects of altered bone metabolism after ovariectomy such as bone marrow adiposity and pathological bone loss were evaluated using μCT and histological assessments. Key findingsBA/NPs exert potential osteogenic efficacy by modulating key osteogenic markers such as RUNX2 and BMP2. Mechanistically BA/NPs regulate osteoblastogenesis through Wnt/β-catenin signaling. Further, BA/NPs showed the potential to inhibit the differentiation of multipotent BMSCs towards adipogenesis while favouring the osteogenic lineage via Wnt/β-catenin pathway. In the in vivo study, increased bone marrow adiposity was reduced in ovariectomized rats after BA/NPs treatment as assessed by histology and μCT analysis. Loss of bone mineral density as a hallmark of pathological bone loss was also abrogated by BA/NPs. Furthermore, increased obesity after OVX was also prevented in BA/NPs treated animals. SignificanceOur findings imply that BA/NPs could be used further as a viable drug lead to counteract various pathophysiological challenges after menopause.

Angioprotective and Follicle-Stimulating Effects of Administration of Bone Marrow Mesenchymal Stromal Cells in Chronic Inflammation of Internal Reproductive Organs.

Induced inflammation of reproductive organs in female Wistar rats was associated with an increase in the diameters of arteries and veins and number of blood vessels in the ovary medulla in combination with an increase in the number of lymphatic vessels; these changes were accompanied by reduction of the ovarian reserve and number of yellow bodies. Intravenous and submucosal injection of bone marrow multipotent mesenchymal stromal cells (BМ-ММSC) led to further increase in the diameters of arteries and veins and number of primordial and primary follicles. The injection of conditioned medium of BМ-ММSC cultures generally produced the same effects, which could demonstrate the secretory mechanisms of their influence on local angiogenesis and folliculogenesis.

Adipocyte-Cancer Cell Interactions in the Bone Microenvironment.

When compared to adipocytes in other anatomical sites, the interaction of bone marrow resident adipocytes with the other cells in their microenvironment is less well understood. Bone marrow adipocytes originate from a resident, self-renewing population of multipotent bone marrow stromal cells which can also give rise to other lineages such as osteoblasts. The differentiation fate of these mesenchymal progenitors can be influenced to favour adipogenesis by several factors, including the administration of thiazolidinediones and increased age. Experimental data suggests that increases in bone marrow adipose tissue volume may make bone both more attractive to metastasis and conducive to cancer cell growth. Bone marrow adipocytes are known to secrete a variety of lipids, cytokines and bioactive signaling molecules known as adipokines, which have been implicated as mediators of the interaction between adipocytes and cancer cells. Recent studies have provided new insight into the impact of bone marrow adipose tissue volume expansion in regard to supporting and exacerbating the effects of bone metastasis from solid tumors, focusing on prostate, breast and lung cancer and blood cancers, focusing on multiple myeloma. In this mini-review, recent research developments pertaining to the role of factors which increase bone marrow adipose tissue volume, as well as the role of adipocyte secreted factors, in the progression of bone metastatic prostate and breast cancer are assessed. In particular, recent findings regarding the complex cross-talk between adipocytes and metastatic cells of both lung and prostate cancer are highlighted.

Bone Marrow Multipotent Mesenchymal Stromal Cells from Patients with Aplastic Anemia Retain Their Ability to Support Hematopoietic Precursors despite Pronounced Changes in Gene Expression.

The properties of bone marrow-derived multipotent mesenchymal stromal cells (MSC) of patients with aplastic anemia at the onset of the disease are studied insufficiently. The aim of this work was to test the ability of MSC from patients with aplastic anemia to maintain hematopoietic precursors and to analyze the expression of genes associated with hematopoiesis and immune response. The ability of MSC to maintain hematopoietic precursors was determined by counting cobblestone area-forming cells; gene expression was analyzed by quantitative PCR. It was shown that MSC of patients with aplastic anemia preserve their ability to maintain hematopoietic precursors. Pronounced changes in the expression of the VEGFA and ANGPT1 genes were found. MSC from aplastic anemia patients with PNH clone significantly differ from those from aplastic anemia patients without PNH clone in terms of the expression of the SDF1, IL1R, and VEGFA genes. Changes in gene expression can be associated with the pathogenesis of the disease.

Multipotent Mesenchymal Stromal Cells from the Bone Marrow of Untreated Aplastic Anemia Patients Preserve Their Ability to Support Hematopoietic Precursors However Display the Pronounced Changes in Gene Expression

Aplastic anemia (AA) is believed to be an autoimmune disorder characterized by the pancytopenia due to the depletion of hematopoietic stem and progenitor cells in the bone marrow. There are three forms of AA depending on the severity of pancytopenia: moderate, or non-severe AA (NAA), severe AA (SAA), and very severe AA (VSAA). Clones of cells typical for paroxysmal nocturnal hemoglobinuria (PNH-clones) are frequently present in AA patients in various proportions.We aimed to study stromal microenvironment of untreated AA patients depending on the AA severity, presence or absence of PNH-clone, and on the response to the therapy after 3 and 6 months of treatment. We analyzed the bone marrow (BM) multipotent mesenchymal stromal cells (MMSCs) in their ability to maintain hematopoietic precursors and examined relative expression levels (REL) of selected genes.The study included 17 patients with NAA (53% females, 47% males, 33.8±2.2 years old), 12 patients with SAA (33% females, 67% males, 29.3±3.8 years old). Among NAA patients 7 had PNH-clone, and among SAA - 6 patients. Control group consisted of 19 donors (42% females, 58% males, 30.4±3.1 years old). The ability to support hematopoietic precursors by MMSCs from the BM of AA patients was measured by cobble stone area forming cells (CAFC) assay, where BM cells from one healthy donor were seeded on different MMSCs; REL of selected genes was analyzed with TaqMan RT-PCR. Only the genes with statistically significant differences are presented. The data are presented as mean ± standard error of measures, the differences were statistically significant when p<0.05 when Student's unpaired t-test or Mann-Whitney test was applied.MMSCs from AA patients preserve their ability to maintain hematopoietic precursors. CAFC 7 frequency reflects the number of late hematopoietic precursors. CAFC 7 frequency was slightly higher on MMSCs from NAA patients (9.92±2.73 per 10 6 healthy BM cells) then on MMSCs from healthy donors (5.56±1.14 per 10 6 healthy BM cells), although the difference was not statistically significant. MMSCs from SAA patients maintained CAFC 7 as well as donors' MMSCs (6.75±1.96 per 10 6 healthy BM cells). The frequency of CAFC 28, reflecting the number of early hematopoietic precursor, displayed similar but more pronounced dynamics. CAFC 28 frequency on NAA patients' MMSCs was significantly higher than on donors' ones (2.17±0.34 versus 1.11±0.31 per 10 6 healthy BM cells, p=0.03), while on SAA patients' MMSCs it was also high (1.92±0.57 per 10 6 healthy BM cells) but the difference was insignificant (Table 1). The presence of PNH-clone does not affect the ability of stromal cells to maintain hematopoiesis. MMSCs from the patients that had responded to the therapy in 90 or 180 days did not differ in their ability to maintain hematopoietic precursors from the MMSCs of treatment resistant the patients. Therefore, we can assume that physiological function of stromal microenvironment is not affected deeply in the debut of AA.Gene expression analysis revealed statistically significant upregulation of FGFR1, PDGFRA, VEGFA and downregulation of ANG1 (in MMSCs from both NAA and SAA patients), and upregulation of FGFR2 and CFH (only in NAA patients' MMSCs) (Table 2). In MMSCs of AA patients (both NAA and SAA) without PNH-clone the upregulation of CFH gene was detected (Table 3). CFH is one of the players in the complement system which is disrupted in PNH. This fact needs to be further scrutinized. In addition, IL1R, SDF1 and VEGFA were statistically significantly downregulated in MMSCs from AA patients with PNH-clone compared with MMSCs from patients without PNH-clone. It seems that the presence of PNH-clone corresponds with the changes in stromal microenvironment. Gene expression of analyzed genes was the same in MMSCs of the patients that had responded or not responded to the treatment in 90 or 180 days since the therapy begun.Thus, MMSCs from the BM of untreated AA patients preserve their ability to support hematopoietic precursors however display the pronounced changes in gene expression.The work is supported by the RFBR, project 19-015-00280. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bone-Forming Peptide-4 Induces Osteogenic Differentiation and VEGF Expression on Multipotent Bone Marrow Stromal Cells.

Bone morphogenetic proteins (BMPs) have been widely used as treatment for bone repair. However, clinical trials on fracture repair have challenged the effectiveness of BMPs and suggested that delivery of multipotent bone marrow stromal cells (BMSCs) might be beneficial. During bone remodeling and bone fracture repair, multipotent BMSCs differentiate into osteoblasts or chondrocytes to stimulate bone formation and regeneration. Stem cell-based therapies provide a promising approach for bone formation. Extensive research has attempted to develop adjuvants as specific stimulators of bone formation for therapeutic use in patients with bone resorption. We previously reported for the first time bone-forming peptides (BFPs) that induce osteogenesis and bone formation. BFPs are also a promising osteogenic factor for prompting bone regeneration and formation. Thus, the aim of the present study was to investigate the underlying mechanism of a new BFP-4 (FFKATEVHFRSIRST) in osteogenic differentiation and bone formation. This study reports that BFP-4 induces stronger osteogenic differentiation of BMSCs than BMP-7. BFP-4 also induces ALP activity, calcium concentration, and osteogenic factors (Runx2 and osteocalcin) in a dose dependent manner in BMSCs. Therefore, these results indicate that BFP-4 can induce osteogenic differentiation and bone formation. Thus, treatment of multipotent BMSCs with BFP-4 enhanced osteoblastic differentiation and displayed greater bone-forming ability than BMP-7 treatment. These results suggest that BFP-4-stimulated cell therapy may be an efficient and cost-effective complement to BMP-7-based clinical therapy for bone regeneration and formation.

Bone Marrow Multipotent Mesenchymal Stromal Cells as Autologous Therapy for Osteonecrosis: Effects of Age and Underlying Causes.

Bone marrow (BM) is a reliable source of multipotent mesenchymal stromal cells (MSCs), which have been successfully used for treating osteonecrosis. Considering the functional advantages of BM-MSCs as bone and cartilage reparatory cells and supporting angiogenesis, several donor-related factors are also essential to consider when autologous BM-MSCs are used for such regenerative therapies. Aging is one of several factors contributing to the donor-related variability and found to be associated with a reduction of BM-MSC numbers. However, even within the same age group, other factors affecting MSC quantity and function remain incompletely understood. For patients with osteonecrosis, several underlying factors have been linked to the decrease of the proliferation of BM-MSCs as well as the impairment of their differentiation, migration, angiogenesis-support and immunoregulatory functions. This review discusses the quality and quantity of BM-MSCs in relation to the etiological conditions of osteonecrosis such as sickle cell disease, Gaucher disease, alcohol, corticosteroids, Systemic Lupus Erythematosus, diabetes, chronic renal disease and chemotherapy. A clear understanding of the regenerative potential of BM-MSCs is essential to optimize the cellular therapy of osteonecrosis and other bone damage conditions.

The possibility of using biomedical cell product in the treatment of chronic glomerulonephritis

The basic principles of rational treatment of glomerulonephritis are considered, based on etiology, clinical manifestations and pathomorphological variants of its different forms. Today it has been established that glomerulonephritis can be primary (the etiology is usually unknown) and secondary, when the disease occurs against the background of concomitant pathology (systemic lupus erythematosus, vasculitis, hepatitis, oncological diseases, chronic viral and bacterial infections). The participation of various pathological, genetic and systemic factors in the development of various forms of glomerulonephritis has been shown. The characteristic clinical picture of manifestations of different forms of the disease and the degree of involvement of different renal structures in them are described. Against the background of the severity of the course and prevalence of various forms of glomerulonephritis, it is especially important to develop therapeutic approaches aimed at the full restoration of kidney function and cure of patients. One of such approaches is the use of biomedical cell products based on allogenic mesenchymal multipotent stromal cells and hematopoietic stromal cells. A number of studies have confirmed that the best results can be achieved with the use of cell products in complex therapies with standard treatment methods (use of cytostatics and steroid anti-inflammatory drugs) and alternative drug approaches (combination of monoclonal antibodies and polyenzyme drugs). At the same time, the use of standard and alternative techniques does not lead to a complete recovery of patients, but only transfers the course of the disease from the exacerbation phase to remission. The relevance of further development of biomedical cell products of allogeneic mesenchymal multipotent bone marrow stromal cells is shown, verification of their safety and efficacy in preclinical and clinical studies.

90. Nanoroughened microstructured orthopedic implant surfaces induce osteogenesis via soluble signaling factors produced by MSCs

BACKGROUND CONTEXT There is a growing clinical need to create implants with material properties that promote accelerated osseointegration and reduced recovery time for interbody fusion healing. We have shown that machined Ti-based implants possessing biomimetic surface topographies can direct osteoblast differentiation of multipotent bone marrow stromal cells (MSCs) in vitro and improve osseointegration in vivo in both animals and in humans following dental implant placement. PURPOSE This study examined the hypothesis that MSCs grown on nanotextured microstructured Ti6Al4V surfaces produce factors that regulate osteogenic differentiation of MSCs not on the surface. STUDY DESIGN/SETTING Not applicable. PATIENT SAMPLE Not applicable. OUTCOME MEASURES Not applicable. METHODS Ti6Al4V discs were grit-blasted and acid-etched, producing complex topography at the macro/micro/nanoscale (MMN; Medtronic/Titan Spine). All cultures were grown in MSC growth media (GM). Co-cultures were used to examine paracrine effects of factors released by human MSCs cultured on MMN surfaces. MSCs were grown on tissue culture polystyrene (TCPS) or MMN discs in 24-well plates. At 24 hours, inserts containing MSCs were placed into the wells and cultured in GM for 7 days, then moved to fresh GM for 24 hours. Alternatively, MSCs were cultured on MMN surfaces and TCPS for 7 days. Inserts with MSCs were added to the wells on day 7 and the co-culture was incubated for another 4 days. Inserts were moved on day 11 and cultured for 24 hours. In each experiment, a group of MMN discs received GM+15µg/mL BMP2 blocking antibody (abBMP2). BMP2, OPN, OPG, OCN, IL6, and VEGF-A in the conditioned media (CM) were assayed by ELISA and normalized to DNA. Athymic mice were divided into 3 groups: inactive demineralized bone matrix (iDBM), iDBM+rhBMP2 (MMN-CM concentration), and iDBM+MMN-CM proteins. MMN-CM was derived by culturing MSCs on MMN surfaces for 10 days and lyophilizing the CM from days 5-10. Gel capsules were implanted bilaterally into intramuscular pockets created in the hind limbs (N=8 capsules/group). Legs were harvested at 35 days. Bone formation was assessed by micro-CT and histomorphometry. RESULTS MSCs grown directly on MMN discs and on inserts above MMN discs increased production of OCN, OPN, BMP2, and VEGF-A compared to MSCs above TCPS, MMN discs+abBMP2, and insert control. Blocking BMP2 after surface mediated differentiation reduced OCN, OPN, and BMP2 compared to MMN groups. Total DNA content was increased on MMN discs+abBMP2. Micro-CT revealed greater mineralized tissue in MMN-CM groups compared to iDBM and iDBM+BMP2. There was more live bone in the MMN-CM group compared to iDBM. CONCLUSIONS MSCs cultured on nanoroughened microtextured biomimetic Ti6Al4V surfaces differentiate and produce local factors that can induce MSC differentiation above the surface through paracrine signaling factors such as BMP2. Reducing BMP2 function via abBMP2 inhibits MSC differentiation in contact with the surface and above the surface, indicating that BMP2 produced by cells on the surface is involved in the surface-dependent osteogenic differentiation process. Factors produced by the MSCs increased bone formation in a mouse model compared to control and BMP2-only treatment groups, suggesting that the engineered biomimetic surface topography may improve bone formation. FDA DEVICE/DRUG STATUS Interbody Fusion Implants (Approved for this indication)

Niches for Skeletal Stem Cells of Mesenchymal Origin.

With very few exceptions, all adult tissues in mammals are maintained and can be renewed by stem cells that self-renew and generate the committed progeny required. These functions are regulated by a specific and in many ways unique microenvironment in stem cell niches. In most cases disruption of an adult stem cell niche leads to depletion of stem cells, followed by impairment of the ability of the tissue in question to maintain its functions. The presence of stem cells, often referred to as mesenchymal stem cells (MSCs) or multipotent bone marrow stromal cells (BMSCs), in the adult skeleton has long been realized. In recent years there has been exceptional progress in identifying and characterizing BMSCs in terms of their capacity to generate specific types of skeletal cells in vivo. Such BMSCs are often referred to as skeletal stem cells (SSCs) or skeletal stem and progenitor cells (SSPCs), with the latter term being used throughout this review. SSPCs have been detected in the bone marrow, periosteum, and growth plate and characterized in vivo on the basis of various genetic markers (i.e., Nestin, Leptin receptor, Gremlin1, Cathepsin-K, etc.). However, the niches in which these cells reside have received less attention. Here, we summarize the current scientific literature on stem cell niches for the SSPCs identified so far and discuss potential factors and environmental cues of importance in these niches in vivo. In this context we focus on (i) articular cartilage, (ii) growth plate cartilage, (iii) periosteum, (iv) the adult endosteal compartment, and (v) the developing endosteal compartment, in that order.

Identifying adipogenic chemicals: Disparate effects in 3T3-L1, OP9 and primary mesenchymal multipotent cell models

3T3-L1 pre-adipocytes are used commonly to identify new adipogens, but this cell line has been shown to produce variable results. Here, potential adipogenic chemicals (identified in the ToxCast dataset using the Toxicological Priority Index) were tested for their ability to induce adipocyte differentiation in 3T3-L1 cells, OP9 cells and primary mouse bone marrow multipotent stromal cells (BM-MSC). Ten of the 36 potential adipogens stimulated lipid accumulation in at least one model (novel: fenthion, quinoxyfen, prallethrin, allethrin, pyrimethanil, tebuconzaole, 2,4,6-tris (tert-butyl)phenol; known: fentin, pioglitazone, 3,3′,5,5′-tetrabromobisphenol A). Only prallethrin and pioglitazone enhanced lipid accumulation in all models. OP9 cells were significantly more sensitive to chemicals known to activate PPARγ through RXR than the other models. Coordinate effects on adipocyte and osteoblast differentiation were investigated further in BM-MSCs. Lipid accumulation was correlated with the ability to stimulate expression of the PPARγ target gene, Plin1. Induction of lipid accumulation also was associated with reduction in alkaline phosphatase activity. Allethrin, prallethrin, and quinoxyfen strongly suppressed osteogenic gene expression. BM-MSCs were useful in coordinately investigating pro-adipogenic and anti-osteogenic effects. Overall, the results show that additional models should be used in conjunction with 3T3-L1 cells to identify a broader spectrum of adipogens and their coordinate effects on osteogenesis.

Neuropeptide Y is involved in the regulation of quiescence of hematopoietic stem cells

Differentiation, self-renewal and quiescence of Hematopoietic stem cells (HSCs) is tightly regulated in order to protect the HSCs from the strain of constant cell division and depletion of the stem cell pool. The neurotransmitter Neuropeptide Y (NPY) is released from sympathetic nerves in the bone marrow and has been shown to indirectly affect HSC function through effects on bone marrow (BM) multipotent Mesenchymal Stromal Cells (MSCs), osteoblasts (OBs) and macrophages. Although the absence of NPY has been shown to be accompanied by severe BM impairment and delayed engraftment of HSCs, the direct effects of NPY on HSCs have never been assessed. Here, we aimed to explore the effect of NPY on the regulation of HSCs. All NPY receptors Y1, Y2, Y4 and Y5 were found to be highly expressed on most HSCs and mature hematopoietic cell subsets. In culture, in particularly expression of the Y1 receptor was shown to decrease in time. Doses of 300 nM NPY suppressed HSC proliferation in cell cultures, as confirmed by an increase of HSCs in G0 phase and an increase in the gene expression levels of FOXO3, DICER1, SMARCA2 and PDK1, which all have been shown to play an important role in the regulation of cell quiescence. These data support the idea that NPY may have a direct effect on the regulation of HSC fate by modulating cell quiescence.

Involvement of Bone Marrow Multipotent Stromal Cells in the Processes Presumably Provoking Vascular Calcification.

During serial transplantation of bone marrow derived from young and aged donor CBA mice to 5-month-old recipients, the counts of multipotent stromal cells (MSC) in transplants from young donors assessed at each passage surpassed those of aged donors by 3.2, 7.8, 3.0, and 2.2 times attesting to the age-related decrease of active pool of bone marrow MSC. The medullary curettage in mouse femur increased the total number of MSC and the number of osteogenic MSC both in the contralateral femur and in the bone marrow transplants attesting to spread of the effects of osteogenic factors after bone injury onto the bone tissue of the body even if this tissue if not topographically related to the skeleton. Combined and simultaneous administration of antigenic complex of S. typhimurium (or LPS) with BMP-2 markedly increased the count of osteogenic medullary MSC by 3.6 or 4.6 times in comparison with intact control or by 2.1 and 2.7 times in comparison with administration of BMP-2 alone, which probably resulted from enlargement of the pool of osteogenesis-inducible MSC due to inflammation. Addition of BMP-2 to the culture of splenic stromal cells where osteogenesis does not occur under normal conditions provoked appearance of MSC colonies with alkaline phosphatase activity attesting to involvement of inducible osteogenic MSC in vascular calcification. It can be hypothesized that the reaction to the age-related changes in the bone tissue and osteoporosis is similar to the reaction to bone marrow injury and includes initiation of systemic inflammation and elevation of blood BMP-2, both of which are prerequisite for vascular calcification.

Defective Proliferation and Osteogenic Potential with Altered Immunoregulatory phenotype of Native Bone marrow-Multipotential Stromal Cells in Atrophic Fracture Non-Union

Bone marrow-Multipotential stromal cells (BM-MSCs) are increasingly used to treat complicated fracture healing e.g., non-union. Though, the quality of these autologous cells is not well characterized. We aimed to evaluate bone healing-related capacities of non-union BM-MSCs. Iliac crest-BM was aspirated from long-bone fracture patients with normal healing (U) or non-united (NU). Uncultured (native) CD271highCD45low cells or passage-zero cultured BM-MSCs were analyzed for gene expression levels, and functional assays were conducted using culture-expanded BM-MSCs. Blood samples were analyzed for serum cytokine levels. Uncultured NU-CD271highCD45low cells significantly expressed fewer transcripts of growth factor receptors, EGFR, FGFR1, and FGRF2 than U cells. Significant fewer transcripts of alkaline phosphatase (ALPL), osteocalcin (BGLAP), osteonectin (SPARC) and osteopontin (SPP1) were detected in NU-CD271highCD45low cells. Additionally, immunoregulation-related markers were differentially expressed between NU- and U-CD271highCD45low cells. Interestingly, passage-zero NU BM-MSCs showed low expression of immunosuppressive mediators. However, culture-expanded NU and U BM-MSCs exhibited comparable proliferation, osteogenesis, and immunosuppression. Serum cytokine levels were found similar for NU and U groups. Collectively, native NU-BM-MSCs seemed to have low proliferative and osteogenic capacities; therefore, enhancing their quality should be considered for regenerative therapies. Further research on distorted immunoregulatory molecules expression in BM-MSCs could potentially benefit the prediction of complicated fracture healing.