3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling

Mesenchymal stem/stromal cells (MSC) are rapidly becoming a leading candidate for use in tissue regeneration, with first generation of therapies being approved for use in orthopaedic repair applications. Capturing the full potential of MSC will likely require the development of novel in vitro culture techniques and devices. Herein we describe the development of a straightforward surface modification of an existing commercial product to enable the efficient study of three dimensional (3D) human bone marrow-derived MSC osteogenic differentiation. Hundreds of 3D microaggregates, of either 42 or 168 cells each, were cultured in osteogenic induction medium and their differentiation was compared with that occurring in traditional two dimensional (2D) monolayer cultures. Osteogenic gene expression and matrix composition was significantly enhanced in the 3D microaggregate cultures. Additionally, BMP-2 gene expression was significantly up-regulated in 3D cultures at day 3 and 7 by approximately 25- and 30-fold, respectively. The difference in BMP-2 gene expression between 2D and 3D cultures was negligible in the more mature day 14 osteogenic cultures. These data support the notion that BMP-2 autocrine signalling is up-regulated in 3D MSC cultures, enhancing osteogenic differentiation. This study provides both mechanistic insight into MSC differentiation, as well as a platform for the efficient generation of microtissue units for further investigation or use in tissue engineering applications.

Dissecting the prostate cancer stem cell niche inside the bone marrow

Prostate cancer frequently metastasizes to bone, which becomes incurable; yet how cancer cells manage to migrate and grow inside the bone remains unknown. In this study I have discovered that both bone and fat cells within the bone marrow actively promote the survival and expansion of prostate cancer cells, and have subsequently developed approaches that can effectively inhibit these processes. Therefore, my work offers opportunities for the development of new prognostic and therapeutic approaches against metastatic prostate cancer and have the potential for improving the treatment outcome of the patients.

Chondrogenesis of adult stem cells from adipose tissue and bone marrow: induction by growth factors and cartilage-derived matrix.

OBJECTIVES: Adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) are multipotent adult stem cells with potential for use in cartilage tissue engineering. We hypothesized that these cells show distinct responses to different chondrogenic culture conditions and extracellular matrices, illustrating important differences between cell types. METHODS: Human ASCs and MSCs were chondrogenically differentiated in alginate beads or a novel scaffold of reconstituted native cartilage-derived matrix with a range of growth factors, including dexamethasone, transforming growth factor beta3, and bone morphogenetic protein 6. Constructs were analyzed for gene expression and matrix synthesis. RESULTS: Chondrogenic growth factors induced a chondrocytic phenotype in both ASCs and MSCs in alginate beads or cartilage-derived matrix. MSCs demonstrated enhanced type II collagen gene expression and matrix synthesis as well as a greater propensity for the hypertrophic chondrocyte phenotype. ASCs had higher upregulation of aggrecan gene expression in response to bone morphogenetic protein 6 (857-fold), while MSCs responded more favorably to transforming growth factor beta3 (573-fold increase). CONCLUSIONS: ASCs and MSCs are distinct cell types as illustrated by their unique responses to growth factor-based chondrogenic induction. This chondrogenic induction is affected by the composition of the scaffold and the presence of serum.

Diet-induced obesity alters the differentiation potential of stem cells isolated from bone marrow, adipose tissue and infrapatellar fat pad: the effects of free fatty acids.

INTRODUCTION: Obesity is a major risk factor for several musculoskeletal conditions that are characterized by an imbalance of tissue remodeling. Adult stem cells are closely associated with the remodeling and potential repair of several mesodermally derived tissues such as fat, bone and cartilage. We hypothesized that obesity would alter the frequency, proliferation, multipotency and immunophenotype of adult stem cells from a variety of tissues. MATERIALS AND METHODS: Bone marrow-derived mesenchymal stem cells (MSCs), subcutaneous adipose-derived stem cells (sqASCs) and infrapatellar fat pad-derived stem cells (IFP cells) were isolated from lean and high-fat diet-induced obese mice, and their cellular properties were examined. To test the hypothesis that changes in stem cell properties were due to the increased systemic levels of free fatty acids (FFAs), we further investigated the effects of FFAs on lean stem cells in vitro. RESULTS: Obese mice showed a trend toward increased prevalence of MSCs and sqASCs in the stromal tissues. While no significant differences in cell proliferation were observed in vitro, the differentiation potential of all types of stem cells was altered by obesity. MSCs from obese mice demonstrated decreased adipogenic, osteogenic and chondrogenic potential. Obese sqASCs and IFP cells showed increased adipogenic and osteogenic differentiation, but decreased chondrogenic ability. Obese MSCs also showed decreased CD105 and increased platelet-derived growth factor receptor α expression, consistent with decreased chondrogenic potential. FFA treatment of lean stem cells significantly altered their multipotency but did not completely recapitulate the properties of obese stem cells. CONCLUSIONS: These findings support the hypothesis that obesity alters the properties of adult stem cells in a manner that depends on the cell source. These effects may be regulated in part by increased levels of FFAs, but may involve other obesity-associated cytokines. These findings contribute to our understanding of mesenchymal tissue remodeling with obesity, as well as the development of autologous stem cell therapies for obese patients.

Treatment of relapse after allogeneic bone marrow transplantation with reduced intensity conditioning (FLAG +/- Ida) and second allogeneic stem cell transplant

Acute leukaemias in relapse after allogeneic stem cell transplantation (SCT) respond poorly to donor leucocyte infusions (DLI) compared with chronic myeloid leukaemia (CML), at least in part because of faster disease kinetics. Fludarabine-containing 'non-myeloablative' chemotherapy followed by further allo SCT may offer more rapid and effective disease control. We report 14 patients with relapse after allo SCT for acute leukaemia [seven acute myeloid leukaemia (AML), five acute lymphoblastic leukaemia (ALL)] or refractory anaemia with excess blasts in transformation (RAEB-t, n = 2) treated with fludarabine, high-dose cytosine arabinoside (ara-C) and granulocyte colony-simulating factor (G-CSF) with (n = 10) or without (n = 2) idarubicin (FLAG +/- Ida) or DaunoXome (FLAG-X) (n = 2) and second allo SCT from the original donor. Donors were fully human leucocyte antigen (HLA) -matched in 13 cases with a single class A mismatch in one. Actuarial overall survival was 60% and disease-free survival was 26% at 58 months. Remissions after the second SCT were longer than those after the first bone marrow transplantation (BMT) in eight of the 13 assessable patients to date. Haematopoietic recovery was rapid. Transplants were well tolerated with no treatment-related deaths. The major complication was graft-versus-host disease (GvHD, acute >/= grade II-2 cases, chronic - eight cases, two limited, six extensive) although there have been no deaths attributable to this. FLAG +/- Ida and second allo SCT is a safe and useful approach and may be more effective than DLI in the treatment of acute leukaemias relapsing after conventional allo SCT.

Evaluation of castor oil-based polyurethane membranes in rat bone-marrow cell culture

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Clinical outcome in chronic myeloid leukemia after allogeneic hematopoietic stem cell transplantation: The experience of the Bone Marrow Transplantation Unit of FUNFARME/BRAZIL using FISH

Investigation of the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in chronic myeloid leukemia patients is essential to predict prognosis and survival. In 20 patients treated at the Bone Marrow Transplantation Unit of São José do Rio Preto (São Paulo, Brazil), we used fluorescence in situ hybridization (FISH) to investigate the frequency of cells with BCR/ABL rearrangement at diagnosis and at distinct intervals after allo-HSCT until complete cytogenetic remission (CCR). We investigated the disease-free survival, overall survival in 3 years and transplant-related mortality rates, too. Bone marrow samples were collected at 1, 2, 3, 4, 6, 12, and 24 months after transplantation and additional intervals as necessary. Success rate of the FISH analyses was 100%. CCR was achieved in 75% of the patients, within on average of 3.9 months; 45% patients showed CCR within 60 days after HSCT. After 3 years of the allo-HSCT, overall survival rate was 60%, disease-free survival was 50% and the transplant-related mortality rate was 40%. The study demonstrated that the BCR-ABL FISH assay is useful for follow-up of chronic myeloid leukemia patients after HSCT and that the clinical outcome parameters in our patient cohort were similar to those described for other bone marrow transplantation units. ©FUNPEC-RP.

In vitro analysis with human bone marrow stem cells on Ti-15Mo alloy for dental and orthopedic implants application

Aim: Nowadays, research on orthopedic and dental implants is focused on titanium alloys for their mechanical properties and corrosion resistance in the human body environment. Another important aspect to be investigated is their surface topography, which is very important to osseointegration. With laser beam irradiation for roughening the implants surface an easier control of the microtopography is achieved, and surface contamination is avoided. The aim of this study was to assess human bone marrow stem cells response to a newly developed titanium alloy, Ti-15Mo, with surface topography modified by laser beam irradiation. Materials and methods: A total of 10 Ti machined disks (control), 10 Ti-15Mo machined disks and 10 Ti-15Mo disks treated by laser beam-irradiation were prepared. To study how Ti-15Mo surface topografy can induce osteoblast differentiation in mesenchymal stem cells, the expression levels of bone related genes and mesenchymal stem cells marker were analyzed, using real time Reverse Transcription-Polymerase Chain Reaction. Results: In Test 1 (comparison between Ti-15Mo machined disks and Ti-machined disks) quantitative real-time RT-PCR showed a significant induction of ALPL, FOSL1 and SPP1, which increase 20% or more. In Test 2 (comparison between Ti-15Mo laser treated disks and Ti-machined disks) all investigated genes were up-regulated. By comparing Test 1 and Test 2 it was detected that COL1A1, COL3A1, FOSL1 and ENG sensibly increased their expression whereas RUNX2, ALPL and SPP1 expression remained substantially unchanged. Conclusion: The present study demonstrated that laser treated Ti-15Mo alloys are promising materials for implants application.

Bone Marrow'S Harvest in The Coxal Tuberosity for Isolation and Culture of Mesenchymal Stem Cells of Buffaloes (Bubalus Bubalis)

Several studies with mesenchymal stem cells (MSCs) have been developed in many species because of its ability to differentiate into other mesoderm lineages, capacity of self-regeneration, low immunogenicity, paracrine, anti-inflamatory, immunomodulatory and antiapoptotic effects which make then a promissory source to be used in therapeutic strategies. The aim of this study is to report the technique of harvest of bone marrow (BM) in the coxal tuberosity (CT) of buffaloes. For this, the animals were selected, identified and contained in a stock. Then trichotomy was performed in the region corresponding to the CT. After identifying the anatomic site it was performed antisepsis, local anesthetic block and introduction of a myelogram's needle (Lang(R)) for BM aspiration. Once the needle was firmly fixed in the CT, the mandril was removed and proceeded to BM aspiration with a syringe (20 mL) containing 1 ml of heparin at 1000 IU / mL and 1 mL of PBS. After the collection, each sample collected was manually homogenized, identified and referred to the LRACT - FMVZ / UNESP-BRAZIL for the correct processing. The anatomical site tested showed to be an alternative site of harvest of BM once provided the appropriate isolation and culture of the mononuclear fraction. Moreover, the procedure was performed without difficulty and with great security. Based on this, it can be conclude that CT is an excellent anatomical site for isolation and culture of MSCs and the proposed technique is viable and feasible to be held in buffaloes.

Cardiomyopathy and cell therapy: ejection fraction improvement and cardiac muscle mass increasing, after a year of bone marrow stem cells transplantation, by magnetic resonance image

The idiopathic dilated cardiomyopathy (IDC) is one of the major public health problems in the western world. Patients with IDC in functional class IV (New York Health Association - NYHA), even after therapeutic optimization, have high mortality. Stem cell therapy has emerged as a potential therapeutic option for cell death-related heart diseases and several positive effects were assigned to cell therapy in cardiomyopathy. The aim of this study was identify short-term result of cell transplantation in idiopathic dilated cardiomyopathy patients (IDC) who were treated by transplantation of autologous bone marrow mononuclear cells (BMMC). Intracoronary injections of autologous BMMC were performed in eight patients with severe ventricle dysfunction (mean of left ventricle ejection fraction – LEVF=20.03%), cardiac mass muscle around 156.2 g and NYHA between III and IV grades, other 8 IDC patients received placebo. The IDCs were followed - up for one and two years, by magnetic resonance imaging (MRI). The results after one year showed significant improvement in LVEF (mean=181.4) and muscle mass increasing (mean=181.4 g), after two years the LVEF continued improving, reaching a mean of 32.69% and the cardiac muscle mass kept stable (mean=179.4 g). Excepted for one patient, all the other had improvement in the NYHA functional class. The placebo group did not show any improvement. We believe that BMMC implant may be a beneficial therapeutic option for IDC patients.

A quantitative proteomic and transcriptomic comparison of human mesenchymal stem cells from bone marrow and umbilical cord vein

Human mesenchymal stem cells (hMSCs) are adult multipotent cells that have high therapeutic potential due to their immunological properties. They can be isolated from several different tissues with bone marrow (BM) being the most common source. Because the isolation procedure is invasive, other tissues such as human umbilical cord vein (UCV) have been considered. However, their interchangeability remains unclear. In the present study, total protein extracts of BM-hMSCs and UCV-hMSCs were quantitatively compared using gel-LC-MS/MS. Previous SAGE analysis of the same cells was re-annotated to enable comparison and combination of these two data sets. We observed a more than 63% correlation between proteomic and transcriptomic data. In silico analysis of highly expressed genes in cells of both origins suggests that they can be modulated by microRNA, which can change protein abundance. Our results showed that MSCs from both tissues shared high similarity in metabolic and functional processes relevant to their therapeutic potential, especially in the immune system process, response to stimuli, and processes related to the delivery of the hMSCs to a given tissue, such as migration and adhesion. Hence, our results support the idea that the more accessible UCV could be a potentially less invasive source of MSCs.

Time-lapse microscopy and classification of 2D human mesenchymal stem cells based on cell shape picks up myogenic from osteogenic and adipogenic differentiation

Current methods to characterize mesenchymal stem cells (MSCs) are limited to CD marker expression, plastic adherence and their ability to differentiate into adipogenic, osteogenic and chondrogenic precursors. It seems evident that stem cells undergoing differentiation should differ in many aspects, such as morphology and possibly also behaviour; however, such a correlation has not yet been exploited for fate prediction of MSCs. Primary human MSCs from bone marrow were expanded and pelleted to form high-density cultures and were then randomly divided into four groups to differentiate into adipogenic, osteogenic chondrogenic and myogenic progenitor cells. The cells were expanded as heterogeneous and tracked with time-lapse microscopy to record cell shape, using phase-contrast microscopy. The cells were segmented using a custom-made image-processing pipeline. Seven morphological features were extracted for each of the segmented cells. Statistical analysis was performed on the seven-dimensional feature vectors, using a tree-like classification method. Differentiation of cells was monitored with key marker genes and histology. Cells in differentiation media were expressing the key genes for each of the three pathways after 21 days, i.e. adipogenic, osteogenic and chondrogenic, which was also confirmed by histological staining. Time-lapse microscopy data were obtained and contained new evidence that two cell shape features, eccentricity and filopodia (= 'fingers') are highly informative to classify myogenic differentiation from all others. However, no robust classifiers could be identified for the other cell differentiation paths. The results suggest that non-invasive automated time-lapse microscopy could potentially be used to predict the stem cell fate of hMSCs for clinical application, based on morphology for earlier time-points. The classification is challenged by cell density, proliferation and possible unknown donor-specific factors, which affect the performance of morphology-based approaches. Copyright © 2012 John Wiley & Sons, Ltd.

Lung on a chip: Co-culture of alveolar epithelial cells and bone marrow derived stromal stem cells in a microfluidic system

Background: Microfluidics system are novel tools to study cell-cell interactions in vitro. This project focuses on the development of a new microfluidic device to co-culture alveolar epithelial cells and mesenchymal stem cells to study cellular interactions involved in healing the injured alveolar epithelium. Methods: Microfluidic systems in polydimethylsiloxane were fabricated by soft lithography. The alveolar A549 epithelial cells were seeded and injury tests were made on the cells by perfusion with media containing H2O2 or bleomycin during 6 or 18hrs. Rat Bone marrow derived stromal cells (BMSC) were then introduced into the system and cell-cell interaction was studied over 24 hrs. Results: A successful co-culture of A549 alveolar epithelial cells and BMS was achieved in the microfluidic system. The seeded alveolar epithelial cells and BMSC adhered to the bottom surface of the microfluidic device and proliferated under constant perfusion. Epithelial injury to mimic mechanisms seen in idiopathic pulmonary fibrosis was induced in the microchannels by perfusing with H2O2 or bleomycin. Migration of BMSC towards the injured epithelium was observed as well as cell-cell interaction between the two cell types was also seen. Conclusion: We demonstrate a novel microfluidic device aimed at showing interactions between different cell types on the basis of a changing microenvironment. Also we were able to confirm interaction between injured alvolar epithelium and BMSC, and showed that BMSC try to heal the injured epitelium.