Behavior Of Mesenchymal Stromal Cells Research Articles

Tailored environments for directed mesenchymal stromal cell proliferation and differentiation using decellularized extracellular matrices in conjunction with substrate modulus

Decellularised extracellular matrix (dECM) produced by mesenchymal stromal cells (MSCs) is a promising biomaterial for improving the ex vivo expansion of MSCs. The dECMs are often deposited on high modulus surfaces such as tissue culture plastic or glass, and subsequent differentiation assays often bias towards osteogenesis. We tested the hypothesis that dECM deposited on substrates of varying modulus will produce cell culture environments that are tailored to promote the proliferation and/or lineage-specific differentiation of MSCs. dECM was produced on type I collagen-functionalised polyacrylamide hydrogels with discrete moduli (∼4, 10, and 40 kPa) or in a linear gradient of modulus that spans the same range, and the substrates were used as culture surfaces for MSCs. Fluorescence spectroscopy and mass spectrometry characterization revealed structural compositional changes in the dECM as a function of substrate modulus. Softer substrates (4 kPa) with dECM supported the largest number of MSCs after 7 days (∼1.6-fold increase compared to glass). Additionally, osteogenic differentiation was greatest on high modulus substrates (40 kPa and glass) with dECM. Nuclear translocation of YAP1 was observed on all surfaces with a modulus of 10 kPa or greater and may be a driver for the increased osteogenesis on the high modulus surfaces. These data demonstrate that dECM technology can be integrated with environmental parameters such as substrate modulus to improve/tailor MSC proliferation and differentiation during ex vivo culture. These results have potential impact in the improved expansion of MSCs for tailored therapeutic applications and in the development of advanced tissue engineering scaffolds. Statement of significanceMesenchymal stromal cells (MSCs) are extensively used in tissue engineering and regenerative medicine due to their ability to proliferate, differentiate, and modulate the immune environment. Controlling MSC behavior is critical for advances in the field. Decellularised extracellular matrix (dECM) can maintain MSC properties in culture, increase their proliferation rate and capacity, and enhance their stimulated differentiation. Substrate stiffness is another key driver of cell function, and previous reports have primarily looked at dECM deposition and function on stiff substrates such as glass. Herein, we produce dECM on substrates of varying stiffness to create tailored environments that enhance desired MSC properties such as proliferation and differentiation. Additionally, we complete mechanistic studies including quantitative mass spec of the ECM to understand the biological function.

Mesenchymal stromal cell response to intervertebral disc-like pH is tissue source dependent.

Intervertebral disc (IVD) degeneration (IVDD) has become increasingly prevalent and is a common contributing factor to low back pain. Current treatment options are limited to either symptom management or surgery. A promising treatment option being explored is intradiscal administration of mesenchymal stromal cells (MSCs). However, there remains a gap in knowledge as to whether MSCs from different tissue sources have similar responses to the low pH microenvironment of the IVD and the possible mechanisms governing these responses. To study this, MSCs from three different tissue sources: adipose (adipose-derived mesenchymal stem cell), bone marrow (bone marrow mesenchymal stem cells), and amnion (amniotic membrane mesenchymal stem cell) were cultured at low pHs representative of IVDD. MSCs were assessed for survival, senescence, apoptosis, metabolic activity, and cytokine release profile. Additionally, western blot was utilized to assess acid sensing ion channel 1 and 3 expression. The results of this study indicated that MSC viability, cell proliferation, senescence, and metabolic activity is negatively affected by low pH and alters MSC cytokine production. This study also demonstrated that MSCs behavior is dependent on tissue source. Understanding how MSC behavior is altered by pH will allow further research aimed at increasing the efficacy of MSC therapy to promote in situ IVDtissue regeneration to combat IVDD.

Engagement of Mesenchymal Stromal Cells in the Remodeling of the Bone Marrow Microenvironment in Hematological Cancers.

Mesenchymal stromal cells (MSCs) are a subset of heterogeneous, non-hematopoietic fibroblast-like cells which play important roles in tissue repair, inflammation, and immune modulation. MSCs residing in the bone marrow microenvironment (BMME) functionally interact with hematopoietic stem progenitor cells regulating hematopoiesis. However, MSCs have also emerged in recent years as key regulators of the tumor microenvironment. Indeed, they are now considered active players in the pathophysiology of hematologic malignancies rather than passive bystanders in the hematopoietic microenvironment. Once a malignant event occurs, the BMME acquires cellular, molecular, and epigenetic abnormalities affecting tumor growth and progression. In this context, MSC behavior is affected by signals coming from cancer cells. Furthermore, it has been shown that stromal cells themselves play a major role in several hematological malignancies' pathogenesis. This bidirectional crosstalk creates a functional tumor niche unit wherein tumor cells acquire a selective advantage over their normal counterparts and are protected from drug treatment. It is therefore of critical importance to unveil the underlying mechanisms which activate a protumor phenotype of MSCs for defining the unmasked vulnerabilities of hematological cancer cells which could be pharmacologically exploited to disrupt tumor/MSC coupling. The present review focuses on the current knowledge about MSC dysfunction mechanisms in the BMME of hematological cancers, sustaining tumor growth, immune escape, and cancer progression.

Synthesis and characterization of a bovine collagen: GAG scaffold with Uruguayan raw material for tissue engineering.

Tissue engineering (TE) and regenerative medicine offer strategies to improve damaged tissues by using scaffolds and cells. The use of collagen-based biomaterials in the field of TE has been intensively growing over the past decades. Mesenchymal stromal cells (MSCs) and dental pulp stem cells (DPSCs) are promising cell candidates for development of clinical composites. In this study, we proposed the development of a bovine collagen type I: chondroitin-6-sulphate (CG) scaffold, obtained from Uruguayan raw material (certified as free bovine spongiform encephalopathy), with CG crosslinking enhancement using different gamma radiation doses. Structural, biomechanical and chemical characteristics of the scaffolds were assessed by Scanning Electron Microscopy, axial tensile tests, FT-IR and Raman Spectroscopy, respectively. Once we selected the most appropriate scaffold for future use as a TE product, we studied the behavior of MSCs and DPSCs cultured on the scaffold by cytotoxicity, proliferation and differentiation assays. Among the diverse porous scaffolds obtained, the one with the most adequate properties was the one exposed to 15kGy of gamma radiation. This radiation dose contributed to the crosslinking of molecules, to the formation of new bonds and/or to the reorganization of the collagen fibers. The selected scaffold was non-cytotoxic for the tested cells and a suitable substrate for cell proliferation. Furthermore, the scaffold allowed MSCs differentiation to osteogenic, chondrogenic, and adipogenic lineages. Thus, this work shows a promising approach to the synthesis of a collagen-scaffold suitable for TE.

The Therapeutic Potential of Mesenchymal Stromal Cells for Regenerative Medicine: Current Knowledge and Future Understandings.

In recent decades, research on the therapeutic potential of progenitor cells has advanced considerably. Among progenitor cells, mesenchymal stromal cells (MSCs) have attracted significant interest and have proven to be a promising tool for regenerative medicine. MSCs are isolated from various anatomical sites, including bone marrow, adipose tissue, and umbilical cord. Advances in separation, culture, and expansion techniques for MSCs have enabled their large-scale therapeutic application. This progress accompanied by the rapid improvement of transplantation practices has enhanced the utilization of MSCs in regenerative medicine. During tissue healing, MSCs may exhibit several therapeutic functions to support the repair and regeneration of injured tissue. The process underlying these effects likely involves the migration and homing of MSCs, as well as their immunotropic functions. The direct differentiation of MSCs as a cell replacement therapeutic mechanism is discussed. The fate and behavior of MSCs are further regulated by their microenvironment, which may consequently influence their repair potential. A paracrine pathway based on the release of different messengers, including regulatory factors, chemokines, cytokines, growth factors, and nucleic acids that can be secreted or packaged into extracellular vesicles, is also implicated in the therapeutic properties of MSCs. In this review, we will discuss relevant outcomes regarding the properties and roles of MSCs during tissue repair and regeneration. We will critically examine the influence of the local microenvironment, especially immunological and inflammatory signals, as well as the mechanisms underlying these therapeutic effects. Importantly, we will describe the interactions of local progenitor and immune cells with MSCs and their modulation during tissue injury. We will also highlight the crucial role of paracrine pathways, including the role of extracellular vesicles, in this healing process. Moreover, we will discuss the therapeutic potential of MSCs and MSC-derived extracellular vesicles in the treatment of COVID-19 (coronavirus disease 2019) patients. Overall, this review will provide a better understanding of MSC-based therapies as a novel immunoregenerative strategy.

The Role of Amino Acids in the Crosstalk Between Mesenchymal Stromal Cells and Neoplastic Cells in the Hematopoietic Niche.

Within the bone marrow hematopoietic cells are in close connection with mesenchymal stromal cells (MSCs), which influence the behavior and differentiation of normal or malignant lymphoid and myeloid cells. Altered cell metabolism is a hallmark of cancer, and changes in nutrient pools and fluxes are important components of the bidirectional communication between MSCs and hematological cancer cells. Among nutrients, amino acids play a significant role in cancer progression and chemo-resistance. Moreover, selected types of cancer cells are extremely greedy for glutamine, and significantly deplete the extracellular pool of the amino acid. As a consequence, this influences the behavior of MSCs in terms of either cytokine/chemokine secretion or differentiation potential. Additionally, a direct nutritional interaction exists between MSCs and immune cells. In particular, selected subpopulations of lymphocytes are dependent upon selected amino acids, such as arginine and tryptophan, for full differentiation and competence. This review describes and discusses the nutritional interactions existing in the neoplastic bone marrow niche between MSCs and other cell types, with a particular emphasis on cancer cells and immune cells. These relationships are discussed in the perspective of potential novel therapeutic strategies based on the interference on amino acid metabolism or intercellular fluxes.

MSC-like cells increase ability of monocyte-derived dendritic cells to polarize IL-17-/IL-10-producing T cells via CTLA-4

SummaryMesenchymal stromal cell-like (MSCl) cells generated from human embryonic stem cells are considered to be an eligible cell line to model the immunomodulatory behavior of mesenchymal stromal cells (MSCs) in vitro. Dendritic cells (DCs) are essential players in the maintenance and restoration of the sensitive balance between tolerance and immunity. Here, the effects of MSCl cells on the in vitro differentiation of human monocytes into DCs were investigated. MSCl cells promote the differentiation of CTLA-4 expressing DCs via the production of all-trans retinoic acid (ATRA) functioning as a ligand of RARα, a key nuclear receptor in DC development. These semi-matured DCs exhibit an ability to activate allogeneic, naive T cells and polarize them into IL-10 + IL-17 + double-positive T helper cells in a CTLA-4-dependent manner. Mapping the molecular mechanisms of MSC-mediated indirect modulation of DC differentiation may help to expand MSCs' clinical application in cell-free therapies.

Impact of the physico-chemical properties of polymeric microspheres functionalized with cell adhesion molecules on the behavior of mesenchymal stromal cells

Polymeric, biodegradable, microspheres (MS) presenting a biomimetic surface of extracellular matrix (ECM) proteins are currently used for transporting cells and/or encapsulated proteins for regenerative medicine studies. They can be made of (lactic-co-glycolic acid) (PLGA) or of a more hydrophilic PLGA-P188 (Poloxamer188)-PLGA polymer allowing for the complete release of the therapeutic proteins. They promote stem cell adhesion, cell survival and differentiation after transplantation. Although the biological effectiveness of these microcarriers is established, a detailed understanding of the protein and cell interactions with the microcarrier surface remain unclear due to a lack of information of their surface properties. The aim of this study was to characterize the physicochemical properties of two polymeric MS systems and determine the effect of laminin and poly-d-lysine coated microcarriers on stem cell adhesion, survival and neuronal differentiation. The hydrophobicity and topography of PLGA MS promoted protein adsorption and the stem cells quickly adhered and spread on the surface of these microcarriers. In contrast less proteins adsorbed onto PLGA-P188-PLGA MS and although cells adhered to these microcarriers, they remained round and did not spread on their surface. Despite these early-stage differences, our results suggest that the nature of the MS does not strongly influence the long-term cell behavior. The cells exhibit the same cell number, differentiation profile and ability to secrete ECM molecules regardless of the type of microcarrier used. Likely the ECM molecules that form a microenvironment around both of these 3D microcarrier/cell constructs over time play a role in this converging cell behavior. We have thus furthered our understanding of the physicochemical properties of polymeric cell carriers affecting stem cell behavior to help tailor suitable microcarriers for neuroregenerative applications.

Multi-peptide presentation and hydrogel mechanics jointly enhance therapeutic duo-potential of entrapped stromal cells

The native extracellular matrix (ECM) contains a host of matricellular proteins and bioactive factors that regulate cell behavior, and many ECM components have been leveraged to guide cell fate. However, the large size and chemical characteristics of these constituents complicate their incorporation into biomaterials without interfering with material properties, motivating the need for alternative approaches to regulate cellular responses. Mesenchymal stromal cells (MSCs) can promote osseous regeneration in vivo directly or indirectly through multiple means including (1) secretion of proangiogenic and mitogenic factors to initiate formation of a vascular template and recruit host cells into the tissue site or (2) direct differentiation into osteoblasts. As MSC behavior is influenced by the properties of engineered hydrogels, we hypothesized that the biochemical and biophysical properties of alginate could be manipulated to promote the dual contributions of encapsulated MSCs toward bone formation. We functionalized alginate with QK peptide to enhance proangiogenic factor secretion and RGD to promote adhesion, while biomechanical-mediated osteogenic cues were controlled by modulating viscoelastic properties of the alginate substrate. A 1:1 ratio of QK:RGD resulted in the highest levels of both proangiogenic factor secretion and mineralization in vitro. Viscoelastic alginate outperformed purely elastic gels in both categories, and this effect was enhanced by stiffness up to 20 kPa. Furthermore, viscoelastic constructs promoted vessel infiltration and bone regeneration in a rat calvarial defect over 12 weeks. These data suggest that modulating viscoelastic properties of biomaterials, in conjunction with dual peptide functionalization, can simultaneously enhance multiple aspects of MSC regenerative potential and improve neovascularization of engineered tissues.

Potential of electrospun cationic BSA fibers to guide osteogenic MSC differentiation via surface charge and fibrous topography

Large or complex bone fractures often need clinical treatments for sufficient bone repair. New treatment strategies have pursued the idea of using mesenchymal stromal cells (MSCs) in combination with osteoinductive materials to guide differentiation of MSCs into bone cells ensuring complete bone regeneration. To overcome the challenge of developing such materials, fundamental studies are needed to analyze and understand the MSC behavior on modified surfaces of applicable materials for bone healing. For this purpose, we developed a fibrous scaffold resembling the bone/bone marrow extracellular matrix (ECM) based on protein without addition of synthetic polymers. With this biomimetic in vitro model we identified the fibrous structure as well as the charge of the material to be responsible for its effects on MSC differentiation. Positive charge was introduced via cationization that additionally supported the stability of the scaffold in cell culture, and acted as nucleation point for mineralization during osteogenesis. Furthermore, we revealed enhanced focal adhesion formation and osteogenic differentiation of MSCs cultured on positively charged protein fibers. This pure protein-based and chemically modifiable, fibrous ECM model allows the investigation of MSC behavior on biomimetic materials to unfold new vistas how to direct cells’ differentiation for the development of new bone regenerating strategies.

Bronchoalveolar lavage fluid gliotoxin induces mesenchymal stromal cell death: a potential explanation to the decreased protective capacity of MSCs in clinical trials

Background & Aim Background Growing evidence demonstrate that human mesenchymal stromal cells (MSCs) tailor their in vivo anti-inflammatory response depending on the specific inflammatory environment they encounter. Understanding the anti-inflammatory mechanisms of systemic or intratracheal administrated MSCs in a range of lung diseases, including cystic fibrosis (CF), is crucial to refine MSC-based cell therapies. The aim of this study was to determine the effects of the in vivo lung environment in CF patients on MSCs behavior. Methods, Results & Conclusion Methods Clinically utilized MSCs were exposed to bronchoalveolar lavage (BAL) fluids, a surrogate for the in vivo environment, obtained from patients with CF and healthy volunteers, or Gliotoxin. Following exposure, cells and conditioned medium were assessed for cytotoxicity, levels of immunomodulatory cytokines, and the presence of apoptotic markers. BAL-fluid samples were assessed for pro- and anti-inflammatory cytokines, osmolality, protease activity, gliotoxin concentrations, and double-stranded DNA content. Results Following five hours of BAL-fluid exposure, we found that a sub-population of BAL-fluid from CF-patients induced cellular death. Importantly, BAL-fluid from healthy volunteers did not induce any apparent MSCs death. Utilizing an RNAseq approach, we were able to identify the BCL-2 apoptotic pathway, which could be activated by Aspergillus fumigatus (Asp) a common infection seen in CF-patients, and found that only BAL-fluid from Asp+ CF-patients induced MSC death. Furthermore, we found that Gliotoxin, the major toxin produced by Asp, induced MSC death, an effect which was inhibited by addition of dithiothreitol (DTT). Finally, gliotoxin-exposed MSCs were shown to have less therapeutic effect compared to untreated MSCs in an acute lung injured mice model. Conclusions MSCs exposed to BAL-fluids from Aspergillus fumigatus infected CF-patients displayed an increased cellular death, and exposure to the major fungal toxin resulted in a decreased therapeutic capacity of MSCs in vivo.These results highlight the need to understand the effect of the in vivo inflammatory environment on systemic or locally administered MSCs.

Effects of Normothermic Machine Perfusion Conditions on Mesenchymal Stromal Cells.

Ex-situ normothermic machine perfusion (NMP) of transplant kidneys allows assessment of kidney quality and targeted intervention to initiate repair processes prior to transplantation. Mesenchymal stromal cells (MSC) have been shown to possess the capacity to stimulate kidney repair. Therefore, the combination of NMP and MSC therapy offers potential to repair transplant kidneys. It is however unknown how NMP conditions affect MSC. In this study the effect of NMP perfusion fluid on survival, metabolism and function of thawed cryopreserved human (h)MSC and porcine (p)MSC in suspension conditions was studied. Suspension conditions reduced the viability of pMSC by 40% in both perfusion fluid and culture medium. Viability of hMSC was reduced by suspension conditions by 15% in perfusion fluid, whilst no differences were found in survival in culture medium. Under adherent conditions, survival of the cells was not affected by perfusion fluid. The perfusion fluid did not affect survival of fresh MSC in suspension compared to the control culture medium. The freeze-thawing process impaired the survival of hMSC; 95% survival of fresh hMSC compared to 70% survival of thawed hMSC. Moreover, thawed MSC showed increased levels of reactive oxygen species, which indicates elevated levels of oxidative stress, and reduced mitochondrial activity, which implies reduced metabolism. The adherence of pMSC and hMSC to endothelial cells was reduced after the thawing process, effect which was particularly profound in in the perfusion fluid. To summarize, we observed that conditions required for machine perfusion are influencing the behavior of MSC. The freeze-thawing process reduces survival and metabolism and increases oxidative stress, and diminishes their ability to adhere to endothelial cells. In addition, we found that hMSC and pMSC behaved differently, which has to be taken into consideration when translating results from animal experiments to clinical studies.

Coculture with monocytes/macrophages modulates osteogenic differentiation of adipose-derived mesenchymal stromal cells on poly(lactic-co-glycolic) acid/polycaprolactone scaffolds.

The effects of immune cells, in particular macrophages, on the behaviour of mesenchymal stromal cells (MSCs) have recently gained much attention for MSCs‐based tissue‐engineered constructs. This study aimed to evaluate the effect of monocytes/macrophages on the osteogenic differentiation of adipose‐derived mesenchymal stromal cells (ADMSCs) in three‐dimensional (3D) cocultures. For this, we cocultured THP‐1 monocytes, M1 macrophages, or M2 macrophages with ADMSCs on 3D poly(lactic‐co‐glycolic) acid (PLGA)/polycaprolactone (PCL) scaffolds using osteogenic medium for up to 42 days. We found that osteogenic differentiation of ADMSCs was inhibited by monocytes and both macrophage subtypes in 3D scaffolds. Furthermore, coculture of monocytes/macrophages with ADMSCs resulted in downregulated secretion of oncostatin M (OSM) and bone morphogenetic protein 2 (BMP‐2) and inhibited expression of osteogenic markers alkaline phosphatase (ALP), bone sialoprotein (BSP), and runt‐related transcription factor 2 (RUNX2). Compared with both macrophage subtypes, monocytes inhibited osteogenic differentiation of ADMSCs more significantly. These data suggest that the mutual interactions between monocytes/macrophages and ADMSCs negatively affect MSC osteogenic differentiation and thus possibly bone healing capacity, which highlights the importance of the micro‐environment in influencing cell‐based constructs to treat bone defects and the potential to improve their performance by resolving the inflammation ahead of treatment.

Human Osteoblast-Derived Extracellular Matrix with High Homology to Bone Proteome Is Osteopromotive.

Efficient osteogenic differentiation of mesenchymal stromal cells (MSCs) is crucial to accelerate bone formation. In this context, the use of extracellular matrix (ECM) as natural 3D framework mimicking in vivo tissue architecture is of interest. The aim of this study was to generate a devitalized human osteogenic MSC-derived ECM and to investigate its impact on MSC osteogenic differentiation to improve MSC properties in bone regeneration. The devitalized ECM significantly enhanced MSC adhesion and proliferation. Osteogenic differentiation and mineralization of MSCs on the ECM were quicker than in standard conditions. The presence of ECM promoted in vivo bone formation by MSCs in a mouse model of ectopic calcification. We analyzed the ECM composition by mass spectrometry, detecting 846 proteins. Of these, 473 proteins were shared with the human bone proteome we previously described, demonstrating high homology to an in vivo microenvironment. Bioinformatic analysis of the 846 proteins showed involvement in adhesion and osteogenic differentiation, confirming the ECM composition as key modulator of MSC behavior. In addition to known ECM components, proteomic analysis revealed novel ECM functions, which could improve culture conditions. In summary, this study provides a simplified method to obtain an in vitro MSC-derived ECM that enhances osteogenic differentiation and could be applied as natural biomaterial to accelerate bone regeneration.

Thrombospondin-1 promotes mesenchymal stromal cell functions via TGFβ and in cooperation with PDGF

Mesenchymal stromal cells (MSC) are characterized by unique tropism for wounded tissues, high differentiating capacity, ability to induce tissue repair, and anti-inflammatory and immunoregulatory activities. This has generated interest in their therapeutic use in severe human conditions as well as in regenerative medicine and tissue engineering. Identification of factors involved in the regulation of MSC proliferation, migration and differentiation could provide insights into the pathophysiological regulation of MSC and be exploited to optimize clinical grade expansion protocols for therapeutic use.Here we identify thrombospondin-1 (TSP-1) as a major regulator of MSC. TSP-1 induced MSC proliferation. This effect was mediated by TSP-1-induced activation of endogenous TGFβ, as shown by the inhibitory effects of anti-TGFβ antibodies and by the lack of activity of TSP-2 — that does not activate TGFβ. Moreover, TSP-1 strongly potentiated the proliferative and migratory activity of PDGF on MSC. TSP-1 directly bound to PDGF, through a site located within the TSP-1 type III repeats, and protected the growth factor from degradation by MSC-derived proteases, hence increasing its stability and bioavailability.The studies presented here identify a more comprehensive picture of the pleiotropic effect of TSP-1 on MSC behavior, setting the basis for further studies aimed at investigating the possible use of PDGF and TSP-1 in the in vitro expansion of MSC for therapeutic applications.

Cisplatin-induced mesenchymal stromal cells-mediated mechanism contributing to decreased antitumor effect in breast cancer cells.

BackgroundCells of the tumor microenvironment are recognized as important determinants of the tumor biology. The adjacent non-malignant cells can regulate drug responses of the cancer cells by secreted paracrine factors and direct interactions with tumor cells.ResultsHuman mesenchymal stromal cells (MSC) actively contribute to tumor microenvironment. Here we focused on their response to chemotherapy as during the treatment these cells become affected. We have shown that the secretory phenotype and behavior of mesenchymal stromal cells influenced by cisplatin differs from the naïve MSC. MSC were more resistant to the concentrations of cisplatin, which was cytotoxic for tumor cells. They did not undergo apoptosis, but a part of MSC population underwent senescence. However, MSC pretreatment with cisplatin led to changes in phosphorylation profiles of many kinases and also increased secretion of IL-6 and IL-8 cytokines. These changes in cytokine and phosphorylation profile of MSC led to increased chemoresistance and stemness of breast cancer cells.ConclusionTaken together here we suggest that the exposure of the chemoresistant cells in the tumor microenvironment leads to substantial alterations and might lead to promotion of acquired microenvironment-mediated chemoresistance and stemness.

Platelet-Rich Plasma Obtained with Different Anticoagulants and Their Effect on Platelet Numbers and Mesenchymal Stromal Cells Behavior In Vitro.

There are promising results in the use of platelet-rich plasma (PRP) for musculoskeletal tissue repair. However, the variability in the methodology for its obtaining may cause different and opposing findings in the literature. Particularly, the choice of the anticoagulant is the first definition to be made. In this work, blood was collected with sodium citrate (SC), ethylenediaminetetraacetic acid (EDTA), or anticoagulant citrate dextrose (ACD) solution A, as anticoagulants, prior to PRP obtaining. Hematological analysis and growth factors release quantification were performed, and the effects on mesenchymal stromal cell (MSC) culture, such as cytotoxicity and cell proliferation (evaluated by MTT method) and gene expression, were evaluated. The use of EDTA resulted in higher platelet yield in whole blood; however, it induced an increase in the mean platelet volume (MPV) following the blood centrifugation steps for PRP obtaining. The use of SC and ACD resulted in higher induction of MSC proliferation. On the other hand, PRP obtained in SC presented the higher platelet recovery after the blood first centrifugation step and a minimal change in MSC gene expression. Therefore, we suggest the use of SC as the anticoagulant for PRP obtaining.

Adipocyte Derived Mesenchymal Stromal Cell and Platelet Lysate: Ideal Cell and Supplement for the Treatment of Immune-Inflammatory Diseases?

Background: Mesenchymal stromal cells (MSC) are being tested for the treatment of immune diseases. MSC are present in several adult tissues which milieu may influence MSC behavior particularly under inflammatory conditions. Additionally, culture conditions also can modify cell function or state of activation. Methods: To address the influence of the MSC source on its characteristics, we studied a xenofree, platelet lysate supplemented MSC from dental pulp, adipose tissue and bone marrow, co-cultured with isolated T cells and PBMC subset, and studied the effect of culture animal or human supplements immunomodulatory effect. Results: All three sources were efficient in inhibiting T cells. Among all MSC sources, as also described by others, adipose MSC was capable to significantly induce Treg phenotype and decrease T CD8+. Furthermore, comparing fetal bovine serum and platelet lysate, results demonstrate that platelet lysate alone is capable to induce immunomodulatory phenotype. Additional studies have to be made to elucidate the PL immunomodulatory effect.

Effect of radio frequency discharge plasma on surface properties and biocompatibility of polycaprolactone matrices

Surface modification of bioresorbable polymer material (polycaprolactone, PCL) with abnormal glow discharge, initiated during radio-frequency magnetron sputtering of a hydroxyapatite target was investigated. Plasma treatment resulted in an increase of surface roughness of PCL, crystallite size, the surface free energy and hydrophilicity. Increased treatment time (30, 60, 150 seconds) provoked the polymer surface saturation with the sputtering target ions (calcium, phosphorus). The assessment of plasma exposure of PCL surface on bone marrow multipotent mesenchymal stromal cells behavior (BM MSCs) has been performed. Modification of the polymer surface with the abnormal glow discharge stimulated adhesion and subsequent proliferation of BM MSCs; thus, maximum values were achieved with the surface treatment for 60 s. This type of plasma modification did not affect cell viability (apoptosis, necrosis). Thus, the surface modification with abnormal glow discharge, initiated during radio-frequency magnetron sputtering of a hydroxyapatite target, appear to be a promising method of surface modification of bioresorbable polymer material (PCL) for tissue engineering.

Labelling and tracking of human mesenchymal stromal cells in preclinical studies and large animal models of degenerative diseases.

Success of stem cell therapies were reported in different medical disciplines, including haematology, rheumatology, orthopaedic surgery, traumatology, and others. Currently, more than 4000 clinical trials using stem cells have been completed or are underway, among which 378 investigated or are at present investigating mesenchymal stromal cells (MSCs). The majority of clinical trials using stem- or progenitor- cells, including hematopoietic stem cells and MSCs, target the immune system. However, therapies based on MSCs are increasingly implemented to treat symptoms in which failure of the resident stem cells in situ, or malfunction of tissues or structures are not associated with immune cells or inflammation, but instead are associated with mechanical or metabolic stress, ageing, developmental or acquired malformations, and other causes. To proceed further in the development of stem cell therapies as a safe and effective treatment for surgical and other medical specialities, the behaviour of MSCs implanted in preclinical models and their impact on the site of application need to be explored in detail. Depending on the pre-clinical model employed, tracking of labelled stem cells in live animals makes an enormous difference for exploration of the mechanisms and kinetics involved in MSC-mediated tissue regeneration. Here we review (pre-)clinically applicable key methods to label human MSCs for short and long-term observations in small and large animal models.