Mesenchymal Stromal Cells Cultures Research Articles

Quantification of chondrogenic differentiation in monolayer cultures of mesenchymal stromal cells

Determining inhibitory or supportive effects of biological, chemical or physical factors on cell fates, including chondrogenic differentiation of mesenchymal stromal cells (MSCs), requires quantification techniques that are rapid, reproducible, and able to monitor these effects over time. Methods currently used to analyze chondrogenic differentiation are either qualitative staining procedures or indirect DNA quantifications. Because of these limitations, further methods are needed to improve determination of chondrogenic differentiation. In the present study, we applied a histological staining method, which is established for investigation of articular cartilage degeneration by use of Safranin O dye, on chondrogenic differentiated cells in monolayer cultures. MSCs were differentiated on 12-well formats, at increasing concentrations of TGF-β3, cell numbers, and incubation times. Quantification was performed by solubilizing the adsorbed dye into isopropanol followed by determining the optical density (O.D.) through spectrophotometry. Our results show that the O.D. is directly related to cell numbers and incubation periods, and that the technique is applicable to study agents which affect chondrogenic differentiation.

EVALUATION OF BIOCOMPATIBILITY AND EFFICIENCY OF PLASMID DNA DELIVERY BY GENE- ACTIVATED HYDROGELS IN VITRO

Injectable forms of biomaterials have significant prospects for development of minimally invasive medical technologies in regenerative medicine. In this study, we evaluated the opportunities of using synthetic and natural hydrogels, including those containing calcium phosphates and hydroxyapatite, to deliver gene constructs, plasmid DNA molecules carrying some genes (encoding vascular endothelial growth factor, green fluorescent protein, or luciferase), to cells in vitro. The study of plasmid DNA delivery was performed in the line of mouse embryonic fibroblasts 3T3 using fluorescence and luminescent methods. Hydrogel biocompatibility was characterized in cultures of multipotent mesenchymal stromal cells. We found that the extracts of investigated hydrogels did not exert cytotoxic effects, however, with direct contact of materials containing either calcium phosphate particles or hydroxyapatite, a cytotoxic effect was observed. In the case of hydrogels based on collagen and hyaluronic acid that showed optimal biocompatibility, the transfection efficiency turned out to be low without using of a transfection agent and when used, it was 24 and 31%, respectively. Other variants of hydrogels did not provide plasmid DNA delivery, possibly due to cytotoxic effects. Further in vivo studies will clarify the features of plasmid DNA delivery with hydrogels, as well as evaluate the effectiveness of gene-activated hydrogels in tissue regeneration of the musculoskeletal system.

STUDY OF BIOCOMPATIBILITY OF THE MATERIAL BASED ON SMALL INTESTINAL SUBMUCOSA IN TESTS IN VITRO AND IN VIVO

The development of barrier membranes for guided tissue regeneration remains an urgent task. A several authors proposed to use for this purpose xenomaterials from the small intestinal submucosa (SIS). The properties of such materials depend on the technology of donor cell removal (decellularization) and the condition of their extracellular matrix after processing (the presence or absence of proinflammatory damaged matrix components). The aim of this work was to study of biological properties of tissue-engineered xenogenic membranes made from porcine SIS by our patented technology (Cardioplant LLC, Russia) in experiments in vitro and in vivo. In vitro experiments was performed on cultures of multipotent mesenchymal stromal cells of the bone marrow and human skin fibroblasts, assessing viability, proliferative and mitotic activity of cells cultured on the surface of materials during 1 -7 days. The lyophilized barrier membrane bioPLATE MEMBRANE Barrier (Cardioplant LLC, Russia) used as control. To study of biocompatibility of experimental membranes in vivo, heterotopic implantation of materials to male Wistar rats was performed. The cell and tissue reactions and the degree of biointegration and the resorption of experimental materials were evaluated by rateover 14, 30, 60, and 90 days of implantation. The results indicate a higher biocompatibility of SIS-membrane compared with pericardial materials, and indicate the promise of using the porcine small intestinal submucosa to develop implants for guided tissue regeneration.

PB2109 NICHEFORMING STRUCTURES OF BONE MARROW OF PATIENTS WITH MULTIPLE MYELOMA BEFORE AND AFTER AUTOHSCT

Background: Autologous transplantation of hematopoietic stem cells (AutoHSCT) is one of the most effective methods of treatment in patients with MM, which can significantly reduce the volume of cells of the pathological clone and improve the quality of the response. However, there remain groups of patients with ineffective AutoHSCT: patients with unsuccessful mobilization and with early relapse after AutoHSCT. AutoHSCT success depends on many factors, including the state of the niche of HSC, which has been subjected to toxic effects of aggressive treatment and damaged by the underlying disease. Aims: To investigate the morphological characteristics of the main stromal components of the niche HSC and cultural characteristics of MSC patients with multiple myeloma (MM) before and after treatment, including AutoHSCT. Methods 32 trepanobiopsy of bone marrow from patients diagnosed with MM the ages of 48 and 68 (median age 56) before and after AutoHSCT were used in the study. The study applied histological, immunohistochemical and cultural methods. 20 patients diagnosed with primary MM included in the study. 12 patients diagnosed with progressive MM: inefficient mobilization of HSC was noted for 5 patients and early relapse for seven. Results: Changes of stromal microenvironment of bone marrow (BM) showed of all patients with MM: increased microvascular density and the number of endosteal stromal cells, strengthening of reticulin fiber in subendosteal and perivascular spaces. Increased angiogenesis correlates with the number of plasma cells in the myelogram (r = 0.58; p < 0.05) and with the type of infiltration of BM (r = 0.85; P < 0,05), as well as with osteodestructive changes in the patient's history (r = 0.65; p < 0.05). Shown significant changes in cultures of mesenchymal stromal cells (MSC), both before and after therapy, including AutoHSCT. In cultural studies of MSC BM MM patients compared to healthy donors showed a decrease in the speed of proliferation (2.4 times on average), a decrease in the total time of passionation from 7 to 5 passages. A marked increase expression of markers associated with myofibroblastic phenotype and aging (smooth-muscle actin, β-galactosidase) - 45-80% of cells in the culture were positively stained with these markers. In MSC cultures of patients after AutoHSCT β-galactosidase were synthesized weaker than before treatment, but nevertheless higher than in the control group. MSCS of healthy donors BM of the enzyme is almost not synthesized. Fibrils smooth-muscle actin was detected in MSCS to AutoHSCT, after AutoHSCT was practically absent. Summary/Conclusion: Thus, the cultures of patients with MSC and BM nicheforming elements of patients with MM possess the features of tumor-associated microenvironment, despite the treatment of the underlying disease.

Chitin Nanofibrils and Nanolignin as Functional Agents in Skin Regeneration.

Chitin and lignin, by-products of fishery and plant biomass, can be converted to innovative high value bio- and eco-compatible materials. On the nanoscale, high antibacterial, anti-inflammatory, cicatrizing and anti-aging activity is obtained by controlling their crystalline structure and purity. Moreover, electropositive chitin nanofibrlis (CN) can be combined with electronegative nanolignin (NL) leading to microcapsule-like systems suitable for entrapping both hydrophilic and lipophilic molecules. The aim of this study was to provide morphological, physico-chemical, thermogravimetric and biological characterization of CN, NL, and CN-NL complexes, which were also loaded with glycyrrhetinic acid (GA) as a model of a bioactive molecule. CN-NL and CN-NL/GA were thermally stable up to 114 °C and 127 °C, respectively. The compounds were administered to in vitro cultures of human keratinocytes (HaCaT cells) and human mesenchymal stromal cells (hMSCs) for potential use in skin contact applications. Cell viability, cytokine expression and effects on hMSC multipotency were studied. For each component, CN, NL, CN-NL and CN-NL/GA, non-toxic concentrations towards HaCaT cells were identified. In the keratinocyte model, the proinflammatory cytokines IL-1α, IL-1 β, IL-6, IL-8 and TNF-α that resulted were downregulated, whereas the antimicrobial peptide human β defensin-2 was upregulated by CN-LN. The hMSCs were viable, and the use of these complexes did not modify the osteo-differentiation capability of these cells. The obtained findings demonstrate that these biocomponents are cytocompatible, show anti-inflammatory activity and may serve for the delivery of biomolecules for skin care and regeneration.

P16INK4A-expressing mesenchymal stromal cells restore the senescence–clearance–regeneration sequence that is impaired in chronic muscle inflammation

BackgroundThe therapeutic benefits of mesenchymal stromal cells (MSCs) include treatment of chronic inflammation. However, given the short-lived engraftment of these cells in vivo, their therapeutic efficacy remains mysterious. Transient induction of cellular senescence contributes to activation of immune cells, which promotes clearance of damaged cells during tissue remodelling. This may occur in tissue-resident mesenchymal progenitor cells during regeneration. Elucidation of the role of senescence in tissue-resident mesenchymal progenitor cells during regeneration would provide insight into the profile of therapeutic MSCs for treatment of chronic inflammatory disease. MethodsWe evaluated multipotent mesenchymal progenitor cells, termed fibro/adipogenic progenitors (FAPs), and immune cells in acute muscle injury (AMI) model mice and mice with myosin-induced experimental autoimmune myositis, a model of chronic inflammatory myopathy (CIM). Human bone marrow MSCs were optimised for the treatment of CIM using placental extract. FindingFAPs in AMI transiently expressed p16INK4A on days 1 and 2 after injury and recruited phagocytic immune cells, whereas in CIM, p16INK4A expression in FAPs was low. Cellular senescence occurs during the natural maturation of the placenta. Therefore, we used human placental extract to induce p16INK4A expression in therapeutic human bone marrow MSCs in culture. Treatment of CIM with p16INK4A-expressing MSCs promoted tissue remodelling by transiently increasing the abundance of engrafted MSCs, inducing cellular senescence in innate FAPs, and recruiting phagocytic immune cells. InterpretationMSCs may exert their effect by remodelling the chronic inflammatory environment via senescence-related regenerative processes.

The comparative study of chondrogenic differentiation of mesenchymal stromal cells allocated from different sources

Introduction. As an alternative to autochondral transplantation, variants of chondrocyte replacement with mesenchymal stromal cells (MSCs) were considered, since these cells are present in all organs and tissues of human body and possess multilinear potential for differentiation. A number of studies demonstrate that the ability for chondrogenic differentiation of MSCs from different tissues varies, however, those studies are few and controversial. In accordance with the ethical principles and the technical ease of obtaining, adipose tissue, Wharton’s jelly (stroma) and dental pulp are the most attractive sources of MSCs for tissue engineering.Aim: to compare the chondrogenic potential of MSCs allocated from adipose tissue, Wharton’s jelly of the umbilical cord, and human deciduous teeth pulp cultured in the composition of microspheres (pellets).Materials and methods. The phenotype of primary cultures of MSCs was studied via flow microscopy. Chondrogenic differentiation was performed with 3D-culture in microspheres in the presence of TGFβ1 for two weeks under standard conditions. Human chondroblasts were used as a positive control. Cell viability was determined by fluorescent staining. Morphological study was performed using histological and immunohistochemical staining methods.Results. Cultures of MSCs from all sources had similar phenotypes CD29+, CD34–, CD44+, CD49b+, CD45–, CD73+, CD90+, HLADR. In the mass of living cells in microspheres only singular dead cells were visualized. A significant production of extracellular matrix (ECM) was observed in the chondroblast and adipose tissue microspheres, whereas in the microspheres of dental pulp and umbilical cord stroma derived MSCs, no significant ECM was detected. Among the MSCs, the highest production of collagen and glycosaminoglycans (GAG) in the ECM was observed in the microspheres of adipose tissue-derived MSCs, and the lowest production – in the microspheres of dental pulp-derived MSCs. Conclusion. A histological analysis of all microspheres after 14 days of culturing in the chondrogenic medium revealed the signs of differentiation in the chondrogenic direction, a progressive increase of the ECM produced by cells, and the presence of total collagen and GAG in it. Of all the MSCs studied, the greatest chondrogenic potential in vitro (the intensity of development of ECM components) is possessed by the adipose tissue-derived MSCs.

Chelidonic Acid and Its Derivatives from Saussurea Controversa: Isolation, Structural Elucidation and Influence on the Osteogenic Differentiation of Multipotent Mesenchymal Stromal Cells In Vitro.

4-oxo-4H-pyran-2.6-dicarboxylic acid (chelidonic acid, ChA) in the native state and in the complex with calcium [Ca(ChA)(H2O)3], named saucalchelin (CaChA), was isolated from the extract of Saussurea controversa leaves for the first time for the Asteraceae family. The structure of ChA was determined by NMR, MS and confirmed by X-ray analysis of its monomethyl ester, and CaChA was described by IR, ICP-MS, CHN analysis. The yield of ChA and CaChA was 45 mg/g and 70 mg/g of extract, respectively. The osteogenic activity of ChA, n-monobutyl ester of chelidonic acid, and CaChA has been studied in vitro in a 21-day culture of human adipose-derived multipotent mesenchymal stromal cells (hAMMSCs) in a standard nutrient medium without osteogenic supplements. CaChA significantly stimulated the growth of cell mass and differentiation of hAMMSCs into osteoblasts with subsequent mineralization of the culture and it may be a promising substance for accelerating bone tissue regeneration and engineering.

Nature vs. Nurture: Defining the Effects of Mesenchymal Stromal Cell Isolation and Culture Conditions on Resiliency to Palmitate Challenge.

As MSC products move from early development to clinical translation, culture conditions shift from xeno- to xeno-free systems. However, the impact of isolation and culture-expansion methods on the long-term resiliency of MSCs within challenging transplant environments is not fully understood. Recent work in our lab has shown that palmitate, a saturated fatty acid elevated in the serum of patients with obesity, causes MSCs to convert from an immunosuppressive to an immunostimulatory state at moderate to high physiological levels. This demonstrated that metabolically-diseased environments, like obesity, alter the immunomodulatory efficacy of healthy donor MSCs. In addition, it highlighted the need to test MSC efficacy not only in ideal conditions, but within challenging metabolic environments. To determine how the choice of xeno- vs. xeno-free media during isolation and expansion would affect future immunosuppressive function, umbilical cord explants from seven donors were subdivided and cultured within xeno- (fetal bovine serum, FBS) or xeno-free (human platelet lysate, PLT) medias, creating 14 distinct MSC preparations. After isolation and primary expansion, umbilical cord MSCs (ucMSC) were evaluated according to the ISCT minimal criteria for MSCs. Following baseline characterization, ucMSC were exposed to physiological doses of palmitate and analyzed for metabolic health, apoptotic induction, and immunomodulatory potency in co-cultures with stimulated human peripheral blood mononuclear cells. The paired experimental design (each ucMSC donor grown in two distinct culture environments) allowed us to delineate the contribution of inherent (nature) vs. environmentally-driven (nurture) donor characteristics to the phenotypic response of ucMSC during palmitate exposure. Culturing MSCs in PLT-media led to more consistent growth characteristics during the isolation and expansion for all donors, resulting in faster doubling times and higher cell yields compared to FBS. Upon palmitate challenge, PLT-ucMSCs showed a higher susceptibility to palmitate-induced metabolic disturbance, but less susceptibility to palmitate-induced apoptosis. Most striking however, was that the PLT-ucMSCs resisted the conversion to an immunostimulatory phenotype better than their FBS counterparts. Interestingly, examining MSC suppression of PBMC proliferation at physiologic doses of palmitate magnified the differences between donors, highlighting the utility of evaluating MSC products in stress-based assays that reflect the challenges MSCs may encounter post-transplantation.

Efficient GMP compliant expansion of mesenchymal stromal cells (MSCs) from umbilical cord, bone marrow and adipose tissue using a closed cultivation system.

Background & Aim Human mesenchymal stem cells (MSCs) hold great promise for clinical use and cell therapy applications and can be isolated from multiple tissue, e.g. bone marrow (BM), umbilical cord (UC) or adipose tissue (AT). As there is a high number of clinical trials and applications using MSCs, quality and safety of the resulting cellular products are indispensable. The xeno-free MSC-Brew GMP Medium following the recommendations of USP on ancillary materials enables a standardized expansion and culture of MSC from different tissue sources. To increase the level of process standardization and product safety we developed the CliniMACS Prodigy® Adherent Cell Culture System for GMP compliant isolation and cultivation of adherent cells using a closed single used tubing set. The process includes the flexible combination of the following modules: - Density Gradient Centrifugation (DGC) - Surface Coating - Inoculation - Culture - Media Change - Harvest Here we show that MSCs from different tissue sources (AT, BM, UC) can be expanded and passaged using the CliniMACS Prodigy® Adherent Cell Culture System from primary tissue or single-cell suspensions combining the process modules in a flexible way. Methods, Results & Conclusion BM-MSCs were isolated via DGC and subsequent adherence to plastic using semi-automatic feeding, harvesting and reseeding procedures. AT-MSCs as well as UC-MSCs were isolated and expanded from single-cell suspensions obtained after enzymatic digestion. UC was digested using an optimized and automated procedure using the gentle MACSTM Dissociator. Subsequently, MSCs were isolated from single cell suspension using the flexible cultivation procedure of the CliniMACS Prodigy® Adherent Cell Culture System. Resulting cells displayed an MSC specific phenotype as defined by the ISCT consortium. Furthermore MSCs revealed their immunomodulatory potential as assessed by in vitro assay and multi-color flow cytometric analysis. With the CliniMACS Prodigy®, MSCs can be expanded in large scales, thus providing high numbers of cells for consecutive clinical applications.

Culture enhanced mesenchymal stromal cells with increased potency

Background & Aim Mesenchymal stromal cells (MSCs) have been pursued for treatment of many inflammatory and fibrotic diseases due to their capacity to modulate immune cell (such as monocyte/macrophage, MΦ) phenotype and to dampen fibrosis. With the goal of developing an MSC product with increased potency, the Viswanathan lab has pioneered a strategy to generate 3D MSC aggregates with enhanced immunomodulatory functions (US62/397,572). The objective of this work is to assess the ability of culture enhanced MSCs to reduce inflammation and fibrosis, and to investigate mechanism of action by probing extracellular vesicle (EV) release and microRNA (miR) expression in 3D versus 2D MSCs. In parallel, hypoxic MSC cultures will also be investigated. Methods, Results & Conclusion MSCs were isolated from human bone marrow and subject to a serum-free 3D cell aggregation protocol (US62/397,572) with conventional 2D culture in serum-free medium or with 10% fetal bovine serum serving as controls. To evaluate MSC potency, gene expression analysis (quantitative PCR) was performed on MSCs licensed with tumour necrosis factor-α (TNFα) or interferon-γ (IFNγ) for 24 h. Results showed that TNFα-licensed 3D MSCs displayed highest expression of the anti-inflammatory marker TNFα-stimulated gene 6 ( TSG6 ) (Fig. 1) which has been correlated to MSC anti-inflammatory function. IFNγ-licensed 3D MSCs displayed reduced transcript levels of pro-inflammatory markers chemokine (C-X-C motif) ligand 8 ( CXCL8 ) and cyclooxygenase-2 ( COX2 ), as well as the pro-fibrotic marker transforming growth factor-β ( TGFβ ). Co-culture of MSCs with human peripheral blood-derived monocytes demonstrated that MSCs promote a shift toward a more homeostatic “M2-like” MΦ phenotype with higher expression of CD163 and CD206 relative to M1 markers CD86 and HLA-DR measured by flow cytometry (Fig. 2). A trend toward increased expression of CD163 was observed in 3D MSC co-cultures relative to serum 2D MSCs (p=0.0802) but no other statistical differences were noted. Taken together, these results demonstrate that 3D MSCs may have improved immunomodulatory capacity and are less fibrotic and inflammatory relative to MSCs cultured in 2D. In ongoing work, we are evaluating EV release and miR expression in enhanced MSCs (3D and hypoxic cultures) and will examine the role of these factors in mediating MΦ polarization and fibroblast phenotype in vitro . The results will aid in developing MSC therapies with increased potency for treatment of inflammatory and fibrotic conditions.

Bach impeller for suspension microcarrier cultures of mesenchymal stromal cells

Background & Aim Mesenchymal stromal cells (MSC) form the majority of very promising late stage clinical trials for indications like stroke, heart failure and osteoarthritis. As the industry seeks to meet the requirements for commercial scale offerings to large patient populace, production of these cell therapies will have to be met in scalable suspension culture vessels. We evaluated the Bach impeller that has an exponential spiral design which operates by creating fluid-fluid motion to culture MSC at 1 and 5 litre scale compared to the conventional axial flow impeller and paddle blade impeller. Simulations indicated that suspension of microcarriers would be rapid (approx. 5 mins) at 100rpm and the Bach would be able to provide 3-4 times the oxygen transfer capability of the axial flow impeller at the equivalent power per unit volume input. Methods, Results & Conclusion Culture of Wharton Jelly MSC at 1 litre with Solohill microcarriers showed 25% improved cell densities of the Bach impeller over the axial flow and paddle impellers: 4 × 10e5 cells/ml vs. 3 × 10e5 cells/ml. This was primarily due to extensive clumping of cell-microcarrier aggregates seen in the conventional impellers from day 3 onwards; whereas the laminar flow prevented aggregation up to the final day of harvesting. Significantly, when MSC were cultured on Cytodex microcarriers at 1 litre, cell yields increased dramatically to 15 × 10e5 cells/ml; viability and identity were equivalent to axial flow or spinner flask cultures. Furthermore, when MSC cultures were scaled further to 5 litre, the Bach yielded 13 × 10e5 cells/ml, and high viability. Thus, the Bach impeller is suitable for microcarrier suspension cultures of both polystyrene as well as hydrogel based microcarriers, and is a promising new impeller to add to the field of regenerative medicine for cell therapy manufacturing.

Human platelet lysate is suitable for the propagation of human bone marrow derived mesenchymal stromal cells

Background & Aim To evaluate the suitability of human platelet lysate (HPL) as a replacement for foetal bovine serum (FBS) for the propagation of human bone marrow derived mesenchymal stromal cells (MSCs). Background FBS is used universally as a cell culture supplement. Currently the use of FBS is still tolerated, however its use in clinical trials is tightly regulated and the US Food and Drug Administration (FDA) is moving towards adopting European guidelines, requiring that xenogeneic materials be eliminated in the preparation of any human therapeutic product containing cultured cells. Therefore, the use of HPL as a replacement for FBS in MSC expansion was evaluated. Methods, Results & Conclusion Methods Two types of HPL were produced by freeze-thawing and pooling either 12 units of expired apheresis platelet concentrates or 10 units of expired pooled platelet concentrates (n=3 batches each). MSCs from two donors were seeded at 5000 cells/cm2 and cultured to approximately 75-80% confluency using DMEM supplemented with either apheresis HPL, pooled HPL or FBS. Similar to FBS, HPL was heat-inactivated for 60 min at 56°C prior to addition to DMEM. The optimum concentration of HPL was determined by titration (2.5%, 5%, 7.5%, or 10%) against MSC culture expansion. Cell proliferation rates and doubling time were determined. Post-thaw immunophenotype and viability (7AAD) of BM-MSCs were determined by flow cytometry. Tri-lineage differentiation capacity was confirmed by microscopy after in vitro stimulation for either 14 days (chondrogenic and adipogenic) or 28 days (osteogenic). Results All 3 batches of HPL demonstrated an optimal culture concentration of 5% for apheresis HPL and 7.5 % for pooled HPL. Both types of HPL induced faster MSC proliferation when compared with matched 10% FBS controls. Apheresis HPL consistently outperformed pooled HPL, presumably as it is richer in growth factors. MSCs expanded under optimum conditions showed >90% expression of CD73, CD90 and CD105, and all were negative for CD45, CD34, CD19, CD11b, and HLA-DR. Mean 7AAD viability was 90% for MSCs grown in apheresis HPL, 88% for pooled HPL, and 84% for FBS. One batch of each type of HPL was used for tri-lineage differentiation capacity analysis. All MSCs were able to differentiate into either chondrogenic, osteogenic, or adipogenic lineages. Conclusion Apheresis and pooled HPL induced higher MSC expansion than FBS. The use of HPL as a substitute for FBS in MSC propagation warrants further investigation.

Nicotinamide mononucleotide promotes osteogenesis and reduces adipogenesis by regulating mesenchymal stromal cells via the SIRT1 pathway in aged bone marrow

Mesenchymal stromal cells (MSCs) can differentiate to various cell types including osteoblasts, chondrocytes, and adipocytes. This cellular flexibility contributes to widespread clinical use of MSCs in tissue repair. However, challenges remain in efficient cellular expansion of MSCs for stem cell therapy. Current MSC culture methods have resulted in reduced self-renewal of MSCs and compromised therapeutic outcomes. This study identifies that nicotinamide mononucleotide (NMN), a key natural NAD+ intermediate, effectively encourages MSC expansion in vitro and in vivo. The in vitro expanded MSCs had heightened osteogenesis, but reduced adipogenesis. Furthermore, NMN supplementation stimulated osteogenesis of endogenous MSCs, and protected bone from aging and irradiation induced damage in mice. Mechanistically, we found that NMN treatment upregulated SIRT1. Genetically overexpressing SIRT1 in MSCs by using Prx1 cre; ColA1flox-stop-flox-SIRT1 mice promoted osteogenesis and reduced adipogenesis in aged mice. Overall, our data demonstrate that NMN promoted MSC self-renewal with strengthened osteogenesis and reduced adipogenesis via upregulating SIRT1 in aged mice.

Sub-confluent culture of human mesenchymal stromal cells on biodegradable polycaprolactone microcarriers enhances bone healing of rat calvarial defect

In the current emerging trend of using human mesenchymal stromal cell (MSCs) for cell therapy, large quantities of cells are needed for clinical testing. Current methods of culturing cells, using tissue culture flasks or cell multilayer vessels, are proving to be ineffective in terms of cost, space and manpower. Therefore, alternatives such as large-scale industrialized production of MSCs in stirred tank bioreactors using microcarriers (MCs) are needed. Moreover, the development of biodegradable MCs for MSC expansion can streamline the bioprocess by eliminating the need for enzymatic cell harvesting and scaffold seeding for bone-healing therapies. Our previous studies described a process of making regulated density (1.06 g/cm3) porous polycaprolactone biodegradable MCs Light Polycarprolactone (LPCL) (MCs), which were used for expanding MSCs from various sources in stirred suspension culture. Here, we use human early MSCs (heMSCs) expanded on LPCL MCs for evaluation of their osteogenic differentiation potential in vitro as well as their use in vivo calvarial defect treatment in a rat model. In summary, (i) in vitro data show that LPCL MCs can be used to efficiently expand heMSCs in stirred cultures while maintaining surface marker expression; (ii) LPCL MCs can be used as scaffolds for cell transfer for transplantation in vivo; (iii) 50% sub-confluency, mid-logarithmic phase, on LPCL MCs (50% confluent) exhibited higher secretion levels of six cytokines (interleukin [IL]-6, IL-8, Vascular endothelial growth factor (VEGF), Monocyte Chemoattractant Protein-1 (MCP-1), growth-regulated oncogene-α (GRO-α) and stromal cell-derived factor-1α (SDF-1α)) as compared with 100% confluent, stationary phase cultures (100% confluent); (iv) these 50% confluent cultures demonstrated better in vitro osteogenic differentiation capacity as compared with 100% confluent cultures (higher levels of calcium deposition and at earlier stage); the improved bone differentiation capacity of these 50% confluent cultures was also demonstrated at the molecular level by higher expression of early osteoblast genes Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP), collagen type I, osterix and osteocalcin); and (v) in vivo implantation of biodegradable LPCL MCs covered with 50% heMSCs into rats with calvarial defect demonstrated significantly better bone formation as compared with heMSCs obtained from monolayer cultures (5.1 ± 1.6 mm3 versus 1.3 ± 0.7 mm3). Moreover, the LPCL MCs covered with 50% heMSCs supported better in vivo bone formation compared with 100% confluent culture (2.1 ± 1.3 mm3). Taken together, our study highlights the potential of implanting 50% confluent MSCs propagated on LPCL MCs as optimal for bone regeneration. This methodology allows for the production of large numbers of MSCs in a three-dimensional (3D) stirred reactor, while supporting improved bone healing and eliminating the need for a 3D matrix support scaffold, as traditionally used in bone-healing treatments.

Erratum: Spheroid Culture of Mesenchymal Stromal Cells Results in Morphorheological Properties Appropriate for Improved Microcirculation.

[This corrects the article DOI: 10.1002/advs.201802104.].

Circulating factors present in the sera of naturally skinny people may influence cell commitment and adipocyte differentiation of mesenchymal stromal cells.

BACKGROUNDResearch on physiopathology of obesity may receive new hints from studies on skinny people (SP). These are individuals who show a poor or null gaining of body weight, in spite of high-calorie intake, by far exceeding the body requirements.AIMTo evaluate how circulating factors present in the SP sera may affect adipogenesis of mesenchymal stromal cells (MSCs).METHODSWe isolated MSCs from bone marrow of healthy donors with both normal body mass index (BMI) and caloric consumption. MSC cultures were primed with sera collected from SP or normal people (NP). Then biomolecular assays were performed to evaluate effect on proliferation, apoptosis, senescence, cell commitment, and differentiation.RESULTSSP priming affected adipocyte cell commitment and reduced spontaneous adipogenesis. Moreover, an in-depth analysis of exogenous-induced adipocyte differentiation showed striking differences between differentiation in SP-primed samples compared with NP ones. In adipocytes from SP cultures we observed a reduced size of lipid droplets, an increased expression of adipose triglyceride lipase, along with high mitochondria content and ability to produce ATP in starvation condition. These data and the expression of UCP1 protein, indicated that SP pretreatment produced a bias toward brown adipocyte differentiation.CONCLUSIONOur data suggest that sera from SP may promote brown adipogenesis rather that white adipocyte differentiation. This finding could explain why SP present normal body composition in spite of an excess of caloric intake. We hypothesize that some circulating components present in the blood of these individuals may favor brown adipogenesis at expense of white adipocyte production.

Spheroid Culture of Mesenchymal Stromal Cells Results in Morphorheological Properties Appropriate for Improved Microcirculation.

Human bone marrow mesenchymal stromal cells (MSCs) are used in clinical trials for the treatment of systemic inflammatory diseases due to their regenerative and immunomodulatory properties. However, intravenous administration of MSCs is hampered by cell trapping within the pulmonary capillary networks. Here, it is hypothesized that traditional 2D plastic‐adherent cell expansion fails to result in appropriate morphorheological properties required for successful cell circulation. To address this issue, a method to culture MSCs in nonadherent 3D spheroids (mesenspheres) is adapted. The biological properties of mesensphere‐cultured MSCs remain identical to conventional 2D cultures. However, morphorheological analyses reveal a smaller size and lower stiffness of mesensphere‐derived MSCs compared to plastic‐adherent MSCs, measured using real‐time deformability cytometry and atomic force microscopy. These properties result in an increased ability to pass through microconstrictions in an ex vivo microcirculation assay. This ability is confirmed in vivo by comparison of cell accumulation in various organ capillary networks after intravenous injection of both types of MSCs in mouse. The findings generally identify cellular morphorheological properties as attractive targets for improving microcirculation and specifically suggest mesensphere culture as a promising approach for optimized MSC‐based therapies.

The effects of cell type and culture condition on the procoagulant activity of human mesenchymal stromal cell-derived extracellular vesicles.

Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have great potential as a cell-free therapy in wound healing applications. Because EV populations are not equivalent, rigorous characterization is needed before clinical use. Although there has been much focus on their RNA composition and regenerative capabilities, relatively less is known regarding the effects of MSC cell type (adipose tissue [Ad-MSCs] or bone marrow [BM-MSCs]) and culture condition (monolayer or spheroid) on MSC-EV performance, including characteristics related to their ability to promote coagulation, which could determine EV safety if administered intravenously. The successful isolation of EVs derived from Ad-MSCs or BM-MSCs cultured in either monolayer or spheroid cultures was confirmed by NanoSight (particle size distribution) and Western blot (surface marker expression). Extracellular vesicle surface expression of procoagulant molecules (tissue factor and phosphatidylserine) was evaluated by flow cytometry. Extracellular vesicle thrombogenicity was tested using calibrated thrombogram, and clotting parameters were assessed using thromboelastography and a flow-based adhesion model simulating blood flow over a collagen-expressing surface. The MSC cell type and culture condition did not impact EV size distribution. Extracellular vesicles from all groups expressed phosphatidylserine and tissue factor on their surfaces were functionally thrombogenic and tended to increase clotting rates compared to the negative control of serum-free media without EVs. On average, EVs did not form significantly larger or stronger clots than the negative control, regardless of cell source or culture condition. Additionally, EVs interfered with platelet adhesion in an in vitro flow-based assay. Adipose-derived EVs were more thrombogenic and expressed higher amounts of phosphatidylserine. Our findings suggest that, like intact MSCs, source variability among EVs is an important factor when considering EVs for potential therapeutic purposes. Therapeutic care management, level II.

CHARACTERISTICS OF BONE MARROW MICROENVIRONMENT IN MULTIPLE MYELOMA BEFORE AND AFTER TREATMENT

The purpose of the study is to evaluate the morphological and functional features of the tumor microenvironment and the cultural characteristics of mesenchymal stromal cells (MSC) of patients with multiple myeloma (MM) before and after treatment. Material and methods. Parenchymal and stromal bone marrow tissue in iliac bone trepanobiopsy of 32 patients with MM aged from 48 to 78 years (median age 56 years) before and after treatment was investigated. The study applied histological, immunohistochemical and cultural methods. Results and discussion. Сhanges of stromal microenvironment of bone marrow in all patients with MM have been revealed: increased microvascular density and the number of endosteal stromal cells, strengthening of reticulin fiber in subendosteal and perivascular spaces. Increased angiogenesis correlates with the number of plasma cells in the myelogram ( r = 0.58; p < 0.05) and with the type of bone marrow infiltration ( r = 0.85; р < 0.05), as well as with osteodestructive changes in the patient’s history ( r = 0.65; p < 0.05). Significant changes in MSC cultures, both before and after therapy, including autologous hematopoietic stem-cell transplantation, were shown. Bone marrow MSC of patients with MM had decreased speed of proliferation (2.4 times on average), total time of passaging from 7 to 5 passages, increased expression of markers associated with myofibroblastic phenotype and aging (smooth muscle actin, β-galactosidase) compared to healthy donors. Conclusion. MSC cultures and BM niche forming elements of patients with MM have signs of tumor-associated microenvironment, despite the treatment of the underlying disease.