Mesenchymal Stromal Cells Cultures Research Articles

Cobblestone Area-forming Cell Assay of Mouse Bone Marrow Hematopoietic Stem Cells.

Bone Marrow Hematopoietic Stem Cells (HSCs) require bone marrow microenvironment for their maintenance and proliferation. Culture of Bone Marrow Mesenchymal Stromal Cells (MSCs) provides appropriate environmental signals for HSCs survival in vitro. Here, we provide a detailed protocol that describes culture conditions for MSCs, flow cytometric isolation of HSCs from mouse bone marrow, and perform co-culture of MSCs and HSCs known as Cobblestone area-forming cell (CAFC) assay. Altogether, CAFC assays can be used as a high-throughput in vitro screening model where efforts are made to understand and develop therapies for complex bone marrow diseases. This protocol needs 3 to 4 weeks starting from culturing MSCs, isolating LSK cells (HSCs), and to performing limited dilution CAFC assay.

Activation of NK Cells in Mixed Cultures of Wharton's Jelly Mesenchymal Stromal Cells and Peripheral Blood Lymphocytes.

Mesenchymal stromal cells possess immunosuppressive properties that might be used for the therapy of inflammatory diseases of various geneses. The effects of mesenchymal stromal cells depend on their lifetime in the recipient tissues. During heterologous transplantation, mesenchymal stromal cells are eliminated by NK cells. We studied NK cell formation in mixed cultures of Wharton's jelly mesenchymal stromal cells and peripheral blood lymphocytes from an autologous donor. Lymphocytes were activated by a mitogen or IL-2. The lifetime of mesenchymal stromal cells was estimated by MTT test. Cytotoxic activity and phenotype of NK cells were evaluated by flow cytometry. It was found that activation of NK cells depended on IL-2 and was registered on day 2 of incubation with IL-2. In cultures with mitogen-activated lymphocytes, cytotoxicity was observed after 5-6 days. Cytotoxicity of NK correlated with significant decrease in CD16+ and increase in CD56+ NK and with reduction of mesenchymal stromal cell viability. Thus, the main mechanism of elimination of mesenchymal stromal cells is cytotoxicity of NK cells that depended on IL-2 production.

Standards for the culture and quality control of umbilical cord mesenchymal stromal cells for neurorestorative clinical application (2017)

Standards for the culture and quality control of umbilical cord mesenchymal stromal cells for neurorestorative clinical application (2017)

Indirect osteoblast differentiation by liposomal clodronate.

Bisphosphonates impair function of osteoclasts and prevent bone resorption, the mechanism of which has been studied extensively. However, the possible effects of bisphosphonates on chondroblast differentiation and calcium deposition by osteoblasts have only been demonstrated recently. Moreover, cells from monocytic lineage are capable of stimulating osteoblast proliferation. Hence, susceptibility of osteoblasts to various factors requires further investigation. A primary culture of bone marrow‐derived stromal cells was treated with liposomal clodronate (0.1, 0.5, or 1.0 mg/ml) or conditioned medium from liposomal clodronate. Liposomal clodronate (0.25 mg) was injected into mouse femur for in vivo experiments. The effects of liposomal clodronate were examined by alkaline phosphatase staining and/or activity assay, and real‐time RT‐PCR was used for studying the effect on osteogenic gene expression. Administration of liposomal clodronate to bone marrow‐derived mesenchymal stromal cell culture enhanced alkaline phosphatase activity and mRNA levels of Runx2 and Dlx5. In addition, conditioned medium from liposomal clodronate also stimulated osteogenic characteristics similar to those of observed in vitro, and the number of exosomes in the conditioned medium was highest when pre‐treated with liposomal clodronate. Western blot analysis revealed the presence of RANK proteins in exosomes collected from conditioned medium of liposomal clodronate. Identical observations were obtained in vivo, as liposomal clodronate‐injected mouse femur showed increased alkaline phosphatase activity and Runx2 and Dlx5 mRNA expressions, even though the numbers of monocytes and macrophages were reduced. In conclusion, osteoblast differentiation was promoted via soluble RANK‐containing exosomes in response to clodronates.

Serum-free human MSC medium supports consistency in human but not in equine adipose-derived multipotent mesenchymal stromal cell culture.

For clinical applications of multipotent mesenchymal stromal cells (MSCs), serum-free culture is preferable to standardize cell products and prevent contamination with pathogens. In contrast to human MSCs, knowledge on serum-free culture of large animal MSCs is limited, despite its relevance for preclinical studies and development of veterinary cellular therapeutics. This study aimed to evaluate the suitability of a commercially available serum-free human MSC medium for culturing equine adipose-derived MSCs in comparison with human adipose MSCs. Enzyme-free isolation by explant technique and expansion of equine and human cells in the serum-free medium were feasible. However, serum-free culture altered the morphology and complicated handling of equine MSCs, with cell aggregation and spontaneous detachment of multilayers, compared to culture in standard medium supplemented with fetal bovine serum. Furthermore, proliferation and the surface immunophenotype of equine cells were more variable compared to the controls and appeared to depend on the lot of the serum-free medium. Particularly the expression of CD90 was different between experimental groups (P < 0.05), with lower percentages of CD90+ cells found in equine MSC samples cultured in serum-free medium (5.21-83.40%) compared to standard medium (86.20-99.50%). Additionally, small subpopulations expressing MSC exclusion markers such as CD14 (0.28-11.60%), CD34 (0.00-9.87%), CD45 (0.35-10.50%), or MHCII (0.00-3.67%) were found in equine samples after serum-free culture. In contrast, human samples displayed a more consistent morphology and a consistent CD29+ (98.60-99.90%), CD73+ (94.60-98.40%), CD90+ (99.60-99.90%), and CD105+ (97.40-99.80%) immunophenotype after culture in serum-free medium. The obtained data demonstrate that the serum-free medium was suitable for human MSC culture but did not lead to entirely satisfactory results in equine MSCs. This underlines that requirements regarding serum-free culture conditions are species-specific, indicating aneed for serum-free media to be optimized for MSCs from relevant animal species. © 2017 International Society for Advancement of Cytometry.

Proliferation of Peripheral Blood Lymphocytes and Mesenchymal Stromal Cells Derived from Wharton's Jelly in Mixed and Membrane-Separated Cultures.

We studied the effect of mesenchymal stromal cells on proliferation of CFSE-stained T cells in mixed and membrane-separated (Transwell) cultures and in 3D culture of mesenchymal stromal cells from Wharton's jelly. The interaction of mesenchymal stromal cells with mitogen-activated peripheral blood lymphocytes from an allogeneic donor was followed by suppression of T-cell proliferation in a wide range of cell proportions. Culturing in the Transwell system showed the absence of suppression assessed by the fraction of proliferating cells and by the cell cycle analysis. In 3D cultures, contact interaction of mesenchymal stromal cells and lymphocytes was demonstrated that led to accumulation of G2/M phase lymphocytes and G0/G1 phase mesenchymal stromal cells. The suppressive effect of mesenchymal stromal cells from Wharton's jelly is mediated by two mechanisms. The effects are realized within 6 days, which suggests that the therapeutic effects of mesenchymal stromal cells persist until their complete elimination from the body.

Shift of EMT gradient in 3D spheroid MSCs for activation of mesenchymal niche function

Despite the wide use of mesenchymal stromal cells (MSCs) for paracrine support in clinical trials, their variable and heterogeneous supporting activity pose major challenges. While three-dimensional (3D) MSC cultures are emerging as alternative approaches, key changes in cellular characteristics during 3D-spheroid formation remain unclear. Here, we show that MSCs in 3D spheroids undergo further progression towards the epithelial-mesenchymal transition (EMT), driven by upregulation of EMT-promoting microRNAs and suppression of EMT-inhibitory miRNAs. The shift of EMT in MSCs is associated with widespread histone modifications mimicking the epigenetic reprogramming towards enhanced chromatin dynamics and stem cell-like properties, but without changes in their surface phenotype. Notably, these molecular shifts towards EMT in 3D MSCs caused enhanced stem cell niche activity, resulting in higher stimulation of hematopoietic progenitor self-renewal and cancer stem cell metastasis. Moreover, miRNA-mediated induction of EMT in 2D MSCs were sufficient to mimic the enhanced niche activity of 3D spheroid MSCs. Thus, the molecular hierarchy in the EMT gradient among phenotypically indistinguishable MSCs revealed the previously unrecognized functional parameters in MSCs, and the EMT-enhanced “naïve” mesenchymal state represents an ‘activated mesenchymal niche’ in 3D spheroid MSCs.

Human Platelet Lysate versus Fetal Calf Serum: These Supplements Do Not Select for Different Mesenchymal Stromal Cells

Culture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology are affected by these supplements – it is therefore important to determine if they favor outgrowth of different subpopulations and thereby impact on the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow-derived MSCs in parallel with HPL or FCS and demonstrated that HPL significantly increases proliferation and leads to dramatic differences in cellular morphology. Remarkably, global DNA-methylation profiles did not reveal any significant differences. Even at the transcriptomic level, there were only moderate changes in pairwise comparison. Furthermore, the effects on proliferation, cytoskeletal organization, and focal adhesions were reversible by interchanging to opposite culture conditions. These results indicate that cultivation of MSCs with HPL or FCS has no systematic bias for specific cell types.

Extracellular vesicles of bone marrow stromal cells rescue chronic lymphocytic leukemia B cells from apoptosis, enhance their migration and induce gene expression modifications.

Interactions between chronic lymphocytic leukemia (CLL) B cells and the bone marrow (BM) microenvironment play a major function in the physiopathology of CLL. Extracellular vesicles (EVs), which are composed of exosomes and microparticles, play an important role in cell communication. However, little is known about their role in CLL / microenvironment interactions. In the present study, EVs purified by ultracentrifugation from BM mesenchymal stromal cell (BM-MSC) cultures were added to CLL B cells. After their integration into CLL B cells, we observed a decrease of leukemic cell spontaneous apoptosis and an increase in their chemoresistance to several drugs, including fludarabine, ibrutinib, idelalisib and venetoclax after 24 hours. Spontaneous (P=0.0078) and stromal cell-derived factor 1α -induced migration capacities of CLL B cells were also enhanced (P=0.0020). A microarray study highlighted 805 differentially expressed genes between leukemic cells cultured with or without EVs. Of these, genes involved in the B-cell receptor pathway such as CCL3/4, EGR1/2/3, and MYC were increased. Interestingly, this signature presents important overlaps with other microenvironment stimuli such as B-cell receptor stimulation, CLL/nurse-like cells co-culture or those provided by a lymph node microenvironment. Finally, we showed that EVs from MSCs of leukemic patients also rescue leukemic cells from spontaneous or drug-induced apoptosis. However, they induce a higher migration and also a stronger gene modification compared to EVs of healthy MSCs. In conclusion, we show that EVs play a crucial role in CLL B cells/BM microenvironment communication.

ПОЛОЖИТЕЛЬНЫЙ ОПЫТ ТРАНСПЛАНТАЦИИ МНОГОКОМПОНЕНТНОГО КОМПОЗИТА, СОДЕРЖАЩЕГО АЛЛОГЕННЫЕ МЕЗЕНХИМАЛЬНЫЕ СТВОЛОВЫЕ КЛЕТКИ, ПОСЛЕ РЕЗЕКЦИИ СТЕНКИ МОЧЕВОГО ПУЗЫРЯ КРОЛИКА

Of late much attention has been paid to tissue engineering by urologists. After successful testing on animals, artificial urinary bladders with self-specific cells were transplanted to humans. Our research is aimed at investigating the opportunity of using cellular technologies if no healthy self-specific material is available. The goal of this experiment is to investigate the opportunity of using a multi-component composite material containing allogeneic cells to replace the defect of urinary wall under experimental conditions. The standard technique was used for isolation and culturing of mesenchymal stromal stem cells from the rabbit's bone marrow. Multi-component composite material based on the polylactide matrix was inoculated by allogeneic cells and transplanted in vivo to the model of partial cystectomy. In 2.5 months the presence of labeled cells in the implantation site was confirmed by objective methods.

Selective laser melting-enabled electrospinning: Introducing complexity within electrospun membranes.

Additive manufacturing technologies enable the creation of very precise and well-defined structures that can mimic hierarchical features of natural tissues. In this article, we describe the development of a manufacturing technology platform to produce innovative biodegradable membranes that are enhanced with controlled microenvironments produced via a combination of selective laser melting techniques and conventional electrospinning. This work underpins the manufacture of a new generation of biomaterial devices that have significant potential for use as both basic research tools and components of therapeutic implants. The membranes were successfully manufactured and a total of three microenvironment designs (niches) were chosen for thorough characterisation. Scanning electron microscopy analysis demonstrated differences in fibre diameters within different areas of the niche structures as well as differences in fibre density. We also showed the potential of using the microfabricated membranes for supporting mesenchymal stromal cell culture and proliferation. We demonstrated that mesenchymal stromal cells grow and populate the membranes penetrating within the niche-like structures. These findings demonstrate the creation of a very versatile tool that can be used in a variety of tissue regeneration applications including bone healing.

Expansion strategies for human mesenchymal stromal cells culture under xeno-free conditions.

Choosing the culture system and culture medium used to produce cells are key steps toward a safe, scalable, and cost-effective expansion bioprocess for cell therapy purposes. The use of AB human serum (AB HS) as an alternative xeno-free supplement for mesenchymal stromal cells (MSC) cultivation has increasingly gained relevance due to safety and efficiency aspects. Here we have evaluated different scalable culture systems to produce a meaningful number of umbilical cord matrix-derived MSC (UCM MSC) using AB HS for culture medium supplementation during expansion and cryopreservation to enable a xeno-free bioprocess. UCM MSC were cultured in a scalable planar (compact 10-layer flasks and roller bottles) and 3-D microcarrier-based culture systems (spinner flasks and stirred tank bioreactor). Ten layer flasks and roller bottles enabled the production of 2.6±0.6×104 and 1.4±0.3×104 cells/cm2 . UCM MSC-based microcarrier expansion in the stirred conditions has enabled the production of higher cell densities (5.5-23.0×104 cells/cm2 ) when compared to planar systems. Nevertheless, due to the moderate harvesting efficiency attained, (80% for spinner flasks and 46.6% for bioreactor) the total cell number recovered was lower than expected. Cells maintained the functional properties after expansion in all the culture systems evaluated. The cryopreservation of cells (using AB HS) was also successfully carried out. Establishing scalable xeno-free expansion processes represents an important step toward a GMP compliant large-scale production platform for MSC-based clinical applications. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1358-1367, 2017.

Human adult mesangiogenic progenitor cells reveal an early angiogenic potential, which is lost after mesengenic differentiation

BackgroundMesangiogenic progenitor cells (MPCs) have shown the ability to differentiate in-vitro toward mesenchymal stromal cells (MSCs) as well as angiogenic potential. MPCs have so far been described in detail as progenitors of the mesodermal lineage and appear to be of great significance in tissue regeneration and in hemopoietic niche regulation. On the contrary, information regarding the MPC angiogenic process is still incomplete and requires further clarification. In particular, genuine MPC angiogenic potential should be confirmed in-vivo.MethodsIn the present article, markers and functions associated with angiogenic cells have been dissected. MPCs freshly isolated from human bone marrow have been induced to differentiate into exponentially growing MSCs (P2-MSCs). Cells have been characterized and angiogenesis-related gene expression was evaluated before and after mesengenic differentiation. Moreover, angiogenic potential has been tested by in-vitro and in-vivo functional assays.ResultsMPCs showed a distinctive gene expression profile, acetylated-low density lipoprotein uptake, and transendothelial migration capacity. However, mature endothelial markers and functions of endothelial cells, including the ability to form new capillaries, were absent, thus suggesting MPCs to be very immature endothelial progenitors. MPCs showed marked 3D spheroid sprouting activating the related molecular machinery, a clear in-vitro indication of early angiogenesis. Indeed, MPCs applied to chicken chorioallantoic membrane induced and participated in neovessel formation. All of these features were lost in mesengenic terminally differentiated P2-MSCs, showing definite separation of the two differentiation lineages.ConclusionOur results confirm the bona-fide angiogenic potential of MPCs and suggest that the high variability reported for MSC cultures, responsible for the controversies regarding MSC angiogenic potential, could be correlated to variable percentages of co-isolated MPCs in the different culture conditions so far used.

Cytokine Production in Mixed Cultures of Mesenchymal Stromal Cells from Wharton's Jelly and Peripheral Blood Lymphocytes.

We compared the production of 19 humoral factors in mixed cultures of mesenchymal stromal cells from Wharton's jelly and allogenic peripheral blood lymphocytes. For evaluation of the specificity of immunosuppressive activity of mesenchymal stromal cells, comparative analysis of the production of these humoral factors in mixed cultures of lymphocytes and epithelial BxPC-3 cells was conducted. The production of soluble factors in both mono- and mixed cultures significantly correlated (p<0.05). The maximum production was found for proinflammatory chemokine IP-10 and IFN-γ and anti-inflammatory cytokine IL-10. The major difference of mesenchymal stromal cells from epithelial BxPC-3 cells was 7-fold higher production of IL-10, which can explain the immunosuppressive effect of mesenchymal stromal cells.

Polymeric film of 6-arm-poly(ethylene glycol) amine graphene oxide with poly (ε-caprolactone): Adherence and growth of adipose derived mesenchymal stromal cells culture on rat bladder

Nanotechnology has been more present in different fields related to health. The need to find a durable material, of easy use, and which does not interfere significantly in the growth and differentiation of stem cells for the construction of a scaffold for use in urologic surgery, with the purpose of reducing infections, regeneration times and even graft rejection during reconstitution in patients with urethral stricture was conducted a broad survey of information about this and came to the consensus of this project: using graphene oxide, a widely studied nanomaterials which has been presenting numerous beneficial results when in contact with the adipose-derived stem cells. Advanced techniques for the growth, differentiation and proliferation of adipose-derived stem cells were used, as well as the characterization of graphene oxide sheets. For this study, it was prepared the graphene oxide/6 ARM-Poly (ethylene glycol) amine films with poly (ε-caprolactone). The graphene suspension in organic solvent was prepared by using an ultrasonicator bath and subsequently, the film was formed by solvent evaporation. Total characterization of graphene oxide/6 ARM-PEG-amine/ poly (ε-caprolactone) film was carried out. It was tested growth and adhesion of adipose-derived stem cells on the film, as well as, were verified the histopathological effects of this scaffold when implanted in the urinary bladder to repair the lesion. Our results demonstrated that this scaffold with adipose-derived stem cells enhanced the repair in rat urinary bladder defect model, resulting in a regular bladder. Improved organized muscle bundles and urothelial layer were observed in animals treated with this scaffold with adipose-derived stem cells compared with those treated only suture thread or scaffold. Thus, our biomaterial could be suitable for tissue engineered urinary tract reconstruction.

Human Umbilical Cord Blood Serum: Effective Substitute of Fetal Bovine Serum for Culturing of Human Multipotent Mesenchymal Stromal Cells.

Optimal conditions for culturing of multipotent mesenchymal stromal cells in the presence of pooled umbilical cord blood serum were determined. It was found that umbilical cord blood serum in a concentration range of 1-10% effectively supported high viability and proliferative activity of cells with unaltered phenotype and preserved multilineage differentiation capacity. The proposed approach allows avoiding the use of xenogenic animal sera for culturing of multipotent mesenchymal stromal cells and creates prerequisites for designing and manufacturing safe cellular and/or acellular products for medical purposes.

Conditions for Collection of Placental Tissue Samples for Culturing of Multipotent Mesenchymal Stromal Cells.

Placentas from women aged 25-32 years with normal course of gestation were studied. It is essential to stick to certain methodological approaches for preparing viable multipotent mesenchymal stromal cell culture and to carry out morphological (macro and micro) evaluation of the chorionic villi, umbilical cords, and placentas. At stage I of the study, patients' histories, labor course, and examinations of the newborns should be analyzed to exclude women with genital and extragenital diseases. At stage II, it is essential to stick to special regulations and methods for collection of specimens of the cord, amnion, and placental tissue proper. Histological control of the placental structures collected for multipotent mesenchymal stromal cell culturing is obligatory.

Тканеинженерные конструкции для регенеративной медицины на основе мезенхимальных клеток пульпы молочного зуба и полимерных матриксов нового поколения

The aim of the present work was to develop an approach for producing tissue engineered constructs based on mesenchymal stromal cell culture and new-generation biodegradable synthetic polymer matrices obtained by the technology of surface-selective laser sintering. Primary mesenchymal stromal cells were derived from the pulp of naturally shed deciduous teeth. Following seeding, the cells were cultured on matrices for ten days. Cell viability was quantitatively assessed using CytoTox Assay (Promega) based on the colorimetric measurement of mitochondrial dehydrogenase activity. Cellular structure and spatial arrangement within a tissue-engineered matrix was visualized by scanning electron microscopy (SEM), using supravital lanthanoid staining. The results obtained showed that matrices under development enable good cell adhesion and proliferation, and hence can be used as a cell carrier in tissue engineering. Thus, the techniques of surface-selective laser sintering, which was developed by us, could find wide use in regenerative medicine.

Molecular Mechanisms of Immunomodulation Properties of Mesenchymal Stromal Cells: A New Insight into the Role of ICAM-1.

Mesenchymal stromal cells (MSC) control excessive inflammation and create a microenvironment for tissue repair protecting from chronic inflammation and tissue fibrosis. We examined the molecular mechanisms of MSC immunomodulatory function in mixed cultures of human adipose-derived MSC with lymphocytes. Our data show that MSC promote unstimulated lymphocyte survival potentially by an increase in antigen presentation. Under inflammatory conditions, mimicked by stimulation of TCR in lymphocytes, MSC suppress activation and proliferation of stimulated T cells. Immunosuppression is accompanied by downregulation of IL-2Rα that negatively affects the survival of activated T cells. MSC upregulate transcription of indolamine-2,3-dioxygenase (IDO) and inducible NO synthase (iNOS), which generate products negatively affecting T cell function. Both MSC and lymphocytes dramatically increase the surface ICAM-1 level in mixed cultures. Antibody-mediated blockage of surface ICAM-1 partially releases MSC-mediated immune suppression in vitro. Our data suggest that MSC have cell-intrinsic molecular programs depending on the inflammatory microenvironment. We speculate that MSC sense soluble factors and respond by surface ICAM-1 upregulation. ICAM-1 is involved in the control of T cell activation leading to immunosuppression or modest stimulation depending on the T cell status. Immunomodulation by MSC ranging from support of naive T cell survival to immunosuppression of activated T cells may affect the tissue microenvironment protecting from aberrant regeneration.

Comparison of the expression profile of surface molecular markers on mesenchymal stromal cell cultures isolated from human endometrium and umbilical cord

Endometrial mesenchymal stromal cells (eMSCs), along with mesenchymal stromal cells (MSCs) isolated from other tissues, are promising for use in regenerative medicine. The benefits of eMSCs include their presence in adults, simplicity of isolation, high proliferative and differentiation capacity. In this study, we have employed the flow cytometry technique to assess expression of 28 molecular markers on the surface of two eMSCs cultures. The culture of MSCs isolated from Wharton's jelly of the umbilical cord (uMSCs) was used as a reference, because uMSCs were studied in details earlier and demonstrated their effectiveness in vivo. Both types of MSCs demonstrated similar expression profiles. They included stem cells surface molecules, cell adhesion molecules and their ligands, some receptor molecules responsible for cell metabolism and proliferation, as well as immunological response molecules.