Mesenchymal Stromal Cells Cultures Research Articles

Incorporation of strontium-containing bioactive particles into PEOT/PBT electrospun scaffolds for bone tissue regeneration

The combination of biomaterials and bioactive particles has shown to be a successful strategy to fabricate electrospun scaffolds for bone tissue engineering. Among the range of bioactive particles, hydroxyapatite and mesoporous bioactive glasses (MBGs) have been widely used for their osteoconductive and osteoinductive properties. Yet, the comparison between the chemical and mechanical characteristics as well as the biological performances of these particle-containing scaffolds have been characterized to a limited extent. In this work, we fabricated PEOT/PBT-based composite scaffolds incorporating either nanohydroxyapatite (nHA), strontium-containing nanohydroxyapatite (nHA_Sr) or MBGs doped with strontium ions up to 15 wt./vol% and 12,5 wt./vol% for nHA and MBG, respectively. The composite scaffolds presented a homogeneous particle distribution. Morphological, chemical and mechanical analysis revealed that the introduction of particles into the electrospun meshes caused a decrease in the fiber diameter and mechanical properties, yet maintaining the hydrophilic nature of the scaffolds. The Sr2+ release profile differed according to the considered system, observing a 35-day slowly decreasing release from strontium-containing nHA scaffolds, whereas MBG-based scaffolds showed a strong burst release in the first week. In vitro, culture of human bone marrow-derived mesenchymal stromal cells (hMSCs) on composite scaffolds demonstrated excellent cell adhesion and proliferation. In maintenance and osteogenic media, all composite scaffolds showed high mineralization as well as expression of Col I and OCN compared to PEOT/PBT scaffolds, suggesting their ability to boost bone formation even without osteogenic factors. The presence of strontium led to an increase in collagen secretion and matrix mineralization in osteogenic medium, while gene expression analysis showed that hMSCs cultured on nHA-based scaffolds had a higher expression of OCN, ALP and RUNX2 compared to cells cultured on nHA_Sr scaffolds in osteogenic medium. Yet, cells cultured on MBGs-based scaffolds showed a higher gene expression of COL1, ALP, RUNX2 and BMP2 in osteogenic medium compared to nHA-based scaffolds, which is hypothesized to lead to high osteoinductivity in long term cultures.

Discriminant Principal Component Analysis of ToF-SIMS Spectra for Deciphering Compositional Differences of MSC-Secreted Extracellular Matrices.

Identifying characteristic extracellular matrix (ECM) variants is a key challenge in mechanistic biology, bioengineering, and medical diagnostics. The reported study demonstrates the potential of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to detect subtle differences between human mesenchymal stromal cell (MSC)-secreted ECM types as induced by exogenous stimulation or emerging pathology. ToF-SIMS spectra of decellularized ECM samples are evaluated by discriminant principal component analysis (DPCA), an advanced multivariate analysis technique, to decipher characteristic compositional features. To establish the approach, signatures of major ECM proteins are determined from samples of pre-defined mixtures. Based on that, sets of ECM variants produced by MSCs in vitro are analyzed. Differences in the content of collagen, fibronectin, and laminin in the ECM resulting from the combined supplementation of MSC cultures with polymers that induce macromolecular crowding and with ascorbic acid are detected from the DPCA of ToF-SIMS spectra. The results are verified by immunostaining. Finally, the comparative ToF-SIMS analysis of ECM produced by MSCs of healthy donors and patients suffering from myelodysplastic syndrome display the potential of the novel methodology to reveal disease-associated alterations of the ECM composition.

Effect of the bone marrow mesenchymal stromal cells cytokine-producing activity on mobilization of hematopoietic stem cells

Hematopoietic stem cell transplantation is used to treat hemoblastoses and some other diseases. Depending on the diagnosis, autologous cells of the patient or allogeneic cells obtained from a healthy related or unrelated donor are used. A sufficient count of harvested hematopoietic progenitor cells is necessary for successful transplantation. Currently, stimulation of their egress from the bone marrow into the peripheral blood by a granulocyte colony-stimulating factor, followed by collection by leukapheresis, is widely used for their preparation. Hematopoietic stem cells leave the bone marrow niche formed by the stromal microenvironment when their holding bonds are interrupted. Mesenchymal stromal cells regulate the release of hematopoietic precursors by producing various cytokines and other biologically active substances. Therefore, the study of the functional properties of bone marrow mesenchymal cells can help in solving the problem of “poor” mobilizations that occur in patients with multiple myeloma.The aim of the study was to estimate the effectiveness of mobilization of hematopoietic stem cells depending on the cytokine-producing ability of mesenchymal stromal cells in the bone marrow of donors and patients with multiple myeloma.The yields of hematopoietic progenitors were studied in 10 donors (median age 27 years) and 18 patients with multiple myeloma (median age 57 years). In donors, the release of hematopoietic stem cells into the peripheral blood was stimulated by subcutaneous administration of G-CSF at a dose of 10 μg/kg/day. Patients with multiple myeloma received vinorelbine at a dose of 35 mg/m2 of body surface, followed by administration of G-CSF (10 μg/kg/day subcutaneously). A culture of mesenchymal stromal cells was obtained from bone marrow taken prior to mobilization and the content of interleukins IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α and IFN-γ in supernatants was studied by enzyme-linked immunosorbent assay using kits of reagents produced by Vector-Best (Novosibirsk).Patients with multiple myeloma were characterized by high secretion of the pro-inflammatory cytokine IL-2 (4.70 pg/ml vs 3.55 pg/ml in healthy individuals, p=0.003) and low IFN-γ (0.41 pg/ml vs 2.14 pg/ml in healthy, p<0.001), but no relationship was found between these cytokines and the hematopoietic stem cells yield. The present study showed that in patients with ineffective harvesting of hematopoietic precursors, secretion of IL-8 by stromal cells was low (308.08 pg/ml vs 561.29 pg/ml in healthy individuals, p=0.04).The obtained results are consistent with the literature data on the important role of IL-8 in the mobilization of hematopoietic stem cells, which allows to consider IL-8 as an informative marker for predicting the insufficient yields of hematopoietic precursors.

Cytokines in equine platelet lysate and related blood products.

In equine medicine, the use of regenerative therapeutics has gained growing attention, but is still a new and complex field with room for improvement. Platelet lysate (PL) can be used as therapeutic agent but is also a promising supplement for the culture of multipotent mesenchymal stromal cells. To enable a targeted use of PL both in clinic and laboratory, it is crucial to learn more details on its effective ingredients. While so far, mainly growth factor components have been analyzed in platelet-based products such as PL, the current study focuses on the content of cytokines in serum, plasma, platelet concentrate and PL. Blood was harvested from 20 clinically healthy horses and subjected to blood count and chemistry analysis, as well as to further processing to PL. Plasma and platelet concentrate were produced by a buffy-coat-based method and PL was produced from the concentrate by freeze-thawing. Samples from each horse were analyzed regarding interleukin (IL)-1β, -4, -6 and -10, interferon-γ and tumor necrosis factor-α concentrations using sandwich ELISAs. Cytokine concentrations in serum, plasma, concentrate and PL were similar and correlated significantly. However, there was a large inter-individual variability in cytokine concentrations between the different donor horses. The samples from some donor animals had overall very high cytokine concentrations, while samples from other donors had no measurable cytokine ingredient. This pattern was observed for all cytokines. There was a noticeable link between high cytokine concentrations in the blood products and abnormal findings in blood chemistry. Cytokine concentrations in samples from horses with abnormal findings were significantly higher than in samples from the remaining horses. The interindividual differences in cytokine concentrations could be highly relevant when using PL for therapy and cell culture, as the mode of action of the PL is likely changed depending on the presence of pro- and anti-inflammatory cytokines. Blood chemistry might be useful to predict cytokine concentrations in blood products.

ВЛИЯНИЕ ПРЕПАРАТА ГУМИНО-ФУЛЬВОВЫХ КИСЛОТ НА КОЛИЧЕСТВЕННЫЙ ВЫХОД ОСТАТОЧНЫХ ФОКУСОВ γH2AX И ПРОЛИФЕРАТИВНУЮ АКТИВНОСТЬ В ОБЛУЧЕННЫХ МЕЗЕНХИМАЛЬНЫХ СТРОМАЛЬНЫХ КЛЕТКАХ ЧЕЛОВЕКА

Purpose: To evaluate the influence of a humic-fulvic acid substance on the quantitative yield of residual foci of the DNA double-strand break (DSB) repair protein-marker - phosphorylated histone H2AX (γH2AX) and proliferation activity in a culture of human mesenchymal stromal cells (MSCs) 24, 48, and 72 h after exposure to X-ray radiation at doses of 2, 4 and 10 Gy. Material and methods: Through 24 hours after incubation of MSCs with a substance of humic-fulvic acids (Humic Complex, OOO Sistema-BioTechnologies, Russia) at a dilution of 1/1000. Cells were irradiated on an X-ray biological device RUB RUST-M1 at a voltage of 200 kV, beam current 2×5 mA, aluminum filter 1.5 mm, absorbed dose rate 0.85 Gy/min. Immunocytochemical staining was used to quantify the residual γH2AX foci and the percentage of proliferating cells using antibodies to γH2AX and Ki-67 (a marker protein for cell proliferation), respectively. Statistical analysis of the obtained data was carried out using the statistical software package Statistica 8.0 (StatSoft). To assess the significance of differences between samples, Student’s t-test was used. Results and conclusion: The conducted studies showed that on the cell model used and under the above experimental conditions, the humic-fulvic acid substance does not affect the efficiency of repair of radiation-induced DNA DSBs, however, it significantly reduces the proliferation activity of both irradiated and non-irradiated MSCs. It is advisable to conduct detailed studies of the molecular and cellular mechanisms of the antiproliferative effect of humic and fulvic acids.

A novel approach for large-scale manufacturing of small extracellular vesicles from bone marrow-derived mesenchymal stromal cells using a hollow fiber bioreactor.

Mesenchymal stromal cells (MSCs) are promising therapeutic candidates in a variety of diseases due to having immunomodulatory and pro-regenerative properties. In recent years, MSC-derived small extracellular vesicles (sEVs) have attracted increasing interest as a possible alternative to conventional cell therapy. However, translational processes of sEVs for clinical applications are still impeded by inconsistencies regarding isolation procedures and culture conditions. We systematically compared different methods for sEV isolation from conditioned media of ex vivo expanded bone marrow-derived MSCs and demonstrated considerable variability of quantity, purity, and characteristics of sEV preparations obtained by these methods. The combination of cross flow filtration with ultracentrifugation for sEV isolation resulted in sEVs with similar properties as compared to isolation by differential centrifugation combined with ultracentrifugation, the latter is still considered as gold standard for sEV isolation. In contrast, sEV isolation by a combination of precipitation with polyethylene glycol and ultracentrifugation as well as cross flow filtration and size exclusion chromatography resulted in sEVs with different characteristics, as shown by surface antigen expression patterns. The MSC culture requires a growth-promoting supplement, such as platelet lysate, which contains sEVs itself. We demonstrated that MSC culture with EV-depleted platelet lysate does not alter MSC characteristics, and conditioned media of such MSC cultures provide sEV preparations enriched for MSC-derived sEVs. The results from the systematic stepwise evaluation of various aspects were combined with culture of MSCs in a hollow fiber bioreactor. This resulted in a strategy using cross flow filtration with subsequent ultracentrifugation for sEV isolation. In conclusion, this workflow provides a semi-automated, efficient, large-scale-applicable, and good manufacturing practice (GMP)-grade approach for the generation of sEVs for clinical use. The use of EV-depleted platelet lysate is an option to further increase the purity of MSC-derived sEVs.

A controllable gelatin-based microcarriers fabrication system for the whole procedures of MSCs amplification and tissue engineering.

Biopolymer microbeads present substantial benefits for cell expansion, tissue engineering, and drug release applications. However, a fabrication system capable of producing homogeneous microspheres with high precision and controllability for cell proliferation, passaging, harvesting and downstream application is limited. Therefore, we developed a co-flow microfluidics-based system for the generation of uniform and size-controllable gelatin-based microcarriers (GMs) for mesenchymal stromal cells (MSCs) expansion and tissue engineering. Our evaluation of GMs revealed superior homogeneity and efficiency of cellular attachment, expansion and harvest, and MSCs expanded on GMs exhibited high viability while retaining differentiation multipotency. Optimization of passaging and harvesting protocols was achieved through the addition of blank GMs and treatment with collagenase, respectively. Furthermore, we demonstrated that MSC-loaded GMs were printable and could serve as building blocks for tissue regeneration scaffolds. These results suggested that our platform held promise for the fabrication of uniform GMs with downstream application of MSC culture, expansion and tissue engineering.

Customizable 3D printed perfusion bioreactor for the engineering of stem cell microenvironments.

Faithful modeling of tissues and organs requires the development of systems reflecting their dynamic 3D cellular architecture and organization. Current technologies suffer from a lack of design flexibility and complex prototyping, preventing their broad adoption by the scientific community. To make 3D cell culture more available and adaptable we here describe the use of the fused deposition modeling (FDM) technology to rapid-prototype 3D printed perfusion bioreactors. Our 3D printed bioreactors are made of polylactic acid resulting in reusable systems customizable in size and shape. Following design confirmation, our bioreactors were biologically validated for the culture of human mesenchymal stromal cells under perfusion for up to 2weeks on collagen scaffolds. Microenvironments of various size/volume (6-12mm in diameter) could be engineered, by modulating the 3D printed bioreactor design. Metabolic assay and confocal microscopy confirmed the homogenous mesenchymal cell distribution throughout the material pores. The resulting human microenvironments were further exploited for the maintenance of human hematopoietic stem cells. Following 1 week of stromal coculture, we report the recapitulation of 3D interactions between the mesenchymal and hematopoietic fractions, associated with a phenotypic expansion of the blood stem cell populations.Our data confirm that perfusion bioreactors fit for cell culture can be generated using a 3D printing technology and exploited for the 3D modeling of complex stem cell systems. Our approach opens the gates for a more faithful investigation of cellular processes in relation to a dynamic 3D microenvironment.

Genomic instability in long-term culture of human adipose-derived mesenchymal stromal cells.

Mesenchymal stromal/stem cells stem (MSC) have been widely studied due to their great potential for application in tissue engineering and regenerative and translational medicine. In MSC-based therapy for human diseases, cell proliferation is required to obtain a large and adequate number of cells to ensure therapeutic efficacy. During in vitro culture, cells are under an artificial environment and manipulative stress that can affect genetic stability. Several regulatory agencies have established guidelines to ensure greater safety in cell-based regenerative and translational medicine, but there is no specific definition about the maximum number of passages that ensure the lowest possible risk in MSC-based regenerative medicine. In this context, the aim of this study was to analyze DNA damage and chromosome alterations in adipose-derived mesenchymal stromal cells (ADMSC) until the eleventh passage and to provide additional subsidies to regulatory agencies related to number of passages in these cells. Thus, two methods in genetic toxicology were adopted: comet assay and micronucleus test. The comet assay results showed an increase in DNA damage from the fifth passage onwards. The micronucleus test showed a statistically significant increase of micronucleus from the seventh passage onwards, indicating a possible mutagenic effect associated with the increase in the number of passages. Based on these results, it is important to emphasize the need to assess genetic toxicology and inclusion of new guidelines by regulatory agencies to guarantee the safety of MSC-based therapies for human diseases.

The inflammation mediated by mesenchymal stem cells enhances blood group antigen-antibody responses <i>via</i> IL-1β

While it is known that the inflammatory state of the recipient may regulate the production of blood group antibodies, the cellular and molecular mechanisms are unclear. Mesenchymal stromal cells (MSCs) can produce inflammatory factors in response to inflammation and regulate inflammatory immunity. To explore how inflammatory MSCs exert influence on blood group antigen-antibody responses, the inflammatory MSC culture solution was added to the blood group antigen-antibody response system. Flow cytometry was used to detect the effect of different concentrations of inflammatory MSC supernatant on the production of blood group antibodies. Western blotting was used to determine whether the IL-1β receptor was expressed on the surface of red blood cells (RBCs). The intensity of antigen-antibody response was detected by serological methods. The results demonstrated that IL-1β produced by inflammation-induced MSCs could directly bind to the surface of erythrocytes and interfere with blood group antigen-antibody responses. This study suggests that inflammatory MSC-derived IL-1β might enhance the strength of blood antigen-antibody responses.

Biodegradable Mg-Sc-Sr Alloy Improves Osteogenesis and Angiogenesis to Accelerate Bone Defect Restoration.

Magnesium (Mg) and its alloys are considered to be biodegradable metallic biomaterials for potential orthopedic implants. While the osteogenic properties of Mg alloys have been widely studied, few reports focused on developing a bifunctional Mg implant with osteogenic and angiogenic properties. Herein, a Mg-Sc-Sr alloy was developed, and this alloy's angiogenesis and osteogenesis effects were evaluated in vitro for the first time. X-ray Fluorescence (XRF), X-ray diffraction (XRD), and metallography images were used to evaluate the microstructure of the developed Mg-Sc-Sr alloy. Human umbilical vein/vascular endothelial cells (HUVECs) were used to evaluate the angiogenic character of the prepared Mg-Sc-Sr alloy. A mix of human bone-marrow-derived mesenchymal stromal cells (hBM-MSCs) and HUVEC cell cultures were used to assess the osteogenesis-stimulating effect of Mg-Sc-Sr alloy through alkaline phosphatase (ALP) and Von Kossa staining. Higher ALP activity and the number of calcified nodules (27% increase) were obtained for the Mg-Sc-Sr-treated groups compared to Mg-treated groups. In addition, higher VEGF expression (45.5% increase), tube length (80.8% increase), and number of meshes (37.9% increase) were observed. The Mg-Sc-Sr alloy showed significantly higher angiogenesis and osteogenic differentiation than pure Mg and the control group, suggesting such a composition as a promising candidate in bone implants.

Single-cell RNA sequencing reveals different signatures of mesenchymal stromal cell pluripotent-like and multipotent populations

Somatic stem cells are advantageous research targets for understanding the properties required to maintain stemness. Human bone marrow-mesenchymal stromal cells (BM-MSCs) were separated into pluripotent-like SSEA-3(+) Muse cells (Muse-MSCs) and multipotent SSEA-3(-) MSCs (MSCs) and were subjected to single-cell RNA sequencing analysis. Compared with MSCs, Muse-MSCs exhibited higher expression levels of the p53 repressor MDM2; signal acceptance-related genes EGF, VEGF, PDGF, WNT, TGFB, INHB, and CSF; ribosomal protein; and glycolysis and oxidative phosphorylation. Conversely, MSCs had higher expression levels of FGF and ANGPT; Rho family and caveola-related genes; amino acid and cofactor metabolism; MHC class I/II, and lysosomal enzyme genes than Muse-MSCs. Unsupervised clustering further divided Muse-MSCs into two clusters stratified by the expression of cell cycle-related genes, and MSCs into three clusters stratified by the expression of cell cycle-, cytoskeleton-, and extracellular matrix-related genes. This study evaluating the differentiation ability of BM-MSC subpopulations provides intriguing insights for understanding stemness.

Iniciation and cultivation of multipotent mesenchimal human umbilical stroma cells in a laboratory experiment

BACKGROUND: Whartons jelly of the human umbilical cord is one of the sources of multipotent mesenchymal stromal cells. The cell population obtained from the postpartum biomaterial is characterized by high proliferative and regenerative properties. Isolation of a culture of multipotent mesenchymal stromal cells from the umbilical cord does not pose a threat to the health and life of the donor.
 AIM: Optimization of the technique for isolating a reproducible population of multipotent mesenchymal stromal cells from Whartons jelly is an urgent task in biomedicine, which can accelerate the process of obtaining donor cells for cell therapy and tissue engineering.
 MATERIALS AND METHODS: In the study, the main techniques and methods for isolating the culture of umbilical cord stroma cells were tested, the cultivation process was optimized to increase its efficiency and reduce the time of growth of cell biomass. The effect of the components of the nutrient medium on the cells obtained from Whartons jelly of the human umbilical cord was studied. Currently, there is no universal composition of the growth medium; in various studies, nutrient media from different manufacturers are used, which differ in composition. The most discussed issue is the selection of serum, which is part of the nutrient medium.
 RESULTS: In the work, a comparative evaluation of five different sera was carried out. It has been shown that the most stable physiological parameters are observed in cell suspension samples with the addition of FBS (SKPK, Russia) and FBS (Capricorn, USA) sera. A study of the effect of hypoxia on cell culture in combination with the most effective sera showed that hypoxic stress acts as an activator of primary cell proliferation. The assessment of the effect of serum and hypoxia on cell culture was carried out visually using microscopy, assessment of changes in cell morphology during cultivation, and the results of testing the action of sera by the intensity of respiration of free and immobilized cells under the action of inhibitors.
 CONCLUSION: As a result of the experiments, the influence of the type of serum on the initiation of cell expansion from primary explants and further cell proliferation in vitro was established. Hypoxia during exposure of primary explants enhances the expansion of cells from tissue fragments of Whartons jelly tissue.

Stage-Dependent Activity and Pro-Chondrogenic Function of PI3K/AKT during Cartilage Neogenesis from Mesenchymal Stromal Cells

Differentiating mesenchymal stromal cells (MSCs) into articular chondrocytes (ACs) for application in clinical cartilage regeneration requires a profound understanding of signaling pathways regulating stem cell chondrogenesis and hypertrophic degeneration. Classifying endochondral signals into drivers of chondrogenic speed versus hypertrophy, we here focused on insulin/insulin-like growth factor 1 (IGF1)-induced phosphoinositide 3-kinase (PI3K)/AKT signaling. Aware of its proliferative function during early but not late MSC chondrogenesis, we aimed to unravel the late pro-chondrogenic versus pro-hypertrophic PI3K/AKT role. PI3K/AKT activity in human MSC and AC chondrogenic 3D cultures was assessed via Western blot detection of phosphorylated AKT. The effects of PI3K inhibition with LY294002 on chondrogenesis and hypertrophy were assessed via histology, qPCR, the quantification of proteoglycans, and alkaline phosphatase activity. Being repressed by ACs, PI3K/AKT activity transiently rose in differentiating MSCs independent of TGFβ or endogenous BMP/WNT activity and climaxed around day 21. PI3K/AKT inhibition from day 21 on equally reduced chondrocyte and hypertrophy markers. Proving important for TGFβ-induced SMAD2 phosphorylation and SOX9 accumulation, PI3K/AKT activity was here identified as a required stage-dependent driver of chondrogenic speed but not of hypertrophy. Thus, future attempts to improve MSC chondrogenesis will depend on the adequate stimulation and upregulation of PI3K/AKT activity to generate high-quality cartilage from human MSCs.

Challenges and opportunities in downstream separation processes for mesenchymal stromal cells cultured in microcarrier-based stirred suspension bioreactors.

Mesenchymal stromal cells (MSC) are a promising platform for regenerative medicine applications because of their multilineage differentiation abilities and ease of collection, isolation, and growth ex vivo. To meet the demand for clinical applications, large-scale manufacturing will be required using three-dimensional culture platforms in vessels such as stirred suspension bioreactors. As MSCs are an adherent cell type, microcarriers are added to the culture to increase the available surface area for attachment and growth. Although extensive research has been performed on efficiently culturing MSCs using microcarriers, challenges persist in downstream processing, including harvesting, filtration, and volume reduction, which all play a critical role in the translation of cell therapies to the clinic. The objective of this review is to assess the current state of downstream technologies available for microcarrier-based MSC cultures. This includes a review of current research within the three stages: harvesting, filtration, and volume reduction. Using this information, a downstream process for MSCs is proposed, which can be applied to a wide range of applications.

Preclinical Evaluation of the Tumorigenic and Immunomodulatory Properties of Human Bone Marrow Mesenchymal Stromal Cell Populations with Clonal Trisomy 5.

Cytogenetic aberrations may emerge in human mesenchymal stromal cells (MSC) during ex vivo expansion for cell therapy. We have detected clonal trisomy 5 in two distinct autologous MSC products expanded from bone marrow which, based on the current quality control criteria, could not be released for clinical use. Although a safety concern, it is still unclear to what extent recurrent aneuploidies detected in MSC products may affect the threshold for neoplastic transformation or the medicinal properties of these cells. We have carried out an exploratory preclinical study to evaluate these MSC products with clonal trisomy 5, regarding their oncogenic and immunomodulatory potential. Cell population growth in vitro was reduced in MSC cultures with clonal trisomy 5 compared with the population growth of their euploid MSC counterparts, based on a lower cumulative population doubling level, reduced cell proliferation index, and increased senescence-associated beta-galactosidase activity. Subcutaneous injection of clinically relevant amount of MSC population, either with or without clonal trisomy 5, did not generate tumors in immunodeficient mice within a follow-up period of six months. Most importantly, MSC population with clonal trisomy 5 kept immunomodulatory properties upon interferon gamma (IFNγ) licensing, displaying overexpression of IDO, CXCL9, CXCL10, and CXCL11, in a similar fashion than that of IFNγ-licensed euploid MSC. Our findings suggest that bone marrow MSC products with clonal trisomy 5 may retain their therapeutic potential, based on poor tumor initiating capability and preserved immunomodulatory potency. This preclinical evidence may further support the definition of release criteria of autologous MSC products for cell therapy under critical clinical scenarios. This trial is registered with Clinical Study registration number: RBR-29x2pr.

Effect of cell culture media on extracellular vesicle secretion from mesenchymal stromal cells and neurons

BackgroundExtracellular vesicles (EVs) secreted by neuronal cells in vitro have promising therapeutic potential for brain diseases. Optimization of cell culture conditions and methodologies for high-yield isolation of EVs for preclinical and clinical applications, however, remains a challenge. ObjectiveTo probe the cell culture conditions required for optimal EV secretion by human-derived neuronal cells. MethodologyFirst, we optimized the EV purification protocol using human mesenchymal stromal cell (MSC) cultures. Next, we compared the effects of different variables in human pluripotent stem cell (hPSC)-derived neuronal cultures on EV secretion. EVs were isolated from cell conditioned media (CCM) and control media with no cells (NCC) using ultrafiltration combined with size-exclusion chromatography (SEC). The hPSC neurons were cultured in 2 different media from which EVs were collected at 2 maturation time-points (days 46 and 60). Stimulation with 25 mM KCl was also evaluated as an activator of EV secretion by neurons. The collected SEC fractions were analyzed by nanoparticle tracking analysis (NTA), protein concentration assay, and blinded transmission electron microscopy (TEM). ResultsA peak in cup-shaped particles was observed in SEC fractions 7–10 of MSC samples, but not corresponding media controls, indicating successful isolation of EVs. Culture medium had no significant effect on EV yield. The EV yield of the samples did not differ significantly according to the culture media used or the cell maturation time-points. Stimulation of neurons with KCl for 3 h reduced rather than increased the EV yield. ConclusionsWe demonstrated successful EV isolation from MSC and neuronal cells using an ultrafiltration-SEC method. The EV yield from MSC and neuronal cultures exhibited a large batch effect, apparently related to the culture media used, highlighting the importance of including NCC as a negative control in all cell culture experiments.

Characteristics of the effect of exosomes isolated from donor plasma and placenta-derived mesenchymal stromal cell-conditioned medium on the paracrine secretion of human peripheral blood mononuclear cells

Extracellular small vesicles – exosomes, attract the attention of researchers as a promising tool for regulating intercellular communication. At the same time, the therapeutic effects achieved through the use of mesenchymal stem cells (MSCs) are traditionally considered by medicine as a time-tested, multi-vector strategy for the treatment of various pathologies. In particular, in addition to the application of MSCs directly, it is important to study the products of their secretion. Aim. To study characteristics of the influence of exosomes isolated from blood plasma of healthy donors and conditioned medium of placental mesenchymal stromal cell (MSC) culture on paracrine secretion of peripheral blood mononuclear cells (PBMCs) in patients with chronic heart failure (CHF) in vitro. Materials and methods. The characteristics of paracrine secretion of PBMCs in patients with CHF by the content of proteins VEGF-A, MCP-1, ICAM-1 in their culture medium were studied in two directions: under the influence of exosomes isolated from plasma of healthy donors and exosomes isolated from placental MSC culture medium. Results. Incubation of PBMCs with plasma exosomes increased VEGF-A secretion in the group of healthy donors by 2.73 times (P ≤ 0.05), in patients with CHF – by 2 times (P ≤ 0.05); but there were a multidirectional effect on the content of ICAM-1 protein: it was 1.8 times (P ≤ 0.05) increased in the group of donors and 1.4 times (P ≤ 0.05) decreased in the group of CHF patients; MCP-1 secretion was insignificantly 10 % reduced in the donor group and did not change significantly in patients. Incubation of РВМС with exosomes isolated from MSC conditioned medium did not cause significant changes in paracrine secretion of PBMCs: there was a decrease in secretion of VEGF-A by 25 %, ICAM-1 by 17 %, MCP-1 by 22 % in the group of healthy volunteers; secretion of these proteins was 19.7 %, 22.0 % and 25.0 % decreased, respectively, in the group of CHF patients. The effects observed in the incubation of PBMCs with exosomes isolated from blood plasma of healthy donors and conditioned medium of placental MSC culture have provided valuable information for the design of future studies in this area of cell biology. Conclusions. Our studies have demonstrated in vitro the effects of exosomes isolated from donor plasma and conditioned medium on the functional properties of human PBMCs.

Longitudinal two-dimensional gas chromatography mass spectrometry as a non-destructive at-line monitoring tool during cell manufacturing identifies volatile features correlative to cell product quality

Background aimsCell therapies have emerged as a potentially transformative therapeutic modality in many chronic and incurable diseases. However, inherent donor and patient variabilities, complex manufacturing processes, lack of well-defined critical quality attributes and unavailability of in-line or at-line process or product analytical technologies result in significant variance in cell product quality and clinical trial outcomes. New approaches for overcoming these challenges are needed to realize the potential of cell therapies. MethodsHere the authors developed an untargeted two-dimensional gas chromatography mass spectrometry (GC×GC-MS)-based method for non-destructive longitudinal at-line monitoring of cells during manufacturing to discover correlative volatile biomarkers of cell proliferation and end product potency. ResultsSpecifically, using mesenchymal stromal cell cultures as a model, the authors demonstrated that GC×GC-MS of the culture medium headspace can effectively discriminate between media types and tissue sources. Headspace GC×GC-MS identified specific volatile compounds that showed a strong correlation with cell expansion and product functionality quantified by indoleamine-2,3-dioxygenase and T-cell proliferation/suppression assays. Additionally, the authors discovered increases in specific volatile metabolites when cells were treated with inflammatory stimulation. ConclusionsThis work establishes GC×GC-MS as an at-line process analytical technology for cell manufacturing that could improve culture robustness and may be used to non-destructively monitor culture state and correlate with end product function.

Mesenchymal Stromal Cell Therapy in Spinal Cord Injury: Mechanisms and Prospects.

Spinal cord injury (SCI) often leads to severe motor, sensory, and autonomic dysfunction in patients and imposes a huge economic cost to individuals and society. Due to its complicated pathophysiological mechanism, there is not yet an optimal treatment available for SCI. Mesenchymal stromal cells (MSCs) are promising candidate transplant cells for use in SCI treatment. The multipotency of MSCs, as well as their rich trophic and immunomodulatory abilities through paracrine signaling, are expected to play an important role in neural repair. At the same time, the simplicity of MSCs isolation and culture and the bypassing of ethical barriers to stem cell transplantation make them more attractive. However, the MSCs concept has evolved in a specific research context to encompass different populations of cells with a variety of biological characteristics, and failure to understand this can undermine the quality of research in the field. Here, we review the development of the concept of MSCs in order to clarify misconceptions and discuss the controversy in MSCs neural differentiation. We also summarize a potential role of MSCs in SCI treatment, including their migration and trophic and immunomodulatory effects, and their ability to relieve neuropathic pain, and we also highlight directions for future research.