Mesenchymal Stromal Cells Cultures Research Articles

Delphi-driven consensus definition for MSCs and clinical reporting guidelines for MSC-based therapeutics

BackgroundDespite promising results in preclinical studies, mesenchymal stromal cells (MSCs) face significant challenges in clinical translation. A scoping review by our group highlighted two key issues contributing to this gap: i) the lack of clear and consensus definition for MSCs and ii) under reporting of critical parameters in MSC clinical studies. To address these issues, we conducted a modified Delphi study to establish and implement a consensus definition for MSCs and develop reporting guidelines for MSC clinical studies. MethodsA steering committee of 22 international experts including stakeholders from different MSC research fields participated in the 3 Delphi rounds. For the first round, to obtain a broad perspective, additional investigators recommended by the steering committee were invited to participate. The first 2 rounds consisted of online surveys, while the 3rd round took the form of a virtual meeting. Participants were asked to rate a series of potential defining characteristics of MSCs and items for reporting guidelines. Consensus was defined as at least 80% of the participants rating the item in the same category of importance. ResultsEighty-seven international participants participated in the first-round survey (Spring 2023), 17 participants in the second online survey (Fall 2023) and 15 participants in the final virtual consensus meeting (January 2024). For the MSC definition, 20 items were considered and 9 reached consensus. Items included terminology (1 item), cell marker expression (5 items), tissue origin (1 item), stemness (1 item) and description of critical quality attributes (1 item). For the reporting guidelines, on the 28 initial items and with additional items suggested during round 1, a total of 33 items to report were included. This included items on MSC intervention group and control (i.e., MSC product, dose, and administration, etc.), MSC characteristics (i.e., MSCs provenance, “fitness and viability, immune compatibility, etc.), and MSC culture conditions (i.e., oxygen environment, culture medium, use of serum, etc.). ConclusionBy applying a Delphi method to establish a consensus definition for MSCs and reporting guidelines for MSC-based clinical, this work represents a significant advance in improving transparency and reproducibility in the conduct and reporting of MSC research.

Therapeutic potential of mesenchymal stromal cells on morphological parameters in the hippocampus of rats with brain ischemia-reperfusion modeling

Ischemic stroke is an extremely important pathology with high mortality, in which more than 50 % of patients with occlusion of the main vessels remain disabled, despite early reperfusion therapy by thrombolysis or thrombectomy. As part of the regenerative strategy, stem cell transplantation in ischemic stroke became a new impetus. Cell therapy with the use of mesenchymal stromal cells demonstrated encouraging results regarding endogenous mechanisms of neuroregeneration in response to ischemic damage to brain structures. The aim of the research is to study the influence of mesenchymal stromal cells of various genesis, lysate of mesenchymal stromal cells obtained from Wharton’s jelly umbilical cord and citicoline on the dynamics of morphological changes in the hippocampal CA1 region of rats with acute cerebral ischemia-reperfusion according to light microscopy and micromorphometry data. The experiment was carried out using 200 male Wistar rats, which were subjected to ischemia-reperfusion by reversible 20-minute bilateral occlusion of the internal carotid arteries. Animals with modeled pathology were intravenously transplanted with mesenchymal stromal cells of various genesis (from Wharton’s jelly of the human umbilical cord, human and rat adipose tissue), and rat embryonic fibroblasts, lysate of mesenchymal stromal cells and citicoline were injected. Histological analysis of rat brain sections was performed on the 7th and 14th day of the experiment. Statistical analysis was performed using “Statistica 6.0” (StatSoft® Snc, USA). The significance of differences was assessed using the Student’s t-test and the nonparametric Mann-Whitney U test. During the study, it was found that modeled ischemia-reperfusion in rats caused almost complete degeneration of the structure of the pyramidal layer of hippocampal CA1 region, gave uniformity to the structure of the radiant layer, infiltration of microglia, contributed to the disruption of the arrangement of apical dendrite bundles and narrowing of blood vessels as a result of perivascular edema. Also, the modeled pathology reduced the total number of neuronal nuclei in the hippocampal CA1 area, the overwhelming majority of which had signs of pathological changes. Transplantation of mesenchymal stromal cells of various origins, lysate of mesenchymal stromal cells and citicoline contributed to a significant increase in the number of neuronal nuclei in the hippocampal CA1 zone and nuclei that did not undergo pathological changes. The most positive effect was found in the transplantation of mesenchymal stromal cells from human Wharton’s jelly-derived cells. Thus, both in the subacute and recovery periods of ischemic stroke in rats, the transplantation of human Wharton’s jelly-derived mesenchymal stromal cells was significantly surpassed the reference drug citicoline in its ability to reduce the number of pathologically changed nuclei by 1.5 times (p<0.05). At the same time, the number of pathologically unchanged nuclei significantly exceeded the number of nuclei with signs of karyorrhexis and karyopyknosis, so it would be advisable to use mesenchymal stromal cells of various genesis, lysate or citicoline in conditions of acute cerebral ischemia-reperfusion, taking into account their ability to reduce the volume of the infarct. In the future, an injectable drug will be created from the most effective culture of mesenchymal stromal cells in terms of cerebroprotective properties for cell therapy of patients with acute ischemic stroke.

Thermosensitive chitosan-based hydrogel: A vehicle for overcoming the limitations of nose-to-brain cell therapy

Cell therapy is a promising strategy for treating neurological pathologies but requires invasive methods to bypass the blood–brain barrier restrictions. The nose-to-brain route has been presented as a direct and less invasive alternative to access the brain. The primary limitations of this route are low retention in the olfactory epithelium and poor cell survival in the harsh conditions of the nasal cavity. Thus, using chitosan-based hydrogel as a vehicle is proposed in this work to overcome the limitations of nose-to-brain cell administration. The hydrogelʼs design was driven to achieve gelification in response to body temperature and a mucosa-interacting chemical structure biocompatible with cells. The hydrogel showed a < 30 min gelation time at 37 °C and >95 % biocompatibility with 2D and 3D cultures of mesenchymal stromal cells. Additionally, the viability, stability, and migration capacity of oligodendrocyte precursor cells (OPCs) within the hydrogel were maintained in vitro for up to 72 h. After the intranasal administration of the OPCs-containing hydrogel, histological analysis showed the presence of viable cells in the nasal cavity for up to 72 h post-administration in healthy athymic mice. These results demonstrate the hydrogel's capacity to increase the residence time in the nasal cavity while providing the cells with a favorable environment for their viability. This study presents for the first time the use of thermosensitive hydrogels in nose-to-brain cell therapy, opening the possibility of increasing the delivery efficiency in future approaches in translational medicine. Statement of significanceThis work highlights the potential of biomaterials, specifically hydrogels, in improving the effectiveness of cell therapy administered through the nose. The nose-to-brain route has been suggested as a non-invasive way to directly access the brain. However, delivering stem cells through this route poses a challenge since their viability must be preserved and cells can be swept away by nasal mucus. Earlier attempts at intranasal cell therapy have shown low efficiency, but still hold promise to the future. The hydrogels designed for this study can provide stem cells with a biocompatible environment and adhesion to the nasal atrium, easing the successful migration of viable cells to the brain.

Heparan Sulfate-Collagen Surface Multilayers Support Serum-Free Microcarrier Culture of Mesenchymal Stem Cells.

The increasing cost of high-volume cultures and dependence on serum and growth factor supplementation limit the affordability of mesenchymal stromal cell (MSC) therapies. This has spurred interest in developing strategies that support adherent cell expansion while reducing raw material costs. Culture surfaces coated with sulfated glycosaminoglycans (GAGs), specifically heparan sulfate (HS), are an alternative to prolong growth factor retention in cell cultures. Unlike heparin, recombinant HS (rHS) offers strong binding affinity for multiple growth factors and extracellular matrix components, such as collagen I, without undesirable anticoagulant effects or xenobiotic health risks. The potential of rHS as a factor reservoir in MSC cultures remains underexplored. This study investigated the impact of rHS on the growth and anti-inflammatory properties of undifferentiated bone marrow MSCs in both planar and microcarrier-based cultures. It was hypothesized that rHS would enable MSC growth with minimal growth factor supplementation in a sulfation level-dependent manner. Cell culture surfaces were assembled via the layer-by-layer (LbL) method, combining alternating collagen I (COL) and rHS. These bilayers support cell adhesion and enable the incorporation of distinct sulfation levels on the culture surface. Examination of pro-mitogenic FGF and immunostimulatory IFN-γ release dynamics confirmed prolonged availability and sulfate level dependencies. Sulfated surfaces supported cell growth in low serum (2% FBS) and serum-free (SF) media at levels equivalent to standard culture conditions. Cell growth on rHS-coated surfaces in SF was comparable to that on heparin-coated surfaces and commercial surface-coated microcarriers in low serum. These growth benefits were observed in both planar and microcarrier (μCs) cultures. Additionally, rHS surfaces reduced β-galactosidase expression relative to uncoated surfaces, delaying cell senescence. Multivariate analysis of cytokines in conditioned media indicated that rHS-containing surfaces enhanced cytokine levels relative to uncoated surfaces during IFN-γ stimulation and correlated with decreased pro-inflammatory macrophage activity. Overall, utilizing highly sulfated rHS with COL reduces the need for exogenous growth factors and effectively supports MSC growth and anti-inflammatory potency on planar and microcarrier surfaces under minimal factor supplementation.

Culture and Immunomodulation of Equine Muscle-Derived Mesenchymal Stromal Cells: A Comparative Study of Innovative 2D versus 3D Models Using Equine Platelet Lysate.

Muscle-derived mesenchymal stromal cells (mdMSCs) hold great promise in regenerative medicine due to their immunomodulatory properties, multipotent differentiation capacity and ease of collection. However, traditional in vitro expansion methods use fetal bovine serum (FBS) and have numerous limitations including ethical concerns, batch-to-batch variability, immunogenicity, xenogenic contamination and regulatory compliance issues. This study investigates the use of 10% equine platelet lysate (ePL) obtained by plasmapheresis as a substitute for FBS in the culture of mdMSCs in innovative 2D and 3D models. Using muscle microbiopsies as the primary cell source in both models showed promising results. Initial investigations indicated that small variations in heparin concentration in 2D cultures strongly influenced medium coagulation with an optimal proliferation observed at final heparin concentrations of 1.44 IU/mL. The two novel models investigated showed that expansion of mdMSCs is achievable. At the end of expansion, the 3D model revealed a higher total number of cells harvested (64.60 ± 5.32 million) compared to the 2D culture (57.20 ± 7.66 million). Trilineage differentiation assays confirmed the multipotency (osteoblasts, chondroblasts and adipocytes) of the mdMSCs generated in both models with no significant difference observed. Immunophenotyping confirmed the expression of the mesenchymal stem cell (MSC) markers CD-90 and CD-44, with low expression of CD-45 and MHCII markers for mdMSCs derived from the two models. The generated mdMSCs also had great immunomodulatory properties. Specific immunological extraction followed by enzymatic detection (SIEFED) analysis demonstrated that mdMSCs from both models inhibited myeloperoxidase (MPO) activity in a strong dose-dependent manner. Moreover, they were also able to reduce reactive oxygen species (ROS) activity, with mdMSCs from the 3D model showing significantly higher dose-dependent inhibition compared to the 2D model. These results highlighted for the first time the feasibility and efficacy of using 10% ePL for mdMSC expansion in novel 2D and 3D approaches and also that mdMSCs have strong immunomodulatory properties that can be exploited to advance the field of regenerative medicine and cell therapy instead of using FBS with all its drawbacks.

Cartilage-Specific Gene Expression and Extracellular Matrix Deposition in the Course of Mesenchymal Stromal Cell Chondrogenic Differentiation in 3D Spheroid Culture.

Articular cartilage damage still remains a major problem in orthopedical surgery. The development of tissue engineering techniques such as autologous chondrocyte implantation is a promising way to improve clinical outcomes. On the other hand, the clinical application of autologous chondrocytes has considerable limitations. Mesenchymal stromal cells (MSCs) from various tissues have been shown to possess chondrogenic differentiation potential, although to different degrees. In the present study, we assessed the alterations in chondrogenesis-related gene transcription rates and extracellular matrix deposition levels before and after the chondrogenic differentiation of MSCs in a 3D spheroid culture. MSCs were obtained from three different tissues: umbilical cord Wharton's jelly (WJMSC-Wharton's jelly mesenchymal stromal cells), adipose tissue (ATMSC-adipose tissue mesenchymal stromal cells), and the dental pulp of deciduous teeth (SHEDs-stem cells from human exfoliated deciduous teeth). Monolayer MSC cultures served as baseline controls. Newly formed 3D spheroids composed of MSCs previously grown in 2D cultures were precultured for 2 days in growth medium, and then, chondrogenic differentiation was induced by maintaining them in the TGF-β1-containing medium for 21 days. Among the MSC types studied, WJMSCs showed the most similarities with primary chondrocytes in terms of the upregulation of cartilage-specific gene expression. Interestingly, such upregulation occurred to some extent in all 3D spheroids, even prior to the addition of TGF-β1. These results confirm that the potential of Wharton's jelly is on par with adipose tissue as a valuable cell source for cartilage engineering applications as well as for the treatment of osteoarthritis. The 3D spheroid environment on its own acts as a trigger for the chondrogenic differentiation of MSCs.

Proposal of Simplified Standardization of the Cell-Growth-Promoting Activity of Human Adipose Tissue Mesenchymal Stromal Cell Culture Supernatants.

The conditioned medium (CM) obtained from mesenchymal stromal cell (MSC) culture has excellent cell growth-promoting activity and is used for cosmetics and healthcare products. Unlike pharmaceuticals, strict efficacy verification is not legally required for these products. However, their efficacy must be substantiated as commercial products. We attempted to simplify CM production and to standardize the evaluation of the growth-promoting activity of CM. CM was obtained through the culturing of two lines of commercially available human adipose tissue-derived MSCs using MEMα with or without 10% fetal bovine serum (FBS) for 24 h. Non-CM control media were produced by the same protocol without MSCs. Growth-promoting activities of the CM were estimated by [3H]-thymidine pulse. CM were subjected to molecular weight fractionation with ultrafiltration using 10 k-, 30 k-, 50 k-, and 100 k-membranes. The FBS-free CMs showed 1.34- to 1.85-fold increases and FBS-containing CMs showed 1.45- to 1.67-fold increases in proliferation-promoting activity compared with non-CM controls, regardless of the source of the cell. The thymidine incorporation levels were approximately three times higher in FBS-containing CMs. Aged cells also showed 1.67- to 2.48-fold increases in the activity due to FBS-containing CM, but not to FBS-free CM. The CM activities were sustained even after 1 year at 4 °C. Molecular weight fractionation showed that the activity was recovered in the fraction above 100 k. Clear and stable cell-growth-promoting activity was confirmed with CMs of commercially available adipose tissue MSCs. The activity was detected in the fraction over 100 k. We propose here the importance of standardizing the production and evaluation of CMs to indicate their specific action.

Differential Smad2/3 linker phosphorylation is a crosstalk mechanism of Rho/ROCK and canonical TGF-β3 signaling in tenogenic differentiation

The transforming growth factor (TGF)-β3 is a well-known inducer for tenogenic differentiation, signaling via the Smad2/3 pathway. Furthermore, other factors like extracellular matrix or mechanical force can induce tenogenic differentiation and possibly alter the response to TGF-β3 by signaling via the Rho/ROCK pathway. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-β3/Smad signaling in tenogenic differentiation, with the Smad2/3 molecule hypothesized as a possible interface. Cultured as monolayers or on collagen I matrices, mesenchymal stromal cells (MSC) were treated with the ROCK inhibitor Y-27632 (10 µM), TGF-β3 (10 ng/ml) or both combined. Control cells were cultured accordingly, without Y-27632 and/or without TGF-β3. At different time points, MSC were analyzed by real-time RT-PCR, immunofluorescence, and Western blot. Cultivation of MSC on collagen matrices and ROCK inhibition supported tenogenic differentiation and fostered the effect of TGF-β3. The phosphorylation of the linker region of Smad2 was reduced by cultivation on collagen matrices, but not by ROCK inhibition. The latter, however, led to increased phosphorylation of the linker region of Smad3. In conclusion, collagen matrices and the Rho/ROCK signaling pathway influence the TGF-β3/Smad2/3 pathway by regulating different phosphorylation sites of the Smad linker region.

Mesenchymal stromal cell chondrogenesis under ALK1/2/3-specific BMP inhibition: a revision of the prohypertrophic signalling network concept

BackgroundIn vitro chondrogenesis of mesenchymal stromal cells (MSCs) driven by the essential chondro-inducer transforming growth factor (TGF)-β is instable and yields undesired hypertrophic cartilage predisposed to bone formation in vivo. TGF-β can non-canonically activate bone morphogenetic protein-associated ALK1/2/3 receptors. These have been accused of driving hypertrophic MSC misdifferentiation, but data remained conflicting. We here tested the antihypertrophic capacity of two highly specific ALK1/2/3 inhibitors – compound A (CompA) and LDN-212854 (LDN21) – in order to reveal potential prohypertrophic contributions of these BMP/non-canonical TGF-β receptors during MSC in vitro chondrogenesis.MethodsStandard chondrogenic pellet cultures of human bone marrow-derived MSCs were treated with TGF-β and CompA (500 nM) or LDN21 (500 nM). Daily 6-hour pulses of parathyroid hormone-related peptide (PTHrP[1–34], 2.5 nM, from day 7) served as potent antihypertrophic control treatment. Day 28 samples were subcutaneously implanted into immunodeficient mice.ResultsAll groups underwent strong chondrogenesis, but GAG/DNA deposition and ACAN expression were slightly but significantly reduced by ALK inhibition compared to solvent controls along with a mild decrease of the hypertrophy markers IHH-, SPP1-mRNA, and Alkaline phosphatase (ALP) activity. When corrected for the degree of chondrogenesis (COL2A1 expression), only pulsed PTHrP but not ALK1/2/3 inhibition qualified as antihypertrophic treatment. In vivo, all subcutaneous cartilaginous implants mineralized within 8 weeks, but PTHrP pretreated samples formed less bone and attracted significantly less haematopoietic marrow than ALK1/2/3 inhibitor groups.ConclusionsOverall, our data show that BMP-ALK1/2/3 inhibition cannot program mesenchymal stromal cells toward stable chondrogenesis. BMP-ALK1/2/3 signalling is no driver of hypertrophic MSC misdifferentiation and BMP receptor induction is not an adverse prohypertrophic side effect of TGF-β that leads to endochondral MSC misdifferentiation. Instead, the prohypertrophic network comprises misregulated PTHrP/hedgehog signalling and WNT activity, and a potential contribution of TGF-β-ALK4/5-mediated SMAD1/5/9 signalling should be further investigated to decide about its postulated prohypertrophic activity. This will help to successfully engineer cartilage replacement tissues from MSCs in vitro and translate these into clinical cartilage regenerative therapies.

The Use of Human Platelet Lysate as a Coating Substance for Adipose-Derived Stem Cell Expansion.

Large-scale production of mesenchymal stromal cells is essential for sufficient therapeutic doses in regenerative medicine. However, long-term cultivation encounters limited cell growth and cellular aging. Therefore, an alternative cell culture approach that promotes proliferation and attenuates cell senescence is required. Human platelet lysate (HPL) is a potent supplement for in vitro cell expansion. Applying HPL as a coating material can potentially improve mesenchymal stromal cell cultures. To examine the capacity of HPL, it was used to pre-coat a tissue culture plate for in vitro adipose-derived mesenchymal stromal cell expansion. Alterations in biological features of adipose-derived stem cells (ADSCs) were investigated, including cell adhesion assays, cell proliferation, population doubling time, and cellular senescence. ADSCs cultured on HPL-coated plates significantly increased cell adhesion rate, shortened population doubling time, and stimulated cell growth. The senescent cells were significantly decreased in ADSCs cultured in an HPL-coated plate, and the expression levels of senescence-associated genes, including p16, p21, and p53, were downregulated. Furthermore, Western blotting analysis revealed that HPL was enriched with fibronectin and vitronectin, essential cell adhesive proteins. HPL was effectively used as a coating material for ADSC expansions. Cellular cultivation on the HPL coating is an alternative approach for producing mesenchymal stromal cells.

Transitioning from static to suspension culture system for large-scale production of xeno-free extracellular vesicles derived from mesenchymal stromal cells.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown increasing therapeutic potential in the last years. However, large production of EV is required for therapeutic purposes. Thereby, scaling up MSC cultivation in bioreactors is essential to allow culture parameters monitoring. In this study, we reported the establishment of a scalable bioprocess to produce MSC-EV in suspension cultures using spinner flasks and human collagen-coated microcarriers (3D culture system). We compared the EV production in this 3D culture system with the standard static culture using T-flasks (2D culture system). The EV produced in both systems were characterized and quantify by western blotting and nanoparticle tracking analysis. The presence of the typical protein markers CD9, CD63, and CD81 was confirmed by western blotting analyses for EV produced in both culture systems. The cell fold-increase was 5.7-fold for the 3D culture system and 4.6-fold for the 2D culture system, signifying a fold-change of 1.2 (calculated as the ratio of fold-increase 3D to fold-increase 2D). Furthermore, it should be noted that the total cell production in the spinner flask cultures was 4.8 times higher than that in T-flask cultures. The total cell production in the spinner flask cultures was 5.2-fold higher than that in T-flask cultures. While the EV specific production (particles/cell) in T-flask cultures (4.40 ± 1.21 × 108 particles/mL, p < 0.05) was higher compared to spinner flask cultures (2.10 ± 0.04 × 108 particles/mL, p < 0.05), the spinner flask culture system offers scalability, making it capable of producing enough MSC-EV at a large scale for clinical applications. Therefore, we concluded that 3D culture system evaluated here serves as an efficient transitional platform that enables the scaling up of MSC-EV production for therapeutic purposes by utilizing stirred tank bioreactors and maintaining xeno-free conditions.

In vitro osteogenic and in ovo angiogenic effects of a family of natural origin P2O5-free bioactive glasses.

Bioactive glasses (BGs) belong to a group of ceramic biomaterials having numerous applications due to their excellent biocompatibility and bioactivity. Depending on their composition, properties of BGs can be finely tuned. In this study, we investigated both angiogenic and osteogenic properties of a novel family of BGs from the SiO2-CaO-Na2O system. Three BGs were synthesised from calcite minerals and silica sands extracted from natural deposits. Silica sands used for the synthesis of each glass were obtained from different depths of the deposit, resulting in a different colour and elemental composition. The composition and structural properties of the obtained BGs were determined. Direct culture of human mesenchymal stromal cells (hMSCs) with BG particles at different concentrations was used to investigate the biocompatibility as well as the osteogenic and angiogenic properties of the BGs. In addition, BGs' effect on angiogenesis was further studied in a chick chorioallantoic membrane (CAM) model. Material characterisation confirmed the amorphous character of BGs. Investigated BGs were biocompatible and stimulated early upregulation of RUNX2, ALPL, COL1A1, OCN, and OPN. All BGs tested in a CAM model positively influenced the number, distribution, and branching of the blood vessels. Furthermore, our study revealed that the depth of sand deposit, at which the raw material was collected, had an impact on the osteogenic and angiogenic properties of the resulting glasses. On the one hand, silica sand collected at the deepest layer of the deposit, featuring a higher content of Fe2O3 and Al2O3, originated BGs with potent stimulative capacity of osteogenic and angiogenic gene expression. On the other hand, sand with high silica content and titanium ions resulted in a glass that better supported vessel structure. The BGs presented in this study showed the potential to promote osteogenesis and angiogenesis during bone tissue regeneration, and thus, they will be further studied as part of composite materials for the development of 3D implantable scaffolds.

Cellular Therapy with Autologous Mesenchymal Stromal Cells in Patients with Drug Resistant Pulmonary Tuberculosis

The objective: to evaluate the effectiveness of treatment with autologous mesenchymal stromal cells (MSCs) in patients with drug resistant pulmonary tuberculosis (DR TB).Subjects and Methods. 120 patients with drug resistant tuberculosis aged 18 to 61 years old were enrolled in the study. They all were treated at the Republican Scientific and Practical Center of Pulmonology and Phthisiology from 2009 to 2018. Against the background of anti-tuberculosis treatment, patients (60 people) from Main Group (MG) underwent bone marrow sampling and then intravenous administration of autologous MSCs.Results. The average dose of administered cells was 1x106 per kg of the patient body weight. The average duration of MSC cultivation made 35 days. Treatment effectiveness in MG made 90% versus 71% in Control Group (CG). All patients who were successfully cured of tuberculosis (TB) using autologous MSCs had no tuberculosis relapses during 5 years of follow-up. In Control Group, 8 (19%) cases of relapse were reported. In Main Group versus Comparison Group, the rates indicating healing of cavities and absence of relapse are statistically significantly better.Conclusion. The use of autologous MSCs in patients with drug resistant tuberculosis has shown its high effectiveness.

Experimental study of transient particle suspension in bioreactors using a light attenuation technique

The culture of mesenchymal stromal cells relies on the use of suspended microcarriers as adhesion supports. During the particle suspension phases, the temporal evolution of the spatial distribution of particle concentrations is generally poorly characterized. In this study, the light attenuation technique was used to access this distribution and the duration of particle homogenization. Considering the minimal agitation rate Nref for which particle suspension was observed for all the microcarrier concentrations 〈C〉 studied, the results showed that using an agitation frequency close to Nref significantly increased the homogenization duration compared to N≃1.7×Nref. The transient microcarrier overconcentration was also determined locally. At N=Nref, the resuspension time was negatively impacted by 〈C〉, which also resulted in longer particle overconcentrations. A time-integral particle overconcentration term was considered to estimate the severity of inter-microcarrier collisions. The results suggested that increased agitation could limit time-cumulative overconcentrations beyond a particle concentration of 3 g/L.

Pre-treatment of granular osteoplastic material for improving the reparative regeneration of jaw bone defects

Aim – to develop a method for preliminary preparation of a granular osteoplastic material (GOM) for improving the quality of reparative osteogenesis of jaw bone defects that can be used in clinical setting.&#x0D; Material and methods. The in vitro study was performed using 9 samples (3 samples in each study group). Group 1 included intact samples of grafting material Cerabone (Botiss biomaterials GmbH, Germany). In group 2, the samples of Cerabone (Botiss biomaterials GmbH, Germany) were subject to degassing and dust extraction in two stages using the original method developed by the author. In group 3, a culture of mesenchymal-stromal cells (MSC) obtained from the human umbilical cord was used as a control. The developed method was assessed for cytotoxicity using human MSC. The proliferative index and cell doubling time were registered. A topographic analysis of the sample surface was performed using scanning electron microscopy. To assess the rate of degassing, the experiment included three replicates. The fluid volume was recorded every 2.5 minute. For statistical data processing, we used SPSS 25.0 software (IBM Corporation, Armonk, New York, USA).&#x0D; Results. In Group 2, that included the samples of GOM fractions with the preliminary degassing and dust extraction, we revealed a decreased content of coarse and fine fraction dust on the outer and inner surfaces of the pores and mouths of interpore channels and voids in all samples. The granular osteoplastic material prepared for the use by the method of degassing and dust extraction proved to have no toxic effect on the growth and viability of human mesenchymal-stromal cells.&#x0D; Conclusion. A preliminary preparation of GOM by the method of degassing and dust extraction developed by the author in clinical conditions ex tempore significantly optimizes the adsorption and drainage properties of the GOM fraction.

Effect of Silicone Rubber/Polyvinyl Alcohol /Zirconium Oxide Compound on Mesenchymal Stromal Cells

Objective: This study aimed to conduct a cytotoxicity test in determining the viability and proliferation profile for novel material of silicone rubber/polyvinyl alcohol (PVA) with or without Zirconium oxide on mesenchymal stromal cell culture. Methods: An in vitro study was carried out on adipose-derived mesenchymal stromal cell culture. Samples were divided into five groups: control, silicone rubber/polyvinyl alcohol (PVA) without Zirconium oxide, silicone rubber/polyvinyl alcohol (PVA) with Zirconium oxide 1 %, 3 %, and 5 %. Each group contained 2×105 seeded MSCs/well stained with MTT for its viability. For proliferation, MTT staining was performed on days 1, 3, and 5 to assess the trend of the percentage of the living cell. Statistical analysis was conducted using ANOVA, or the Kruskal-Walli’s test with a CI of 95%.

Local manufacturing processes contribute to variability in human mesenchymal stromal cell expansion while growth media supplements contribute to variability in gene expression and cell function: a Biomedical Excellence for Safer Transfusion (BEST) collaborative study

Background aimsCulture-derived mesenchymal stromal cells (MSCs) exhibit variable characteristics when manufactured using different methods, source material and culture media. The purpose of this multicenter study was to assess the impact on MSC expansion, gene expression and other characteristics when different laboratories expanded MSCs from cultures initiated with bone marrow−MSC aliquots derived from the same donor source material yet with different growth media. MethodsEight centers expanded MSCs using four human platelet lysate (HPL) and one fetal bovine serum (FBS) products as media supplements. The expanded cells were taken through two passages then assessed for cell count, viability, doubling time, immunophenotype, cell function, immunosuppression and gene expression. Results were analyzed by growth media and by center. ResultsCenter methodologies varied by their local seeding density, feeding regimen, inoculation density, base media and other growth media features (antibiotics, glutamine, serum). Doubling times were more dependent on center than on media supplements. Two centers had appropriate immunophenotyping showing all MSC cultures were positive for CD105, CD73, CD90 and negative for CD34, CD45, CD14, HLA-DR. MSCs cultured in media supplemented with FBS compared with HPL featured greater T-cell inhibition potential. Gene expression analysis showed greater impact of the type of media supplement (HPL versus FBS) than the manufacturing center. Specifically, nine genes were decreased in expression and six increased when combining the four HPL-grown MSCs versus FBS (false discovery rate [FDR] <0.01), however, without significant difference between different sources of HPL (FDR <0.01). ConclusionsLocal manufacturing process plays a critical role in MSC expansion while growth media may influence function and gene expression. All HPL and FBS products supported cell growth.

Extracellular Matrix Deposition Defines the Duration of Cell Sheet Assembly from Human Adipose-Derived MSC

Cell sheet (CS) engineering using mesenchymal stromal cells (MSC) draws significant interest for regenerative medicine and this approach translates to clinical use for numerous indications. However, little is known of factors that define the timing of CS assembly from primary cultures. This aspect is important for planning CS delivery in autologous and allogeneic modes of use. We used a comparative in vitro approach with primary donors’ (n = 14) adipose-derived MSCs and evaluated the impact of healthy subject’s sex, MSC culture features (population doubling time and lag-phase), and extracellular matrix (ECM) composition along with factors related to connective tissue formations (α-SMA and FAP-α) on CS assembly duration. Using qualitative and quantitative analysis methods, we found that, in seeded MSCs, high contents of collagen I and collagen IV had a direct correlation with longer CS assembly duration. We found that short lag-phase cultures faster turned to a ready-to-use CS, while age, sex, fibronectin, laminin, α-SMA, and FAP-α failed to provide a significant correlation with the timing of assembly. In detachable CSs, FAP-α was negatively correlated with the duration of assembly, suggesting that its concentration rose over time and contributed to MSC activation, transitioning to α-SMA-positive myofibroblasts and ECM turnover. Preliminary data on cell density and collagen I deposition suggested that the TGF-β1 signaling axis is of pivotal importance for ECM composition and construct maturation.

An Intravital Imaging Protocol to Visualize and Phenotype the Hematopoietic Niche of Clonal Hematopoiesis

Clonal Hematopoiesis of indeterminate potential (CHIP) is a condition where blood cells are produced from a few clones of hematopoietic stem cells (HSCs) carrying leukemia-associated driver mutations 1. Although selective pressures from low-grade inflammation of the bone marrow microenvironment have been shown to promote mutant cell expansion 2-7, we focus on the disease-initiating niche, which is defined as a subset of marrow cells surrounding rare HSCs. Indeed, as we have recently described, clonal expansion of acute myeloid leukemia and activated HSCs 8,9 was not uniform, and only occurred in a subset of marrow spaces harboring bone remodeling activities. To identify the disease-initiating niche for therapeutic targeting, we developed an intravital imaging protocol to visualize the rare pre-malignant clones in the minimally perturbed microenvironment and phenotype their niche. The standard practice to generate CHIP models relies on transplanting syngeneic or allogeneic cells into a sub-lethally irradiated recipient. However, concerns arise as irradiation compromises the microenvironment 10. Successful engraftment in non-conditioned mice can be achieved by injecting a high number of cells (1.5 x 10 7 cells) 11,12, but modeling early clonal expansion events from single cells remain challenging, as a crowd of cells is often observed in proximity. Here, we established a low dose (0.5-1.5 Gy), whole body or local irradiation regimen that preserves the integrity of the bone marrow microenvironment. We performed high-resolution in-vivo imaging to capture dynamics of single cells and cells undergoing early expansion. Specifically, as little as 0.5Gy irradiation enabled survival of the transplanted cells (2x10 6 GFP + healthy whole bone marrow) in non-irradiated side, and allowed direct visualization of early clonal expansion in vivo through 16 weeks . Engraftment was negligible in non-irradiated mice.Through in-vivo, video-rate tracking of Rhodamine dextran dye (70kDa) leakage from vessels into the extravascular space showed no increase in permeability 13 or signs of vessel dilation, suggesting minimal inflammation after the radiation insult (N= 3 mice, data points are individual vessel segments. Mann-Whitney U test). Preliminary findings with in-vitro cultures of mesenchymal stromal cells (MSCs) have also suggested that the MSCs retain their ability for tri-lineage differentiation after the low-dose irradiation. With this imaging protocol, we showed that hot spots of cell expansion exist in the Tet2 +/- murine model. To capture the highly localized niche factors responsible for these hot spots, we implemented image-guided live-cell labeling adapted from the previously published Image-seq protocols 9. As illustrated in Figure 1, a small channel is etched in the bone with plasma-mediated laser ablation. The micropipette was then inserted to the region of interest for delivery of fluorescent antibodies under image guidance. By administering FITC-conjugated anti-CD45 antibodies into the marrow cavities, we can label and recover ~10,000 live cells with 98% viability from two regions of interest, suitable for next generation single cell sequencing. The technique can be expanded to employ antibody cocktails to visualize and enrich particular cell lineages of interest during cell isolation, while preserving spatial information. Its compatibility with standard cell isolation protocols for FACS may help collect cellular targets that are geometrically inaccessible for cell aspiration. In conclusion, we established a working model to visualize expansion of healthy and Tet2 +/- hematopoietic cells in vivo. This can be followed by image-assisted live-cell tagging to isolate local microenvironment cells and study cell-niche coordination at high spatial precision. The imaging protocol may also be broadly applied to study microenvironment regulations in non-malignant clonal disorders.

Presence of Recurrent Somatic Mutations in Mesenchymal Stromal Cell Fractions Isolated from Acute Myeloid Leukemia As an Evidence of Clonality

Introduction: Acute myeloid leukemia (AML) involves somatic mutations in hematopoietic stem cells (HSCs). Recent evidence suggests the bone marrow microenvironment, particularly mesenchymal stromal cells (MSCs), also influences AML development. Abnormalities in MSCs from AML patients have been observed (von der Heide et al., 2016), potentially impacting leukemia progression. This study aims to identify MSC somatic mutations and their consequences, further exploring their role in AML. Understanding MSCs' impact on the leukemic microenvironment may shed light on AML pathophysiology and guide targeted therapies, particularly benefiting patients with adverse risk disease. Methods: A total of 28 bone marrow cases were collected from AML patients with adverse risk, including those with complex karyotype, monosomy karyotype, chromosome 5/7/17 abnormalities, TP53 mutation, or relapsed/refractory AML. From these cases, 14 paired MSC and leukemic cell (LC) fractions were analyzed and are presented here. Magnetic bead sorting was used to isolate CD33+ LCs, while the plastic adherence method was employed for MSC culture. Following magnetic isolation, CD33- cells were plated in DMEM-LG with FBS and pen/strep, and MSC colonies formed within 48 hours. Contaminating cells were removed through media changes, and passaging was performed at 70-80% confluency. Flow cytometry at passage 3 characterized MSCs based on cell surface expression of CD105, CD73, and CD90, in accordance with the ISCT guideline. DNA and RNA were extracted from MSCs at passage 3 and subjected to whole exome sequencing (WES) using the Illumina platform, along with the LC fractions. WES aimed for 200X depth for the paired DNA samples. A standard bioinformatics pipeline was used for sequence alignment and variant calling, with variants requiring sufficient read depth (≥30x) and variant allele frequency in the case (&amp;gt;5%) and in the control (&amp;lt;5%). Synonymous variants were filtered out, and gene ontology and pathway enrichment analyses explored the biological significance and potential involvement in leukemogenesis of the identified mutations. Whole transcriptomics for gene expression analyses was also conducted for both MSC and LC fractions. Results: In 14 analyzed cases, a total of 548 somatic variants were identified in the MSC fraction, affecting 480 genes, including 518 nonsynonymous single nucleotide variants (SNVs), 7 stop-gain SNVs, 1 stoploss, 1 non-frameshift insertion, 2 frameshift deletions, 1 frameshift insertion, and 2 non-frameshift deletions, with 16 unknown variants. The average read depth was 75 (range: 15 - 887), and the average variant allele frequency was 50% (range: 11% - 90%). On average, 38 genes were mutated per MSC case, with 8 genes detected in at least 3 cases, 25 in exactly 2 cases, and 447 genes found in only one case. In the leukemic fraction, 686 somatic variants in 595 genes were identified, with an average read depth of approximately 75 (range: 10 - 2596) and an average variant allele frequency of 47% (range: 8% - 85%). On average, 48 mutations were detected per leukemic case, with 12 mutations found in at least 3 cases, 40 in 2 cases, and 543 in only one case. Among the 14 cases, 10 cases (71%) had mutations in known driver genes for AML ( FLT3, NPM1, IDH1, IDH2, DNMT3A, RUNX1, TP53) exclusively in the leukemic fraction. As seen in Figure 1., of the 480 genes mutated in the MSC fraction, 69 overlapped with the 595 genes mutated in the leukemic fraction, while 411 genes were uniquely mutated in the MSC fraction. Further analysis is ongoing, and pathway analysis will be conducted once the remaining 14 cases are processed. Conclusions: This study identifies distinct somatic mutations in AML patients' MSC and leukemic cell fractions, revealing genomic complexity and crosstalk impacting leukemia progression. Understanding the functional implications of these mutations is crucial for unraveling their roles in leukemogenesis and developing personalized therapeutic interventions targeting MSC somatic mutations.