Mesenchymal Stromal Cells Cultures Research Articles

Xeno-Free Spheroids of Human Gingiva-Derived Progenitor Cells for Bone Tissue Engineering.

Gingiva has been identified as a minimally invasive source of multipotent progenitor cells (GPCs) for use in bone tissue engineering (BTE). To facilitate clinical translation, it is important to characterize GPCs in xeno-free cultures. Recent evidence indicates several advantages of three-dimensional (3D) spheroid cultures of mesenchymal stromal cells (MSCs) over conventional 2D monolayers. The present study aimed to characterize human GPCs in xeno-free 2D cultures, and to test their osteogenic potential in 3D cultures, in comparison to bone marrow MSCs (BMSCs). Primary GPCs and BMSCs were expanded in human platelet lysate (HPL) or fetal bovine serum (FBS) and characterized based on in vitro proliferation, immunophenotype and multi-lineage differentiation. Next, 3D spheroids of GPCs and BMSCs were formed via self-assembly and cultured in HPL. Expression of stemness- (SOX2, OCT4, NANOG) and osteogenesis-related markers (BMP2, RUNX2, OPN, OCN) was assessed at gene and protein levels in 3D and 2D cultures. The cytokine profile of 3D and 2D GPCs and BMSCs was assessed via a multiplex immunoassay. Monolayer GPCs in both HPL and FBS demonstrated a characteristic MSC-like immunophenotype and multi-lineage differentiation; osteogenic differentiation of GPCs was enhanced in HPL vs. FBS. CD271+ GPCs in HPL spontaneously acquired a neuronal phenotype and strongly expressed neuronal/glial markers. 3D spheroids of GPCs and BMSCs with high cell viability were formed in HPL media. Expression of stemness- and osteogenesis-related genes was significantly upregulated in 3D vs. 2D GPCs/BMSCs; the latter was independent of osteogenic induction. Synthesis of SOX2, BMP2 and OCN was confirmed via immunostaining, and in vitro mineralization via Alizarin red staining. Finally, secretion of several growth factors and chemokines was enhanced in GPC/BMSC spheroids, while that of pro-inflammatory cytokines was reduced, compared to monolayers. In summary, monolayer GPCs expanded in HPL demonstrate enhanced osteogenic differentiation potential, comparable to that of BMSCs. Xeno-free spheroid culture further enhances stemness- and osteogenesis-related gene expression, and cytokine secretion in GPCs, comparable to that of BMSCs.

Discarded plasma obtained after cord blood volume reduction as an alternative for fetal calf serum in mesenchymal stromal cells cultures.

Utilization of the fetal calf serum (FCS) carries a potential health risk and raises growing economic and ethical problems. Umbilical cord blood volume reduction, required for banking, provides clinical-grade umbilical cord blood plasma (UCBP) discarded as a waste. The aim of this study was to test whether serum derived from UCBP could replace FCS for the amplification of mesenchymal stromal cells (MSCs). To this end, the amplification of the MSCs and mesenchymal progenitors was estimated in the presence of serum derived from UCBP and its cytokine content was determined by cytometric bead array and enzyme-linked immunosorbent assay techniques. As a comparison, other sources of clinical-grade human serum were tested in parallel: serum derived from solvent/detergent-treated fresh-frozen plasma (S/D-FFP) and from platelet (PLT)-rich and PLT-poor umbilical plasma. Serum derived from UCBP-supplemented culture sustains identical amplification of MSCs and their progenitors as in the case of FCS addition. Furthermore, the assays reveal the presence in the serum derived from UCBP of cytokines influencing the properties of MSCs (basic fibroblast growth factor, transforming growth factor-β, vascular endothelial growth factor, and interleukin-8) or involved in the development of the myeloid lineage (thrombopoietin, erythropoietin, granulocyte-colony-stimulating factor, and granulocyte-macrophage-colony-stimulating factor). Also, our study indicates important differences between neonatal and adult-derived serum. Poor cytokine content in the S/D-FFP makes a less efficient replacement of FCS comparing to other human blood-derived supplements. Our work shows that the discarded human cord blood plasma from volume reduction is an easily obtainable and greatly available, xeno-free source of serum that is a highly efficient replacement of FCS in sustaining MSC growth.

PH Monitoring: Probing the pH Microenvironment of Mesenchymal Stromal Cell Cultures on Additive‐Manufactured Scaffolds (Small 34/2020)

pH Monitoring: Probing the pH Microenvironment of Mesenchymal Stromal Cell Cultures on Additive‐Manufactured Scaffolds (Small 34/2020)

Probing the pH Microenvironment of Mesenchymal Stromal Cell Cultures on Additive-Manufactured Scaffolds.

Despite numerous advances in the field of tissue engineering and regenerative medicine, monitoring the formation of tissue regeneration and its metabolic variations during culture is still a challenge and mostly limited to bulk volumetric assays. Here, a simple method of adding capsules-based optical sensors in cell-seeded 3D scaffolds is presented and the potential of these sensors to monitor the pH changes in space and time during cell growth is demonstrated. It is shown that the pH decreased over time in the 3D scaffolds, with a more prominent decrease at the edges of the scaffolds. Moreover, the pH change is higher in 3D scaffolds compared to monolayered 2D cell cultures. The results suggest that this system, composed by capsules-based optical sensors and 3D scaffolds with predefined geometry and pore architecture network, can be a suitable platform for monitoring pH variations during 3D cell growth and tissue formation. This is particularly relevant for the investigation of 3D cellular microenvironment alterations occurring both during physiological processes, such as tissue regeneration, and pathological processes, such as cancer evolution.

Recent Approaches to Isolating and Culturing Mouse Bone Marrowderived Mesenchymal Stromal Stem Cells.

The isolation and culture of murine Bone Marrow-derived Mesenchymal stromal Stem Cells (mBMSCs) have attracted great interest in terms of the pre-clinical applications of stem cells in tissue engineering and regenerative medicine. In addition, culturing mBMSCs is important for studying the molecular mechanisms of bone remodeling using relevant transgenic mice. Several factors have created challenges in the isolation and high-yield expansion of homogenous mBMSCs; these factors include low frequencies of Bone Marrow-derived mesenchymal stromal Stem Cells (BMSCs) in bone marrow, variation among inbred mouse strains, contamination with Haematopoietic Progenitor Cells (HPCs), the replicative senescence phenotype and cellular heterogeneity. In this review, we provide an overview of nearly all protocols used for isolating and culturing mBMSCs with the aim of clarifying the most important guidelines for culturing highly purified mBMSC populations retaining in vitro and in vivo differentiation potential.

Enhanced proliferative capacity of human preadipocytes achieved by an optimized cultivating method that induces transient activity of hTERT

Human mesenchymal stromal cells (MSC) are an important tool for basic and translational research. Large amounts of MSC are required for in vitro and in vivo studies, however, the limited life-span and differentiation ability in vitro hamper their optimal use. Here we report that 1:1 mixture of L15 and mTeSR1 culture media increased the life-span of IPI-SA3-C4, a normal non-immortalized human subcutaneous preadipocyte strain by 20% while retaining their adipogenic capacity and stable karyotype. The increased proliferative capacity was accompanied by increased expression of the stem markers POU5F1, SOX2, MYC and hTERT, and inhibition of hTERT activity abolished the growth advantage of L15-mTeSR1. Consequently, the described MSC culture would considerably enhance the utility of MSC for in vitro studies.

Novel delivery of cellular therapy to reduce ischemia reperfusion injury in kidney transplantation.

Ex vivo normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for direct delivery of cellular therapeutics to optimize organ quality prior to transplantation. Multipotent Adult Progenitor Cells (MAPC® ) possess potent immunomodulatory properties that could minimize ischemia reperfusion injury. We investigated the potential capability of MAPC cells in kidney NMP. Pairs (5) of human kidneys, from the same donor, were simultaneously perfused for 7 hours. Kidneys were randomly allocated to receive MAPC treatment or control. Serial samples of perfusate, urine, and tissue biopsies were taken for comparison. MAPC-treated kidneys demonstrated improved urine output (P=.009), decreased expression of injury biomarker NGAL (P=.012), improved microvascular perfusion on contrast-enhanced ultrasound (cortex P=.019, medulla P=.001), downregulation of interleukin (IL)-1β (P=.050), and upregulation of IL-10 (P<.047) and Indolamine-2, 3-dioxygenase (P=.050). A chemotaxis model demonstrated decreased neutrophil recruitment when stimulated with perfusate from MAPC-treated kidneys (P<.001). Immunofluorescence revealed prelabeled MAPC cells in the perivascular space of kidneys during NMP. We report the first successful delivery of cellular therapy to a human kidney during NMP. Kidneys treated with MAPC cells demonstrate improvement in clinically relevant parameters and injury biomarkers. This novel method of cell therapy delivery provides an exciting opportunity to recondition organs prior to transplantation.

Development of chitosan, gelatin and liposome film and analysis of its biocompatibility in vitro

A film of chitosan, gelatin and liposome has been designed for dermatological applications. Several adaptations were required throughout development to facilitate in vitro analysis, physicochemical characterization and biocompatibility evaluation. The final version of the film was characterized by differential scanning calorimetry, evaluation of swelling and scanning electron microscopy. The biocompatibility of the film was assessed by investigating cellular parameters of three types of human cells by direct contact or through films extracts: I) primary culture of adipose-derived mesenchymal stromal cells (ADCSs) and melanoma cell lines were used to test cell adhesion and morphology by direct cell culture on the material; II) ADSCs and immortalized keratinocytes were used in cell viability assay using different films extracts. The film showed physicochemical characteristics that favored cellular input, being suitable for in vitro analysis, which allowed its biocompatible characteristics such as the absence of toxicity to be verified without causing significant morphological changes in ADSCs and melanoma cell line. Altogether, these results suggest that the material has a potential application for drug delivery and promotion of skin tissue repair and is therefore worthwhile for further investigations using preclinical models to cover dermal lesions.

Experimental Study of the Possibility of Culturing of Mesenchymal Stromal Cell and Induction of Osteogenic Differentiation on Collagen-Based Scaffolds of Various Modifications.

We analyzed the suitability of various collagen-based scaffolds for culturing and osteogenic differentiation of mesenchymal stromal cells (MSC). Decellularized and lyophilized swine intestinal submucosa (SIS) and porous collagen sponge made from reconstructed bovine derma (PCS) were the most effective in promoting MSC adhesion, survival, and growth. MSC from rat and mouse bone marrow and rat adipose tissue successfully adhered to the scaffold surface and penetrated into its deep layers. These scaffolds were also the most effective in inducing osteogenesis. These results indicate that microarchitectonics of PCS and SIS is optimal for support of MSC growth and osteogenic differentiation.

Investigating lipid mechanisms of MSC immune modulation using metabolic and phenotypic single cell profiling

Background & Aim The immunosuppressive capabilities of human mesenchymal stromal cells (MSCs) have led researchers to explore their potential as therapeutics targeting numerous autoimmune diseases. However, MSC therapies have had limited success so far in clinical trials. Their immune effects are mediated through a complex combination of direct receptor-mediated interactions, vesicle communication and cytokine release mechanisms, making it difficult to identify a unifying underlying biological mechanism of action that could be leveraged to monitor and enhance cell potency. Here, we investigate the role of membrane lipids, a key cell membrane component, associated with these immunosuppressive mechanisms. We aim to evaluate the lipid metabolic profiles of MSCs with known variability in immune regulation and explore the phenotypic changes that occur as a result of differences in lipid signaling. We use longitudinal imaging strategies to directly monitor the associations of cell-cell receptor binding, vesicle release, cytoskeletal network and secretory pathways with immune-related cell activities. Overall, this study aims to directly link lipid metabolism with MSC immunosuppressive behavior using mass spectrometry and image-based approaches, which could be further used to monitor and predict immune response during biomanufacturing of therapeutic cells. Methods, Results & Conclusion MSCs were obtained from cultures with varying immune suppressive activity and secretory profiles. Lipidomic analysis conducted on bulk MSC populations using HPLC/MS/MS revealed several key differences between high and low potency donors. High potency lines showed a negative correlation between sphingomyelin (SM) and ceramide (Cer) whereas the low potency line had a positive correlation indicating that conversion between SM and Cer was associated with cell immune suppression. Multivariate phenotypic imaging analysis of cytoskeletal and vesicle markers showed a strong correlation to cell SM conversion rates. To study heterogeneity of the culture subpopulations, we used Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging to obtain single cell lipid profiles and used advanced deep learning on single cell fluorescent imaging features to understand phenotypic outcomes of lipid signaling. Our findings could have profound implications in understanding immunosuppressive activity of MSCs that could be tuned to enhance the therapeutic efficacy of MSC cultures.

Comparability of MSC manufacturing protocols for clinical trials

Background & Aim Mesenchymal stromal cells (MSC) are clinically applied for their immunomodulatory and regenerative capacities. Despite promising results, comparison of the many different trials is hampered by the heterogeneity in MSC manufacturing protocols. Understanding and delineation of the impact of different production methods by harmonized QC-tests and in-process controls in this respect, will be crucial to optimize MSC therapy for use in larger groups of patients. Methods, Results & Conclusion In our center, MSC are generated from bone marrow mononuclear cells (BM-MNC) using ficoll density centrifugation and subsequent expansion steps in T-flasks. After passaging 3 times, MSC are harvested and cryopreserved in a typical product size of 300 × 106 cells. Previously, changes in our clinical MSC manufacturing protocol involved changes in antibiotics and providers of raw materials such as ficoll and albumin. For GMP-conform change of control validation, comparability studies were performed and >30 real-time manufacturing procedures before and after these changes were compared for classical release criteria such as identity, purity and potency as well as by in-process tests and controls. Notwithstanding the seemingly small changes, the change in ficoll e.g. modulated the BM-MNC subsets composition of the initial MSC culture. Moreover, changing the antibiotics-regimen significantly decreased the population doubling level and decreased the drug product yield at second passage. Despite these differences, identity and purity of the drug products were comparable for both manufacturing protocols. In conclusion, even minor changes in a clinical manufacturing protocol can have a significant impact on MSC production and possibly also on its therapeutic characteristics. Our results indicate that for proper comparisons between trial results, detailed harmonization of production methods but also verification of this by standardized QC testing is needed. This will be the key for optimizing and and proper comparison of cell therapies.

Interest of interleukin-1β priming on various sources of MSC for wound healing: role of interleukin-6, a MSC secreted factor

Background & Aim Deep and extensive skin wounds and burns are treated with autologous Split-Thickness Skin Grafts, with a possible combination of adjuvant therapy, or grafted with Cultured Epidermal Autografts (CEAs) when donor sites are limited. Despite many years of use and the allowing of patient survival, CEA still fail to provide satisfactory clinical outcomes due to poor engraftment rates and reduced skin functionality. In the past few decades, Mesenchymal Stromal Cells (MSCs) therapy have been proposed as a novel therapeutic tool to promote wound healing through anti-inflammatory, pro-trophic and pro-remodeling effects. MSCs are also able to interact with their environment and modify their secretory profile depending on cellular, molecular or physical environment. Our team recently shown that, IL-1β-primed gingival MSCs promoted wound healing in vitro, and in vivo through the secretion of a mix of secreted products and extracellular vesicles. Paracrine mechanism involving MMPs and TGF-βs signaling has been shown to be implicated in MSC primed effect on wound healing. Preliminary results also indicated that IL-6 could also been involved. Therefore, in the present work, we investigated whether other source of MSC could respond similarly to Interleukin 1β to favor wound healing through the secretion of extracellular vesicles and secreted products. Methods, Results & Conclusion Naive and IL-1β-primed MSCs from gingival or bone marrow sources were evaluated in in vitro models of migration, inflammation and Dermal-Epidermal Junction (DEJ) formation. Extracellular vesicles and IL-6 expression were analyzed by NTA and ELISA respectively in the Conditioned Media (CM) of MSC culture. To investigate the mechanism of action of IL-6 in particular, we used human recombinant IL-6 at the concentration found in IL-CM by ELISA and specific blocking antibody in our different in vitro models. Preliminary results indicated that both MSC sources responds similarly to Interleukin-1β priming and have been shown to promote skin cell migration, favor extracellular matrix deposition and reduce inflammation. In vivo studies will be performed to confirm the results observed in vitro. Overall, these results underline the benefit of the IL-1β priming strategy in the treatment of severe skin defects using MSC and epidermal substitute. We hope that this approach could soon improve the current medical treatment of full-thickness burn wounds.

Desirability profiling to rank scalable mesenchymal stromal cell enhancement methods according to multimodal mechanisms

Background & Aim Mesenchymal stromal cells (MSCs) are widely investigated due to their anti-inflammatory, angiogenic, and anti-fibrotic properties. However, insufficient potency, MSC donor variability, and deficient knowledge on therapeutic mechanisms can limit MSC treatment efficacy. The Viswanathan lab has pioneered a novel, non-genetic proprietary strategy to culture MSCs in 3D aggregates under serum-free conditions to augment anti-inflammatory functions. Hypoxic culture has also been shown to enhance MSC paracrine functions. Our objective is to systematically characterize the anti-inflammatory, angiogenic, and anti-fibrotic properties of 3D and hypoxic MSCs, and to apply desirability profiling to empirically rank the potency of the MSCs according to these different properties. Methods, Results & Conclusion METHODS: Human adipose tissue (AT) or bone marrow (BM) MSCs were expanded with serum-free medium. MSCs were then cultured for 24-h under 3D (static culture or in 0.1 L vertical-wheel bioreactors) or 2D hypoxic (5% O2) conditions, with 2D normoxic MSCs as controls. Gene expression was measured by RT-qPCR and using a NanoString 50-gene panel, while microRNAs (miR) were examined by miR-sequencing. In ongoing work, desirability profiling will be performed on these data and on additional functional read-outs in MSC co-cultures with human monocytes, endothelial cells, or fibroblasts. RESULTS 3D MSC culture under static and bioreactor conditions resulted in similar high viable cell yields (>80%). 3D MSCs had increased expression of immunomodulatory genes (e.g. IDO, TSG6 with 4- to 10-fold increased expression vs. 2D normoxic control) while hypoxic MSCs had greater expression of angiogenic genes (e.g. VEGF with 2-fold greater expression vs. control). Preliminary investigation into miR expression revealed that AT-MSCs displayed a distinct miR profile relative to BM-MSCs, and that 3D enhanced AT-MSCs had a differential miR profile vs. controls. The miRs and their target pathways are being investigated to identify mechanisms underlying the enhanced MSC functionality. CONCLUSION MSCs act through multimodal mechanisms that are enhanced through 3D or hypoxic cultures. Statistical modeling is being used to systematically rank the potency attributes of enhanced MSCs in terms of their anti-inflammatory, angiogenic, and anti-fibrotic functionality in vitro and in inflammatory mouse models. Ultimately, this work will generate methods for improved MSC products with therapeutic profiles that are “fit-for-purpose.”

Effect of culture medium and cell size on functional heterogeneity of canine adipose-derived mesenchymal stromal cells

Background & Aim Introduction Mesenchymal stromal cells (MSCs) are being investigated in clinical trials for treatment of a variety of veterinary conditions based on their anti-inflammatory actions and potential to induce functional tissue repair. However, outcomes of clinical trials have often fallen short of pre-clinical expectations. Heterogeneity of MSC preparations and lack of adequate characterization may be partial contributors to the variability in clinical outcomes. Purpose Identification of subpopulations within heterogenous MSC cultures may improve selection of cells with the desired functionality. Therefore, the functional heterogeneity of different subpopulations of MSCs that vary in size was evaluated, as size is hypothesized to relate to function. Methods, Results & Conclusion Methods A high-throughput, biophysical, label-free microfluidic sorting approach was used to separate subpopulations of canine adipose-derived MSCs (Ad-MSCs) based on size. Size sorted MSCs were then characterized and evaluated for the impact of culture conditions on functional heterogeneity. Results We found that larger subpopulations of MSCs with a mean diameter of ∼18 μm proliferate more slowly, senesce at earlier passages, and are inclined to form adipocytes compared to smaller MSC subpopulations (mean diameter of ∼15 μm). Most importantly, size-dependent functions are affected by culture conditions as well as time in culture. Cell surface staining for MSC-specific CD44 & CD90 antigens showed different sized subpopulations of MSCs had comparable expression levels suggesting that this criterion is not relevant to defining subpopulations of MSCs. However, transcriptome analysis showed that differently sized subpopulations of canine Ad-MSCs have distinct gene expression profiles, and culture medium and time in culture further affect gene expression. Conclusion This work provides a strategy for identifying functional subpopulations of canine MSCs based on size that could facilitate the development of quality cellular therapies through a better understanding of functional heterogeneity.

Identification by next generation sequencing analysis of secreted small rnas from IL-1β-primed gingival mesenchymal stromal cells with pro-healing properties

Background & Aim In spite of several research advances in the field of skin tissue engineering and regenerative medicine, severe burns clinical management remains a major therapeutic issue. During the past few years, Mesenchymal Stromal Cells (MSCs) have become an attractive therapeutic option to improve tissue repair such as skin disorders. Several preclinical studies have highlighted their potential to reduce inflammation and promote reepithelialization through the secretion of bioactive molecules and extracellular vesicles containing proteins, mRNA and microRNAs. Recent discovery of their high plasticity to external stimuli has fostered the development of new targeted therapies known as “priming strategies”, that could orientate secretory products of MSCs. IL-1β is a molecule released early after an injury and can persist at high levels long after severe skin traumas such as burn. Therefore, IL-1β was used in a previous study as priming molecule and we found that IL-1β-primed gingival MSCs were superior to naive MSCs to improve wound healing and epidermal engraftment in vitro and in vivo. In the present study, we investigated the mechanism by which IL-1β-primed MSC promote a better wound healing. In particular, we wondered if some small RNAs are implicated in the activities of MSC. Methods, Results & Conclusion Therefore, we analyzed small RNAs with Next Generation Sequencing technic and PCR in conditioned media prepared from IL-1β-primed or naive gingival MSC culture. This analysis showed the down regulation and upregulation of several kind of small RNAs. We found that IL-1β-primed MSCs secrete higher level of miRNAs playing a role in inflammation such as miR-210-3p, and miR146-5p. This analysis also highlights a great number of other small RNAs that can be implicated in the pro-healing effect of primed MSC observed in our in vitro and in vivo models. To investigate their involvement in the wound healing process, mimics RNA corresponding to miRNA and other small RNA candidates will be used in models of inflammation, migration and dermal-epidermal junction formation in vitro. This study aims to better understand the mechanism of action of IL-1β primed MSC on wound healing, and the impact of the priming on the RNA secretion.

A Protocol for the Isolation, Culture, and Cryopreservation of Umbilical Cord-Derived Canine Mesenchymal Stromal Cells: Role of Cell Attachment in Long-Term Maintenance.

Mesenchymal stromal cells (MSCs) hold great promise in the field of regenerative medicine due to their ability to create a variable localized anti-inflammatory effect in injuries such as Crohn's disease and osteoarthritis or by incorporation in tissue engineered constructs. Currently, the MSC literature uses rodents for preclinical disease models. There is growing interest in using naturally occurring disease in large animals for modeling human disease. By review of the canine MSCs literature, it appears that canine MSCs can be difficult to maintain in culture for extended passages and this greatly varies between tissue sources, compared with human and rodent MSCs, and limited lifespan is an obstacle for preclinical investigation and therapeutic use. Research using canine MSCs has been focused on cells derived from bone marrow or adipose tissue, and the differences in manufacturing MSCs between laboratories are problematic due to lack of standardization. To address these issues, here, a stepwise process was used to optimize canine MSCs isolation, expansion, and cryopreservation utilizing canine umbilical cord-derived MSCs. The culture protocol utilizes coating of tissue culture surfaces that increases cellular adherence, increases colony-forming units-fibroblast efficiency, and decreases population doubling times. Canine MSCs isolated with our protocol could be maintained longer than published canine MSCs methods before senescing. Our improved cryopreservation protocols produce on average >90% viable MSCs at thaw. These methods enable master-bank and working-bank scenarios for allogeneic MSC testing in naturally occurring disease in dogs.

Human Platelet Lysate Can Replace Fetal Calf Serum as a Protein Source to Promote Expansion and Osteogenic Differentiation of Human Bone-Marrow-Derived Mesenchymal Stromal Cells.

Fetal calf serum (FCS) is frequently used as a growth factor and protein source in bone-marrow-derived mesenchymal stromal cell (BMSC) culture media, although it is a xenogenic product presenting multiple disadvantages including but not limited to ethical concerns. A promising alternative for FCS is human platelet lysate (hPL), which is produced out of human platelet concentrates and happens to be a stable and reliable protein source. In this study, we investigated the influence of hPL in an expansion medium (ESM) and an osteogenic differentiation medium (ODM) on the proliferation and osteogenic differentiation capacity of human BMSC. Therefore, we assessed population doublings during cell expansion, performed alizarin red staining to evaluate the calcium content in the extracellular matrix and determined the activity of alkaline phosphatase (ALP) as osteogenic differentiation correlates. The proliferation rate of BMSC cultured in ESM supplemented with hPL exceeded the proliferation rate of BMSC cultured in the presence of FCS. Furthermore, the calcium content and ALP activity was significantly higher in samples incubated in hPL-supplemented ODM, especially in the early phases of differentiation. Our results show that hPL can replace FCS as a protein supplier in cell culture media and does not negatively affect the osteogenic differentiation capacity of BMSC.

Human mesenchymal stromal cell source and culture conditions influence extracellular vesicle angiogenic and metabolic effects on human endothelial cells in vitro.

Mesenchymal stem/stromal cell (MSC)-derived extracellular vesicles (EVs) are a possible cell-free alternative to MSCs because they retain the regenerative potential of MSCs, while still mitigating some of their limitations (such as the possible elicitation of host immune responses). The promotion and restoration of angiogenesis, however, is an important component in treating trauma-related injuries, and has not been fully explored with EVs. Herein, we describe the effects of monolayer adipose-derived EVs, spheroid adipose-derived EVs (SAd-EVs), monolayer bone marrow-derived EVs (MBM-EVs), and spheroid bone marrow-derived EVs (SBM-EVs) on human umbilical vein endothelial cell (HUVEC) tube formation and mitochondrial respiration. The successful isolation of EVs derived from adipose MSCs or bone marrow MSCs in monolayer or spheroid cultures was confirmed by NanoSight (particle size distribution) and Western blot (surface marker expression). The EV angiogenic potential was measured using a 24-hour HUVEC tube formation assay. The EV effects on HUVEC mitochondrial function were evaluated using the Seahorse respirometer machine. The number of junctions, branches, and the average length of branches formed at 24 hours of tube formation were significantly affected by cell and culture type; overall adipose-derived EVs outperformed bone marrow-derived EVs, and spheroid-derived EVs outperformed monolayer-derived EVs. Additionally, adipose-derived EVs resulted in significantly increased HUVEC mitochondrial maximal respiration and adenosine triphosphate (ATP) production, while only MBM-EVs negatively impacted HUVEC proton leak. Adipose-derived EVs promoted HUVEC tube formation significantly more than bone marrow-derived EVs, while also maximizing HUVEC mitochondria function. Results demonstrate that, as with MSC therapies, it is possible to tailor EV culture and production to optimize therapeutic potential. Basic or Foundational Research.

ALCAM (CD166) as a gene expression marker for human mesenchymal stromal cell characterisation.

BackgroundHuman mesenchymal stromal cells (MSCs) phenotypically share their positive expression of the International Society for Cell and Gene Therapy (ISCT) markers CD73, CD90 and CD105 with fibroblasts. Fibroblasts are often co-isolated as an unwanted by-product from biopsy and they can rapidly overgrow the MSCs in culture. Indeed, many other surface markers have been proposed, though no unique MSC specific marker has been identified yet. Quantitative PCR (qPCR) is a precise, efficient and rapid method for gene expression analysis. To identify a marker suitable for accurate MSC characterisation, qPCR was exploited.Methods and resultsTwo commercially obtained bone marrow (BM) derived MSCs and an hTERT immortalised BM-MSC line (MSC-TERT) have been cultured for different days and at different oxygen levels before RNA extraction. Together with RNA samples previous extracted from umbilical cord derived MSCs and MSC-TERT cells cultured in 2D or 3D, this heterogeneous sample set was quantitatively analysed for the expression levels of 18 candidate MSC marker genes. The expression levels in MSCs were compared with the expression levels in fibroblasts to verify the differentiation capability of these genes between MSCs and fibroblasts. None of the ISCT markers could differentiate between fibroblasts and MSCs. A total of six other genes (ALCAM, CLIC1, EDIL3, EPHA2, NECTIN2, and TMEM47) were identified as possible biomarkers for accurate identification of MSCs.ConclusionJustified by considerations on expression level, reliability and specificity, Activated-Leukocyte Cell Adhesion Molecule (ALCAM) was the best candidate for improving the biomarker set of MSC identification.

Coronary corium, a new source of equine mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) have attracted great attention for therapeutic applications. Since cells derived from different tissues have different properties, using the right tissue source may impact their efficiency in regenerative medicine. This study describes for the first time the isolation and characterization of MSCs derived from the equine coronary corium, which may be useful for treating diseases such as laminitis. Seven coronary corium samples were used for isolation of cells (ccMSCs). Adherent cells were characterized for morphology, immunophenotype, proliferation and differentiation potential, in vitro migration and colony-forming capacity. The cells displayed the characteristic fibroblastoid morphology, with population doubling time increasing until passage 7 and reaching a plateau in passage 10. Cells were negative for CD14 and CD45, and positive for CD73 and CD90. ccMSCs showed chondrogenic and osteogenic, but not adipogenic differentiation, and migrated with nearly total closing of the empty area in 48h, in the scratch assay. The clonogenic potential was in average 18% to 23%. This study describes for the first time the establishment of mesenchymal stromal cell cultures from the equine coronary corium. The results are similar to MSCs isolated from many other equine tissues, except for restricted differentiation potential. As coronary corium stem cell regulation may contribute to the pathogenesis of equine chronic laminitis, the use of ccMSCs in cell therapy for this significantly debilitating disease should be further investigated.