scholarly journals Impact of Three Different Serum Sources on Functional Properties of Equine Mesenchymal Stromal Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Lynn Pezzanite ◽  
Lyndah Chow ◽  
Gregg Griffenhagen ◽  
Steven Dow ◽  
Laurie Goodrich

Culture and expansion of equine mesenchymal stromal cells (MSCs) are routinely performed using fetal bovine serum (FBS) as a source of growth factors, nutrients, and extracellular matrix proteins. However, the desire to minimize introduction of xenogeneic bovine proteins or pathogens and to standardize cellular products intended for clinical application has driven evaluation of alternatives to FBS. Replacement of FBS in culture for several days before administration has been proposed to reduce antigenicity and potentially prolong survival after injection. However, the functional consequences of MSC culture in different serum types have not been fully evaluated. The objective of this study was to compare the immunomodulatory and antibacterial properties of MSCs cultured in three serum sources: FBS or autologous or allogeneic equine serum. We hypothesized that continuous culture in FBS would generate MSCs with improved functionality compared to equine serum and that there would not be important differences between MSCs cultured in autologous vs. allogeneic equine serum. To address these questions, MSCs from three healthy donor horses were expanded in medium with FBS and then switched to culture in FBS or autologous or allogeneic equine serum for 72 h. The impact of this 72-h culture period in different sera on cell viability, cell doubling time, cell morphology, bactericidal capability, chondrogenic differentiation, and production of cytokines and antimicrobial peptides was assessed. Altering serum source did not affect cell viability or morphology. However, cells cultured in FBS had shorter cell doubling times and secreted more interleukin 4 (IL-4), IL-5, IL-17, RANTES, granulocyte–macrophage colony-stimulating factor, fibroblast growth factor 2, eotaxin, and antimicrobial peptide cathelicidin/LL-37 than cells cultured in either source of equine serum. Cells cultured in FBS also exhibited greater spontaneous bactericidal activity. Notably, significant differences in any of these parameters were not observed when autologous vs. allogeneic equine serum was used for cell culture. Chondrogenic differentiation was not different between different serum sources. These results indicate that MSC culture in FBS will generate more functional cells based on a number of parameters and that the theoretical risks of FBS use in MSC culture should be weighed against the loss of MSC function likely to be incurred from culture in equine serum.

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Ramana Vaka ◽  
Saad Khan ◽  
Bin Ye ◽  
Yousef Risha ◽  
Sandrine Parent ◽  
...  

Abstract Background Although 90% of infections with the novel coronavirus 2 (COVID-19) are mild, many patients progress to acute respiratory distress syndrome (ARDS) which carries a high risk of mortality. Given that this dysregulated immune response plays a key role in the pathology of COVID-19, several clinical trials are underway to evaluate the effect of immunomodulatory cell therapy on disease progression. However, little is known about the effect of ARDS associated pro-inflammatory mediators on transplanted stem cell function and survival, and any deleterious effects could undermine therapeutic efficacy. As such, we assessed the impact of inflammatory cytokines on the viability, and paracrine profile (extracellular vesicles) of bone marrow-derived mesenchymal stromal cells, heart-derived cells, and umbilical cord-derived mesenchymal stromal cells. Methods All cell products were manufactured and characterized to established clinical release standards by an accredited clinical cell manufacturing facility. Cytokines and Extracellular vesicles in the cell conditioned media were profiled using proteomic array and nanoparticle tracking analysis. Using a survey of the clinical literature, 6 cytotoxic cytokines implicated in the progression of COVID-19 ARDS. Flow cytometry was employed to determine receptor expression of these 6 cytokines in three cell products. Based on clinical survey and flow cytometry data, a cytokine cocktail that mimics cytokine storm seen in COVID-19 ARDS patients was designed and the impact on cytokine cocktail on viability and paracrine secretory ability of cell products were assessed using cell viability and nanoparticle tracking analysis. Results Flow cytometry revealed the presence of receptors for all cytokines but IL-6, which was subsequently excluded from further experimentation. Despite this widespread expression, exposure of each cell type to individual cytokines at doses tenfold greater than observed clinically or in combination at doses associated with severe ARDS did not alter cell viability or extracellular vesicle character/production in any of the 3 cell products. Conclusions The paracrine production and viability of the three leading cell products under clinical evaluation for the treatment of severe COVID-19 ARDS are not altered by inflammatory mediators implicated in disease progression.


2018 ◽  
Vol 39 (8) ◽  
pp. 954-959 ◽  
Author(s):  
Jason T. Bariteau ◽  
Rishin J. Kadakia ◽  
Brian C. Traub ◽  
Manjula Viggeswarapu ◽  
Nick J. Willett

Background: Vancomycin is frequently applied locally to the operative site during foot and ankle procedures to help prevent infection. Although the efficacy of locally applied vancomycin has been demonstrated in spine surgery, there is no consensus on dosing and indication within foot and ankle surgery. Osteogenic differentiation of human mesenchymal stromal cells (hMSCs) is key to healing of both fractures and arthrodesis. The purpose of this research was to determine the impact of vancomycin on human hMSCs during the process of osteogenic differentiation. Methods: hMSCs were cultured in osteogenic differentiation media to promote osteogenic differentiation. Cells were treated with vancomycin at differing concentrations of 0, 50, 500, and 5000 µg/mL. Viability and cell growth were assessed via LIVE/DEAD viability/cytotoxicity kit (Invitrogen, Waltham, MA) after 1, 3, and 7 days of vancomycin treatment. Differentiation and mineralization was assessed via alizarin red staining after 21 days of treatment. Mean cell viability, cell number, and mineralization were compared between treatment groups using 1-way analysis of variance and the Tukey-Kramer method for post hoc pairwise comparisons. Results: At the highest concentrations of vancomycin, there was a significant reduction in cell viability and proliferation after 3 days compared with all other treatment groups. Mineralization was also significantly decreased with higher doses of vancomycin. Conclusion: At high concentrations, vancomycin may impair hMSC viability and osteogenic differentiation. Clinical Relevance: Surgeons should exercise caution and consider the limited soft tissue envelope when applying vancomycin locally during foot and ankle surgery, especially during arthrodesis procedures.


2021 ◽  
Vol 10 (13) ◽  
pp. 2991
Author(s):  
Raquel Sanabria-de la Torre ◽  
María I. Quiñones-Vico ◽  
Ana Fernández-González ◽  
Manuel Sánchez-Díaz ◽  
Trinidad Montero-Vílchez ◽  
...  

The well-known immunomodulatory and regenerative properties of mesenchymal stromal cells (MSCs) are the reason why they are being used for the treatment of many diseases. Because they are considered hypoimmunogenic, MSCs treatments are performed without considering histocompatibility barriers and without anticipating possible immune rejections. However, recent preclinical studies describe the generation of alloantibodies and the immune rejection of MSCs. This has led to an increasing number of clinical trials evaluating the immunological profile of patients after treatment with MSCs. The objective of this systematic review was to evaluate the generation of donor specific antibodies (DSA) after allogeneic MSC (allo-MSC) therapy and the impact on safety or tolerability. Data from 555 patients were included in the systematic review, 356 were treated with allo-MSC and the rest were treated with placebo or control drugs. A mean of 11.51% of allo-MSC-treated patients developed DSA. Specifically, 14.95% of these patients developed DSA and 6.33% of them developed cPRA. Neither the production of DSA after treatment nor the presence of DSA at baseline (presensitization) were correlated with safety and/or tolerability of the treatment. The number of doses administrated and human leucocyte antigen (HLA) mismatches between donor and recipient did not affect the production of DSA. The safety of allo-MSC therapy has been proved in all the studies and the generation of alloantibodies might not have clinical relevance. However, there are very few studies in the area. More studies with adequate designs are needed to confirm these results.


BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Lizhen Liu ◽  
Kaimin Hu ◽  
Jingjing Feng ◽  
Huafang Wang ◽  
Shan Fu ◽  
...  

Abstract Background Isocitrate dehydrogenase (IDH1/2) gene mutations are the most frequently observed mutations in cartilaginous tumors. The mutant IDH causes elevation in the levels of R-enantiomer of 2-hydroxylglutarate (R-2HG). Mesenchymal stromal cells (MSCs) are reasonable precursor cell candidates of cartilaginous tumors. This study aimed to investigate the effect of oncometabolite R-2HG on MSCs. Methods Human bone marrow MSCs treated with or without R-2HG at concentrations 0.1 to 1.5 mM were used for experiments. Cell Counting Kit-8 was used to detect the proliferation of MSCs. To determine the effects of R-2HG on MSC differentiation, cells were cultured in osteogenic, chondrogenic and adipogenic medium. Specific staining approaches were performed and differentiation-related genes were quantified. Furthermore, DNA methylation status was explored by Illumina array-based arrays. Real-time PCR was applied to examine the signaling component mRNAs involved in. Results R-2HG showed no influence on the proliferation of human MSCs. R-2HG blocked osteogenic differentiation, whereas promoted adipogenic differentiation of MSCs in a dose-dependent manner. R-2HG inhibited chondrogenic differentiation of MSCs, but increased the expression of genes related to chondrocyte hypertrophy in a lower concentration (1.0 mM). Moreover, R-2HG induced a pronounced DNA hypermethylation state of MSC. R-2HG also improved promotor methylation of lineage-specific genes during osteogenic and chondrogenic differentiation. In addition, R-2HG induced hypermethylation and decreased the mRNA levels of SHH, GLI1and GLI2, indicating Sonic Hedgehog (Shh) signaling inhibition. Conclusions The oncometabolite R-2HG dysregulated the chondrogenic and osteogenic differentiation of MSCs possibly via induction of DNA hypermethylation, improving the role of R-2HG in cartilaginous tumor development.


Stroke ◽  
2013 ◽  
Vol 44 (suppl_1) ◽  
Author(s):  
Kate Xie* ◽  
Quynh Vu* ◽  
Mark Eckert ◽  
Weian Zhao ◽  
Steven C Cramer

INTRODUCTION: Mesenchymal stromal cells (MSC) are multipotent cells that support numerous restorative processes after stroke. The ease of isolation and immunoprivileged status of MSC have stimulated numerous preclinical stroke studies. We performed a meta-analysis to estimate study quality, size of behavioral effects, and the impact of variables such as timing of MSC administration in relation to stroke onset. METHODS: Studies of MSC and stroke were identified through PubMed and Web of Science. Studies of hemorrhage, not in English, or using modified MSC were excluded. A Quality Score was determined for each study, estimating methodological quality using 10 criteria derived from STAIR guidelines, with higher Quality Scores reflecting greater compliance with issues such as randomization and outcome blinding. Outcome data extracted for MSC and control arms were used to derive estimates of effect size using Cohen’s d. RESULTS: A total of 46 studies met criteria, with 39 studying rats, 6 mice, and 1 primates. There were 61 treatment groups, as some studies had >1 independent MSC treatment arms; MSC were introduced intravenously in 41, intracerebrally in 15, and intraarterially in 6. MSC source was rat in 24, human in 16, and mouse in 6. Time of MSC administration ranged from 5 weeks pre- to 1 month post-stroke. MSC dose ranged from 1x10^4 to 3.25x10^7. The median Quality Score was 6 (IQR 5-7). Quality Score was not related to time of MSC administration relative to stroke or to behavioral effect size. Median effect size was 2.05 for the Modified Neurological Severity Scale (n=23), 1.88 for Adhesive Removal Test (n=19), and 2.70 for the Rotarod Test (n=14). Effect sizes were substantial across all routes of administration and differed only for the mNSS (p<0.04), favoring the IC route. Effect size did not vary with time of MSC administration relative to stroke for any behavioral measure. CONCLUSIONS: The quality of preclinical MSC stroke studies has generally been good. MSC consistently provide very large behavioral benefits, across scales and routes of administration. The magnitude of behavioral effects was not related to the Quality Score or to the time of MSC administration relative to stroke. These findings support translation of MSC to human trials.


2018 ◽  
Vol 1859 ◽  
pp. e45
Author(s):  
Sergiu Dumitrescu ◽  
Adelheid Weidinger ◽  
Asmita Banerjee ◽  
Susanne Wolbank ◽  
Karlheinz Hilber ◽  
...  

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