scholarly journals Cardiac Migration of Endogenous Mesenchymal Stromal Cells in Patients with Inflammatory Cardiomyopathy

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Caroline Schmidt-Lucke ◽  
Felicitas Escher ◽  
Sophie Van Linthout ◽  
Uwe Kühl ◽  
Kapka Miteva ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Multivariate Analysis ◽  
Mesenchymal Stromal Cells ◽  
Coronary Sinus ◽  
Stromal Cells ◽  
Real Time Pcr ◽  
Strong Correlation ◽  
Inflammatory Cardiomyopathy ◽  
Cardiac Inflammation ◽  
Peripheral Mononuclear Cell

Introduction. Mesenchymal stromal cells (MSC) have immunomodulatory features. The aim of this study was to investigate the migration and homing potential of endogenous circulating MSC in virus negative inflammatory cardiomyopathy (CMi).Methods. In 29 patients withn=23or withoutn=6CMi undergoing endomyocardial biopsies (EMB), transcardiac gradients (TCGs) of circulating MSC were measured by flow cytometry from blood simultaneously sampled from aorta and coronary sinus. The presence of MSC in EMB, cardiac inflammation, and SDF-1αmRNA expression were detected via immunohistochemistry and real-time PCR.Results. MSC defined as CD45−CD34−CD11b−CD73+CD90+cells accounted for 0.010 [0.0025–0.048]%/peripheral mononuclear cell (PMNC) and as CD45−CD34−CD11b−CD73+CD105+cells for 0.019 [0.0026–0.067]%/PMNC, both with similar counts in patients with or without cardiac inflammation. There was a 29.9%P<0.01transcardiac reduction of circulating MSC in patients with CMi, correlating with the extent of cardiac inflammation (P<0.05, multivariate analysis). A strong correlation was found between the TCG of circulating MSC and numbers of MSC (CD45−CD34−CD90+CD105+) in EMB (r=-0.73,P<0.005). SDF-1αwas the strongest predictor for increased MSC in EMB (P<0.005, multivariate analysis).Conclusions. Endogenous MSC continuously migrate to the heart in patients with CMi triggered by cardiac inflammation.

scholarly journals Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Weichao Zhai ◽  
Jerome Tan ◽  
Tobias Russell ◽  
Sixun Chen ◽  
Dennis McGonagle ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Preclinical Model ◽  
Label Free ◽  
Current Standard ◽  
Differentiation Potential ◽  
Human Mesenchymal Stromal Cells ◽  
Endogenous Fluorophores

AbstractHuman mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difficult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture’s viability, differentiation potential and ultimately leads to reduced therapeutic efficacies. Histochemical $$\upbeta $$ β -galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label-free. In this study, we have investigated alternatives in quantification of hMSCs senescence, which included flow cytometry methods that are based on a combination of cell size measurements and fluorescence detection of SA-$$\upbeta $$ β -galactosidase activity using the fluorogenic substrate, C$${_{12}}$$ 12 FDG; and autofluorescence methods that measure fluorescence output from endogenous fluorophores including lipopigments. For identification of senescent cells in the hMSC batches produced, the non-destructive and label-free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the final output of the therapeutic-grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced flow cytometry and autofluorescence methods, which were benchmarked with the current standard in $$\upbeta $$ β -galactosidase staining. Both the advanced methods demonstrated statistically significant values, (r = 0.76, p $$\le $$ ≤ 0.001 for the fluorogenic C$${_{12}}$$ 12 FDG method, and r = 0.72, p $$\le $$ ≤ 0.05 for the forward scatter method), and good fold difference ranges (1.120–4.436 for total autofluorescence mean and 1.082–6.362 for lipopigment autofluorescence mean) between early and senescent passage hMSCs. Our autofluroescence imaging and spectra decomposition platform offers additional benefit in label-free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers.

scholarly journals Vadadustat, a HIF Prolyl Hydroxylase Inhibitor, Improves Immunomodulatory Properties of Human Mesenchymal Stromal Cells

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2396
Author(s):  
Katarzyna Zielniok ◽  
Anna Burdzinska ◽  
Beata Kaleta ◽  
Radoslaw Zagozdzon ◽  
Leszek Paczek
Keyword(s):  
Real Time ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Real Time Pcr ◽  
Mononuclear Cells ◽  
Therapeutic Potential ◽  
Human Peripheral Blood ◽  
Immunomodulatory Activity ◽  
Peripheral Blood Mononuclear ◽  
Immunomodulatory Properties

The therapeutic potential of mesenchymal stromal cells (MSCs) is largely attributed to their immunomodulatory properties, which can be further improved by hypoxia priming. In this study, we investigated the immunomodulatory properties of MSCs preconditioned with hypoxia-mimetic Vadadustat (AKB-6548, Akebia). Gene expression analysis of immunomodulatory factors was performed by real-time polymerase chain reaction (real-time PCR) on RNA isolated from six human bone-marrow derived MSCs populations preconditioned for 6 h with 40 μM Vadadustat compared to control MSCs. The effect of Vadadustat preconditioning on MSCs secretome was determined using Proteome Profiler and Luminex, while their immunomodulatory activity was assessed by mixed lymphocyte reaction (MLR) and Culturex transwell migration assays. Real-time PCR revealed that Vadadustat downregulated genes related to immune system: IL24, IL1B, CXCL8, PDCD1LG1, PDCD1LG2, HIF1A, CCL2 and IL6, and upregulated IL17RD, CCL28 and LEP. Vadadustat caused a marked decrease in the secretion of IL6 (by 51%), HGF (by 47%), CCL7 (MCP3) (by 42%) and CXCL8 (by 40%). Vadadustat potentiated the inhibitory effect of MSCs on the proliferation of alloactivated human peripheral blood mononuclear cells (PBMCs), and reduced monocytes-enriched PBMCs chemotaxis towards the MSCs secretome. Preconditioning with Vadadustat may constitute a valuable approach to improve the therapeutic properties of MSCs.

scholarly journals Human levator veli palatini muscle: a novel source of mesenchymal stromal cells for use in the rehabilitation of patients with congenital craniofacial malformations

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Daniela Franco Bueno ◽  
Gerson Shigueru Kabayashi ◽  
Carla Cristina Gomes Pinheiro ◽  
Daniela Y. S. Tanikawa ◽  
Cassio Eduardo Raposo-Amaral ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cleft Palate ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Osteogenic Potential ◽  
Flow Cytometry Analysis ◽  
Non Invasive ◽  
Levator Veli Palatini

Abstract Background Bone reconstruction in congenital craniofacial differences, which affect about 2–3% of newborns, has long been the focus of intensive research in the field of bone tissue engineering. The possibility of using mesenchymal stromal cells in regenerative medicine protocols has opened a new field of investigation aimed at finding optimal sources of multipotent cells that can be isolated via non-invasive procedures. In this study, we analyzed whether levator veli palatini muscle fragments, which can be readily obtained in non-invasive manner during palatoplasty in cleft palate patients, represent a novel source of MSCs with osteogenic potential. Methods We obtained levator veli palatini muscle fragments (3–5 mm3), during surgical repair of cleft palate in 5 unrelated patients. Mesenchymal stromal cells were isolated from the muscle using a pre-plating technique and other standard practices. The multipotent nature of the isolated stromal cells was demonstrated via flow cytometry analysis and by induction along osteogenic, adipogenic, and chondrogenic differentiation pathways. To demonstrate the osteogenic potential of these cells in vivo, they were used to reconstruct a critical-sized full-thickness calvarial defect model in immunocompetent rats. Results Flow cytometry analysis showed that the isolated stromal cells were positive for mesenchymal stem cell antigens (CD29, CD44, CD73, CD90, and CD105) and negative for hematopoietic (CD34 and CD45) or endothelial cell markers (CD31). The cells successfully underwent osteogenic, chondrogenic, and adipogenic cell differentiation under appropriate cell culture conditions. Calvarial defects treated with CellCeram™ scaffolds seeded with the isolated levator veli palatini muscle cells showed greater bone healing compared to defects treated with acellular scaffolds. Conclusion Cells derived from levator veli palatini muscle have phenotypic characteristics similar to other mesenchymal stromal cells, both in vitro and in vivo. Our findings suggest that these cells may have clinical relevance in the surgical rehabilitation of patients with cleft palate and other craniofacial anomalies characterized by significant bone deficit.

scholarly journals Immunoregulatory Properties of Apoptotic Human Amniontic Mesenchymal Stromal Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5678-5678
Author(s):  
Ying Xu ◽  
Ya Gao ◽  
Yan chun Yang ◽  
Dongmao Zhu ◽  
Yintian Zhang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Natural Science ◽  
Guangdong Province ◽  
Morphological Features ◽  
Cell Contact ◽  
Tnf Α ◽  
Ifn Γ ◽  
Tgf Β1

Abstract Objective Mesenchymal stromal cells (MSCs) have been used in preventing and treating acute graft-versus-host disease (aGVHD), but the mechanism is not fully understood. Apoptotic bone marrow mesenchymal stromal cells (BMSCs) were showed could induce vivo recipient-mediated immunomodulation in mice GVHD model. We had demonstrated that, similar to BM-MSCs, human amniontic mesenchymal stromal cells (hAMSCs) exhibit potent immunosuppressive and anti-inflammatory activities but possess a higher proliferation activity and clearer stem cell properties in vitro. This study focuses on the immunoregulatory properties of apoptotic human amniontic mesenchymal stromal cells (apo-hAMSCs) in an inflammatory microenvironment. Methods hAMSCs from human amniotic membrane were cultured with tissue mass cell culture. The cell phenotype of the 3rd passage were detected by flow cytometry. Transwell co-culture experiments and cell-cell contact co-culture experiments were conducted, consisting of hAMSCs and peripheral blood mononuclear cells stimulated with phytohemagglutinin (PHA-PBMCs), as the positive control group. While other groups were PBMCs without PHA and hAMSCs(PBMCs+hAMSCs), PBMCs and PHA (PHA-PBMCs), hAMSCs and PBMCs. For apoptosis evalution, the morphological features of hAMSCs were recorded in different groups, and apo-hAMSCs were analyzed by flow cytometry at 24 hours. The production of Interferon-γ (IFN-γ), transforming growth factor-β1 (TGF-β1), prostaglandin E2 (PGE-2), soluble human leukocyte antigen G (sHLA-G), Tumor necrosis factor-α(TNF-α) and interleukin-17A (IL-17A) in the co-culture supernatant was detected using enzyme-linked immunosorbent assay (ELISA), and kynurenine were dectected by spectrophotometer. CD4+CD25+FOXP3+ regulatory T cells (Tregs) in PBMC were analyaed by flow cytometry. Result hAMSCs expressed CD105, CD73, CD90, while not CD19, CD34, CD45, CD11b, HLA-DR. In the group of hAMSCs and PHA-PBMCs, the number of hAMSCs reduced. The morphological features were that cells shrinked, turned round, separated from the bottle and suspended in supernatant. However, hAMSCs in the groups of hAMSCs+PBMCs and hAMSCs stayed the same. Apoptosis in hAMSCs cultivated with PHA-PBMCs via transwell or cell-cell contact experiment increased compared with the group of hAMSCs+PBMCs (P<0.05) and hAMSCs (P<0.05). In the two co-culture experments, the secretion level of PGE-2, TGF-β1, sHLA-G, and KYN significantly increased in hAMSCs with PHA-PBMCs compared with hAMSC (P<0.05) and hAMSCs with PBMCs (P<0.05). The level of IFN-γ and TNF-α decreased in hAMSCs with PHA-PBMCs compared with PBMCs with PHA (P<0.05). While the level of IL-17A was significantly increase in hAMSCs with PHA-PBMCs compared with hAMSCs (P<0.05), hAMSCs with PBMCs (P<0.05) and PHA-PBMCs (P>0.05). Evident difference of CD4+CD25+FOXP3+ Tregs was shown between hAMSCs with PHA-PBMCs and PHA-PBMCs (P<0.05). Conclusion Activated, but not resting, PBMCs induce extensive early apoptosis in hAMSCs. And apoptosis in hAMSCs need inflammatory microenvirenment. Apoptotic hAMSCs still have immunoregulatory effects in cytokines and immune cells. Funding This work was supported by Natural Science Foundation of China (81701243), Key Sci-Tech Research Projects of Guangdong Province (2014A02021102), Natural Science Foundation of Guangdong Province, China (2014A030310373), the Pearl River S&T Nova Program of Guangzhou (201710010047). Disclosures No relevant conflicts of interest to declare.

scholarly journals Latent tri-lineage potential of human menstrual blood-derived mesenchymal stromal cells revealed by specific in vitro culture conditions

2021 ◽  
Author(s):  
Diana Quintero-Espinosa ◽  
Viviana Soto-Mercado ◽  
Catherine Quintero-Quinchia ◽  
Miguel Mendivil-Perez ◽  
Carlos Velez-Pardo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Culture Conditions ◽  
Menstrual Blood ◽  
Neurologic Disorders ◽  
Cellular Source ◽  
Timely Fashion ◽  
Direct Transdifferentiation

Abstract Human menstrual blood-derived mesenchymal stromal cells (MenSCs) have become not only an important source of stromal cells for cell therapy but also a cellular source for neurologic disorders in vitro modeling. By using culture protocols originally developed in our laboratory, we show that MenSCs can be converted into floating neurospheres (NSs) using the Fast-N-Spheres medium for 24-72h, and can be transdifferentiated into functional dopaminergic-like (DALNs, ~26% TH+/DAT+ flow cytometry) and cholinergic-like neurons (ChLNs, ~46% ChAT+/VAChT flow cytometry) which responded to dopamine- and acetylcholine- triggered neuronal Ca 2+ inward stimuli when cultured with the NeuroForsk and the Cholinergic-N-Run medium , respectively in a timely fashion (i.e., 4-7 days). Here, we also report a direct transdifferentiation method to induce MenSCs into functional astrocyte-like cells (ALCs) by incubation of MenSCs in commercial Gibco® Astrocyte Medium in 7-days. The MSCs-derived ALCs (~59% GFAP+/S100b+) were found to respond to glutamate-induced Ca 2+ inward stimuli. Altogether these results show that MenSCs are a reliable source to obtain functional neurogenic cells to further investigate the neurobiology of neurologic disorders.

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