Vimentin-Induced Cardiac Mesenchymal Stem Cells Proliferate in the Acute Ischemic Myocardium

2018 ◽  
Vol 206 (1-2) ◽  
pp. 35-45 ◽  
Author(s):  
Christian Klopsch ◽  
Ralf Gaebel ◽  
Heiko Lemcke ◽  
Martin Beyer ◽  
Praveen Vasudevan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Fate ◽  
Cell Tracking ◽  
Fluorescent Protein ◽  
Ischemic Heart ◽  
Border Zone ◽  
Differentiation Potential

In-depth knowledge of the mechanisms induced by early postischemic cardiac endogenous mesenchymal stem cells (MSCs) in the acutely ischemic heart could advance our understanding of cardiac regeneration. Herein, we aimed to identify, isolate, and initially characterize the origin, kinetics and fate of cardiac MSCs. This was facilitated by in vivo genetic cell fate mapping through green fluorescent protein (GFP) expression under the control of vimentin induction after acute myocardial infarction (MI). Following permanent ligation of the left anterior descending coronary artery in CreER+ mTom/mGFP+ mice, vimentin/GFP+ cells revealed ischemia-responsive activation, survival, and local enrichment inside the peri-infarction border zone. Fluorescence-activated cell sorting (FACS)-isolated vimentin/GFP+ cells could be strongly expanded in vitro with clonogenic precursor formation and revealed MSC-typical cell morphology. Flow-cytometric analyses demonstrated an increase in cardiac vimentin/GFP+ cells in the ischemic heart, from a 0.6% cardiac mononuclear cell (MNC) fraction at 24 h to 1.6% at 72 h following MI. Sca-1+CD45– cells within the vimentin/GFP+ subtype of this MNC fraction increased from 35.2% at 24 h to 74.6% at 72 h after MI. The cardiac postischemic vimentin/GFP+ MNC subtype showed multipotent adipogenic, chondrogenic, and osteogenic differentiation potential, which is distinctive for MSCs. In conclusion, we demonstrated a seemingly proliferative first response of vimentin- induced cardiac endogenous MSCs in the acutely ischemic heart. Genetically, GFP-targeted in vivo cell tracking, isolation, and in vitro expansion of this cardiac MSC subtype could help to clarify their reparative status in inflammation, fibrogenesis, cell turnover, tissue homeostasis, and myocardial regeneration.

scholarly journals Effect of the 3D Artificial Nichoid on the Morphology and Mechanobiological Response of Mesenchymal Stem Cells Cultured In Vitro

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1873 ◽  
Author(s):  
Andrea Remuzzi ◽  
Barbara Bonandrini ◽  
Matteo Tironi ◽  
Lorena Longaretti ◽  
Marina Figliuzzi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Cultured Cells ◽  
Three Dimensions ◽  
Nuclear Pore ◽  
Cells Cultured

Stem cell fate and behavior are affected by the bidirectional communication of cells and their local microenvironment (the stem cell niche), which includes biochemical cues, as well as physical and mechanical factors. Stem cells are normally cultured in conventional two-dimensional monolayer, with a mechanical environment very different from the physiological one. Here, we compare culture of rat mesenchymal stem cells on flat culture supports and in the “Nichoid”, an innovative three-dimensional substrate micro-engineered to recapitulate the architecture of the physiological niche in vitro. Two versions of the culture substrates Nichoid (single-layered or “2D Nichoid” and multi-layered or “3D Nichoid”) were fabricated via two-photon laser polymerization in a biocompatible hybrid organic-inorganic photoresist (SZ2080). Mesenchymal stem cells, isolated from rat bone marrow, were seeded on flat substrates and on 2D and 3D Nichoid substrates and maintained in culture up to 2 weeks. During cell culture, we evaluated cell morphology, proliferation, cell motility and the expression of a panel of 89 mesenchymal stem cells’ specific genes, as well as intracellular structures organization. Our results show that mesenchymal stem cells adhered and grew in the 3D Nichoid with a comparable proliferation rate as compared to flat substrates. After seeding on flat substrates, cells displayed large and spread nucleus and cytoplasm, while cells cultured in the 3D Nichoid were spatially organized in three dimensions, with smaller and spherical nuclei. Gene expression analysis revealed the upregulation of genes related to stemness and to mesenchymal stem cells’ features in Nichoid-cultured cells, as compared to flat substrates. The observed changes in cytoskeletal organization of cells cultured on 3D Nichoids were also responsible for a different localization of the mechanotransducer transcription factor YAP, with an increase of the cytoplasmic retention in cells cultured in the 3D Nichoid. This difference could be explained by alterations in the import of transcription factors inside the nucleus due to the observed decrease of mean nuclear pore diameter, by transmission electron microscopy. Our data show that 3D distribution of cell volume has a profound effect on mesenchymal stem cells structure and on their mechanobiological response, and highlight the potential use of the 3D Nichoid substrate to strengthen the potential effects of MSC in vitro and in vivo.

Impact of Indium-111 Oxine Labelling on Viability of Human Mesenchymal Stem Cells In Vitro, and 3D Cell-Tracking Using SPECT/CT In Vivo

2010 ◽  
Vol 13 (6) ◽  
pp. 1204-1214 ◽  
Author(s):  
Franz Josef Gildehaus ◽  
Florian Haasters ◽  
Inga Drosse ◽  
Erika Wagner ◽  
Christian Zach ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Tracking ◽  
Human Mesenchymal Stem Cells ◽  
Indium 111

scholarly journals Treprostinil indirectly regulates endothelial colony forming cell angiogenic properties by increasing VEGF-A produced by mesenchymal stem cells

2015 ◽  
Vol 114 (10) ◽  
pp. 735-747 ◽  
Author(s):  
Marilyne Levy ◽  
Lan Huang ◽  
Elisa Rossi ◽  
Adeline Blandinières ◽  
Dominique Israel-Biet ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cord Blood ◽  
Proliferative Potential ◽  
Differentiation Potential ◽  
Endothelial Colony Forming Cells ◽  
Pulmonary Vasodilators ◽  
High Level

SummaryPulmonary vasodilators and prostacyclin therapy in particular, have markedly improved the outcome of patients with pulmonary hypertension (PH). Endothelial dysfunction is a key feature of PH, and we previously reported that treprostinil therapy increases number and proliferative potential of endothelial colony forming cells (ECFC) isolated from PH patients’ blood. In the present study, the objective was to determine how treprostinil contributes to the proangiogenic functions of ECFC. We examined the effect of treprostinil on ECFC obtained from cord blood in terms of colony numbers, proliferative and clonogenic properties in vitro, as well as in vivo vasculogenic properties. Surprisingly, treprostinil inhibited viability of cultured ECFC but did not modify their clonogenic properties or the endothelial differentiation potential from cord blood stem cells. Treprostinil treatment significantly increased the vessel-forming ability of ECFC combined with mesenchymal stem cells (MSC) in Matrigel implanted in nude mice. In vitro, ECFC proliferation was stimulated by conditioned media from treprostinil-pretreated MSC, and this effect was inhibited either by the use of VEGF-A blocking antibodies or siRNA VEGF-A in MSC. Silencing VEGF-A gene in MSC also blocked the pro-angiogenic effect of treprostinil in vivo. In conclusion, increased VEGF-A produced by MSC can account for the increased vessel formation observed during treprostinil treatment. The clinical relevance of these data was confirmed by the high level of VEGF-A detected in plasma from patients with paediatric PH who had been treated with treprostinil. Moreover, our results suggest that VEGF-A level in patients could be a surrogate biomarker of treprostinil efficacy.

scholarly journals Manufacturing and Banking Canine Adipose-Derived Mesenchymal Stem Cells for Veterinary Clinical Application

2021 ◽  
Author(s):  
Huina Luo ◽  
Dongsheng Li ◽  
Zhisheng Chen ◽  
Bingyun Wang ◽  
Shengfeng Chen
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Normal Karyotype ◽  
Clinical Applications ◽  
Therapeutic Application ◽  
Differentiation Potential ◽  
Safety And Efficacy

Abstract BACKGROUND: Mesenchymal stem cells (MSCs) have generated a great amount of interest in recent years as a novel therapeutic application for improving the quality of pet life and helping them free from painful conditions and diseases. It has now become critical to address the challenges related to the safety and efficacy of MSCs expanded in vitro. In this study, we establish a standardized process for manufacture of canine adipose-derived MSCs (AD-MSCs), including tissue sourcing, cell isolation and culture, cryopreservation, thawing and expansion, quality control and testing, and evaluate the safety and efficacy of those cells for clinical applications. RESULTS: After expansion, the viability of AD-MSCs manufactured under our standardized process was above 90 %. Expression of surface markers and differentiation potential was consistent with ISCT standards. Sterility, mycoplasma, and endotoxin tests were consistently negative. AD-MSCs presented normal karyotype, and did not form in vivo tumors. No adverse events were noted in two cases treated with intravenously AD-MSCs. CONCLUSION: Herein we demonstrated the establishment of a feasible bioprocess for manufacturing and banking canine AD-MSCs for veterinary clinical use.

scholarly journals Identification of WNT16 as a Predictable Biomarker for Accelerated Osteogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells In Vitro

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Yu-Hee Kim ◽  
Kyung-Ah Cho ◽  
Hyun-Ji Lee ◽  
Minhwa Park ◽  
Han Su Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Selection Criterion ◽  
Osteogenic Potential ◽  
Differentiation Potential ◽  
Real Time Quantitative Pcr ◽  
Single Cell Cloning

The application of mesenchymal stem cells (MSCs) for treating bone-related diseases shows promising outcomes in preclinical studies. However, cells that are isolated and defined as MSCs comprise a heterogeneous population of progenitors. This heterogeneity can produce variations in the performance of MSCs, especially in applications that require differentiation potential in vivo, such as the treatment of osteoporosis. Here, we aimed to identify genetic markers in tonsil-derived MSCs (T-MSCs) that can predict osteogenic potential. Using a single-cell cloning method, we isolated and established several lines of nondifferentiating (ND) or osteoblast-prone (OP) clones. Next, we performed transcriptome sequencing of three ND and three OP clones that maintained the characteristics of MSCs and determined the top six genes that were upregulated in OP clones. Upregulation of WNT16 and DCLK1 expression was confirmed by real-time quantitative PCR, but only WNT16 expression was correlated with the osteogenic differentiation of T-MSCs from 10 different donors. Collectively, our findings suggest that WNT16 is a putative genetic marker that predicts the osteogenic potential of T-MSCs. Thus, examination of WNT16 expression as a selection criterion prior to the clinical application of MSCs may enhance the therapeutic efficacy of stem cell therapy for bone-related complications, including osteoporosis.

scholarly journals The Hypoxic Preconditioning Effect On Senescence Cell Process In Cultured Adipose-Derived Mesenchymal Stem Cells (AMSCs)

2019 ◽  
Vol 39 (3) ◽  
Author(s):  
Nadiar Dwi Nuarisa ◽  
I Gde Rurus Suryawan ◽  
Andrianto Andrianto
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Life Span ◽  
Hypoxic Preconditioning ◽  
Differentiation Potential ◽  
Hypoxic Precondition ◽  
Significant Difference

Introduction : Stem cell therapy for myocardial regeneration is expected to increase cardiomyocyte proliferation and trigger neovascularization to improve cardiomyocytes. Mesenchymal Stem Cells (MSCs) are ideal candidates for regenerative medicine and immunotherapy. But low viability of MSCs is a major challenge in this alternative therapy. Therefore, a cytoprotective strategy is needed, one of them is hypoxic preconditioning which can significantly increase survival stem cells after being transplanted. MSCs are known to have a limited life span, after experiencing several splits MSC will enter the senescence process. It is known that hypoxia can also increase cell proliferation and differentiation potential in vitro and in vivo through the role of Octamer-4 (Oct-4) as a regulator of the pluripotency gene. Methods : Experimental laboratory studies (in vitro studies) using human-AMSCs which were given hypoxic preconditioning, observed as a immunocytochemistry. Results : The results showed that hypoxic precondition (1% O2) inhibited the senescence process. It can be seen in the lower expression of senescence in hypoxic conditions at P6, P7, P8, P9, P10 compared to normoxic ((p=0,004, p=0,001, p=0,009, p=0,013, p=0,024. There is a significant difference in the senescence expression of each passage in hypoxic and normoxic conditions with the highest expression at P10. In addition, we also observed AMSCs differentiation through the Oct-4 expression. It is showed that Oct-4 expression were higher in hypoxia compared to normoxia on P7, P8, P9, P10 (p=0,009, p=0,009, p=0,030, p=0,0001). Conclusions : Hypoxic preconditioning have the effect of inhibiting the senescence process on Adipose-derived MSCs (AMSCs) or prolonging their life span. The longer life span of AMSCs is also seen by higher cell differentiation potential from increased expression of Oct-4. However, the mechanism of inhibiting the senescence process in hypoxia in stem cells is still remain unknown. Keywords: human-Adipose derived Mesenchymal Stem Cell Cultures (h-AMSCs), Hypoxic Preconditioning, Senescence cell, Oct-4.

CD10/Neprilysin Enrichment in Infrapatellar Fat Pad–Derived Mesenchymal Stem Cells Under Regulatory-Compliant Conditions: Implications for Efficient Synovitis and Fat Pad Fibrosis Reversal

2020 ◽  
Vol 48 (8) ◽  
pp. 2013-2027 ◽  
Author(s):  
Dimitrios Kouroupis ◽  
Annie C. Bowles ◽  
Thomas M. Best ◽  
Lee D. Kaplan ◽  
Diego Correa
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Ex Vivo ◽  
Infrapatellar Fat Pad ◽  
Therapeutic Effects ◽  
Fat Pad ◽  
In Vitro Proliferation ◽  
Differentiation Potential

Background: Synovitis and infrapatellar fat pad (IFP) fibrosis participate in various conditions of the knee. Substance P (SP), a neurotransmitter secreted within those structures and historically associated with nociception, also modulates local neurogenic inflammatory and fibrotic responses. Exposure of IFP mesenchymal stem cells (IFP-MSCs) to a proinflammatory/profibrotic environment (ex vivo priming with TNFα, IFNγ, and CTGF) induces their expression of CD10/neprilysin, effectively degrading SP in vitro and in vivo. Purpose/Hypothesis: The purpose was to test the therapeutic effects of IFP-MSCs processed under regulatory-compliant protocols, comparing them side-by-side with standard fetal bovine serum (FBS)–grown cells. The hypothesis was that when processed under such protocols, IFP-MSCs do not require ex vivo priming to acquire a CD10-rich phenotype efficiently degrading SP and reversing synovitis and IFP fibrosis. Study Design: Controlled laboratory study. Methods: Human IFP-MSCs were processed in FBS or either of 2 alternative conditions—regulatory-compliant pooled human platelet lysate (hPL) and chemically reinforced medium (Ch-R)—and then subjected to proinflammatory/profibrotic priming with TNFα, IFNγ, and CTGF. Cells were assessed for in vitro proliferation, stemness, immunophenotype, differentiation potential, transcriptional and secretory profiles, and SP degradation. Based on a rat model of acute synovitis and IFP fibrosis, the in vivo efficacy of cells degrading SP plus reversing structural signs of inflammation and fibrosis was assessed. Results: When compared with FBS, IFP-MSCs processed with either hPL or Ch-R exhibited a CD10High phenotype and showed enhanced proliferation, differentiation, and immunomodulatory transcriptional and secretory profiles (amplified by priming). Both methods recapitulated and augmented the secretion of growth factors seen with FBS plus priming, with some differences between them. Functionally, in vitro SP degradation was more efficient in hPL and Ch-R, confirmed upon intra-articular injection in vivo where CD10-rich IFP-MSCs also dramatically reversed signs of synovitis and IFP fibrosis even without priming or at significantly lower cell doses. Conclusion: hPL and Ch-R formulations can effectively replace FBS plus priming to induce specific therapeutic attributes in IFP-MSCs. The resulting fine-tuned, regulatory-compliant, cell-based product has potential future utilization as a novel minimally invasive cell therapy for the treatment of synovitis and IFP fibrosis. Clinical Relevance: The therapeutic enhancement of IFP-MSCs manufactured under regulatory-compliant conditions suggests that such a strategy could accelerate the time from preclinical to clinical phases. The therapeutic efficacy obtained at lower MSC numbers than currently needed and the avoidance of cell priming for efficient results could have a significant effect on the design of clinical protocols to potentially treat conditions involving synovitis and IFP fibrosis.

scholarly journals Cell heterogeneity, rather than the cell storage solution, affects the behavior of mesenchymal stem cells in vitro and in vivo

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yong-Hong Wang ◽  
Ya-Chao Tao ◽  
Dong-Bo Wu ◽  
Meng-Lan Wang ◽  
Hong Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Survival Rates ◽  
Phosphate Buffer Solution ◽  
Hepatic Differentiation ◽  
Differentiation Potential ◽  
Normal Ranges ◽  
Storage Solutions

Abstract Background Mesenchymal stem cells (MSCs) have to be expanded in vitro to reach a sufficient cell dose for the treatment of various diseases. During the process of expansion, some obstacles remain to be overcome. The purpose of this study was to investigate the effects of storage solutions and heterogeneity on the behavior of MSCs in vitro and in vivo. Methods Umbilical cord MSCs (UC-MSCs) of similar sizes within normal ranges were suspended in three different storage solutions, phosphate buffer solution, normal saline, and Dulbecco’s modified Eagle medium. Then, the ultrastructure, viability, and safety of these cells were compared. Other two UC-MSC populations of different sizes were categorized based on their mean diameters. The ultrastructure, proliferation, immunosuppression, hepatic differentiation potential, and number of senescent cells were investigated and compared. The survival rates of mice after the infusion of UC-MSCs of different sizes were compared. Results For UC-MSCs suspended in different storage solutions, the cell apoptosis rates, ultrastructure, and survival rates of mice were similar, and no differences were observed. Cells with a diameter of 19.14 ± 4.89 μm were categorized as the larger UC-MSC population, and cells with a diameter of 15.58 ± 3.81 μm were categorized as the smaller population. The mean diameter of the larger UC-MSC population was significantly larger than that of the smaller UC-MSC population (p < 0.01). Smaller UC-MSCs had more powerful proliferation and immunosuppressive potential and a higher nucleus-cytoplasm ratio than those of large UC-MSCs. The number of cells positive for β-galactosidase staining was higher in the larger UC-MSC population than in the smaller UC-MSC population. The survival rates of mice receiving 1 × 106 or 2 × 106 smaller UC-MSCs were 100%, both of which were higher than those of mice receiving the same amounts of larger UC-MSCs (p < 0.01). The cause of mouse death was explored and it was found that some larger UC-MSCs accumulated in the pulmonary capillary in dead mice. Conclusion Different storage solutions showed no significant effects on cell behavior, whereas heterogeneity was quite prevalent in MSC populations and might limit cells application. Hence, it is necessary to establish a more precise standardization for culture-expanded MSCs.

scholarly journals GFP Labeling and Hepatic Differentiation Potential of Human Placenta-Derived Mesenchymal Stem Cells

2015 ◽  
Vol 35 (6) ◽  
pp. 2299-2308 ◽  
Author(s):  
Jiong Yu ◽  
Xiaoru Su ◽  
Chengxing Zhu ◽  
Qiaoling Pan ◽  
Jinfeng Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Human Placenta ◽  
Fluorescent Protein ◽  
Gene Transduction ◽  
Hepatic Differentiation ◽  
Differentiation Potential ◽  
Gfp Gene ◽  
Cell Based Therapy

Background: Stem cell-based therapy in liver diseases has received increasing interest over the past decade, but direct evidence of the homing and implantation of transplanted cells is conflicting. Reliable labeling and tracking techniques are essential but lacking. The purpose of this study was to establish human placenta-derived mesenchymal stem cells (hPMSCs) expressing green fluorescent protein (GFP) and to assay their hepatic functional differentiation in vitro. Methods: The GFP gene was transduced into hPMSCs using a lentivirus to establish GFP+ hPMSCs. GFP+ hPMSCs were analyzed for their phenotypic profile, viability and adipogenic, osteogenic and hepatic differentiation. The derived GFP+ hepatocyte-like cells were evaluated for their metabolic, synthetic and secretory functions, respectively. Results: GFP+ hPMSCs expressed high levels of HLA I, CD13, CD105, CD73, CD90, CD44 and CD29, but were negative for HLA II, CD45, CD31, CD34, CD133, CD271 and CD79. They possessed adipogenic, osteogenic and hepatic differentiation potential. Hepatocyte-like cells derived from GFP+ hPMSCs showed typical hepatic phenotypes. Conclusions: GFP gene transduction has no adverse influences on the cellular or biochemical properties of hPMSCs or markers. GFP gene transduction using lentiviral vectors is a reliable labeling and tracking method. GFP+ hPMSCs can therefore serve as a tool to investigate the mechanisms of MSC-based therapy, including hepatic disease therapy.

Sign in / Sign up

Export Citation Format

Share Document