scholarly journals Impact of isolation method on cellular activation and presence of specific tendon cell subpopulations during in vitro culture

2021 ◽  
Author(s):  
Anne E.C. Nichols ◽  
Sarah E. Miller ◽  
Luke J. Green ◽  
Michael S. Richards ◽  
Alayna E. Loiselle
Keyword(s):  
Cell Biology ◽  
Tendon Healing ◽  
Marker Genes ◽  
Cell Models ◽  
Isolation Method ◽  
Tendon Cell ◽  
Tendon Cells ◽  
Tissue Systems

AbstractTendon injuries are common and heal poorly, due in part to a lack of understanding of fundamental tendon cell biology. A major impediment to the study of tendon cells is the absence of robust, well-characterized in vitro models. Unlike other tissue systems, current tendon cell models do not account for how differences in isolation methodology may affect the activation state of tendon cells or the presence of various tendon cell sub-populations. The objective of this study was to characterize how common isolation methods affect the behavior, fate, and lineage composition of tendon cell cultures. Tendon cells isolated by explant exhibited reduced proliferative capacity, decreased expression of tendon marker genes, and increased expression of genes associated with fibroblast activation compared to digested cells. Consistently, explanted cells also displayed an increased propensity to differentiate to myofibroblasts compared to digested cells. Explanted cultures from multiple different tendons were substantially enriched for the presence of scleraxis-lineage (Scx-lin+) cells compared to digested cultures, while the overall percentage of S100a4-lineage (S100a4-lin+) cells was dependent on both isolation method and tendon of origin. Neither isolation method preserved the ratios of Scx-lin+ or S100a4-lin+ to non-lineage cells seen in tendons in vivo. Combined, these data indicate that further refinement of in vitro cultures models is required in order to more accurately understand the effects of various stimuli on tendon cell behavior.Statement of clinical significanceThe development of informed in vitro tendon cell models will facilitate enhanced screening of potential therapeutic candidates to improve tendon healing.

Dysregulated Bone Wound Repair and Marrow Functions in Senescence Accelerated Mice (SAMP6),

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3415-3415
Author(s):  
Michael W. Epperly ◽  
Regina P. O'Sullivan ◽  
Shaonan Cao ◽  
Tracy M. Dixon ◽  
Julie P. Goff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Biology ◽  
Wound Repair ◽  
Stromal Cell ◽  
Constitutive Expression ◽  
Marker Genes ◽  
Unexpected Finding ◽  
Semisolid Medium

Abstract Abstract 3415 Introduction: The Senescence Accelerated-Prone mouse (SAMP6) shows normal growth followed by rapid aging, development of osteopenia and shortened lifespan, compared with control R1 mice. The bone defect has been attributed to reduced osteoblast potential of marrow stromal cells. We compared in vivo repair of tibial bone wounds. We compared in vitro hematopoiesis, irradiation sensitivity, stromal cell biology, and osteoblastogenesis with SamP6 and R1 marrow. We tested the hypothesis that SAMP6 mice have poor healing of osseous wounds and that in vitro properties of SAMP6 marrow are abnormal. Methods: As a model for spontaneous bone repair, a 2-mm unicortical wound was made in each proximal tibia. At intervals, wound diameter was measured on radiographs. Long term bone marrow cultures (LTBMCs) were established from other SAMP6 and R1 mice to measure hematopoiesis and to establish marrow stromal cell lines (MSCLs) for analysis. MSCL gene expression and sensitivity to irradiation were measured; osteoblastogenic potential was determined by culture in osteoblastogenic medium (1% FBS, 10 nM dexamathasone, 5 mM b-glycerophosphate, 50 mg/mL ascorbate-2-phosphate) and gene expression analysis for osteoblast markers alkaline phosphatase (ALP), PTH-receptor-1 (PTHR1), and Osteocalcin (OC). Results: There was a faster rate of closure of the tibial wounds in R1 than in SAMP6 mice; at day 21, wounds in R1 mice were 47% the size of those in SAMP6 mice (p = 0.032). For the first 24 weeks, SAMP6 LTBMCs had significantly greater hematopoiesis than R1, shown by more cobblestone islands (sites of stem cell activity) and by more multilineage colonies from nonadherent cells transferred to semisolid medium. After 24 weeks, however, hematopoiesis ceased in SAMP6 cultures but continued in the SAMR1 cultures for 40 weeks. There was constitutive upregulation of TGF-b, an inhibitor of hematopoiesis, in SAMP6 MSCLs, compared with R1 cells. SAMP6 MSCLs were more sensitive to radiation (Do of 1.5 ± 0.1 Gy), compared with R1 (Do of 4.0 ± 0.4 Gy; p = 0.0080). There was unexpected constitutive expression of ALP, PTHR1, and OC in SAMP6 MSCLs, compared with R1. In osteoblastogenic medium, there was greater expression of osteoblast marker genes in the SAMP6 line compared to R1. Conclusion: SAMP6 showed delayed bone wound repair. Marrow from SAMP6 mice showed shortened in vitro hematopoiesis and greater radiation sensitivity, indicative of greater oxidative stress, but the unexpected finding of constitutive expression of osteoblast genes suggest that in vivo factors are more important than innate cellular defects in marrow to account for impaired bone healing. Acknowledgments: This project was supported by NIAID U191A168021-06 and NIA R21AG034254. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Dedifferentiated follicular granulosa cells derived from pig ovary can transdifferentiate into osteoblasts

2012 ◽  
Vol 447 (2) ◽  
pp. 239-248 ◽  
Author(s):  
Yoshinao Oki ◽  
Hiromasa Ono ◽  
Takeharu Motohashi ◽  
Nobuki Sugiura ◽  
Hiroyuki Nobusue ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Cell Biology ◽  
Cell Lineage ◽  
Marker Genes ◽  
Specific Cell ◽  
Specific Marker ◽  
Matrix Mineralization ◽  
Differentiated Cells

Transdifferentiation is the conversion of cells from one differentiated cell type into another. How functionally differentiated cells already committed to a specific cell lineage can transdifferentiate into other cell types is a key question in cell biology and regenerative medicine. In the present study we show that porcine ovarian follicular GCs (granulosa cells) can transdifferentiate into osteoblasts in vitro and in vivo. Pure GCs isolated and cultured in Dulbecco's modified Eagle's medium supplemented with 20% FBS (fetal bovine serum) proliferated and dedifferentiated into fibroblast-like cells. We referred to these cells as DFOG (dedifferentiated follicular granulosa) cells. Microarray analysis showed that DFOG cells lost expression of GC-specific marker genes, but gained the expression of osteogenic marker genes during dedifferentiation. After osteogenic induction, DFOG cells underwent terminal osteoblast differentiation and matrix mineralization in vitro. Furthermore, when DFOG cells were transplanted subcutaneously into SCID mice, these cells formed ectopic osteoid tissue. These results indicate that DFOG cells derived from GCs can differentiate into osteoblasts in vitro and in vivo. We suggest that GCs provide a useful model for studying the mechanisms of transdifferentiation into other cell lineages in functionally differentiated cells.

scholarly journals MBRS-48. IDENTIFICATION OF NOVEL THERAPEUTIC APPROACHES FOR MYC-DRIVEN MEDULLOBLASTOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii406-iii406
Author(s):  
Kübra Taban ◽  
David Pauck ◽  
Mara Maue ◽  
Viktoria Marquardt ◽  
Hua Yu ◽  
...  
Keyword(s):  
New Drugs ◽  
Current Therapy ◽  
Cancer Drug ◽  
Mrna Levels ◽  
Cell Models ◽  
Therapeutic Approaches ◽  
Group 3 ◽  
Novel Therapeutic

Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children and is frequently metastatic at diagnosis. Treatment with surgery, radiation and multi-agent chemotherapy may leave survivors of these brain tumors with long-term deficits as a consequence. One of the four consensus molecular subgroups of MB is the MYC-driven group 3 MB, which is the most malignant type and has a poor prognosis under current therapy. Thus, it is important to discover more effective targeted therapeutic approaches. We conducted a high-throughput drug screening to identify novel compounds showing efficiency in group 3 MB using both clinically established inhibitors (n=196) and clinically-applicable compounds (n=464). More than 20 compounds demonstrated a significantly higher anti-tumoral effect in MYChigh (n=7) compared to MYClow (n=4) MB cell models. Among these compounds, Navitoclax and Clofarabine showed the strongest effect in inducing cell cycle arrest and apoptosis in MYChigh MB models. Furthermore, we show that Navitoclax, an orally bioavailable and blood-brain barrier passing anti-cancer drug, inhibits specifically Bcl-xL proteins. In line, we found a significant correlation between BCL-xL and MYC mRNA levels in 763 primary MB patient samples (Data source: “R2 https://hgserver1.amc.nl”). In addition, Navitoclax and Clofarabine have been tested in cells obtained from MB patient-derived-xenografts, which confirmed their specific efficacy in MYChigh versus MYClow MB. In summary, our approach has identified promising new drugs that significantly reduce cell viability in MYChigh compared to MYClow MB cell models. Our findings point to novel therapeutic vulnerabilities for MB that need to be further validated in vitro and in vivo.

scholarly journals Expression Profile of New Marker Genes Involved in Differentiation of Canine Adipose-Derived Stem Cells into Osteoblasts

2021 ◽  
Vol 22 (13) ◽  
pp. 6663
Author(s):  
Maurycy Jankowski ◽  
Mariusz Kaczmarek ◽  
Grzegorz Wąsiatycz ◽  
Claudia Dompe ◽  
Paul Mozdziak ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Osteogenic Differentiation ◽  
Molecular Mechanisms ◽  
Marker Genes ◽  
P Value ◽  
Illumina Platform

Next-generation sequencing (RNAseq) analysis of gene expression changes during the long-term in vitro culture and osteogenic differentiation of ASCs remains to be important, as the analysis provides important clues toward employing stem cells as a therapeutic intervention. In this study, the cells were isolated from adipose tissue obtained during routine surgical procedures and subjected to 14-day in vitro culture and differentiation. The mRNA transcript levels were evaluated using the Illumina platform, resulting in the detection of 19,856 gene transcripts. The most differentially expressed genes (fold change >|2|, adjusted p value < 0.05), between day 1, day 14 and differentiated cell cultures were extracted and subjected to bioinformatical analysis based on the R programming language. The results of this study provide molecular insight into the processes that occur during long-term in vitro culture and osteogenic differentiation of ASCs, allowing the re-evaluation of the roles of some genes in MSC progression towards a range of lineages. The results improve the knowledge of the molecular mechanisms associated with long-term in vitro culture and differentiation of ASCs, as well as providing a point of reference for potential in vivo and clinical studies regarding these cells’ application in regenerative medicine.

scholarly journals Fabrication of 3D-Printed Interpenetrating Hydrogel Scaffolds for Promoting Chondrogenic Differentiation

Polymers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 2146
Author(s):  
Jian Guan ◽  
Fu-zhen Yuan ◽  
Zi-mu Mao ◽  
Hai-lin Zhu ◽  
Lin Lin ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Elastic Modulus ◽  
Chondrogenic Differentiation ◽  
Marker Genes ◽  
Self Healing ◽  
Polyethylene Glycol Diacrylate ◽  
3D Microenvironment ◽  
3D Printed

The limited self-healing ability of cartilage necessitates the application of alternative tissue engineering strategies for repairing the damaged tissue and restoring its normal function. Compared to conventional tissue engineering strategies, three-dimensional (3D) printing offers a greater potential for developing tissue-engineered scaffolds. Herein, we prepared a novel photocrosslinked printable cartilage ink comprising of polyethylene glycol diacrylate (PEGDA), gelatin methacryloyl (GelMA), and chondroitin sulfate methacrylate (CSMA). The PEGDA-GelMA-CSMA scaffolds possessed favorable compressive elastic modulus and degradation rate. In vitro experiments showed good adhesion, proliferation, and F-actin and chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) on the scaffolds. When the CSMA concentration was increased, the compressive elastic modulus, GAG production, and expression of F-actin and cartilage-specific genes (COL2, ACAN, SOX9, PRG4) were significantly improved while the osteogenic marker genes of COL1 and ALP were decreased. The findings of the study indicate that the 3D-printed PEGDA-GelMA-CSMA scaffolds possessed not only adequate mechanical strength but also maintained a suitable 3D microenvironment for differentiation, proliferation, and extracellular matrix production of BMSCs, which suggested this customizable 3D-printed PEGDA-GelMA-CSMA scaffold may have great potential for cartilage repair and regeneration in vivo.

scholarly journals O23: CHARACTERISING PATIENT-DERIVED COLORECTAL CANCER TISSUE-ORIGINATED ORGANOIDAL SPHEROIDS FOR HIGH-THROUGHPUT MICROFLUIDIC APPLICATIONS

2021 ◽  
Vol 108 (Supplement_1) ◽  
Author(s):  
MI Khot ◽  
M Levenstein ◽  
R Coppo ◽  
J Kondo ◽  
M Inoue ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Fluid Flow ◽  
High Throughput ◽  
Microfluidic Devices ◽  
In Vitro Models ◽  
Fluorescent Imaging ◽  
Cancer Tissue ◽  
Cell Models

Abstract Introduction Three-dimensional (3D) cell models have gained reputation as better representations of in vivo cancers as compared to monolayered cultures. Recently, patient tumour tissue-derived organoids have advanced the scope of complex in vitro models, by allowing patient-specific tumour cultures to be generated for developing new medicines and patient-tailored treatments. Integrating 3D cell and organoid culturing into microfluidics, can streamline traditional protocols and allow complex and precise high-throughput experiments to be performed with ease. Method Patient-derived colorectal cancer tissue-originated organoidal spheroids (CTOS) cultures were acquired from Kyoto University, Japan. CTOS were cultured in Matrigel and stem-cell media. CTOS were treated with 5-fluorouracil and cytotoxicity evaluated via fluorescent imaging and ATP assay. CTOS were embedded, sectioned and subjected to H&E staining and immunofluorescence for ABCG2 and Ki67 proteins. HT29 colorectal cancer spheroids were produced on microfluidic devices using cell suspensions and subjected to 5-fluorouracil treatment via fluid flow. Cytotoxicity was evaluated through fluorescent imaging and LDH assay. Result 5-fluorouracil dose-dependent reduction in cell viability was observed in CTOS cultures (p&lt;0.01). Colorectal CTOS cultures retained the histology, tissue architecture and protein expression of the colonic epithelial structure. Uniform 3D HT29 spheroids were generated in the microfluidic devices. 5-fluorouracil treatment of spheroids and cytotoxic analysis was achieved conveniently through fluid flow. Conclusion Patient-derived CTOS are better complex models of in vivo cancers than 3D cell models and can improve the clinical translation of novel treatments. Microfluidics can streamline high-throughput screening and reduce the practical difficulties of conventional organoid and 3D cell culturing. Take-home message Organoids are the most advanced in vitro models of clinical cancers. Microfluidics can streamline and improve traditional laboratory experiments.

scholarly journals Targeting a Lipid Desaturation Enzyme, SCD1, Selectively Eliminates Colon Cancer Stem Cells through the Suppression of Wnt and NOTCH Signaling

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 106
Author(s):  
Yeongji Yu ◽  
Hyejin Kim ◽  
SeokGyeong Choi ◽  
JinSuh Yu ◽  
Joo Yeon Lee ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Notch Signaling ◽  
Cultured Cells ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Dependent Manner ◽  
Lipid Desaturation ◽  
Novel Target

The elimination of the cancer stem cell (CSC) population may be required to achieve better outcomes of cancer therapy. We evaluated stearoyl-CoA desaturase 1 (SCD1) as a novel target for CSC-selective elimination in colon cancer. CSCs expressed more SCD1 than bulk cultured cells (BCCs), and blocking SCD1 expression or function revealed an essential role for SCD1 in the survival of CSCs, but not BCCs. The CSC potential selectively decreased after treatment with the SCD1 inhibitor in vitro and in vivo. The CSC-selective suppression was mediated through the induction of apoptosis. The mechanism leading to selective CSC death was investigated by performing a quantitative RT-PCR analysis of 14 CSC-specific signaling and marker genes after 24 and 48 h of treatment with two concentrations of an inhibitor. The decrease in the expression of Notch1 and AXIN2 preceded changes in the expression of all other genes, at 24 h of treatment in a dose-dependent manner, followed by the downregulation of most Wnt- and NOTCH-signaling genes. Collectively, we showed that not only Wnt but also NOTCH signaling is a primary target of suppression by SCD1 inhibition in CSCs, suggesting the possibility of targeting SCD1 against colon cancer in clinical settings.

Cellular and immunological basis of the host-parasite relationship during infection withNeospora caninum

Parasitology ◽  
2006 ◽  
Vol 133 (3) ◽  
pp. 261-278 ◽  
Author(s):  
A. HEMPHILL ◽  
N. VONLAUFEN ◽  
A. NAGULESWARAN
Keyword(s):  
Host Cell ◽  
Cell Biology ◽  
Neospora Caninum ◽  
Host Cells ◽  
Term Survival ◽  
Prevention Of Infection ◽  
Parasite Relationship ◽  
Potential Applications

Neospora caninumis an apicomplexan parasite that is closely related toToxoplasma gondii, the causative agent of toxoplasmosis in humans and domestic animals. However, in contrast toT. gondii, N. caninumrepresents a major cause of abortion in cattle, pointing towards distinct differences in the biology of these two species. There are 3 distinct key features that represent potential targets for prevention of infection or intervention against disease caused byN. caninum. Firstly, tachyzoites are capable of infecting a large variety of host cellsin vitroandin vivo. Secondly, the parasite exploits its ability to respond to alterations in living conditions by converting into another stage (tachyzoite-to-bradyzoite orvice versa). Thirdly, by analogy withT. gondii, this parasite has evolved mechanisms that modulate its host cells according to its own requirements, and these must, especially in the case of the bradyzoite stage, involve mechanisms that ensure long-term survival of not only the parasite but also of the host cell. In order to elucidate the molecular and cellular bases of these important features ofN. caninum, cell culture-based approaches and laboratory animal models are being exploited. In this review, we will summarize the current achievements related to host cell and parasite cell biology, and will discuss potential applications for prevention of infection and/or disease by reviewing corresponding work performed in murine laboratory infection models and in cattle.

scholarly journals Histone/protein deacetylases and T-cell immune responses

Blood ◽  
2012 ◽  
Vol 119 (11) ◽  
pp. 2443-2451 ◽  
Author(s):  
Tatiana Akimova ◽  
Ulf H. Beier ◽  
Yujie Liu ◽  
Liqing Wang ◽  
Wayne W. Hancock
Keyword(s):  
T Cell ◽  
Cell Biology ◽  
T Regulatory Cells ◽  
Hdac Inhibitors ◽  
Experimental Studies ◽  
Cell Subset ◽  
Histone Protein ◽  
Anti Inflammatory

Abstract Clinical and experimental studies show that inhibition of histone/protein deacetylases (HDAC) can have important anti-neoplastic effects through cytotoxic and proapoptotic mechanisms. There are also increasing data from nononcologic settings that HDAC inhibitors (HDACi) can exhibit useful anti-inflammatory effects in vitro and in vivo, unrelated to cytotoxicity or apoptosis. These effects can be cell-, tissue-, or context-dependent and can involve modulation of specific inflammatory signaling pathways as well as epigenetic mechanisms. We review recent advances in the understanding of how HDACi alter immune and inflammatory processes, with a particular focus on the effects of HDACi on T-cell biology, including the activation and functions of conventional T cells and the unique T-cell subset, composed of Foxp3+ T-regulatory cells. Although studies are still needed to tease out details of the various biologic roles of individual HDAC isoforms and their corresponding selective inhibitors, the anti-inflammatory effects of HDACi are already promising and may lead to new therapeutic avenues in transplantation and autoimmune diseases.

scholarly journals A cellular endolysosome-modulating pore-forming protein from a toad is negatively regulated by its paralog under oxidizing conditions

2020 ◽  
Vol 295 (30) ◽  
pp. 10293-10306 ◽  
Author(s):  
Qiquan Wang ◽  
Xianling Bian ◽  
Lin Zeng ◽  
Fei Pan ◽  
Lingzhen Liu ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Cell Biology ◽  
Biochemical Properties ◽  
Bacterial Toxin ◽  
Cell Physiology ◽  
Dependent Manner ◽  
Membrane Pore ◽  
Intracellular Vesicles

Endolysosomes are key players in cell physiology, including molecular exchange, immunity, and environmental adaptation. They are the molecular targets of some pore-forming aerolysin-like proteins (ALPs) that are widely distributed in animals and plants and are functionally related to bacterial toxin aerolysins. βγ-CAT is a complex of an ALP (BmALP1) and a trefoil factor (BmTFF3) in the firebelly toad (Bombina maxima). It is the first example of a secreted endogenous pore-forming protein that modulates the biochemical properties of endolysosomes by inducing pore formation in these intracellular vesicles. Here, using a large array of biochemical and cell biology methods, we report the identification of BmALP3, a paralog of BmALP1 that lacks membrane pore-forming capacity. We noted that both BmALP3 and BmALP1 contain a conserved cysteine in their C-terminal regions. BmALP3 was readily oxidized to a disulfide bond-linked homodimer, and this homodimer then oxidized BmALP1 via disulfide bond exchange, resulting in the dissociation of βγ-CAT subunits and the elimination of biological activity. Consistent with its behavior in vitro, BmALP3 sensed environmental oxygen tension in vivo, leading to modulation of βγ-CAT activity. Interestingly, we found that this C-terminal cysteine site is well conserved in numerous vertebrate ALPs. These findings uncover the existence of a regulatory ALP (BmALP3) that modulates the activity of an active ALP (BmALP1) in a redox-dependent manner, a property that differs from those of bacterial toxin aerolysins.

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