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Journal of Tissue Engineering and... Mar 2018Osteoarthritis (OA) is a painful disease, characterized by progressive surface erosion of articular cartilage. The use of human articular chondrocytes (hACs) sourced...
Osteoarthritis (OA) is a painful disease, characterized by progressive surface erosion of articular cartilage. The use of human articular chondrocytes (hACs) sourced from OA patients has been proposed as a potential therapy for cartilage repair, but this approach is limited by the lack of scalable methods to produce clinically relevant quantities of cartilage-generating cells. Previous studies in static culture have shown that hACs co-cultured with human mesenchymal stem cells (hMSCs) as 3D pellets can upregulate proliferation and generate neocartilage with enhanced functional matrix formation relative to that produced from either cell type alone. However, because static culture flasks are not readily amenable to scale up, scalable suspension bioreactors were investigated to determine if they could support the co-culture of hMSCs and OA hACs under serum-free conditions to facilitate clinical translation of this approach. When hACs and hMSCs (1:3 ratio) were inoculated at 20,000 cells/ml into 125-ml suspension bioreactors and fed weekly, they spontaneously formed 3D aggregates and proliferated, resulting in a 4.75-fold increase over 16 days. Whereas the apparent growth rate was lower than that achieved during co-culture as a 2D monolayer in static culture flasks, bioreactor co-culture as 3D aggregates resulted in a significantly lower collagen I to II mRNA expression ratio and more than double the glycosaminoglycan/DNA content (5.8 vs. 2.5 μg/μg). The proliferation of hMSCs and hACs as 3D aggregates in serum-free suspension culture demonstrates that scalable bioreactors represent an accessible platform capable of supporting the generation of clinical quantities of cells for use in cell-based cartilage repair.
Topics: Bioreactors; Cell Aggregation; Cell Culture Techniques; Cell Proliferation; Cells, Cultured; Chondrocytes; Coculture Techniques; Culture Media; Glycosaminoglycans; Humans; Kinetics; Mesenchymal Stem Cells; Osteoarthritis; Serum; Suspensions
PubMed: 28752579
DOI: 10.1002/term.2531 -
Osteoarthritis and Cartilage Oct 2021Growth of mandibular condylar cartilage (MCC) is associated with cell proliferation within the polymorphic cell layer and subsequent differentiation into chondrocytes...
OBJECTIVE
Growth of mandibular condylar cartilage (MCC) is associated with cell proliferation within the polymorphic cell layer and subsequent differentiation into chondrocytes that reside along the condylar surface and along the cartilage/subchondral bone interface. We examined whether cells in the polymorphic layer would proliferate and repopulate toxin-induced cell-depleted areas in MCCs of adult mice.
METHOD
We induced diphtheria toxin (DTA) expression (ROSA26) to cell-autonomously kill large fractions of MCC chondrocytes throughout the cartilage or along the articular cartilage surface with Aggrecan-CreERt2 (Acan) or Lubricin-CreERt2 (Prg4) Cre-recombinase-inducible mice, respectively. We examined MCCs from these mice shortly after cell killing or several months later with histology and confocal microscopy for evidence of chondrocyte proliferation and repopulation.
RESULTS
Acan-induced DTA expression killed an average of 53% MCC chondrocytes in adult mice after 1 week (39-66%, 95% confidence interval (CI)). Twelve weeks later, surviving chondrocytes had proliferated but not migrated to cell depleted areas. Prg4-induced DTA expression killed an average of 24% surface chondrocytes in mice after 5 weeks (14-34% CI). After thirteen weeks there was 34% fewer surface chondrocytes (4-63% CI) in Prg4 DTA-induced mice compared to controls.
CONCLUSION
In adult mice, after diphtheria toxin-mediated chondrocyte killing, cell depleted areas within MCC cartilage are not repopulated by new cells.
Topics: Animals; Apoptosis; Cartilage, Articular; Cell Proliferation; Chondrocytes; Diphtheria Toxin; Mandibular Condyle; Mice, Inbred C57BL; Microscopy, Confocal; Mice
PubMed: 34166809
DOI: 10.1016/j.joca.2021.06.003 -
Cell May 1978The steroid hormone estradiol, and the glycoprotein hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are known to be essential for the growth...
The steroid hormone estradiol, and the glycoprotein hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are known to be essential for the growth and differentiation of follicles in the ovary. The present study was conducted to determine quantitatively the effects of estradiol, FSH and LH on proliferation of different ovarian cell types (granulosa and theca cells). The immature female hypophysectomized rate sequentially primed with estradiol, FSH and LH was used as the experimental model. Proliferation was assessed by examining changes in total DNA, incorporation of 3H-thymidine into DNA and labeling index in specific cell types. Estradiol and FSH each acted on follicles at different stages of development to stimulate proliferative activity of both granulosa and theca cells. Continued administration of either hormone caused a decrease in the proliferative activity of both cell types. These observations have been interpreted to indicate that estradiol and FSH can each alter the length of the specific phases of the cell cycle. A luteinizing dose of LH caused a cessation of proliferation in luteinizing granulosa cells while stimulating a limited proliferation of theca cells. Absence of the appropriate hormonal stimulus caused both granulosa and theca cells to stop proliferating and the follicles to undergo atresia. These results indicate that, depending upon the state of differentiation of granulosa and theca cells, estradiol, FSH and LH can stimulate or inhibit the ability of these cells to proliferate.
Topics: Cell Division; DNA; Estradiol; Female; Follicle Stimulating Hormone; Granulosa Cells; Luteinizing Hormone; Theca Cells
PubMed: 566619
DOI: 10.1016/0092-8674(78)90302-1 -
Immunology Nov 2021Ligation of the inhibitory receptor PD-1 on T cells results in the inhibition of numerous cellular functions. Despite the overtly inhibitory outcome of PD-1 signalling,...
Ligation of the inhibitory receptor PD-1 on T cells results in the inhibition of numerous cellular functions. Despite the overtly inhibitory outcome of PD-1 signalling, there are additionally a collection of functions that are activated. We have observed that CD4 T cells stimulated through the T-cell receptor and PD-1 primarily do not proliferate; however, there is a population of cells that proliferates more than T-cell receptor stimulation alone. These highly proliferating cells could potentially be associated with PD-1-blockade unresponsiveness in patients. In this study, we have performed RNA sequencing and found that following PD-1 ligation proliferating and non-proliferating T cells have distinct transcriptional signatures. Remarkably, the proliferating cells showed an enrichment of genes associated with an activated state despite PD-1 signalling. Additionally, circulating follicular helper T cells were significantly more prevalent in the non-proliferating population, demonstrated by enrichment of the associated genes CXCR5, CCR7, TCF7, BCL6 and PRDM1 and validated at the protein level. Translationally, we also show that there are more follicular helper T cells in patients that respond favourably to PD-1 blockade. Overall, the presence of transcriptionally and functionally distinct T cell populations responsive to PD-1 ligation may provide insights into the clinical differences observed following therapeutic PD-1 blockade.
Topics: CD4-Positive T-Lymphocytes; Cell Proliferation; Cells, Cultured; Datasets as Topic; Humans; Immunophenotyping; Lymphocyte Activation; Primary Cell Culture; Programmed Cell Death 1 Receptor; RNA-Seq; Receptors, Antigen, T-Cell; Single-Cell Analysis; T-Lymphocyte Subsets; Transcriptome
PubMed: 34164813
DOI: 10.1111/imm.13388 -
Immunology May 2002Listeria monocytogenes infection of mice leads to a rapid expansion of activated T cells, followed by a decline in specific cells once the bacteria are eliminated. In...
Listeria monocytogenes infection of mice leads to a rapid expansion of activated T cells, followed by a decline in specific cells once the bacteria are eliminated. In order to define the relationship between T-cell proliferation and activation, and to investigate the role of apoptosis in limiting the expansion, the expression of activation markers, uptake of 5-bromo-2'-deoxyuridine (BrdU) in vivo and the incidence of apoptosis was investigated. Increased numbers of T cells expressing the activated phenotype CD25+, CD44hi and CD62Llo were detected 4 days after infection. Expression of CD25 (IL-2Ralpha chain) on CD4+ and CD8+ T cells peaked at this time and returned to normal by day 7. In contrast, CD44hi and CD62Llo persisted, with the maximum proportion occurring at 7 days after infection. This was accompanied by a burst of in vivo proliferation of CD4+ and CD8+ T cells occurring between day 5 and 7. Apoptosis, which is presumably needed to control this expansion of T cells, also peaked at 7 days after infection. Apoptosis occurred preferentially amongst T cells which had proliferated. Most but not all proliferating T cells had down-regulated their CD62L marker. While most apoptotic T cells were CD62Llo, again not all had down-regulated this marker. Hence, CD25 expression peaked early, but expression of other activation markers, in vivo proliferation and apoptosis coincided after Listeria infection. T cells that had proliferated were over-represented in the apoptotic population.
Topics: Animals; Apoptosis; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Division; Cells, Cultured; Hyaluronan Receptors; L-Selectin; Listeriosis; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Receptors, Interleukin-2; T-Lymphocyte Subsets
PubMed: 11972636
DOI: 10.1046/j.1365-2567.2002.01408.x -
Molecular Biotechnology Nov 2023NKD inhibitor of WNT signaling pathway 2 (NKD2) is an emerging player in cancer onset and progression. Here, it was confirmed that THCA patients have robustly expressed...
NKD inhibitor of WNT signaling pathway 2 (NKD2) is an emerging player in cancer onset and progression. Here, it was confirmed that THCA patients have robustly expressed NKD2, which was linked to an advanced pathologic stage. The prognosis was worse for those with high NKD2 levels. Functionally, ectopically produced NKD2 promotes THCA cell proliferation, whereas NKD2 knockdown impairs the ability of THCA cells to proliferate. Mechanically, ectopically expressed NKD2 activated NF-κB transcriptional activity, whereas NKD2-deficient THCA cells showed lower NF-κB transcriptional activity. As a result, NKD2 activates the NF-κB signaling pathway, encouraging the growth of THCA cells.
Topics: Humans; Calcium-Binding Proteins; Adaptor Proteins, Signal Transducing; NF-kappa B; Wnt Signaling Pathway; Cell Proliferation; Thyroid Neoplasms
PubMed: 36820951
DOI: 10.1007/s12033-023-00665-7 -
Characterization of Proliferating Lesion-Resident Cells During All Stages of Atherosclerotic Growth.Journal of the American Heart... Aug 2016Monocyte recruitment leads to accumulation of macrophage foam cells and contributes to atherosclerotic lesion growth. Recent studies have reported that lesion-resident...
BACKGROUND
Monocyte recruitment leads to accumulation of macrophage foam cells and contributes to atherosclerotic lesion growth. Recent studies have reported that lesion-resident macrophages can proliferate and represent a major cellular component during lesion development. This study was designed to assess whether the rate of macrophage proliferation changes during well-established stages of lesion growth and to characterize other populations of proliferating cells within these lesions.
METHODS AND RESULTS
Using murine models of atherosclerosis (Apoe(-/-) and LDLr(-/-) mice) and human coronary artery lesions, in situ proliferation of lesion-resident cells at different stages of growth was assessed by staining for Ki67 and bromodeoxyuridine (BrdU). In early lesions, close to half of all actively growing macrophages were proliferating in situ. BrdU pulse labeling allowed for accurate identification of in situ proliferating macrophages compared to those derived from monocyte recruitment. Local macrophage proliferation declined as lesions advanced. Interestingly, intimal inflammatory cell infiltrates containing proliferating T lymphocytes were identified during the active phase of lesion growth and correlated with apoptotic cell death. Inflammatory cell infiltrates were completely resolved in advanced lesions and replaced with the necrotic core.
CONCLUSIONS
Our findings indicate that atherosclerotic lesions contain locally proliferating macrophages primarily during early and intermediate stages of lesion growth. Furthermore, T-lymphocyte-enriched inflammatory cell infiltrates represent a novel subset of proliferating cells within the atherosclerotic lesion that correlate with apoptosis and precede the necrotic core. These findings have novel implications in understanding the pathogenesis of atherosclerosis and may implicate proliferating T lymphocytes as a contributing factor to lesion progression and stability.
Topics: Animals; Aorta; Apoptosis; Cell Proliferation; Coronary Artery Disease; Coronary Thrombosis; Coronary Vessels; Disease Models, Animal; Female; Humans; Ki-67 Antigen; Macrophages; Male; Mice, Knockout, ApoE; T-Lymphocytes
PubMed: 27528409
DOI: 10.1161/JAHA.116.003945 -
The Journal of Experimental Medicine Mar 2020Cancer cells often proliferate under hypoxia and reprogram their metabolism. However, how to find targets to effectively block the hypoxia-associated metabolic pathways...
Cancer cells often proliferate under hypoxia and reprogram their metabolism. However, how to find targets to effectively block the hypoxia-associated metabolic pathways remains unclear. Here, we developed a tool to conveniently calculate electrons dissipated in metabolic transformations. Based on the law of conservation of electrons in chemical reactions, we further built up an electron balance model for central carbon metabolism, and it can accurately outline metabolic plasticity under hypoxia. Our model specifies that glutamine metabolism reprogrammed for biosynthesis of lipid and/or proline actually acts as the alternative electron bin to enable electron transfer in proliferating cells under hypoxia. Inhibition of both proline biosynthesis and lipogenesis can synergistically suppress cancer cell growth under hypoxia and in vivo tumor onset. Therefore, our model helps to reveal combinations of potential targets to inhibit tumor growth by blocking hypoxia-rewired metabolism and provides a useful tool for future studies on cancer metabolism.
Topics: A549 Cells; Animals; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Female; Glutamine; HeLa Cells; Hep G2 Cells; Humans; Lipogenesis; MCF-7 Cells; Metabolic Networks and Pathways; Mice; Mice, Nude; Neoplasms; Proline
PubMed: 31961917
DOI: 10.1084/jem.20191226 -
Blood May 1984To study the influence of a biologic environment on cultured human leukemia cells, KG-1, KG-1a, and HL-60 cells were inoculated subcutaneously into newborn nude mice....
To study the influence of a biologic environment on cultured human leukemia cells, KG-1, KG-1a, and HL-60 cells were inoculated subcutaneously into newborn nude mice. The cells developed myelosarcomas at the site of inoculation and in lungs and kidneys. KG-1 and HL-60 myelosarcomas were successfully passaged through adult nude mice, whereas KG-1a tumors proliferated only after transplantation into newborn hosts. The human nature of the cells forming myelosarcomas in mice was assessed by chromosomal analyses and detection of cross-reactivity with an antibody to the human leukemia cell line K562. We undertook electron microscopic and cytochemical examinations of the cells proliferating in vitro and in the mice. The granules of KG-1 cells in vivo did not react for acid phosphatase, as observed in vitro, and the HL-60 cells proliferating in mice lost the perinuclear myeloperoxidase (MPO) demonstrated in cultured cells. Although the influence of an in vivo selection of cell subpopulations cannot be ruled out, the enzymatic changes are compatible with induced cell differentiation. Conclusive evidence of differentiation in vivo was observed in the KG-1a cell subline. The undifferentiated KG-1a blasts developed cytoplasmic granules and synthesized MPO during proliferation in vivo. These observations indicate that human leukemia cells from established cell lines proliferate in nude mice and may acquire new differentiated properties in response to the in vivo environment.
Topics: Animals; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Cytoplasmic Granules; Histocytochemistry; Humans; Karyotyping; Leukemia, Myeloid, Acute; Mice; Mice, Nude; Neoplasm Transplantation; Peroxidase
PubMed: 6324924
DOI: No ID Found -
The Anatomical Record Sep 1998Previous studies of colonic epithelial cell kinetics in mice and rats revealed a pattern similar to small intestine, where basally located stem cells proliferate,...
Previous studies of colonic epithelial cell kinetics in mice and rats revealed a pattern similar to small intestine, where basally located stem cells proliferate, differentiating as they migrate towards the surface epithelium. Vacuolated and goblet cells are assumed to co-migrate at the same rate. The present study indicates that rabbit distal colon has more complicated epithelial cell kinetics. The zone of proliferation was detected immunohistochemically using proliferating cell nuclear antigen (PCNA) and confirmed with the use of colchicine to arrest dividing cells in metaphase. Migrating cells were tracked from the zero-hour position (PCNA labeling, mitosis) to positions 24, 48, 72 hrs by monitoring cell migration with the thymidine analog 5-Bromo-2-Deoxyuridine (BrdU). PCNA revealed a major proliferative zone in the upper third of the crypt column and the presence of mitotic figures after colchicine corroborated these results. Differentiated vacuolated cell proliferation was detected at three crypt sites: base, middle, and top of the crypt, while columnar cells arose from a population of dividing cells at the top of the crypt. Turnover of columnar and vacuolated cells occurred within 72 hrs. Goblet cells exhibited maximal proliferation at the crypt base and migrated at a much slower rate than the other cell types. In rabbit distal colon, populations of proliferating cells exist at multiple levels of the crypt column. Vacuolated and goblet cells differ in their labeling indices and migration rates, suggesting that the two cell types arise and migrate independently.
Topics: Animals; Antimetabolites; Bromodeoxyuridine; Cell Division; Colchicine; Colon; Female; Goblet Cells; Proliferating Cell Nuclear Antigen; Rabbits; Specific Pathogen-Free Organisms; Vacuoles
PubMed: 9737743
DOI: 10.1002/(SICI)1097-0185(199809)252:1<41::AID-AR5>3.0.CO;2-H