scholarly journals Production of Injectable Marine Collagen-Based Hydrogel for the Maintenance of Differentiated Chondrocytes in Tissue Engineering Applications

2020 ◽  
Vol 21 (16) ◽  
pp. 5798
Author(s):  
Salvatrice Rigogliuso ◽  
Monica Salamone ◽  
Enza Barbarino ◽  
Maria Barbarino ◽  
Aldo Nicosia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Biochemical Properties ◽  
Cell Phenotype ◽  
Cytoskeleton Organization ◽  
Polymer Precursors ◽  
Main Challenge ◽  
Phenyl Propionic Acid ◽  
Ability To Regenerate ◽  
Expression Program

Cartilage is an avascular tissue with limited ability of self-repair. The use of autologous chondrocyte transplants represent an effective strategy for cell regeneration; however, preserving the differentiated state, which ensures the ability to regenerate damaged cartilage, represents the main challenge during in vitro culturing. For this purpose, we produced an injectable marine collagen-based hydrogel, by mixing native collagen from the jellyfish Rhizostoma pulmo with hydroxy-phenyl-propionic acid (HPA)-functionalized marine gelatin. This biocompatible hydrogel formulation, due to the ability of enzymatically reticulate using horseradish peroxidase (HPR) and H2O2, gives the possibility of trap cells inside, in the absence of cytotoxic effects, during the cross-linking process. Moreover, it enables the modulation of the hydrogel stiffness merely varying the concentration of H2O2 without changes in the concentration of polymer precursors. The maintenance of differentiated chondrocytes in culture was then evaluated via morphological analysis of cell phenotype, GAG production and cytoskeleton organization. Additionally, gene expression profiling of differentiation/dedifferentiation markers provided evidence for the promotion of the chondrogenic gene expression program. This, combined with the biochemical properties of marine collagen, represents a promising strategy for maintaining in vitro the cellular phenotype in the aim of the use of autologous chondrocytes in regenerative medicine practices.

scholarly journals Asthmatic Bronchial Matrices Determine the Gene Expression and Behavior of Smooth Muscle Cells in a 3D Culture Model

2021 ◽  
Vol 2 ◽  
Author(s):  
Selma Ben Hamouda ◽  
Maria Angélica Miglino ◽  
Gustavo de Sá Schiavo Matias ◽  
Guy Beauchamp ◽  
Jean-Pierre Lavoie
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Control Cell ◽  
3D Culture ◽  
Matrix Group ◽  
Cell Phenotype ◽  
Cell Control ◽  
Control Matrix ◽  
And Control

Asthma is associated with increased deposition and altered phenotype of airway smooth muscle (ASM) cells. However, little is known about the processes responsible for these changes. It has been suggested that alterations of the extracellular matrix (ECM) contribute to the remodeling of ASM cells in asthma. Three-dimensional matrices allow the in vitro study of complex cellular responses to different stimuli in a close-to-natural environment. Thus, we investigated the ultrastructural and genic variations of ASM cells cultured on acellular asthmatic and control bronchial matrices. We studied horses, as they spontaneously develop a human asthma-like condition (heaves) with similarities to chronic pulmonary changes observed in human asthma. Primary bronchial ASM cells from asthmatic (n = 3) and control (n = 3) horses were cultured on decellularized bronchi from control (n = 3) and asthmatic (n = 3) horses. Each cell lineage was used to recellularize six different bronchi for 41 days. Histomorphometry on HEPS-stained-recellularized matrices revealed an increased ASM cell number in the control cell/control matrix (p = 0.02) and asthmatic cell/control matrix group (p = 0.04) compared with the asthmatic cell/asthmatic matrix group. Scan electron microscopy revealed a cell invasion of the ECM. While ASM cells showed high adhesion and proliferation processes on the control ECM, the presence of senescent cells and cellular debris in the asthmatic ECM with control or asthmatic ASM cells suggested cell death. When comparing asthmatic with control cell/matrix combinations by targeted next generation sequencing, only AGC1 (p = 0.04), MYO10 (p = 0.009), JAM3 (p = 0.02), and TAGLN (p = 0.001) were differentially expressed out of a 70-gene pool previously associated with smooth muscle remodeling. To our knowledge, this is the first attempt to evaluate the effects of asthmatic ECM on an ASM cell phenotype using a biological bronchial matrix. Our results indicate that bronchial ECM health status contributes to ASM cell gene expression and, possibly, its survival.

scholarly journals Reciprocal regulation of pancreatic ductal adenocarcinoma growth and molecular subtype by HNF4α and SIX1/4

2019 ◽  
Author(s):  
Soledad A. Camolotto ◽  
Veronika K. Belova ◽  
Luke Torre-Healy ◽  
Jeffery M. Vahrenkamp ◽  
Kristofer C. Berrett ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Molecular Subtype ◽  
Ductal Adenocarcinoma ◽  
Reciprocal Regulation ◽  
Expression Of Genes ◽  
Response To Chemotherapy ◽  
Expression Program

AbstractPancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a five-year survival of less than 5%. Transcriptomic analysis has identified two clinically relevant molecular subtypes of PDAC: Classical and Basal-like. The Classical subtype is characterized by a more favorable prognosis and better response to chemotherapy than the Basal-like subtype. The Classical subtype also expresses higher levels of lineage specifiers that regulate endodermal differentiation, including the nuclear receptor HNF4α. Using in vitro and in vivo PDAC models, we show that HNF4α restrains tumor growth and drives tumor cells toward an epithelial identity. Gene expression analysis from murine models and human tumors shows that HNF4α activates expression of genes associated with the Classical subtype. Although HNF4α loss is not sufficient for complete conversion to the Basal-like subtype gene expression profile, HNF4α directly represses SIX4 and SIX1, mesodermal lineage specifiers expressed in the Basal-like subtype. Finally, HNF4α-negative PDAC cells rely on expression of SIX4 and SIX1 for proliferation in vitro and in vivo. Overall, our data show that HNF4α regulates the growth and molecular subtype of PDAC by multiple mechanisms, including activation of the Classical gene expression program and repression of SIX4 and SIX1, which may represent novel dependencies of the Basal-like subtype.

scholarly journals Evidence for a Novel Gene Expression Program in Peripheral Blood Mononuclear Cells from Mycobacterium avium subsp. paratuberculosis-Infected Cattle

2003 ◽  
Vol 71 (11) ◽  
pp. 6487-6498 ◽  
Author(s):  
Paul M. Coussens ◽  
Christopher J. Colvin ◽  
Guilherme J. M. Rosa ◽  
Juliana Perez Laspiur ◽  
Michael D. Elftman
Keyword(s):  
Gene Expression ◽  
Mycobacterium Avium ◽  
Mononuclear Cells ◽  
Expression Patterns ◽  
Gene Expression Patterns ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Expression Program ◽  
Compare Gene Expression

ABSTRACT A bovine-specific cDNA microarray system was used to compare gene expression profiles of peripheral blood mononuclear cells (PBMCs) from control uninfected (n = 4) and Johne's disease-positive (n = 6) Holstein cows. Microarray experiments were designed so that for each animal, a direct comparison was made between PBMCs stimulated in vitro with Mycobacterium avium subsp. paratuberculosis and PBMCs stimulated with phosphate-buffered saline (nil-stimulated PBMCs). As expected, M. avium subsp. paratuberculosis stimulation of infected cow PBMCs enhanced expression of gamma interferon transcripts. In addition, expression of 15 other genes was significantly affected (>1.25-fold change; P < 0.05) by in vitro stimulation with M. avium subsp. paratuberculosis. Similar treatment of control cow PBMCs with M. avium subsp. paratuberculosis resulted in significant changes in expression of 13 genes, only 2 of which were also affected in PBMCs from the infected cow PBMCs. To compare gene expression patterns in the two cow infection groups (infected cows and uninfected cows), a mixed-model analysis was performed with the microarray data. This analysis indicated that there were major differences in the gene expression patterns between cells isolated from the two groups of cows, regardless of in vitro stimulation. A total of 86 genes were significantly differentially expressed (P < 0.01) in M. avium subsp. paratuberculosis-stimulated PBMCs from infected cows compared to expression in similarly treated PBMCs from control cows. Surprisingly, a larger number of genes (110 genes) were also found to be significantly differentially expressed (P < 0.01) in nil-stimulated cells from the two infection groups. The expression patterns of selected genes were substantiated by quantitative real-time reverse transcriptase PCR. Flow cytometric analysis indicated that there were no gross differences in the relative populations of major immune cell types in PBMCs from infected and control cows. Thus, data presented in this report indicate that the gene expression program of PBMCs from M. avium subsp. paratuberculosis-infected cows is inherently different from that of cells from control uninfected cows.

scholarly journals Extensive effects of in vitro oocyte maturation on rhesus monkey cumulus cell transcriptome

2011 ◽  
Vol 301 (1) ◽  
pp. E196-E209 ◽  
Author(s):  
Young S. Lee ◽  
Catherine A. VandeVoort ◽  
John P. Gaughan ◽  
Uros Midic ◽  
Zoran Obradovic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rhesus Monkey ◽  
Oocyte Maturation ◽  
Gene Array ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Cell Phenotype ◽  
Marker Genes

The elaboration of a quality oocyte is integrally linked to the correct developmental progression of cumulus cell phenotype. In humans and nonhuman primates, oocyte quality is diminished with in vitro maturation. To determine the changes in gene expression in rhesus monkey cumulus cells (CC) that occur during the final day prior to oocyte maturation and how these changes differ between in vitro (IVM) and in vivo maturation (VVM), we completed a detailed comparison of transcriptomes using the Affymetrix gene array. We observed a large number of genes differing in expression when comparing IVM-CC and VVM-CC directly but a much larger number of differences when comparing the transitions from the prematuration to the post-IVM and post-VVM states. We observed a truncation or delay in the normal pattern of gene regulation but also remarkable compensatory changes in gene expression during IVM. Among the genes affected by IVM are those that contribute to productive cell-cell interactions between cumulus cell and oocyte and between cumulus cells. Numerous genes involved in lipid metabolism are incorrectly regulated during IVM, and the synthesis of sex hormones appears not to be suppressed during IVM. We identified a panel of 24 marker genes, the expression of which should provide the foundation for understanding how IVM can be improved for monitoring IVM conditions and for diagnosing oocyte quality.

scholarly journals Engrailed 1 coordinates cytoskeletal reorganization to induce myofibroblast differentiation

2021 ◽  
Vol 218 (9) ◽  
Author(s):  
Andrea-Hermina Györfi ◽  
Alexandru-Emil Matei ◽  
Maximilian Fuchs ◽  
Chunguang Liang ◽  
Aleix Rius Rigau ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Myofibroblast Differentiation ◽  
Dependent Manner ◽  
Cytoskeleton Organization ◽  
Fibrotic Diseases ◽  
Sp Transcription Factors

Transforming growth factor-β (TGFβ) is a key mediator of fibroblast activation in fibrotic diseases, including systemic sclerosis. Here we show that Engrailed 1 (EN1) is reexpressed in multiple fibroblast subpopulations in the skin of SSc patients. We characterize EN1 as a molecular amplifier of TGFβ signaling in myofibroblast differentiation: TGFβ induces EN1 expression in a SMAD3-dependent manner, and in turn, EN1 mediates the profibrotic effects of TGFβ. RNA sequencing demonstrates that EN1 induces a profibrotic gene expression profile functionally related to cytoskeleton organization and ROCK activation. EN1 regulates gene expression by modulating the activity of SP1 and other SP transcription factors, as confirmed by ChIP-seq experiments for EN1 and SP1. Functional experiments confirm the coordinating role of EN1 on ROCK activity and the reorganization of cytoskeleton during myofibroblast differentiation, in both standard fibroblast culture systems and in vitro skin models. Consistently, mice with fibroblast-specific knockout of En1 demonstrate impaired fibroblast-to-myofibroblast transition and are partially protected from experimental skin fibrosis.

scholarly journals Hypoxia modulates the gene expression profile of immunoregulatory receptors in human mature dendritic cells: identification of TREM-1 as a novel hypoxic marker in vitro and in vivo

Blood ◽  
2011 ◽  
Vol 117 (9) ◽  
pp. 2625-2639 ◽  
Author(s):  
Maria Carla Bosco ◽  
Daniele Pierobon ◽  
Fabiola Blengio ◽  
Federica Raggi ◽  
Cristina Vanni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dendritic Cells ◽  
Cell Phenotype ◽  
Surface Receptors ◽  
Immunoregulatory Receptors ◽  
Antigen Presenting ◽  
And Behavior ◽  
The Impact

Abstract Dendritic cells (DCs) are a heterogeneous group of professional antigen-presenting cells functioning as sentinels of the immune system and playing a key role in the initiation and amplification of innate and adaptive immune responses. DC development and functions are acquired during a complex differentiation and maturation process influenced by several factors present in the local milieu. A common feature at pathologic sites is represented by hypoxia, a condition of low pO2, which creates a unique microenvironment affecting cell phenotype and behavior. Little is known about the impact of hypoxia on the generation of mature DCs (mDCs). In this study, we identified by gene expression profiling a significant cluster of genes coding for immune-related cell surface receptors strongly up-regulated by hypoxia in monocyte-derived mDCs and characterized one of such receptors, TREM-1, as a new hypoxia-inducible gene in mDCs. TREM-1 associated with DAP12 in hypoxic mDCs, and its engagement elicited DAP12-linked signaling, resulting in ERK-1, Akt, and IκBα phosphorylation and proinflammatory cytokine and chemokine secretion. Finally, we provided the first evidence that TREM-1 is expressed on mDCs infiltrating the inflamed hypoxic joints of children affected by juvenile idiopathic arthritis, representing a new in vivo marker of hypoxic mDCs endowed with proinflammatory properties.

scholarly journals Potential Endogenous Cell Sources for Retinal Regeneration in Vertebrates and Humans: Progenitor Traits and Specialization

Biomedicines ◽  
2020 ◽  
Vol 8 (7) ◽  
pp. 208
Author(s):  
Eleonora N. Grigoryan
Keyword(s):  
Gene Expression ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cell Phenotype ◽  
Cell Populations ◽  
Specific Gene ◽  
Epigenetic Landscape ◽  
Retinal Regeneration ◽  
Cell Sources

Retinal diseases often cause the loss of photoreceptor cells and, consequently, impairment of vision. To date, several cell populations are known as potential endogenous retinal regeneration cell sources (RRCSs): the eye ciliary zone, the retinal pigment epithelium, the iris, and Müller glia. Factors that can activate the regenerative responses of RRCSs are currently under investigation. The present review considers accumulated data on the relationship between the progenitor properties of RRCSs and the features determining their differentiation. Specialized RRCSs (all except the ciliary zone in low vertebrates), despite their differences, appear to be partially “prepared” to exhibit their plasticity and be reprogrammed into retinal neurons due to the specific gene expression and epigenetic landscape. The “developmental” characteristics of RRCS gene expression are predefined by the pathway by which these cell populations form during eye morphogenesis; the epigenetic features responsible for chromatin organization in RRCSs are under intracellular regulation. Such genetic and epigenetic readiness is manifested in vivo in lower vertebrates and in vitro in higher ones under conditions permissive for cell phenotype transformation. Current studies on gene expression in RRCSs and changes in their epigenetic landscape help find experimental approaches to replacing dead cells through recruiting cells from endogenous resources in vertebrates and humans.

Effects of sub-toxic Cadmium concentrations on bone gene expression program: Results of an in vitro study

2010 ◽  
Vol 24 (6) ◽  
pp. 1670-1680 ◽  
Author(s):  
Maria Bodo ◽  
Stefania Balloni ◽  
Eleonora Lumare ◽  
Mauro Bacci ◽  
Mario Calvitti ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Study ◽  
Vitro Study ◽  
Gene Expression Program ◽  
Expression Program

Differential expression of non-allelic insulin genes in rodent islet tumour cells

1993 ◽  
Vol 11 (3) ◽  
pp. 305-318 ◽  
Author(s):  
K Lund ◽  
N Blume ◽  
B K Michelsen ◽  
D Bucchini ◽  
O D Madsen
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Islet Cell ◽  
Tumour Cells ◽  
Insulin Gene ◽  
Cell Phenotype ◽  
Β Cell ◽  
C Peptide ◽  
Insulin Gene Expression

ABSTRACT We have compared the expression patterns of the non-allelic insulin 1 and 2 genes during prolonged in-vitro culture of the mouse islet cell line β-TC3, where transformation by the SV40 T oncoprotein is targeted to the differentiated β-cell phenotype, and the rat islet cell line NHI-6F, in which the β-cell phenotype is induced by transient in-vivo passage. The NHI-6F clone carries, in addition, a single copy of a transfected silent human insulin gene which contains 3 kb of regulatory sequences known to confer β-cell-specific expression. Insulin gene expression was measured by an assay based on a reverse transcription-polymerase chain reaction, to determine whether the ancestral rodent insulin 2 genes (and the human homologue in the NHI-6F cells) are regulated differently from the duplicated rat and mouse insulin 1 genes. We have shown that activation of insulin gene expression in the NHI-6F cells includes transcriptional activation of all three genes, but that extended propagation of tumour cells in vitro leads to a selective and equal decline in the quantities of transcripts from the rat 2 and human genes relative to transcripts from the rat 1 gene. In the later passages, insulin transcripts were derived almost exclusively from the rat 1 gene. In early in-vitro passages of the mouse endocrine cell line β-TC3, the expression pattern of the mouse 1 and 2 insulin genes resembled that seen in isolated mouse islets. After more than 45 in-vitro passages, expression of the duplicated mouse 1 gene decreased tenfold when compared with the ancestral mouse 2 gene. As previously shown for NHI-6F cells, the differential expression of non-allelic insulin genes in the β-TC3 line was also clearly evident at the cellular level, where a subpopulation of cells selectively expressed readily detectable levels of mouse C-peptide 2 immunoreactivity while devoid of C-peptide 1. Our results suggest that the maintenance of insulin gene expression in rodent tumour cells is influenced by enhancer sequences which are not shared by the ancestral and duplicated insulin genes, and that either species-specific conditions or transformation-related differences exist between the rat and mouse cell lines that govern which gene remains active during prolonged in-vitro propagation.

scholarly journals The Menin-MLL1 interaction is a molecular dependency in NUP98-rearranged AML

Blood ◽  
2021 ◽  
Author(s):  
Emily B Heikamp ◽  
Jill A Henrich ◽  
Florian Perner ◽  
Eric M Wong ◽  
Charles Hatton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Hoxa Cluster ◽  
Critical Set ◽  
Leukemia Cell Lines ◽  
Expression Program

Translocations involving the NUP98 gene produce NUP98-fusion proteins and are associated with a poor prognosis in acute myeloid leukemia (AML). MLL1 is a molecular dependency in NUP98-fusion leukemia, and therefore we investigated the efficacy of therapeutic blockade of the Menin-MLL1 interaction in NUP98-fusion leukemia models. Using mouse leukemia cell lines driven by NUP98-HOXA9 and NUP98-JARID1A fusion oncoproteins, we demonstrate that NUP98-fusion driven leukemia is sensitive to the Menin-MLL1 inhibitor VTP50469, with an IC50 similar to what we have previously reported for MLL-rearranged and NPM1c leukemia cells. Menin-MLL1 inhibition upregulates markers of differentiation such as CD11b and downregulates expression of pro-leukemogenic transcription factors such as Meis1 in NUP98-fusion transformed leukemia cells. We demonstrate that MLL1 and the NUP98 fusion protein itself are evicted from chromatin at a critical set of genes that are essential for maintenance of the malignant phenotype. In addition to these in vitro studies, we established patient-derived xenograft (PDX) models of NUP98-fusion driven AML to test the in vivo efficacy of Menin-MLL1 inhibition. Treatment with VTP50469 significantly prolongs survival of mice engrafted with NUP98-NSD1 and NUP98-JARID1A leukemias. Gene expression analysis revealed that Menin-MLL1 inhibition simultaneously suppresses a pro-leukemogenic gene expression program, including downregulation of the HOXA cluster, and upregulates tissue-specific markers of differentiation. These preclinical results suggest that Menin-MLL1 inhibition may represent a rational, targeted therapy for patients with NUP98-rearranged leukemias.

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