scholarly journals Trichostatin A-Assisted Epigenomic Modulation Affects the Expression Profiles of Not Only Recombinant Human α1,2-Fucosyltransferase and α-Galactosidase A Enzymes But Also Galα1→3Gal Epitopes in Porcine Bi-Transgenic Adult Cutaneous Fibroblast Cells

2021 ◽  
Vol 22 (3) ◽  
pp. 1386
Author(s):  
Jerzy Wiater ◽  
Marcin Samiec ◽  
Maria Skrzyszowska ◽  
Daniel Lipiński
Keyword(s):  
Genetic Resource ◽  
Ex Vivo ◽  
Trichostatin A ◽  
Expression Profiles ◽  
Fibroblast Cells ◽  
Control Groups ◽  
Transgenic Pigs ◽  
Cell Tissue ◽  
Donor Cells ◽  
Lower Expression

This study was conducted to explore whether trichostatin A-assisted epigenomic modulation (TSA-EM) can affect the expression of not only recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) immune system enzymes but also Galα1→3Gal epitopes in ex vivo proliferating adult cutaneous fibroblast cells (ACFCs) derived from hFUT2×hGLA bi-transgenic pigs that had been produced for the needs of future xenotransplantation efforts. The ACFC lines were treated with 50 nM TSA for 24 h and then the expression profiles of rhα1,2-FT and rhα-Gal A enzymes were analyzed by Western blot and immunofluorescence. The expression profiles of the Galα1→3Gal epitope were determined by lectin blotting and lectin fluorescence. The ACFCs derived from non-transgenic (nTG) pigs were served as the negative (TSA−) and positive (TSA+) control groups. For both hFUT2×hGLA and nTG samples, the expression levels of α1,2-FT and α-Gal A proteins in TSA+ cells were more than twofold higher in comparison to TSA− cells. Moreover, a much lower expression of the Galα1→3Gal epitopes was shown in TSA− hFUT2×hGLA cells as compared to the TSA− nTG group. Interestingly, the levels of Galα1→3Gal expression in TSA-treated hFUT2×hGLA and nTG ACFCs were significantly higher than those noticed for their TSA-untreated counterparts. Summing up, ex vivo protection of effectively selected bi-transgenic ACFC lines, in which TSA-dependent epigenetic transformation triggered the enhancements in reprogrammability and subsequent expression of hFUT2 and hGLA transgenes and their corresponding transcripts, allows for cryopreservation of nuclear donor cells, nuclear-transferred female gametes, and resultant porcine cloned embryos. The latter can be used as a cryogenically conserved genetic resource of biological materials suitable for generation of bi-transgenic cloned offspring in pigs that is targeted at biomedical research in the field of cell/tissue xenotransplantation.

42 DEVELOPMENT OF PIG EMBRYOS CLONED FROM DONOR CELLS TREATED WITH TRICHOSTATIN A

2008 ◽  
Vol 20 (1) ◽  
pp. 101 ◽  
Author(s):  
J. Li ◽  
Y. Du ◽  
P. M. Kragh ◽  
S. Purup ◽  
K. Villemoes ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Embryonic Genome ◽  
Donor Cells ◽  
Deacetylase Inhibitor ◽  
Vitro Development

Development to the blastocyst stage following nuclear transfer is dependent on the donor cell's ability to reprogram its genome to a totipotent state. Reprogramming of the transferred somatic nuclei must be completed by the time normal activation of the embryonic genome occurs (Solter 2000 Nat. Rev. Genet. 1, 199–207). Recently, Enright et al. (2003 Biol. Reprod. 69, 896–901) reported that in vitro development of cloned cow embryos was improved by treatment of donor cells with a histone deacetylase inhibitor, TrichostatinA (TSA). So far, there are no reports available for adult pig fibroblast cells treated with TSA. The objective of this study was to investigate whether the development of handmade cloned embryos in pig could be improved by using TSA-treated donor cells. Adult pig fibroblast cells were treated with 100, 150, or 200 nm TSA for 24 h, compared to untreated controls, and were then used as donor cells. The cells were electrofused with handmade enucleated pig oocytes separately and were activated with calcium ionophore and cycloheximide. They were subsequently cultured in porcine zygote medium 3 (PZM-3; Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) using the well of the well system (WOW; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Experiments were repeated 4 times and the data were analyzed with AVEDEV and t-test in Excel (Microsoft Excel 2007). The cleavage rates and the total cell numbers per blastocyst were similar between groups (P > 0.05), as shown in Table 1. However, the cloned blastocyst rate using donor cells treated with 100 nm TSA was higher than in the other groups (69.9 ± 4.7% v. 43.6 ± 4.3%, 43.1 ± 5.8%, or 46.6 ± 3.6%; P < 0.05), as shown in Table 1. These data suggest that proper TSA treatment for donor cells before somatic cloning improves the rate of development of porcine handmade cloned embryos to the blastocyst stage. Further research is needed to examine the in vivo development of embryos reconstructed with TSA-treated donor cells. Table 1. Developmental ability of cloned pig embryos derived fromTSA-treated donor cells

scholarly journals Characterization of Mono- and Bi-Transgenic Pig-Derived Epidermal Keratinocytes Expressing Human FUT2 and GLA Genes—In Vitro Studies

2021 ◽  
Vol 22 (18) ◽  
pp. 9683
Author(s):  
Jerzy Wiater ◽  
Marcin Samiec ◽  
Kamil Wartalski ◽  
Zdzisław Smorąg ◽  
Jacek Jura ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Expression Profiles ◽  
Ex Situ ◽  
Epidermal Keratinocytes ◽  
Phylogenetic Distance ◽  
Cell Nuclei ◽  
Transgenic Pigs ◽  
Xenograft Rejection ◽  
Transgenic Cell

Pig-to-human xenotransplantation seems to be the response to the contemporary shortage of tissue/organ donors. Unfortunately, the phylogenetic distance between pig and human implies hyperacute xenograft rejection. In this study, we tested the hypothesis that combining expression of human α1,2-fucosyltransferase (hFUT2) and α-galactosidase A (hGLA) genes would allow for removal of this obstacle in porcine transgenic epidermal keratinocytes (PEKs). We sought to determine not only the expression profiles of recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) proteins, but also the relative abundance (RA) of Galα1→3Gal epitopes in the PEKs stemming from not only hFUT2 or hGLA single-transgenic and hFUT2×hGLA double-transgenic pigs. Our confocal microscopy and Western blotting analyses revealed that both rhα1,2-FT and rhα-Gal A enzymes were overabundantly expressed in respective transgenic PEK lines. Moreover, the semiquantitative levels of Galα1→3Gal epitope that were assessed by lectin fluorescence and lectin blotting were found to be significantly diminished in each variant of genetically modified PEK line as compared to those observed in the control nontransgenic PEKs. Notably, the bi-transgenic PEKs were characterized by significantly lessened (but still detectable) RAs of Galα1→3Gal epitopes as compared to those identified for both types of mono-transgenic PEK lines. Additionally, our current investigation showed that the coexpression of two protective transgenes gave rise to enhanced abrogation of Galα→3Gal epitopes in hFUT2×hGLA double-transgenic PEKs. To summarize, detailed estimation of semiquantitative profiles for human α-1,2-FT and α-Gal A proteins followed by identification of the extent of abrogating the abundance of Galα1→3Gal epitopes in the ex vivo expanded PEKs stemming from mono- and bi-transgenic pigs were found to be a sine qua non condition for efficiently ex situ protecting stable lines of skin-derived somatic cells inevitable in further studies. The latter is due to be focused on determining epigenomic reprogrammability of single- or double-transgenic cell nuclei inherited from adult cutaneous keratinocytes in porcine nuclear-transferred oocytes and corresponding cloned embryos. To our knowledge, this concept was shown to represent a completely new approach designed to generate and multiply genetically transformed pigs by somatic cell cloning for the needs of reconstructive medicine and dermoplasty-mediated tissue engineering of human integumentary system.

scholarly journals Dynamic integration of enteric neural stem cells in ex vivo organotypic colon cultures

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Georgina Navoly ◽  
Conor J. McCann
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Organotypic Culture ◽  
Ex Vivo ◽  
Donor Cell ◽  
Colonic Tissue ◽  
Cell Therapies ◽  
Enteric Ganglia ◽  
Donor Cells

AbstractEnteric neural stem cells (ENSC) have been identified as a possible treatment for enteric neuropathies. After in vivo transplantation, ENSC and their derivatives have been shown to engraft within colonic tissue, migrate and populate endogenous ganglia, and functionally integrate with the enteric nervous system. However, the mechanisms underlying the integration of donor ENSC, in recipient tissues, remain unclear. Therefore, we aimed to examine ENSC integration using an adapted ex vivo organotypic culture system. Donor ENSC were obtained from Wnt1cre/+;R26RYFP/YFP mice allowing specific labelling, selection and fate-mapping of cells. YFP+ neurospheres were transplanted to C57BL6/J (6–8-week-old) colonic tissue and maintained in organotypic culture for up to 21 days. We analysed and quantified donor cell integration within recipient tissues at 7, 14 and 21 days, along with assessing the structural and molecular consequences of ENSC integration. We found that organotypically cultured tissues were well preserved up to 21-days in ex vivo culture, which allowed for assessment of donor cell integration after transplantation. Donor ENSC-derived cells integrated across the colonic wall in a dynamic fashion, across a three-week period. Following transplantation, donor cells displayed two integrative patterns; longitudinal migration and medial invasion which allowed donor cells to populate colonic tissue. Moreover, significant remodelling of the intestinal ECM and musculature occurred upon transplantation, to facilitate donor cell integration within endogenous enteric ganglia. These results provide critical evidence on the timescale and mechanisms, which regulate donor ENSC integration, within recipient gut tissue, which are important considerations in the future clinical translation of stem cell therapies for enteric disease.

scholarly journals Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. e66-e73 ◽  
Author(s):  
Chih-Wen Ni ◽  
Haiwei Qiu ◽  
Amir Rezvan ◽  
Kihwan Kwon ◽  
Douglas Nam ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Gene Expression Profiles ◽  
Disturbed Flow ◽  
A Genome ◽  
Pattern Comparison

Abstract Recently, we showed that disturbed flow caused by a partial ligation of mouse carotid artery rapidly induces atherosclerosis. Here, we identified mechanosensitive genes in vivo through a genome-wide microarray study using mouse endothelial RNAs isolated from the flow-disturbed left and the undisturbed right common carotid artery. We found 62 and 523 genes that changed significantly by 12 hours and 48 hours after ligation, respectively. The results were validated by quantitative polymerase chain reaction for 44 of 46 tested genes. This array study discovered numerous novel mechanosensitive genes, including Lmo4, klk10, and dhh, while confirming well-known ones, such as Klf2, eNOS, and BMP4. Four genes were further validated for protein, including LMO4, which showed higher expression in mouse aortic arch and in human coronary endothelium in an asymmetric pattern. Comparison of in vivo, ex vivo, and in vitro endothelial gene expression profiles indicates that numerous in vivo mechanosensitive genes appear to be lost or dysregulated during culture. Gene ontology analyses show that disturbed flow regulates genes involved in cell proliferation and morphology by 12 hours, followed by inflammatory and immune responses by 48 hours. Determining the functional importance of these novel mechanosensitive genes may provide important insights into understanding vascular biology and atherosclerosis.

scholarly journals FLAMs: A self-replicating ex vivo model of alveolar macrophages for functional genetic studies

2021 ◽  
Author(s):  
Sean Thomas ◽  
Kathryn Wierenga ◽  
James Pestka ◽  
Andrew Olive
Keyword(s):  
Alveolar Macrophages ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Expression Profiles ◽  
Surface Expression ◽  
Specific Gene ◽  
Ex Vivo Model ◽  
A Genome ◽  
Underlying Mechanisms ◽  
And Function

Alveolar macrophages (AMs) are tissue resident cells in the lungs derived from the fetal liver that maintain lung homeostasis and respond to inhaled stimuli. While the importance of AMs is undisputed, they remain refractory to standard experimental approaches and high-throughput functional genetics as they are challenging to isolate and rapidly lose AM properties in standard culture. This limitation hinders our understanding of key regulatory mechanisms that control AM maintenance and function. Here, we describe the development of a new model, fetal liver-derived alveolar-like macrophages (FLAMs), which maintains cellular morphologies, expression profiles, and functional mechanisms similar to murine AMs. FLAMs combine treatment with two key cytokines for AM maintenance, GM-CSF and TGFβ. We leveraged the long-term stability of FLAMs to develop functional genetic tools using CRISPR-Cas9-mediated gene editing. Targeted editing confirmed the role of AM-specific gene Marco and the IL-1 receptor Il1r1 in modulating the AM response to crystalline silica. Furthermore, a genome-wide knockout library using FLAMs identified novel genes required for surface expression of the AM marker Siglec-F, most notably those related to the peroxisome. Taken together, our results suggest that FLAMs are a stable, self-replicating model of AM function that enables previously impossible global genetic approaches to define the underlying mechanisms of AM maintenance and function.

scholarly journals Fibroblast Protection of Borrelia burgdorferi from Doxycycline, Cefuroxime and Daptomycin Combination is Eliminated by Oregano or Carvacrol Essential Oil

2019 ◽  
Author(s):  
Chunxiang Bai ◽  
Hua Yang ◽  
Peng Cui ◽  
Rong Quan ◽  
Ying Zhang
Keyword(s):  
Lyme Disease ◽  
Essential Oil ◽  
Borrelia Burgdorferi ◽  
Drug Combination ◽  
Defense Mechanisms ◽  
Ex Vivo ◽  
Fibroblast Cells ◽  
Murine Fibroblast ◽  
Drug Regimens

AbstractBorrelia burgdorferi could be occasionally recovered from patients after antibiotic treatment, which indicates it may resist eradication by antibiotic and host defense mechanisms. Skin fibroblast cells have previously been shown to protect the killing of B. burgdorferi by ceftriaxone, a powerful antibiotic commonly used to treat Lyme disease. In this study, we evaluated if fibroblast cells could also protect against the doxycycline+ cefuroxime+ daptomycin drug combination which has previously been shown to completely eradicate highly persistent biofilm-like microcolonies of B. burgdorferi. To do so, we utilized a GFP-labeled B. burgdorferi for infection of murine fibroblast cells and assessed the effect of the drug combination on killing the bacteria in the presence or absence of the fibroblast cells. Surprisingly, we found that fibroblasts could protect B. burgdorferi from being completely killed by the drug combination doxycycline, cefuroxime and daptomycin, which eradicated B. burgdorferi completely in the absence of fibroblast cells. Interestingly, addition of essential oil carvacrol or oregano at 0.1% could enhance the activity of the doxycycline+ cefuroxime+ daptomycin drug combination and led to complete eradication of B. burgdorferi even in the presence of fibroblast cells. Further studies are needed to determine if the essential oil drug combinations could eradicate persistent B. burgdorferi infection in vivo in animal models. Our study provides a useful and convenient ex vivo model for evaluating different drug regimens needed for developing more effective treatment of persistent Lyme disease in the future.

scholarly journals Manipulating the Mitochondrial Genome To Enhance Cattle Embryo Development

2017 ◽  
Vol 7 (7) ◽  
pp. 2065-2080 ◽  
Author(s):  
Kanokwan Srirattana ◽  
Justin C St. John
Keyword(s):  
Embryo Development ◽  
Nuclear Transfer ◽  
Bos Taurus ◽  
Trichostatin A ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Genetic Integrity ◽  
Rna Seq ◽  
Mtdna Copy Number ◽  
Donor Cells

Abstract The mixing of mitochondrial DNA (mtDNA) from the donor cell and the recipient oocyte in embryos and offspring derived from somatic cell nuclear transfer (SCNT) compromises genetic integrity and affects embryo development. We set out to generate SCNT embryos that inherited their mtDNA from the recipient oocyte only, as is the case following natural conception. While SCNT blastocysts produced from Holstein (Bos taurus) fibroblasts were depleted of their mtDNA, and oocytes derived from Angus (Bos taurus) cattle possessed oocyte mtDNA only, the coexistence of donor cell and oocyte mtDNA resulted in blastocysts derived from nondepleted cells. Moreover, the use of the reprogramming agent, Trichostatin A (TSA), further improved the development of embryos derived from depleted cells. RNA-seq analysis highlighted 35 differentially expressed genes from the comparison between blastocysts generated from nondepleted cells and blastocysts from depleted cells, both in the presence of TSA. The only differences between these two sets of embryos were the presence of donor cell mtDNA, and a significantly higher mtDNA copy number for embryos derived from nondepleted cells. Furthermore, the use of TSA on embryos derived from depleted cells positively modulated the expression of CLDN8, TMEM38A, and FREM1, which affect embryonic development. In conclusion, SCNT embryos produced by mtDNA depleted donor cells have the same potential to develop to the blastocyst stage without the presumed damaging effect resulting from the mixture of donor and recipient mtDNA.

scholarly journals Dexamethasone-mediated oncogenicity in vitro and in an animal model of glioblastoma

2018 ◽  
Vol 129 (6) ◽  
pp. 1446-1455 ◽  
Author(s):  
Markus M. Luedi ◽  
Sanjay K. Singh ◽  
Jennifer C. Mosley ◽  
Islam S. A. Hassan ◽  
Masumeh Hatami ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Expression Profiles ◽  
Clinical Information ◽  
Gene Signature ◽  
The Cancer Genome Atlas ◽  
Boyden Chamber ◽  
Ki 67 ◽  
Marker Selection

OBJECTIVEDexamethasone, a known regulator of mesenchymal programming in glioblastoma (GBM), is routinely used to manage edema in GBM patients. Dexamethasone also activates the expression of genes, such as CEBPB, in GBM stem cells (GSCs). However, the drug’s impact on invasion, proliferation, and angiogenesis in GBM remains unclear. To determine whether dexamethasone induces invasion, proliferation, and angiogenesis in GBM, the authors investigated the drug’s impact in vitro, in vivo, and in clinical information derived from The Cancer Genome Atlas (TCGA) cohort.METHODSExpression profiles of patients from the TCGA cohort with mesenchymal GBM (n = 155) were compared with patients with proneural GBM by comparative marker selection. To obtain robust data, GSCs with IDH1 wild-type (GSC3) and with IDH1 mutant (GSC6) status were exposed to dexamethasone in vitro and in vivo and analyzed for invasion (Boyden chamber, human-specific nucleolin), proliferation (Ki-67), and angiogenesis (CD31). Ex vivo tumor cells from dexamethasone-treated and control mice were isolated by fluorescence activated cell sorting and profiled using Affymetrix chips for mRNA (HTA 2.0) and microRNAs (miRNA 4.0). A pathway analysis was performed to identify a dexamethasone-regulated gene signature, and its relationship with overall survival (OS) was assessed using Kaplan-Meier analysis in the entire GBM TCGA cohort (n = 520).RESULTSThe mesenchymal subgroup, when compared with the proneural subgroup, had significant upregulation of a dexamethasone-regulated gene network, as well as canonical pathways of proliferation, invasion, and angiogenesis. Dexamethasone-treated GSC3 demonstrated a significant increase in invasion, both in vitro and in vivo, whereas GSC6 demonstrated a modest increase. Furthermore, dexamethasone treatment of both GSC3 and GSC6 lines resulted in significantly elevated cell proliferation and angiogenesis in vivo. Patients with mesenchymal GBM had significant upregulation of dexamethasone-regulated pathways when compared with patients with proneural GBM. A prognostic (p = 0.0007) 33-gene signature was derived from the ex vivo expression profile analyses and used to dichotomize the entire TCGA cohort by high (median OS 12.65 months) or low (median OS 14.91 months) dexamethasone signature.CONCLUSIONSThe authors present evidence that furthers the understanding of the complex effects of dexamethasone on biological characteristics of GBM. The results suggest that the drug increases invasion, proliferation, and angiogenesis in human GSC-derived orthotopic tumors, potentially worsening GBM patients’ prognoses. The authors believe that careful investigation is needed to determine how to minimize these deleterious dexamethasone-associated side effects in GBM.

scholarly journals Premature differentiation of nephron progenitor cell and dysregulation of gene pathways critical to kidney development in a model of preterm birth

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aleksandra Cwiek ◽  
Masako Suzuki ◽  
Kimberly deRonde ◽  
Mark Conaway ◽  
Kevin M. Bennett ◽  
...  
Keyword(s):  
Preterm Birth ◽  
Progenitor Cells ◽  
Kidney Development ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Neonatal Morbidity ◽  
Expression Of Genes ◽  
Nephron Progenitor ◽  
Lower Expression ◽  
Nephrogenic Zone

AbstractPreterm birth is a leading cause of neonatal morbidity. Survivors have a greater risk for kidney dysfunction and hypertension. Little is known about the molecular changes that occur in the kidney of individuals born preterm. Here, we demonstrate that mice delivered two days prior to full term gestation undergo premature cessation of nephrogenesis, resulting in a lower glomerular density. Kidneys from preterm and term groups exhibited differences in gene expression profiles at 20- and 27-days post-conception, including significant differences in the expression of fat-soluble vitamin-related genes. Kidneys of the preterm mice exhibited decreased proportions of endothelial cells and a lower expression of genes promoting angiogenesis compared to the term group. Kidneys from the preterm mice also had altered nephron progenitor subpopulations, early Six2 depletion, and altered Jag1 expression in the nephrogenic zone, consistent with premature differentiation of nephron progenitor cells. In conclusion, preterm birth alone was sufficient to shorten the duration of nephrogenesis and cause premature differentiation of nephron progenitor cells. These candidate genes and pathways may provide targets to improve kidney health in preterm infants.

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