In Vitro Culture of Cryptosporidium parvum Using Stem Cell-Derived Intestinal Epithelial Monolayers

2019 ◽  
pp. 351-372 ◽  
Author(s):  
Georgia Wilke ◽  
Yi Wang ◽  
Soumya Ravindran ◽  
Thaddeus Stappenbeck ◽  
William H. Witola ◽  
...  
Keyword(s):  
Stem Cell ◽  
In Vitro Culture ◽  
Cryptosporidium Parvum ◽  
Intestinal Epithelial ◽  
Epithelial Monolayers

scholarly journals Activation of two distinct Sox9-EGFP-expressing intestinal stem cell populations during crypt regeneration after irradiation

2012 ◽  
Vol 302 (10) ◽  
pp. G1111-G1132 ◽  
Author(s):  
Laurianne Van Landeghem ◽  
M. Agostina Santoro ◽  
Adrienne E. Krebs ◽  
Amanda T. Mah ◽  
Jeffrey J. Dehmer ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Populations ◽  
Intestinal Epithelial ◽  
P53 Signaling ◽  
Reporter Mice ◽  
Radiation Induced ◽  
Molecular Phenotypes ◽  
Stem Cell Populations ◽  
Induced Changes

Recent identification of intestinal epithelial stem cell (ISC) markers and development of ISC reporter mice permit visualization and isolation of regenerating ISCs after radiation to define their functional and molecular phenotypes. Previous studies in uninjured intestine of Sox9-EGFP reporter mice demonstrate that ISCs express low levels of Sox9-EGFP (Sox9-EGFP Low), whereas enteroendocrine cells (EEC) express high levels of Sox9-EGFP (Sox9-EGFP High). We hypothesized that Sox9-EGFP Low ISCs would expand after radiation, exhibit enhanced proliferative capacities, and adopt a distinct gene expression profile associated with rapid proliferation. Sox9-EGFP mice were given 14 Gy abdominal radiation and studied between days 3 and 9 postradiation. Radiation-induced changes in number, growth, and transcriptome of the different Sox9-EGFP cell populations were determined by histology, flow cytometry, in vitro culture assays, and microarray. Microarray confirmed that nonirradiated Sox9-EGFP Low cells are enriched for Lgr5 mRNA and mRNAs enriched in Lgr5-ISCs and identified additional putative ISC markers. Sox9-EGFP High cells were enriched for EEC markers, as well as Bmi1 and Hopx, which are putative markers of quiescent ISCs. Irradiation caused complete crypt loss, followed by expansion and hyperproliferation of Sox9-EGFP Low cells. From nonirradiated intestine, only Sox9-EGFP Low cells exhibited ISC characteristics of forming organoids in culture, whereas during regeneration both Sox9-EGFP Low and High cells formed organoids. Microarray demonstrated that regenerating Sox9-EGFP High cells exhibited transcriptomic changes linked to p53-signaling and ISC-like functions including DNA repair and reduced oxidative metabolism. These findings support a model in which Sox9-EGFP Low cells represent active ISCs, Sox9-EGFP High cells contain radiation-activatable cells with ISC characteristics, and both participate in crypt regeneration.

scholarly journals The Antibiotic Bacitracin Protects Human Intestinal Epithelial Cells and Stem Cell-Derived Intestinal Organoids from Clostridium difficile Toxin TcdB

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Ziyu Zhu ◽  
Leonie Schnell ◽  
Bastian Müller ◽  
Martin Müller ◽  
Panagiotis Papatheodorou ◽  
...  
Keyword(s):  
Stem Cell ◽  
Epithelial Cells ◽  
Inhibitory Effect ◽  
Target Cells ◽  
Intestinal Epithelial ◽  
Intestinal Organoids ◽  
Human Intestinal Epithelial Cells ◽  
Protein Toxins ◽  
Dependent Transport

Bacitracin is an established antibiotic for local application and inhibits the cell wall synthesis of Gram-positive bacteria. Recently, we discovered a completely different mode of action of bacitracin and reported that this drug protects human cells from intoxication by a variety of medically relevant bacterial protein toxins including CDT, the binary actin ADP-ribosylating toxin of Clostridium (C.) difficile. Bacitracin prevents the transport of CDT into the cytosol of target cells, most likely by inhibiting the transport function of the binding subunit of this toxin. Here, we tested the effect of bacitracin towards TcdB, a major virulence factor of C. difficile contributing to severe C. difficile-associated diseases (CDAD) including pseudomembranous colitis. Bacitracin protected stem cell-derived human intestinal organoids as well as human gut epithelial cells from intoxication with TcdB. Moreover, it prevented the TcdB-induced disruption of epithelia formed by gut epithelium cells in vitro and maintained the barrier function as detected by measuring transepithelial electrical resistance (TEER). In the presence of bacitracin, TcdB was not able reach its substrate Rac1 in the cytosol of human epithelial cells, most likely because its pH-dependent transport across cell membranes into the cytosol is decreased by bacitracin. In conclusion, in addition to its direct antibiotic activity against C. difficile and its inhibitory effect towards the toxin CDT, bacitracin neutralizes the exotoxin TcdB of this important pathogenic bacterium.

scholarly journals Comparison of the Donor Age-Dependent and In Vitro Culture-Dependent Mesenchymal Stem Cell Aging in Rat Model

2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Katarzyna Siennicka ◽  
Aleksandra Zołocińska ◽  
Tomasz Dębski ◽  
Zygmunt Pojda
Keyword(s):  
Stem Cell ◽  
In Vitro Culture ◽  
Growth Potential ◽  
Cell Aging ◽  
Donor Age ◽  
Aging Effects ◽  
Age Limit ◽  
Clinical Experiments

Clinical experiments suggest that mesenchymal stem cells (MSCs) may be useful for tissue repair therapies or treatment of the autoimmune disorders. There is still lack of consensus concerning the age limit of MSC donors, majority of researchers suggest the autologous MSC therapies of patients not exceeding age limit of 55-60 yrs. The purpose of our study was to compare the selected parameters of MSCs from adipose tissue (adipose stem cell, ASC) collected from young and old rats of ages corresponding to patient’s ages 25 yrs. and 80 yrs., respectively. The differences of parameters of ASCs from young and old animals were compared with the differences between ASCs from short-term (3 passage) and long-term (30 passage) in vitro culture. Cell morphology, surface marker expression, growth potential, metabolic activity, β-galactosidase activity, clonogenic potential, angiogenic potential, and differentiation ability of ASCs from young and aged animals and from in vitro cultures at 3rd and 30th passages were compared and analyzed. It may be concluded that ASCs may be applied for autologous transplantations in aged patients. Comparison of ASC aging dynamics depending on host aging or in vitro culture duration suggests that long-term in vitro culture may affect ASCs more than natural aging process of their host. We suggest that ASCs expanded in vitro prior to their clinical use must be carefully screened for the possible aging effects resulting not only from donor age, but from the duration of their in vitro culture.

scholarly journals Characterization of limbal explant sites: Optimization of stem cell outgrowth in in vitro culture

PLoS ONE ◽  
2020 ◽  
Vol 15 (5) ◽  
pp. e0233075
Author(s):  
Pattama Ekpo ◽  
Naharuthai Inthasin ◽  
Sutthicha Matamnan ◽  
Patimaporn Wongprompitak ◽  
Methichit Wattanapanitch ◽  
...  
Keyword(s):  
Stem Cell ◽  
In Vitro Culture ◽  
Cell Outgrowth

scholarly journals Transforming Growth Factor β1 Ameliorates Intestinal Epithelial Barrier Disruption by Cryptosporidium parvum In Vitro in the Absence of Mucosal T Lymphocytes

2000 ◽  
Vol 68 (10) ◽  
pp. 5635-5644 ◽  
Author(s):  
James K. Roche ◽  
Clovis A. P. Martins ◽  
Rosana Cosme ◽  
Ronald Fayer ◽  
Richard L. Guerrant
Keyword(s):  
Epithelial Cell ◽  
Barrier Function ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Epithelial Barrier Function ◽  
Epithelial Monolayers ◽  
Tgf Β1

ABSTRACT Exposure to oocysts of the protozoan Cryptosporidium parvum causes intestinal epithelial cell dysfunction in vivo and in vitro, but effective means by which mucosal injury might be prevented remain unclear. We examined the ability of transforming growth factor β1 (TGF-β1)—a cytokine synthesized and released by cells in the intestine—to preserve the barrier function of human colonic epithelia when challenged with C. parvum oocysts and then studied the mechanisms involved. Epithelial barrier function was monitored electrophysiologically, receptors for TGF-β1 were localized by confocal microscopy, and TGF-β1-induced protein kinase C activation was detected intracellularly by translocation of its α isozyme. TGF-β1 alone enhanced intestinal epithelial barrier function, while exposure to C. parvum oocysts (≥105/monolayer) markedly reduced barrier function to ≤40% of that of the control. When epithelial monolayers were pretreated with TGF-β1 at 5.0 ng/ml, the barrier-disrupting effect ofC. parvum oocysts was almost completely abrogated for 96 h. Further investigation showed that (i) the RI and RII receptors for TGF-β1 were present on 55 and 65% of human epithelial cell line cells, respectively, over a 1-log-unit range of receptor protein expression, as shown by flow cytometry and confirmed by confocal microscopy; (ii) only basolateral and not apical TGF-β1 exposure of the polarized epithelial monolayer resulted in a protective effect; and (iii) TGF-β1 had no direct effect on the organism in reducing its tissue-disruptive effects. In exploring mechanisms to account for the barrier-preserving effects of TGF-β1 on epithelium, we found that the protein kinase C pathway was activated, as shown by translocation of its 80-kDa α isozyme within 30 s of epithelial exposure to TGF-β1; the permeability of epithelial monolayers to passage of macromolecules was reduced by 42% with TGF-β1, even in the face of active protozoal infection; and epithelial cell necrosis monitored by lactate dehydrogenase release was decreased by 50% 70 h after oocyst exposure. Changes in epithelial function, initiated through an established set of surface receptors, likely accounts for the remarkable barrier-sparing effect of nanogram-per-milliliter concentrations of TGF-β1 when human colonic epithelium is exposed to an important human pathogen, C. parvum.

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