scholarly journals Clonal Populations of Amniotic Cells by Dilution and Direct Plating: Evidence for Hidden Diversity

2012 ◽  
Vol 2012 ◽  
pp. 1-15 ◽  
Author(s):  
Patricia G. Wilson ◽  
Lorna Devkota ◽  
Tiffany Payne ◽  
Laddie Crisp ◽  
Allison Winter ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Amniotic Fluid ◽  
Ex Vivo ◽  
Cell Types ◽  
Cell Populations ◽  
Mixed Cell ◽  
Differentiation Potential ◽  
Direct Plating ◽  
Fetal Cells ◽  
Amniotic Cells

Fetal cells are widely considered a superior cell source for regenerative medicine; fetal cells show higher proliferative capacity and have undergone fewer replicative cycles that could generate spontaneous mutations. Fetal cells in amniotic fluid were among the first normal primary cells to be cultured ex vivo, but the undefined composition of amniotic fluid has hindered advance for regenerative applications. We first developed a highly efficient method to generate clonal populations by dilution of amniocentesis samples in media and direct plating without intervening refrigeration, centrifugation, or exposure of cells to the paracrine effects in mixed cell cultures. More than 40 clonal populations were recovered from 4 amniocentesis samples and representative clones were characterized by flow cytometry, conventional assays for differentiation potential, immunofluorescence imaging, and transcript analysis. The results revealed previously unreported diversity among stromal and epithelial cell types and identified unique cell types that could be lost or undetected in mixed cell populations. The differentiation potential of amniotic cells proved to be uncoupled from expression of definitive cell surface or cytoplasmic markers for stromal and epithelial cells. Evidence for diversity among stromal and epithelial cells in amniotic fluid bears on interpretations applied to molecular and functional tests of amniotic cell populations.

186 BOVINE AMNIOTIC FLUID MESENCHYMAL STEM CELLS CHARACTERIZATION AFTER CULTURE IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 207
Author(s):  
B. Rossi ◽  
B. Merlo ◽  
E. Iacono ◽  
P. P. Pagliaro ◽  
P. L. Tazzari ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Amniotic Fluid ◽  
Cell Types ◽  
Calcium Deposition ◽  
Cell Populations ◽  
Rt Pcr ◽  
Differentiation Potential ◽  
Phenotypic Characterisation

In recent years, fetal adnexa and fluids have been recognised as important sources of mesenchymal stem cells (MSC). The aim of this study was to characterise cell populations of bovine amniotic fluid, studying phenotypic characterisation, RNA expression, and differentiation potential of samples after in vitro culture for different lengths of time following trypsinization and expansion (passage). Amniotic fluid samples were recovered at the slaughterhouse from 25 pregnant cows and harvested cells were cultured in DMEM-TCM199 (1 : 1) plus 10% fetal bovine serum (FBS) in 5% CO2 at 38.5°C. At passages P3 and P7, a sample for each of the 4 population found was characterised. Immunophenotypic characterisation was performed for MSC (CD90, CD105, CD44) and haematopoietic (CD14, CD34) markers by flow cytometry (FACS). Immunocytochemistry (ICC) was performed for Oct4, SSEA4, and α-SMA and the ratio between positive cells and total nuclei was evaluated. Gene expression profile was analysed by RT–PCR for pluripotency markers (Oct4, Nanog, Sox2). At the same passages chondrogenic, osteogenic and adipogenic differentiation were induced and evaluated morphologically and cytologically using, respectively, Alcian blue to identify cartilage matrix, Von Kossa for extracellular calcium deposition, and Oil Red O for intracellular lipid droplets. Cell population appeared heterogeneous and we could identify 2 main cell types: round (R) and spindle-shaped (S) cells. Each isolated sample was classified into one of the following 4 types depending on percentages of R or S cells: prevalence of S-cells (S), prevalence of R-cells (R), and samples showing both morphologies with ~10% of S-cells (S10) or 40% S-cells (S40). S-cells percentage decreased with passages in S10 and S40. After FACS, all lines were positive for CD90, CD105, CD44, and CD34 and negative for CD14 both at P3 and at P7. After ICC, Oct4 was negative in all samples analysed, few S cells stained for SSEA4 (8%) at P3 but increased at P7 to 22%; R, S10, and S40 did not express SSEA4 both at P3 and at P7. α-SMA was expressed in all samples at P3 (9.4% S; 0.9% R; 2.5% S10; 27% S40) but not at P7 (27.5% S; 0% R; 0% S10; 0% S40). After RT–PCR analyses, Oct4 was negative in all samples; at P3, Nanog was clearly positive in S-cells, weak in S40, and negative in R and S10, but all samples turned negative at P7. Sox2 was weakly expressed (S) or not expressed (S10, S40, R) at P3 and it was negative in all cells at P7. Only S showed high differentiation potential into all 3 lineages at both P3 and P7, R had the lowest differentiation potential, whereas S10 and S40 were intermediate at both end points. In conclusion, bovine amniotic fluid showed heterogeneous cell populations and S-type had the characteristics of MSCs. S10 and S40 showed more MSC markers at P3, when S cells were still present, and this aspect suggests that S population is the presumptive MSC one. Although prevalent, R-type showed only some MSC characteristics. Further studies are under way to improve S-type isolation, purification, and culture, and to determine the lifespan of these cell types. This work was supported by grant PRIN2009.

scholarly journals Chemogenetic Tools for Causal Cellular and Neuronal Biology

2018 ◽  
Vol 98 (1) ◽  
pp. 391-418 ◽  
Author(s):  
Deniz Atasoy ◽  
Scott M. Sternson
Keyword(s):  
G Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Cellular Processes ◽  
Distinct Cell ◽  
G Protein Coupled ◽  
Pharmacological Control

Chemogenetic technologies enable selective pharmacological control of specific cell populations. An increasing number of approaches have been developed that modulate different signaling pathways. Selective pharmacological control over G protein-coupled receptor signaling, ion channel conductances, protein association, protein stability, and small molecule targeting allows modulation of cellular processes in distinct cell types. Here, we review these chemogenetic technologies and instances of their applications in complex tissues in vivo and ex vivo.

The analysis of spatial distributions in mixed cell populations: a statistical method for detecting sorting out

Development ◽  
1971 ◽  
Vol 26 (1) ◽  
pp. 135-156
Author(s):  
R. A. Elton ◽  
C. A. Tickle
Keyword(s):  
Plant Communities ◽  
Quantitative Measure ◽  
Cell Types ◽  
Limb Bud ◽  
Cell Populations ◽  
Spatial Distributions ◽  
Mixed Cell ◽  
Mixed Aggregates ◽  
Chick Heart ◽  
Gyratory Shaker

1. This work presents a quantitative measure, α, of the degree of segregation of two cell types in sections of aggregates, and some results obtained with the measure relating to ‘sorting out’. The method is designed particularly for the case where labelling of one type of cell is incomplete, and the importance of this effect is assessed. Possible problems in formulating such a model are discussed. The measure α is compared with methods used in investigations of segregation in plant communities. 2. Segregation of chick heart and limb-bud cells in mixed aggregates has been analysed using α. In control aggregates of mixtures of labelled and unlabelled cells of one type, α is near to its random value of 1, and we suggest that the departure from random can be adequately accounted for by cell division. In mixed aggregates, significant segregation is consistently found, even in aggregates formed after 2 and 4 h. Both disaggregation procedures (EDTA, trypsin or trypsin + EDTA) and reaggregation methods (reciprocating or gyratory shaker) are found to have an effect on the degree of segregation. Possible reasons for these findings are discussed. 3. Positioning of the cells relative to the outside of aggregates is also investigated for some of the aggregates.

scholarly journals Human Mesenchymal Stem Cells Display Reduced Expression of CD105 after Culture in Serum-Free Medium

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Peter Mark ◽  
Mandy Kleinsorge ◽  
Ralf Gaebel ◽  
Cornelia A. Lux ◽  
Anita Toelk ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Ex Vivo ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Growth Media ◽  
Clinical Use ◽  
Differentiation Potential ◽  
Serum Free ◽  
The Impact

Human Mesenchymal Stem Cells (hMSCs) present a promising tool for regenerative medicine. However,ex vivoexpansion is necessary to obtain sufficient cells for clinical therapy. Conventional growth media usually contain the critical component fetal bovine serum. For clinical use, chemically defined media will be required. In this study, the capability of two commercial, chemically defined, serum-free hMSC growth media (MSCGM-CD and PowerStem) for hMSC proliferation was examined and compared to serum-containing medium (MSCGM). Immunophenotyping of hMSCs was performed using flow cytometry, and they were tested for their ability to differentiate into a variety of cell types. Although the morphology of hMSCs cultured in the different media differed, immunophenotyping displayed similar marker patterns (high expression of CD29, CD44, CD73, and CD90 cell surface markers and absence of CD45). Interestingly, the expression of CD105 was significantly lower for hMSCs cultured in MSCGM-CD compared to MSCGM. Both groups maintained mesenchymal multilineage differentiation potential. In conclusion, the serum-free growth medium is suitable for hMSC culture and comparable to its serum-containing counterpart. As the expression of CD105 has been shown to positively influence hMSC cardiac regenerative potential, the impact of CD105 expression onto clinical use after expansion in MSCGM-CD will have to be tested.

scholarly journals Epithelial cells of the intestine acquire cell-intrinsic inflammation signatures during ageing

2021 ◽  
Author(s):  
Maja C Funk ◽  
Jan G Gleixner ◽  
Florian Heigwer ◽  
Erica Valentini ◽  
Zeynep Aydin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mhc Class Ii ◽  
Ex Vivo ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Extrinsic Factors ◽  
Intestinal Organoids ◽  
Persistent Inflammation ◽  
A Cell

During ageing, cell-intrinsic and extrinsic factors lead to the decline of tissue function and organismal health. Disentangling these factors is important for developing effective strategies to prolong organismal healthspan. Here, we addressed this question in the mouse intestinal epithelium, which forms a dynamic interface with its microenvironment and receives extrinsic signals affecting its homeostasis and tissue ageing. We systematically compared transcriptional profiles of young and aged epithelial cells in vivo and ex vivo in cultured intestinal organoids. We found that all cell types of the aged epithelium exhibit an inflammation phenotype, which is marked by MHC class II upregulation and most pronounced in enterocytes. This was accompanied by elevated levels of the immune tolerance markers PD-1 and PD-L1 in the aged tissue microenvironment, indicating dysregulation of immunological homeostasis. Intestinal organoids from aged mice still showed an inflammation signature after weeks in culture, which was concurrent with increased chromatin accessibility of inflammation-associated loci. Our results reveal a cell-intrinsic, persistent inflammation phenotype in aged epithelial cells, which might contribute to systemic inflammation observed during ageing.

scholarly journals Boar sperm tyrosine phosphorylation patterns in the presence of oviductal epithelial cells: in vitro, ex vivo, and in vivo models

Reproduction ◽  
2013 ◽  
Vol 146 (4) ◽  
pp. 315-324 ◽  
Author(s):  
Victoria Luño ◽  
Rebeca López-Úbeda ◽  
Francisco Alberto García-Vázquez ◽  
Lydia Gil ◽  
Carmen Matás
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Phosphorylation ◽  
Ex Vivo ◽  
Cell Populations ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Experimental Conditions ◽  
In Vivo Models

Spermatozoa transport through the oviduct is a controlled process that regulates sperm capacitation. A crucial event involved in capacitation is protein tyrosine phosphorylation (TP). This study was undertaken to determine whether similarities exist in protein TP distribution between spermatozoa bound or unbound to oviductal epithelial cells (OEC) in three different conditions: i)in vitro, spermatozoa coincubated with OEC cultures; ii)ex vivo, spermatozoa deposited in porcine oviductal explants from slaughtered animals; iii)in vivo, in which sows were inseminated and the oviduct was recovered. The localization of phosphotyrosine protein was determined using indirect immunofluorescence. The distribution of protein TP was significantly (P<0.05) different between bound and unbound cell populations in all experiments. In sows inseminated close to ovulation, spermatozoa were found mainly in the utero–tubal junction, where spermatozoa exhibited higher proportion of flagellum phosphorylation. Spermatozoa not bound to OEC exhibited high levels of protein phosphorylation (phosphorylated equatorial subsegment and acrosome and/or phosphorylated flagellum) in theex vivoandin vivoexperiments (P<0.05). However, unbound spermatozoa coincubated with OEC inin vitroconditions tended to show intermediate levels of TP (equatorial subsegment with or without phosphorylated flagellum). In spermatozoa bound to OEC, protein TP was located in the equatorial subsegment or presented no phosphorylation (P<0.05). Although sperm capacitation conditionsin vivowere not reproduciblein vitroin our experimental conditions, sperm and OEC binding seemed to be a mechanism for selecting spermatozoa with a low level of TP inin vivo,ex vivo, andin vitroexperiments.

scholarly journals Ex vivo gut culture for studying differentiation and migration of small intestinal epithelial cells

Open Biology ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 170256 ◽  
Author(s):  
Xiaofei Sun ◽  
Xing Fu ◽  
Min Du ◽  
Mei-Jun Zhu
Keyword(s):  
Epithelial Cells ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Cell Types ◽  
Regulatory Function ◽  
Gut Health ◽  
Gut Development ◽  
Epithelial Migration ◽  
Organoid Culture

Epithelial cultures are commonly used for studying gut health. However, due to the absence of mesenchymal cells and gut structure, epithelial culture systems including recently developed three-dimensional organoid culture cannot accurately represent in vivo gut development, which requires intense cross-regulation of the epithelial layer with the underlying mesenchymal tissue. In addition, organoid culture is costly. To overcome this, a new culture system was developed using mouse embryonic small intestine. Cultured intestine showed spontaneous peristalsis, indicating the maintenance of the normal gut physiological structure. During 10 days of ex vivo culture, epithelial cells moved along the gut surface and differentiated into different epithelial cell types, including enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We further used the established ex vivo system to examine the role of AMP-activated protein kinase (AMPK) on gut epithelial health. Tamoxifen-induced AMPK α 1 knockout vastly impaired epithelial migration and differentiation of the developing ex vivo gut, showing the crucial regulatory function of AMPK α 1 in intestinal health.

scholarly journals A High-Affinity Native Human Antibody Neutralizes Human Cytomegalovirus Infection of Diverse Cell Types

2014 ◽  
Vol 59 (3) ◽  
pp. 1558-1568 ◽  
Author(s):  
Lawrence M. Kauvar ◽  
Keyi Liu ◽  
Minha Park ◽  
Neal DeChene ◽  
Robert Stephenson ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Ex Vivo ◽  
Virus Transmission ◽  
Passive Immunization ◽  
Cell Types ◽  
Human Antibody ◽  
Viral Glycoprotein ◽  
High Affinity

ABSTRACTHuman cytomegalovirus (HCMV) is the most common infection causing poor outcomes among transplant recipients. Maternal infection and transplacental transmission are major causes of permanent birth defects. Although no active vaccines to prevent HCMV infection have been approved, passive immunization with HCMV-specific immunoglobulin has shown promise in the treatment of both transplant and congenital indications. Antibodies targeting the viral glycoprotein B (gB) surface protein are known to neutralize HCMV infectivity, with high-affinity binding being a desirable trait, both to compete with low-affinity antibodies that promote the transmission of virus across the placenta and to displace nonneutralizing antibodies binding nearby epitopes. Using a miniaturized screening technology to characterize secreted IgG from single human B lymphocytes, 30 antibodies directed against gB were previously cloned. The most potent clone, TRL345, is described here. Its measured affinity was 1 pM for the highly conserved site I of the AD-2 epitope of gB. Strain-independent neutralization was confirmed for 15 primary HCMV clinical isolates. TRL345 prevented HCMV infection of placental fibroblasts, smooth muscle cells, endothelial cells, and epithelial cells, and it inhibited postinfection HCMV spread in epithelial cells. The potential utility for preventing congenital transmission is supported by the blockage of HCMV infection of placental cell types central to virus transmission to the fetus, including differentiating cytotrophoblasts, trophoblast progenitor cells, and placental fibroblasts. Further, TRL345 was effective at controlling anex vivoinfection of human placental anchoring villi. TRL345 has been utilized on a commercial scale and is a candidate for clinical evaluation.

scholarly journals Amniotic-Fluid Stem Cells: Growth Dynamics and Differentiation Potential after a CD-117-Based Selection Procedure

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
S. Arnhold ◽  
S. Glüer ◽  
K. Hartmann ◽  
O. Raabe ◽  
K. Addicks ◽  
...  
Keyword(s):  
Stem Cells ◽  
Amniotic Fluid ◽  
Cell Sorting ◽  
Growth Dynamics ◽  
Selection Procedure ◽  
Cell Types ◽  
Differentiation Potential ◽  
Cd 117 ◽  
Fetal Stem Cells ◽  
Hypoxic Conditions

Amniotic fluid (AF) has become an interesting source of fetal stem cells. However, AF contains heterogeneous and multiple, partially differentiated cell types. After isolation from the amniotic fluid, cells were characterized regarding their morphology and growth dynamics. They were sorted by magnetic associated cell sorting using the surface marker CD 117. In order to show stem cell characteristics such as pluripotency and to evaluate a possible therapeutic application of these cells, AF fluid-derived stem cells were differentiated along the adipogenic, osteogenic, and chondrogenic as well as the neuronal lineage under hypoxic conditions. Our findings reveal that magnetic associated cell sorting (MACS) does not markedly influence growth characteristics as demonstrated by the generation doubling time. There was, however, an effect regarding an altered adipogenic, osteogenic, and chondrogenic differentiation capacity in the selected cell fraction. In contrast, in the unselected cell population neuronal differentiation is enhanced.

Effect of Calcium on the Growth and Differentiation of Cultured Rat Esophageal Epithelial Cells

1982 ◽  
Vol 40 ◽  
pp. 132-133
Author(s):  
W.T. Gunning ◽  
M.R. Marino ◽  
M.S. Babcock ◽  
G.D. Stoner
Keyword(s):  
Epithelial Cells ◽  
Cell Types ◽  
Replication Rate ◽  
Enzymatic Dissociation ◽  
Growth Assay ◽  
Epidermal Growth ◽  
Fetal Bovine ◽  
Esophageal Epithelial Cells ◽  
Cellular Replication

The role of calcium in modulating cellular replication and differentiation has been described for various cell types. In the present study, the effects of Ca++ on the growth and differentiation of cultured rat esophageal epithelial cells was investigated.Epithelial cells were isolated from esophagi taken from 8 week-old male CDF rats by the enzymatic dissociation method of Kaighn. The cells were cultured in PFMR-4 medium supplemented with 0.25 mg/ml dialyzed fetal bovine serum, 5 ng/ml epidermal growth factor, 10-6 M hydrocortisone 10-6 M phosphoethanolamine, 10-6 M ethanolamine, 5 pg/ml insulin, 5 ng/ml transferrin, 10 ng/ml cholera toxin and 50 ng/ml garamycin at 36.5°C in a humidified atmosphere of 3% CO2 in air. At weekly intervals, the cells were subcultured with a solution containing 1% polyvinylpyrrolidone, 0.01% EGTA, and 0.05% trypsin. After various passages, the replication rate of the cells in PFMR-4 medium containing from 10-6 M to 10-3 M Ca++ was determined using a clonal growth assay.

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