In vitro senescence of Syrian hamster mesenchymal cells of fetal to aged adult origin. Inverse relationship between in vivo donor age and in vitro proliferative capacity

Abstract Tumor necrosis factor (TNF) is a highly expressed cytokine in inflammatory bowel disease (IBD). Although TNF can induce colonic epithelial dysfunction and apoptosis, recent studies suggest that TNF signalling promotes epithelial wound repair and stem cell function. Here we investigated the role of TNF receptor 1 (TNFR1) in mediating TNF’s effects on colonic epithelial stem cells, integral to mucosal healing in colitis. We demonstrate that Tnfr1-/- mice exhibit loss in Lgr5 expression (-52%, p<0.02; N=6) compared to wildtype (WT) controls. However, the opposite result was found in vitro, wherein murine Tnfr1-/- colonoids demonstrated a significant increase in Lgr5 expression (66%, p<0.007; N=6) compared to WT colonoids. Similarly, human colonoids treated with an anti-TNFR1 antibody also demonstrated an increase in Lgr5 expression, relative to IgG controls. To resolve the contradiction in the in vivo versus in vitro environment, we hypothesized that mesenchymal TNFR1 expression regulates the epithelial stem cell niche. To determine the relationships between these cell types, we co-cultured WT or Tnfr1-/- colonoids with WT or Tnfr1-/- colonic myofibroblasts (CMFs). We found that epithelial Lgr5 expression was significantly higher (by 52%, p<0.05; N=3) when co-cultured with WT compared to TNFR1-/- myofibroblasts. The loss of TNFR1 expression in vivo increases the number of αSMA+ mesenchymal cells by nearly 56% (N=6) but considerably reduces the pericryptal PDGFRα+ cells, suggesting modifications in mesenchymal populations that contribute to the epithelial stem cell niche. Functionally, primary Tnfr1-/--CMFs displayed PI3k (p<0.001; N=3) and MAPK (p<0.01; N=3)-dependent increases in migration, proliferation, and differentiation, but RNA profiling demonstrated by diminished levels of stem cell niche factors, Rspo3 (-80%, p<0.0001; N=6) and Wnt2b (-63%, p<0.008; N=6) compared to WT-CMFs. Supplementation with 50ng recombinant Rspo3 for 5 d to Lgr5-GFP organoids co-cultured with TNFR1-/--CMFs restored Lgr5 expression to wildtype levels. Therefore, TNFR1-mediated TNF signalling in mesenchymal cells promotes their ability to support an epithelial stem cell niche. These results should motivate future studies of the stem cell niche in the context of long-term treatment with anti-TNF therapies.

Download Full-text

FGF-4 maintains polarizing activity of posterior limb bud cells in vivo and in vitro

Development ◽

10.1242/dev.119.1.199 ◽

1993 ◽

Vol 119 (1) ◽

pp. 199-206 ◽

Cited By ~ 21

Author(s):

A. Vogel ◽

C. Tickle

Keyword(s):

Posterior Margin ◽

Anterior Margin ◽

Marked Decrease ◽

Mesenchymal Cells ◽

Apical Ectodermal Ridge ◽

Limb Bud ◽

Posterior Limb ◽

Vertebrate Limb

The polarizing region is a major signalling tissue involved in patterning the tissues of the vertebrate limb. The polarizing region is located at the posterior margin of the limb bud and can be recognized by its ability to induce additional digits when grafted to the anterior margin of a chick limb bud. The signal from the polarizing region operates at the tip of the bud in the progress zone, a zone of undifferentiated mesenchymal cells, maintained by interactions with the apical ectodermal ridge. A number of observations have pointed to a link between the apical ectodermal ridge and signalling by the polarizing region. To test this possibility, we removed the posterior apical ectodermal ridge of chick wing buds and assayed posterior mesenchyme for polarizing activity. When the apical ectodermal ridge is removed, there is a marked decrease in polarizing activity of posterior cells. The posterior apical ectodermal ridge is known to express FGF-4 and we show that the decrease in polarizing activity of posterior cells of wing buds that normally follows ridge removal can be prevented by implanting a FGF-4-soaked bead. Furthermore, we show that both ectoderm and FGF-4 maintain polarizing activity of limb bud cells in culture.

Download Full-text

Neoplastic conversion of preneoplastic Syrian hamster cells: rate estimation by fluctuation analysis

Molecular and Cellular Biology ◽

10.1128/mcb.3.5.931-945.1983 ◽

1983 ◽

Vol 3 (5) ◽

pp. 931-945

Author(s):

B D Crawford ◽

J C Barrett ◽

P O Ts'o

Keyword(s):

Syrian Hamster ◽

Neoplastic Transformation ◽

Solid Substrate ◽

Established Cell Line ◽

Fluctuation Analysis ◽

Neoplastic Progression ◽

Early Passage

Analysis of the role of gene mutations in the multistep process of neoplastic transformation requires that the discrete steps in carcinogenesis first be dissected. Toward this end, we have isolated and characterized preneoplastic Syrian hamster cells which exhibit in vitro a trait highly correlated with neoplastic conversion in vivo. Previous findings (J. C. Barrett, Cancer Res. 40:91-94, 1980) indicate that spontaneous neoplastic transformation of Syrian hamster cells occurs in at least two steps. An intermediate stage, characterized by an aneuploid established cell line which has a propensity to become neoplastic spontaneously upon further growth in vitro, has been described. These preneoplastic cells differ from diploid early-passage Syrian hamster cells in becoming capable of anchorage-independent growth in semisolid agar, as well as becoming neoplastic in vivo when attached to a solid substrate. Evidence presented here demonstrates that anchorage-independent conversion in vitro is a reliable marker for neoplastic conversion in this cell system. Fluctuation analyses, patterned after those described by Luria and Delbruck for microbial genetics, demonstrate that anchorage-independent variants are generated randomly from clonally derived preneoplastic cells at the rate of 10(-8) to 10(-7) variants per cell per generation. These results establish a multistep stochastic process for transformation in vitro and indicate that conversion to anchorage independence may be necessary for Syrian hamster cells to become tumorigenic. The possible role of gene mutation in this step during neoplastic progression is discussed.

Download Full-text

The SARS-CoV-2 Omicron (B.1.1.529) variant exhibits altered pathogenicity, transmissibility, and fitness in the golden Syrian hamster model

10.1101/2022.01.12.476031 ◽

2022 ◽

Author(s):

Shuofeng Yuan ◽

Zi-Wei Ye ◽

Ronghui Liang ◽

Kaiming Tang ◽

Anna Jinxia Zhang ◽

...

Keyword(s):

Syrian Hamster ◽

Neutralizing Antibodies ◽

Selection Pressure ◽

Immune Selection ◽

Hamster Model ◽

Golden Syrian Hamster ◽

New Variant ◽

Contact Transmission

The newly emerging SARS-CoV-2 Omicron (B.1.1.529) variant first identified in South Africa in November 2021 is characterized by an unusual number of amino acid mutations in its spike that renders existing vaccines and therapeutic monoclonal antibodies dramatically less effective. The in vivo pathogenicity, transmissibility, and fitness of this new Variant of Concerns are unknown. We investigated these virological attributes of the Omicron variant in comparison with those of the currently dominant Delta (B.1.617.2) variant in the golden Syrian hamster COVID-19 model. Omicron-infected hamsters developed significantly less body weight losses, clinical scores, respiratory tract viral burdens, cytokine/chemokine dysregulation, and tissue damages than Delta-infected hamsters. The Omicron and Delta variant were both highly transmissible (100% vs 100%) via contact transmission. Importantly, the Omicron variant consistently demonstrated about 10-20% higher transmissibility than the already-highly transmissible Delta variant in repeated non-contact transmission studies (overall: 30/36 vs 24/36, 83.3% vs 66.7%). The Delta variant displayed higher fitness advantage than the Omicron variant without selection pressure in both in vitro and in vivo competition models. However, this scenario drastically changed once immune selection pressure with neutralizing antibodies active against the Delta variant but poorly active against the Omicron variant were introduced, with the Omicron variant significantly outcompeting the Delta variant. Taken together, our findings demonstrated that while the Omicron variant is less pathogenic than the Delta variant, it is highly transmissible and can outcompete the Delta variant under immune selection pressure. Next-generation vaccines and antivirals effective against this new VOC are urgently needed.

Download Full-text

Preeclampsia: Сardiotonic Steroids, Fibrosis and a Hint to Cancerogenesis

10.20944/preprints202012.0320.v1 ◽

2020 ◽

Author(s):

Alexei Bagrov ◽

Nikolai Kolodkin ◽

C. David Adair ◽

Natalia Agalakova ◽

Alexandr Trashkov

Keyword(s):

Inverse Relationship ◽

Therapeutic Strategy ◽

Transcriptional Factor ◽

Negative Regulator ◽

Novel Therapy ◽

Mortality And Morbidity ◽

Maternal Mortality And Morbidity ◽

Leading Position

Despite prophylaxis and attempts to select a therapy, the frequency of preeclampsia does not decrease, and it still takes the leading position in the structure of maternal mortality and morbidity worldwide. In this review, we present a new theory of the etiology and pathogenesis of preeclampsia which is based on the interaction of Na/K-ATPase and its endogenous ligands including marinobufagenin. The signaling pathway of marinobufagenin involves an inhibition of transcriptional factor Fli1, a negative regulator of collagen synthesis, followed by deposition of collagen in the vascular tissues and altered vascular functions. Moreover, in vitro and in vivo neutralization of marinobufagenin is associated with restoration of Fli1. The inverse relationship between marinobufagenin and Fli1 opens new possibilities in the treatment of cancer: since Fli1 is a proto-oncogene, a hypothesis on suppression of Fli1 by cardiotonic steroids as potential anti-tumor therapeutic strategy is discussed as well. We propose a novel therapy of preeclampsia which is based on immunoneutralization of the marinobufagenin by monoclonal antibodies, which is capable to impair marinobufagenin-Na/K-ATPase interactions.

Download Full-text

Vitamin A alters the internal viscosity of fragments of limb-bud mesenchyme

Development ◽

10.1242/dev.59.1.325 ◽

1980 ◽

Vol 59 (1) ◽

pp. 325-339

Author(s):

T. E. Kwasigroch ◽

D. M. Kochhar

Keyword(s):

Retinoic Acid ◽

Vitamin A ◽

Mesenchymal Cells ◽

Limb Bud ◽

Mouse Limb ◽

Vitamin A Acid ◽

Internal Viscosity ◽

Fusion Experiments

Two techniques were used to examine the effect of vitamin A compounds (vitamin A acid = retinoic acid and vitamin A acetate) upon the relative strengths of adhesion among mouse limb-bud mesenchymal cells. Treatment with retinoic acid in vivo and with vitamin A acetate in vitro reduced the rate at which the fragments of mesenchyme rounded-up when cultured on a non-adhesive substratum, but these compounds did not alter the behavior of tissues tested in fragment-fusion experiments. These conflicting results indicate that the two tests measure different activities of cells and suggest that treatment with vitamin A alters the property(ies) of cells which regulate the internal viscosity of tissues.

Download Full-text

Analysis of elongating morphogenesis of quail anterior submaxillary gland: absence of localized cell proliferation

Development ◽

10.1242/dev.62.1.229 ◽

1981 ◽

Vol 62 (1) ◽

pp. 229-239

Author(s):

Hiroyuki Nogawa

Keyword(s):

Cell Proliferation ◽

Basement Membrane ◽

Regional Differences ◽

Submaxillary Gland ◽

Mesenchymal Cells ◽

Submaxillary Glands ◽

Parallel Direction ◽

Recombination Experiments

Quail anterior submaxillary glands elongated extensively without branching (more than sevenfold) from 8 to 10 incubation days. Investigation of mitotic activity of the rudiments in vivo showed no localized cell proliferation throughout the rudiments, and recombination experiments in vitro to examine regional differences in mitogenic activity of the surrounding mesenchyme also showed that no mesenchymal region specifically stimulates the epithelial cell proliferation. Histological observation of the rudiments showed that epithelial cells did not lengthen in a parallel direction to the long axis of the rudiment, and that mesenchymal cells encircled the epithelial cord perpendicularly to its axis. The basement membrane was obscure in the distal end of the rudiments, while it was easily detected in the other part of the rudiments. These results suggest that the elongating morphogenesis of the anterior submaxillary rudiments is not achieved by localized cell proliferation but by almost uniformly distributed cell proliferation, and mesenchymal cells surrounding the rudiment or the basement membrane may be involved in the controlling mechanisms of the elongating morphogenesis.

Download Full-text

Adipose Tissue-Derived Mesenchymal Cells Support Skin Reepithelialization through Secretion of KGF-1 and PDGF-BB: Comparison with Dermal Fibroblasts

Cell Transplantation ◽

10.3727/096368912x637064 ◽

2012 ◽

Vol 21 (11) ◽

pp. 2441-2454 ◽

Cited By ~ 17

Author(s):

Vassilia-Ismini Alexaki ◽

Despoina Simantiraki ◽

Marianna Panayiotopoulou ◽

Olga Rasouli ◽

Maria Venihaki ◽

...

Keyword(s):

Wound Healing ◽

Adipose Tissue ◽

Normal Skin ◽

Human Adipose Tissue ◽

Mesenchymal Cells ◽

Dermal Fibroblasts ◽

Skin Substitutes ◽

Keratinocyte Proliferation

Epidermal organization and homeostasis are regulated by mesenchymal influences through paracrine actions. Until today, dermal fibroblasts (DFs) are used in the “dermal” layer to support keratinocyte growth in vitro in dermal and skin substitutes. In the present work, we used human adipose tissue-derived mesenchymal cells (ADMCs) as a support of keratinocyte growth in vitro (in monolayer culture and in 3D skin cell culture models) and in vivo (mouse wound healing models) and compared our findings with those obtained using dermal fibroblasts. ADMCs induce reepithelialization during wound healing more efficiently than DFs, by enhancing keratinocyte proliferation through cell cycle progression, and migration. This effect is mediated (at least partially) by a paracrine action of KGF-1 and PDGF-BB, which are more prominently expressed in ADMCs than in DFs. Furthermore, replacement of DFs by ADMCs in the dermal compartment of organotypic skin cultures leads to an artificial epidermis resembling to that of normal skin, concerning the general histology, although with a higher expression of cytokeratins 5 and 19. In Rag1 knockout mice, ADMCs induced a more rapid reepithelialization and a more effective wound healing, compared to dermal fibroblasts. In conclusion, we provide evidence that ADMCs can serve as supportive cells for primary keratinocyte cultures. In addition, because of their abundance and the great cell yield achieved during ADMC isolation, they represent an interesting cell source, with potential aspects for clinical use.

Download Full-text