Human fetal/tumor metakaryotic stem cells: pangenomic homologous pairing and telomeric end-joining of chromatids

Metakaryotic cells and syncytia with large, hollow, bell-shaped nuclei demonstrate symmetrical and asymmetrical amitotic nuclear fissions in microanatomical positions and numbers expected of stem cell lineages in tissues of all three primordial germ layers and their derived tumors. Using fluorescence in situ hybridization, mononuclear metakaryotic interphase cells have been found with only 23 centromeric and 23 telomeric staining regions. Syncytial bell-shaped nuclei found approximately during weeks 5–12 of human gestation display 23 centromeric and either 23 or 46 telomeric staining regions. These images suggest that (1) homologous chromatids pair at centromeres and telomeres, (2) all paired telomeres join end-to-end with other paired telomeres in all mononuclear and some syncytial metakaryotic cells, and (3) telomere junctions may open and close during the syncytial phase of development. Twenty-three telomeric joining figures could be accounted by 23 rings of one chromatid pair each, a single pangenomic ring of 23 joined chromatid pairs, or any of many possible sets of oligo-chromatid pair rings. As telomeric end-joining may affect peri-telomeric gene expression, a programmed sequence of telomeric end-joining associations in metakaryotic stem cells could guide developmental arboration and errors in, or interruptions of, this program could contribute to carcinogenesis.National Institute of Environmental Health SciencesUnited Therapeutics, Inc

The study of calcified atherosclerotic arteries: an alternative to evaluate the composition of a problematic tissue reveals new insight including metakaryotic cells

Background Calcifications of atherosclerotic plaques represent a controversial issue as they either lead to the stabilization or rupture of the lesion. However, the cellular key players involved in the progression of the calcified plaques have not yet been described. The primary reason for this lacuna is that decalcification procedures impair protein and nucleic acids contained in the calcified tissue. The aim of our study was to preserve the cellular content of heavily calcified plaques with a new rapid fixation in order to simplify the study of calcifications. Methods Here we applied a fixation method for fresh calcified tissue using the Carnoy’s solution followed by an enzymatic tissue digestion with type II collagenase. Immunohistochemistry was performed to verify the preservation of nuclear and cytoplasmic antigens. DNA content and RNA preservation was evaluated respectively with Feulgen staining and RT-PCR. A checklist of steps for successful image analysis was provided. To present the basic features of the F-DNA analysis we used descriptive statistics, skewness and kurtosis. Differences in DNA content were analysed with Kruskal-Wallis and Dunn’s post tests. The value of P < 0.05 was considered significant. Results Twenty-four vascular adult tissues, sorted as calcified (14) or uncalcified (10), were processed and 17 fetal tissues were used as controls (9 soft and 8 hard). Cells composing the calcified carotid plaques were positive to Desmin, Vimentin, Osteocalcin or Ki-67; the cellular population included smooth muscle cells, osteoblasts and osteoclasts-like cells and metakaryotic cells. The DNA content of each cell type found in the calcified carotid artery was successfully quantified in 7 selected samples. Notably the protocol revealed that DNA content in osteoblasts in fetal control tissues exhibits about half (3.0 ng) of the normal nuclear DNA content (6.0 ng). Conclusion Together with standard histology, this technique could give additional information on the cellular content of calcified plaques and help clarify the calcification process during atherosclerosis.United Therapeutics Corporatio

Mutator/Hypermutable Fetal/Juvenile Metakaryotic Stem Cells and Human Colorectal Carcinogenesis

Adult age-specific colorectal cancer incidence rates increase exponentially from maturity, reach a maximum, then decline in extreme old age. Armitage and Doll (1) postulated that the exponential increase resulted from “n” mutations occurring throughout adult life in normal “cells at risk” that initiated the growth of a preneoplastic colony in which subsequent “m” mutations promoted one of the preneoplastic “cells at risk” to form a lethal neoplasia. We have reported cytologic evidence that these “cells at risk” are fetal/juvenile organogenic, then preneoplastic metakaryotic stem cells. Metakaryotic cells display stem-like behaviors of both symmetric and asymmetric nuclear divisions and peculiarities such as bell shaped nuclei and amitotic nuclear fission that distinguish them from embryonic, eukaryotic stem cells. Analyses of mutant colony sizes and numbers in adult lung epithelia supported the inferences that the metakaryotic organogenic stem cells are constitutively mutator/hypermutable and that their contributions to cancer initiation are limited to the fetal/juvenile period. We have amended the two-stage model of Armitage and Doll and incorporated these several inferences in a computer program CancerFit v.5.0. We compared the expectations of the amended model to adult (15–104 years) age-specific colon cancer rates for European-American males born 1890–99 and observed remarkable concordance. When estimates of normal colonic fetal/juvenile APC and OAT gene mutation rates (∼2–5 × 10[superscript −5] per stem cell doubling) and preneoplastic colonic gene loss rates (∼8 × 10[superscript −3]) were applied, the model was in accordance only for the values of n = 2 and m = 4 or 5.United Therapeutics Corporatio

Pathogenic Activation of Mesenchymal Stem Cells Is Induced by the Disease Microenvironment in Systemic Sclerosis

Objective: In systemic sclerosis (SSc), a persistent tissue repair process leads to progressive fibrosis of the skin and internal organs. The role of mesenchymal stem cells (MSCs), which characteristically initiate and regulate tissue repair, has not been fully evaluated. We undertook this study to investigate whether dividing metakaryotic MSCs are present in SSc skin and to examine whether exposure to the disease microenvironment activates MSCs and leads to transdifferentiation. Methods: Skin biopsy material from patients with recent-onset diffuse SSc was examined by collagenase spread of 1-mm–thick surface-parallel sections, in order to identify dividing metakaryotic stem cells in each tissue plane. Adipose-derived MSCs from healthy controls were treated with dermal blister fluid (BF) from patients with diffuse SSc and profiled by next-generation sequencing, or they were evaluated for phenotypic changes relevant to SSc. Differential responses of dermal fibroblasts were studied in parallel. Results: MSC-like cells undergoing active metakaryotic division were identified in SSc sections (but not control sections) most prominently in the deep dermis and adjacent to damaged microvessels, in both clinically involved and uninvolved skin. Furthermore, exposure to SSc BF caused selective MSC activation, inducing a myofibroblast signature, while reducing signatures of vascular repair and adipogenesis and enhancing migration and contractility. Microenvironmental factors implicated in inducing transdifferentiation included the profibrotic transforming growth factor β, the presence of lactate, and mechanosensing, while the microenvironment Th2 cytokine, interleukin-31, enhanced osteogenic commitment (calcinosis). Conclusion: Dividing MSC-like cells are present in the SSc disease microenvironment where multiple factors, likely acting in concert, promote transdifferentiation and lead to a complex and resistant disease state

Metakaryotic stem cell nuclei use pangenomic dsRNA/DNA intermediates in genome replication and segregation

Bell shaped nuclei of metakaryotic cells double their DNA content during and after symmetric and asymmetric amitotic fissions rather than in the separate, pre-mitotic S-phase of eukaryotic cells. A parsimonious hypothesis was tested that the two anti-parallel strands of each chromatid DNA helix were first segregated as ssDNA-containing complexes into sister nuclei then copied to recreate a dsDNA genome. Metakaryotic nuclei that were treated during amitosis with RNase A and stained with acridine orange or fluorescent antibody to ssDNA revealed large amounts of ssDNA. Without RNase treatment metakaryotic nuclei in amitosis stained strongly with an antibody complex specific to dsRNA/DNA. Images of amitotic figures co-stained with dsRNA/DNA antibody and DAPI indicated that the entire interphase dsDNA genome (B-form helices) was transformed into two dsRNA/DNA genomes (A-form helices) that were segregated in the daughter cell nuclei then retransformed into dsDNA. As this process segregates DNA strands of opposite polarity in sister cells it hypothetically offers a sequential switching mechanism within the diverging stem cell lineages of development

Sirolimus-eluting stents: opposite in vitro effects on the clonogenic cell potential on a long-term exposure

Copyright: © Vasuri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. We evaluated the long-term effects of sirolimus on three different cell in vitro models, cultured in physiological conditions mimicking sirolimus-eluted stent, in order to clarify the effectiveness of sirolimus in blocking cell proliferation and survival. Three cells lines (WPMY-1 myofibroblasts, HT-29 colorectal adenocarcinoma, and U2OS osteosarcoma) were selected and growth in 10 ml of Minimum Essential Medium for 5 weeks with serial dilutions of sirolimus. The number of colonies and the number of cells per colony were counted. As main result, the number of WPMY-1 surviving colonies increased in a dose-dependent manner when treated with sirolimus (p = 0.0011), while the number of U2OS colonies progressively decreased (p = 0.0011). The clonal capacity of HT-29 was not modified by the exposure to sirolimus (p = 0.6679). In conclusion sirolimus showed the well-known cytostatic effect, but with an effect on clonogenic potential different among the different cell types. In the practice, the plaque typology and composition may influence the response to sirolimus and thus the effectiveness of eluted stent

Metakaryotic stem cell nuclei use pangenomic dsRNA/DNA intermediates in genome replication and segregation

Bell shaped nuclei of metakaryotic cells double their DNA content during and after symmetric and asymmetric amitotic fissions rather than in the separate, pre-mitotic S-phase of eukaryotic cells. A parsimonious hypothesis was tested that the two anti-parallel strands of each chromatid DNA helix were first segregated as ssDNA-containing complexes into sister nuclei then copied to recreate a dsDNA genome. Metakaryotic nuclei that were treated during amitosis with RNase A and stained with acridine orange or fluorescent antibody to ssDNA revealed large amounts of ssDNA. Without RNase treatment metakaryotic nuclei in amitosis stained strongly with an antibody complex specific to dsRNA/DNA. Images of amitotic figures co-stained with dsRNA/DNA antibody and DAPI indicated that the entire interphase dsDNA genome (B-form helices) was transformed into two dsRNA/DNA genomes (A-form helices) that were segregated in the daughter cell nuclei then retransformed into dsDNA. As this process segregates DNA strands of opposite polarity in sister cells it hypothetically offers a sequential switching mechanism within the diverging stem cell lineages of development