A NEW METHOD FOR THE ESTIMATION OF CELL CYCLE PHASES

A method is described, which is applicable to cell renewal systems with an anatomical structure in which all cell locations may be uniquely mapped. Its use is demonstrated on the rat incisor inner enamel epithelium, which forms a one cell thick column in the sagittally sectioned tooth. Cells born in the apical part of the column migrate toward the distal end of the tooth, where they mature. As the cells migrate along the column, they traverse the various cell cycle phases. The present study has been designed to estimate the probability of a cell being in a given phase; all cells touching the basement membrane were numbered, and the number of cells separating any two cells was taken as a measure of distance. Since generally all cells move in one direction (lateral cell migration may occur), it is possible to solve the problem with the aid of functions describing the renewal counting stochastic process in which cell distance serves as an independent variable. The method predicts labelled cell and mitotic rates which agree with those estimated in the usual way. It was then utilized to estimate the fraction of cells in G₂.     

The adaptation of a two-compartment cell renewal system to external demands

Summary The inner enamel epithelium (IEE) covers the labial tooth aspect as a one cell layer which, when cut sagittally, appears as a longitudinal cell column extending from the tooth origin toward the periphery. Following sudden tooth shortening, the IEE responds by an increased cell production which later declines below normal values. The perturbation affects all cell kinetic parameters; the progenitor compartment, which initially increases, diminishes in size toward end of the experiment. The cell cycle transition times, which initially decline, rise toward the end of the experiment. The mean normal daily cell production rate of 70 cell % (i.e. 70 cells are produced by 100 progenitors) increases to 111 cell % and then declines to a low of 51 cell %. The IEE response typifies the behavior of other cell renewal systems such as intestinal epithelium and epidermis.     

On the progenitor cell migration velocity

An attempt is presented to extract cell kinetic information from histomorphological features. It is applicable to rapidly proliferating tissues like the intestinal epithelium. Each replicating tissue has an origin where cells are formed and a periphery toward which cells migrate. The migration path along which they move is denominated as tissue radius on which all cell positions are mapped. Cell migration on the radius is associated with cell proliferation at tissue origin. Each mitosis there is associated with the displacement of all cells distal to it by one cell position. The more mitoses positioned between a cell and tissue origin, the greater its migration velocity. It is possible therefore to derive the cell migration velocity v(x) from the cumulative mitotic distribution on the radius, N(x). v(x) = N(x)/tm (tm = mitotic time). In this form v(x) represents also cell production at any point on the radius and may serve for the computation of other cell kinetic parameters like generation time. These arguments are illustrated on the rat incisor tooth inner enamel epithelium which has been studied in the normal and rapidly erupting tooth.     

FIBROBLAST CELL KINETICS IN THE PERIODONTAL LIGAMENT OF THE MOUSE

Twelve male mice were injected intraperitoneally with tritiated thymidine. Six were sacrificed after 1 hr and six after 7 days. The right manibular incisors were dissected, cut sagittally and dipped in liquid emulsion. In the exposed and stained slides, observation was restricted to the lingual side of the periodontal ligament. Cells were evaluated sagittally from the basal tooth end up to the distance of 5 mm and up to the depth of 100 μm in the direction of socket wall. Cell and grain count was evaluated separately in 100 × 10 μm rectangles, creating a two-dimensional array onto which the periodontal ligament was mapped. The progenitor compartment extends up to the distance of 2400 μm from origin. Fibroblasts leaving this compartment migrate at different velocities, creating a velocity profile across the ligament. Adjacent to the socket wall cell movement is sluggish, whereas the fastest cell movement is exhibited by cells located 20–30 μm from the tooth. The existence of such a profile indicates a continuous renewal of intercellular bonds, consistent with a process of actively pulling the incisor from its socket by the migrating fibrocytes.     

ON THE PROGENITOR CELL MIGRATION VELOCITY

An attempt is presented to extract cell kinetic information from histomorphological features. It is applicable to rapidly proliferating tissues like the intestinal epithelium. Each replicating tissue has an origin where cells are formed and a periphery toward which cells migrate. The migration path along which they move is denominated as tissue radius on which all cell positions are mapped. Cell migration on the radius is associated with cell proliferation at tissue origin. Each mitosis there is associated with the displacement of all cells distal to it by one cell position. The more mitoses positioned between a cell and tissue origin, the greater its migration velocity. It is possible therefore to derive the cell migration velocity v(x) from the cumulative mitotic distribution on the radius, N(x). v(x) = N(x)/t_m (t_m= mitotic time). In this form v(x) represents also cell production at any point on the radius and may serve for the computation of other cell kinetic parameters like generation time. These arguments are illustrated on the rat incisor tooth inner enamel epithelium which has been studied in the normal and rapidly erupting tooth.     

ON THE DERIVATION OF CELL KINETICS FROM HISTOMORPHOLOGY, OBSERVED IN THE RAT INCISOR ODONTOBLAST

A method to extract cell kinetic information from histomorphology is presented. Each replicating tissue is essentially an ordered structure with an origin where cells are formed and a periphery toward which they are displaced. the displacement path is called the tissue radius. the tissue variables may be studied in two domains, space and time. the first embraces all the states a cell may assume while the second specifies the cell transition rates. During steady state both domains are related linearly. These ideas are illustrated in the rat incisor odontoblast population whose life expectation is determined by the tooth wall shape. the odontoblast cell population paves the interior of the tooth wall delimiting a cone-shaped pulp. Near the root apex the dentine wall is barely visible. As one proceeds distally, the wall thickens while the pulp narrows. Pulp narrowing is associated with odontoblast cell loss whose magnitude may be deduced from the change of the pulp circumference CI(x) (x is the distance from tooth origin). the odontoblast force of mortality μ(x) may be calculated from the instantaneous perimeter change: μ(x) = -CI' (x)/CI(x); where CI'(x) stands for the derivative of CI(x). This equation serves for the construction of the odontoblast life table which may be studied in space and time.     

E-cadherin regulates the behavior and fate of epithelial stem cells and their progeny in the mouse incisor

Stem cells are essential for the regeneration and homeostasis of many organs, such as tooth, hair, skin, and intestine. Although human tooth regeneration is limited, a number of animals have evolved continuously growing teeth that provide models of stem cell-based organ renewal. A well-studied model is the mouse incisor, which contains dental epithelial stem cells in structures known as cervical loops. These stem cells produce progeny that proliferate and migrate along the proximo-distal axis of the incisor and differentiate into enamel-forming ameloblasts. Here, we studied the role of E-cadherin in behavior of the stem cells and their progeny. Levels of E-cadherin are highly dynamic in the incisor, such that E-cadherin is expressed in the stem cells, downregulated in the transit-amplifying cells, re-expressed in the pre-ameloblasts and then downregulated again in the ameloblasts. Conditional inactivation of E-cadherin in the cervical loop led to decreased numbers of label-retaining stem cells, increased proliferation, and decreased cell migration in the mouse incisor. Using both genetic and pharmacological approaches, we showed that Fibroblast Growth Factors regulate E-cadherin expression, cell proliferation and migration in the incisor. Together, our data indicate that E-cadherin is an important regulator of stem cells and their progeny during growth of the mouse incisor.     

Intercalary muscle cell renewal in planarian pharynx

 Planarian cell renewal is achieved as a result of proliferation and differentiation of totipotent undifferentiated cells called neoblasts. The absence of mitosis within the planarian pharynx raises the question as to how cell renewal and growth occur within this organ. Two explanations have been advanced: one proposes that new cells remain close to the base of the pharynx, which then grows by distal displacement of older cells, and the other suggests that the new cells are intercalated between older cells throughout the pharynx. The second alternative, however, does not explain how new cells enter the pharynx or how they reach their final destination. In this study of myosin heavy-chain gene expression within planarian pharynx, a row of differentiating myocytes was detected all along the pharynx parenchyma. According to the hybridization pattern, all these myocytes appeared to be at early stages of differentiation. These data favour an intercalary model for muscle cell renewal within the pharynx. According to this model, neoblasts at the base of the pharynx would enter the pharynx, where they would start differentiation to myocytes, move to the subepithelial musculature and intercalate between the old muscle cells. The possible application of this intercalary model to other pharynx cell types is also discussed. Received: 30 July 1998 / Accepted: 20 November 1998     

Tooth shape formation and tooth renewal: evolving with the same signals

Teeth are found in almost all vertebrates, and they therefore provide a general paradigm for the study of epithelial organ development and evolution. Here, we review the developmental mechanisms underlying changes in tooth complexity and tooth renewal during evolution, focusing on recent studies of fish, reptiles and mammals. Mammals differ from other living vertebrates in that they have the most complex teeth with restricted capacity for tooth renewal. As we discuss, however, limited tooth replacement in mammals has been compensated for in some taxa by the evolution of continuously growing teeth, the development of which appears to reuse the regulatory pathways of tooth replacement.     

Importance and regulation of adult stem cell migration

Cell migration is an essential process throughout the life of vertebrates, beginning during embryonic development and continuing throughout adulthood. Stem cells have an inherent ability to migrate, that is as important as their capacity for self‐renewal and differentiation, enabling them to maintain tissue homoeostasis and mediate repair and regeneration. Adult stem cells reside in specific tissue niches, where they remain in a quiescent state until called upon and activated by tissue environmental signals. Cell migration is a highly regulated process that involves the integration of intrinsic signals from the niche and extrinsic factors. Studies using three‐dimensional in vitro models have revealed the astonishing plasticity of cells in terms of the migration modes employed in response to changes in the microenvironment. These same properties can, however, be subverted during the development of some pathologies such as cancer. In this review, we describe the response of adult stem cells to migratory stimuli and the mechanisms by which they sense and transduce intracellular signals involved in migratory processes. Understanding the molecular events underlying migration may help develop therapeutic strategies for regenerative medicine and to treat diseases with a cell migration component.     

Does heme oxygenase-1 have a role in Caco-2 cell cycle progression?

Intestinal epithelium undergoes a rapid self-renewal process characterized by the proliferation of the crypt cells, their differentiation into mature enterocytes as they migrate up to the villi, followed by their shedding as they become senescent villus enterocytes. The exact mechanism that regulates the intestinal epithelium renewal process is not well understood, but the differential expression of regulatory genes along the crypt-villus axis may have a role. Heme oxygenase-1 (HO-1) is involved in endothelial cell cycle progression, but its role in the intestinal epithelial cell turnover has not been explored. With its effects on cell proliferation and its differential expression along the crypt-villus axis, HO-1 may play a role in the intestinal epithelial cell renewal process. In this study, we examined the role of HO-1 in the proliferation and differentiation of Caco-2 cells, a well-established in vitro model for human enterocytes. After confluence, Caco-2 cells undergo spontaneous differentiation and mimic the crypt to villus maturation observed in vivo. In preconfluent and confluent Caco-2 cells, HO-1 protein expression was determined with the immunoblot. HO-1 activity was determined by the ability of the enzyme to generate bilirubin from hemin. The effect of a HO-1 enzyme activity inhibitor, tin protoporphyrin (SnPP), on Caco-2 cell proliferation and differentiation was examined. In preconfluent cells, cell number was determined periodically as a marker of proliferation. Cell viability was measured with MTT assay. Cell differentiation was assessed by the expression of a brush border enzyme, alkaline phophatase (ALP). HO-1 was expressed in subconfluent Caco-2 cells and remained detectable until 2 days postconfluency. This timing was consistent with cells starting their differentiation and taking the features of normal intestinal epithelial cells. HO-1 was inducible in confluent Caco-2 cells by the enzyme substrate, hemin in a dose- and time-dependent manner. SnPP decreased the cell number and viability of preconfluent cells and delayed the ALP enzyme activity of confluent cells. HO-1 may be involved in intestinal cell cycle progression.     

Apoptosis as a mechanism of skin renewal: Ley-antigen expression is involved in an early event of a cell's commitment to apoptosis

Skin renewal is a typical example of the active participation of a cell in its own death process. Cells arising from mitotic activity in the stratum germinativum of the epidermis continuously migrate upwards to the stratum corneum, where dead cells are eventually desquamated. Recent studies have suggested that apoptosis is involved in the dynamic process of skin renewal. However, this still remains to be further elucidated. In this paper, we investigated the involvement of apoptosis in the skin renewal process. Changes in the morphology of cells in different epidermal layers were compared with histochemical analyses of the extent of DNA fragmentation, as determined by nick end-labelling, and of the reactivities to a monoclonal antibody directed to Le^y-antigen, difucosylated type 2 chain determinant, which has a close association with apoptosis, and to a monoclonal antibody directed to the proliferating cell nuclear antigen. The results show that apoptosis proceeds concomitantly with cell movement in the epidermis. It seems likely that commitment of a cell to death by apoptosis occurs in the epidermal tissue immediately after completion of cell proliferation, and that Le^y-antigen expression may be involved in the entire apoptotic process including this early event.     

Spatial distribution and differentiation potential of stem cells in hatchlings and adults in the marine platyhelminth macrostomum sp.: a bromodeoxyuridine analysis

Stem cells (neoblasts) in Platyhelminthes are pluripotent, and likely totipotent, undifferentiated cells which retain throughout adult life the capacity to proliferate and from which all somatic cells as well as the germ cells derive. However, basic data on the pool and heterogeneity of neoblasts, their rates of differentiation into sets and subsets of differentiated cells, and their migration to different body regions are still lacking. To fill this gap, S-phase cells in the macrostomid Macrostomum sp. were labeled with the thymidine analog 5-bromo-2'-deoxyuridine (BrdU). S-phase cells were found to be neoblasts and to be distributed in two bands along the lateral sides of the body leaving unlabeled the median axis of the body and the region anterior to the eyes. This distribution is parallel to that of mitotic cells demonstrated using an antibody to phosphorylated histone H3. At different chase times, clusters of BrdU-labeled cells appear, labeled cells migrate to formerly unlabeled areas, and they differentiate into several somatic cell types and into germ cells. Finally, continuous exposure to BrdU shows an extensive renewal of the epithelial cells. Altogether, these results strengthen the idea of platyhelminth neoblasts as an unparalleled stem-cell system within the Animal Kingdom calling for further investigation.     

Cell renewal in the epithelium of the alimentary tract of the larval lamprey,Petromyzon marinus L.

Light microscopic autoradiography with ³H-thymidine demonstrates that the three regions of the alimentary tract in the larval (ammocoete) lamprey, Petromyzon marinus L., possess different patterns for renewing their epithelium. In the oesophagus, columnar and mucous cells originate from stem cells located at the bases of folds and migrate to the tops of the folds where they are apparently extruded. Ciliated cells, located only at the tops of the folds, seem to differentiate from migrating columnar cells. In the anterior intestine, stem cells are present throughout the epithelium so that there is limited migration of cells and their extrusion occurs randomly. In the posterior intestine, the stem cells located at the bases of the typhlosole provide a continuous population that differentiates and migrates to the top of the typhlosole and to the opposite epithelial wall where they are presumably extruded. The rates of cell renewal in all three epithelial regions of the alimentary tract are slower in animals maintained at 10 ± 1°C compared with those kept at 21 ± 1°C. Comparatively, ammocoetes have the least specialized system for cell renewal known in the alimentary tract of a vertebrate.     

Growth factor-extracellular matrix synergy in the control of trophoblast invasion

At the periphery of the human placenta, trophoblast attaches to the uterine wall. The tissue interface contains many anchoring sites, with cytotrophoblast columns that form bridges between the overlying extraembryonic (villous) mesenchyme and the maternal decidual stroma beneath. From the periphery of these columns, large numbers of trophoblast cells detach, migrate through the decidua and eventually colonize and transform maternal arteries. In this way the placenta increases and gives priority to the maternal blood supply to the conceptus. We have shown that when early villous tissue is explanted on a collagen gel in serum-free medium, anchoring-site morphogenesis occurs. Thus, in the presence of placental mesenchyme but in the absence of maternal cells, contact with a permissive extracellular matrix (ECM) is necessary and sufficient for cytotrophoblast column development. Proliferation of trophoblast occurs, followed by differentiation into a columnar cell phenotype in which cells remain attached to one another and to the ECM. At this stage, interaction between fibronectin and integrin alpha5beta1 at the cell surface stabilizes the column and the cells remain as a contiguous multilayered sheet. However, the addition of serum-free conditioned medium from first-trimester placental fibroblasts stimulates cytotrophoblast to detach from the distal column and migrate in streams across the ECM. The removal of insulin-like growth factor I (IGF-I) from the fibroblast medium decreases streaming activity, whereas the addition of exogenous IGF-I (10 ng/ml) to serum-free medium produces a streaming phenotype. In contrast, transforming growth factor beta1 (10 ng/ml) maintains the cells in a tight sheet. These results suggest the possibility of a paracrine interaction between villous mesenchyme and cytotrophoblast in anchoring sites to stimulate the infiltration of the maternal ECM by trophoblast. Such a mechanism would be self-limiting because the signal diminishes with distance from the placenta.     

Old colony Mennonites in Mexico: Migration and inbreeding

Abstract

Among Canadian Mennonites whose ancestors left Flanders in the sixteenth century, one group separated as the Old Colony and part migrated in the 1920's from Canada to Mexico, where they constitute a large inbred isolate. In 1967, survey data including migration histories were collected on one‐third of the households in one subdivision, the “M Colony.” Church records were copied that gave vital statistics and surnames of almost all families in the Colony since the migration. Recent migration patterns show marriage restricted by distance and 37 per cent of resident married men remaining in their village of birth; but male migration history for those who migrate within the M Colony shows almost no effect of distance. Analysis of surnames gives an estimate of cumulative inbreeding of F = 0.0096, which is consistent with the individually estimated components, namely, founder effect, historical population constrictions, and slow genetic drift.     

BM stem cells and cardiac repair: where do we stand in 2004?

Adult BM stem cells are being investigated for their potential to regenerate injured tissues by a process referred to as plasticity or transdifferentiation. Although data supporting stem cell plasticity is extensive, a controversy has emerged based on findings that propose cell-cell fusion as a more appropriate interpretation for this phenomenon. A major focus of this controversy is the claim that acutely infarcted myocardium in adult hearts can be regenerated by BM stem cells. Many researchers consider the adult heart to be a post-mitotic organ, whereas others believe that a low level of cardiomyocyte renewal occurs throughout life. If renewal occurs, it may be in response to cardiac stem cell activity or to stem cells that migrate from distant tissues. Post-mortem microscopic analysis of experimentally induced myocardial infarctions in several rodent models suggests that cardiomyocyte renewal is achieved by stem cells that infiltrate the damaged tissue. For a better understanding of the possible involvement of stem cells in myocardial regeneration, it is important to develop appropriate technologies to monitor myocardial repair over time with an emphasis on large animal models. Studies on non-human primate, swine and canine models of acute myocardial infarctions would enable investigators to utilize clinical quality cell-delivery devices, track labeled donor cells after precision transplantation and utilize non-invasive imaging for functional assays over time with clinical accuracy. In addition, if stem cell plasticity is to reach the next level of acceptance, it is important to identify the environmental cues needed for stem cell trafficking and to define the genetic and cellular mechanisms that initiate transdifferentiation. Only then will it be possible to determine if, and to what extent, BM stem cells are involved in myocardial regeneration and to begin to regulate precisely tissue repair.