Stimulation of cellular proliferation and differentiation in the intact and regenerating planarian Dugesia(G) tigrina by the neuropeptide substance P

The neuropeptide substance P (SP) is shown to be a potent mitogen for intact and regenerating planarians. At nanomolar concentrations, SP markedly enhances cellular proliferation causing an increase in the mitotic index and in the number of blastema cells. Moreover, albeit to a lower extent, SP enhances cellular differentiation as shown by the increases in eye and pharynx length in regenerating organisms. On the basis of these observations, we hypothesize that SP may be one of the postulated growth factors necessary for the stimulation of proliferation, and to a lesser extent differentiation of cells in intact and regenerating planarians.     

DNA reproduction in the organism of planarians in post-injury recovery]

No mitotic figures were found during histological observations of intact as well as transsected planarians Dendrocoelum lacteum within 15 days after the operation. DNA content in intact and regenerating worms was measured. It has been shown, that the posttraumatic repair in planarians does not involve DNA synthesis or cell reproduction.     

In vitro development of blastema cells in axolotl limb regeneration: effect of insulin and nerve extracts on cellular proliferation

For the purpose of investigating the nature of the nervous factor which controls cell proliferation in limb blastema of Newts, we have cultured primary mesenchymous cells from limb blastemas of Axolotl. The cultures were carried out in Petri dishes (Primaria, Falcon) with a basal medium with contained diluted MEM supplemented with hormones (insulin, somatotropin, hydrocortisone and thyroxine). In this medium, the cells disperse from the explant from the 4th day of culture and begin to divide from the 7th day; 3 weeks later the culture begins to decline. During the course of culture, beginning at the 8th day, differentiation of myotubes and chondrogenesis occur. The mitotic index, measured on the 16th day after 48 hr of colchicine treatment, is about 1.6%. Addition of foetal calf serum to the basal medium favours cell migration and survival and stimulates proliferation (mitotic: index 6%); beef embryo extract has no effect on cell migration and a small effect on proliferation (mitotic index: 2.3%). Addition to the basal medium of insulin or nerve extracts (brain and spinal cord of adult newts, brain of 12 days chick embryos) 6 days before we measure the mitotic index stimulates proliferation in proportion to the dose, up to 6 times the mitotic index in basal medium. These results are discussed with respect to the problem of cell proliferation control during limb regeneration.     

Stimulation in cell culture of mesenchymal cells of newt limb blastemas by EDGF I or II (basic or acidic FGF)

After amputation of a newt limb, a blastema forms on the amputation plane and later differentiates to regenerate all the missing parts of the limb. Proliferation of blastema cells is under the control of severed nerves which deliver a 'neurotrophic factor' (NTF) of unknown nature. In order to characterize this factor we use a primary culture of blastema mesenchymal cells; changes in mitotic index after 48-h colchicine treatment indicate mitogenic activity of potential growth substances. These cells, which are stimulated by nerve extracts (mitotic index X 6), were tested with two purified growth factors extracted from bovine retina or brain (EDGF I = basic FGF and EDGF II = acidic FGF). We show that these two growth factors stimulate proliferation of blastema cell cultures in a dose-dependent manner. Maximal stimulation was obtained at 3 pM for EDGF I (mitotic index X 5.7) or 300 pM for EDGF II (mitotic index X 4.9). So it appears that these two growth factors have a mitogenic activity on blastema mesenchymal cells similar to that obtained with nerve extracts. The fact that two different growth factors can stimulate these cells raises the question of whether both are present in NTF and/or whether there are receptors to both EDGF I and EDGF II on mesenchymal cell membranes.     

Soluble age-related factors from skeletal muscle which influence muscle development

Successful regeneration of damaged striated muscle in adult mice is dependent on the regeneration of newly differentiated myofibers from proliferating satellite cells and inhibition of scar tissue formation by fibroblasts. As with most tissues, the ability of skeletal muscle to regenerate decreases in older animals. In this study, we have analysed soluble extracts from intact and regenerating skeletal muscle from mice of different ages for their ability to affect avian myogenesis in tissue culture. We were interested in determining whether an age-dependent difference could be detected with this tissue culture bioassay system. Total cell proliferation in the cultures, measured by [3H]thymidine incorporation was increased equally by muscle extracts from both young and older mice but the resulting cell populations differed in proportion of cell types. The ratio of myoblasts to fibroblasts was significantly greater in cultures exposed to extracts from younger mouse muscle as compared with cultures exposed to extracts from older animals. This age-related activity was found to reside in a low molecular weight (MW) (greater than 12 kD) component of the extract. This fraction had dissimilar effects on myoblasts and fibroblasts. Relative to saline controls, myoblast proliferation was increased and fibroblast proliferation decreased. The low MW fraction from younger mouse muscle extracts stimulated myogenic cell proliferation and myotube formation to a greater extent than the similar fraction prepared from older mouse muscle. Conversely, younger mouse muscle fractions had significantly greater inhibitory activity against fibroblast proliferation than did older mouse muscle fractions.     

Selective control of fibroblast proliferation and its effect on cardiac muscle differentiation in vitro

The stability of the differentiated state of cardiac myocytes in vitro was examined under culture conditions which selectively stimulated or inhibited proliferation of fibroblasts. Regulation of fibroblast proliferation in cultures of myocardial cells from 8-day embryonic chicks was achieved by adjustment of the glutamine (Gln) concentration in the culture medium (Ham's F-12 medium containing 2 x amino acids and 5% fetal calf serum). Myocardial cells, when plated at 80 cells/mm2 in Gln- medium, maintained a stable density of approximately 40% of the plating density for more than 30 days. When Gln was added to the medium (292 micrograms/ml) fibroblast proliferation was stimulated, and by 5-6 days after this addition cell densities had increased to confluency. The selective action of glutamine on fibroblast proliferation was determined by labeling cultures with tritiated thymidine ([3H]TdR) and scoring its incorporation into myocytes and fibroblasts by radioautography. After 2 weeks in Gln- medium, the mitotic index was 0.3% and the [3H]TdR-labeling index (1.5-hr pulse) was 6.4%. In addition, the proportion of myocytes in the population was constant at 64.2% for at least 30 days in vitro, and contractile activity was observed for up to 6 months. After 5 days of Gln replacement, the cells exhibited a labeling index of 25%, the proportion of myocytes decreased to less than 10% and contractile activity was rarely observed. Although the [3H]TdR-labeling index of fibroblasts and myocytes was nearly identical in Gln- medium, the addition of Gln produced a fivefold stimulation in the fibroblast labeling index, but did not affect myocyte proliferation or DNA synthesis. A unique phenomenon of myocyte congregation was observed only in Gln- medium which resulted in the formation of myocyte colonies from which fibroblasts were largely absent. It is suggested that this process with the resultant establishment of a functional electrical syncytium plays a significant role in the development and stabilization of myocyte differentiation in vitro.     

Role of c-Jun N-terminal kinase activation in blastema formation during planarian regeneration

The robust regenerative abilities of planarians absolutely depend on a unique population of pluripotent stem cells called neoblasts, which are the only mitotic somatic cells in adult planarians and are responsible for blastema formation after amputation. Little is known about the molecular mechanisms that drive blastema formation during planarian regeneration. Here we found that treatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 blocked the entry of neoblasts into the M-phase of the cell cycle, while allowing neoblasts to successfully enter S-phase in the planarian Dugesia japonica. The rapid and efficient blockage of neoblast mitosis by treatment with the JNK inhibitor provided a method to assess whether temporally regulated cell cycle activation drives blastema formation during planarian regeneration. In the early phase of blastema formation, activated JNK was detected prominently in a mitotic region (the "postblastema") proximal to the blastema region. Furthermore, we demonstrated that undifferentiated mitotic neoblasts in the postblastema showed highly activated JNK at the single cell level. JNK inhibition by treatment with SP600125 during this period caused a severe defect of blastema formation, which accorded with a drastic decrease of mitotic neoblasts in regenerating animals. By contrast, these animals still retained many undifferentiated neoblasts near the amputation stump. These findings suggest that JNK signaling plays a crucial role in feeding into the blastema neoblasts for differentiation by regulating the G2/M transition in the cell cycle during planarian regeneration.     

Does proliferation capacity of lymphocytes depend on human blood types?

In vitro human lymphocyte culture methodology is well established yet certain confounding factors such as age, medical history as well as individual’s blood type may potentially modulate in vitro proliferation response. These factors have to be carefully evaluated to release reliable test report in routine cytogenetic evaluation for various genetic conditions, radiation biodosimetry, etc. With this objective, the current study was focused on analyzing the proliferation response of lymphocytes drawn from 90 individuals (21-29 years) with different blood types. The proliferation response was assessed in the cultured lymphocytes by cell cycle, mitotic index (MI), and nuclear division index (NDI) after stimulation with phytohaemagglutinin (PHA). To investigate the toxic effect on proliferation, MI was calculated in representative samples of each blood type were X-irradiated. The results showed that there was no significant difference among the cell cycle phases of lymphocytes in different blood types (P > 0.05). Similarly, both MI and NDI of lymphocytes derived from different blood types also did not show significant difference ( P > 0.05). The extensive interindividual variation within and among the blood types is likely responsible for the lack of significant difference in lymphocyte proliferation. Although spontaneous proliferation efficiency of lymphocytes of different blood types after PHA stimulation was grossly similar, the MI observed after radiation exposure showed a significant difference ( P < 0.05) indicating a differential proliferation response among the blood types. Our results suggest that the blood types did not have any impact on PHA-induced proliferation; however, a specific differential lymphocyte proliferation observed after radiation exposure needs to be considered.     

Influence of pinealectomy on the mitotic activity of regenerating adrenal cortex in rats

There is some evidence that the pineal gland may influence the proliferation of both normal and neoplastic cells. The adrenal cortex has very high capacity for regeneration. Therefore, the effects of pinealectomy on the mitotic activity of regenerating adrenal cortex of rats were studied on the second, seventh and twelfth days following the enucleation of adrenals. Pinealectomy caused a significant decrease in the mitotic index of regenerating adrenal cortex after 2 and 12 days in comparison to sham operated controls.     

Involvement of plasma membrane glycoproteins in the contact-dependent inhibition of growth of human fibroblasts

The human embryonal lung fibroblasts used in this study showed a pronounced inhibition of growth when reaching a critical cell density. This effect has been mimicked by the addition of glutaraldehyde-fixed human fibroblasts to sparsely seeded growing cells. Inhibition of growth was not observed when glutaraldehyde-fixed cells were pretreated with galactosidase or with galactose-specific lectins, or when glutaraldehyde-fixed human or rabbit erythrocytes were added to the proliferating fibroblasts. In addition, glutaraldehyde-fixed mitotic cells were without effect on the proliferation, while cells prepared from sparse culture had lesser potency than cells prepared from confluent cultures. Plasma membranes, isolated from cells of confluent cultures, when added to growing cultures of human fibroblasts inhibited DNA synthesis in a concentration-dependent manner. On the other hand, plasma membranes isolated from sparsely seeded cells had only minor inhibitory potency. When the plasma membranes were isolated from cells treated previously with tunicamycin, an antibiotic which inhibits the synthesis of the oligosaccharide portion of asparagine-linked glycoproteins, the inhibitory effect was abolished. The same effect was observed when plasma membranes were pretreated with galactosidase. These data indicate that the growth of cells in vitro is regulated by specific cell-cell contacts. They also show that one of the molecular reactants in this process are membrane glycoproteins with asparagine-linked oligosaccharides.     

Growth-promoting effects of iron- and cobalt- protoporphyrins on cultured embryonic cells

The effects of hemin (Fe-protoporphyrin) and Co-protoporphyrin on cellular growth have been investigated principally in cultured fibroblasts, but also in myoblasts and hepatocytes from chick embryos. In the presence of horse serum in the culture medium, which by itself did not stimulate cell growth appreciably, Co-protoporphyrin stimulated cell attachment while hemin stimulated cell proliferation of fibroblasts. When Co-protoporphyrin and hemin were added together, the most potent stimulation of cell growth, consisting of increases in cell attachment and rapid cell proliferation, was observed. These findings indicate that the two metalloporphyrins have differential and complementary effects on cellular growth in culture, with synthetic Co-protoporphyrin principally affecting cellular attachment and Fe-protoporphyrin stimulating cellular proliferation.     

Evidence for the presence of inhibitors of mitotic factors during G1 period in mammalian cells

Our earlier studies indicated that the mitotic factors, which induce germinal vesicle breakdown and chromosome condensation when injected into fully grown Xenopus oocytes, are preferentially associated with metaphase chromosomes and that they bind to chromatin as soon as they are synthesized during the G2 phase. In this study, we attempted to determine the fate of these factors as the cell completes mitosis and enters G1. Extracts from HeLa cells at different points during G1, S, and G2 periods were mixed with mitotic extracts in various proportions, incubated, and then injected into Xenopus oocytes to determine their maturation-promoting activity. The maturation-promoting activity of the mitotic extracts was neutralized by extracts of G1 cells during all stages of G1 but not by those of late S and G2 phase cells. Extracts of quiescent (G0) human diploid fibroblasts exhibited very little inhibitory activity. However, UV irradiation of G0 cells, which is known to cause decondensation of chromatin, significantly enhanced the inhibitory activity of extracts of these cells. These factors are termed inhibitors of mitotic factors (IMF). They seem to be activated, rather than newly synthesized, as the cell enters telophase when chromosomes begin to decondense. The IMF are nondialyzable, nonhistone proteins with a molecular weight of greater than 12,000. Since mitotic factors are known to induce chromosome condensation, it is possible that IMF, which are antagonistic to mitotic factors, may serve the reverse function of the mitotic factors, i.e., regulation of chromosome decondensation.     

The power of regeneration and the stem-cell kingdom: freshwater planarians (Platyhelminthes)

The great powers of regeneration shown by freshwater planarians, capable of regenerating a complete organism from any tiny body fragment, have attracted the interest of scientists throughout history. In 1814, Dalyell concluded that planarians could "almost be called immortal under the edge of the knife". Equally impressive is the developmental plasticity of these platyhelminthes, including continuous growth and fission (asexual reproduction) in well-fed organisms, and shrinkage (degrowth) during prolonged starvation. The source of their morphological plasticity and regenerative capability is a stable population of totipotent stem cells--"neoblasts"; this is the only cell type in the adult that has mitotic activity and differentiates into all cell types. This cellular feature is unique to planarians in the Bilateria clade. Over the last fifteen years, molecular studies have begun to reveal the role of developmental genes in regeneration, although it would be premature to propose a molecular model for planarian regeneration. Genomic and proteomic data are essential in answering some of the fundamental questions concerning this remarkable morphological plasticity. Such information should also pave the way to understanding the genetic pathways associated with metazoan somatic stem-cell regulation and pattern formation.     

Analyses for in vitro growth conditions,cytokinetics, and sister chromatid exchanges in lymphocytes cultured from the Sprague-Dawley rat

Summary Peripheral blood samples from Sprague-Dawley rats gave successful lymphocyte growth in GIBCO: IA, RPMI 1640, and Eagle's minimum essential medium (MEM) culture media. Various growth conditions, cytokinetics, and sister chromatic exchange (SCE) induction were studied using reconstituted GIBCO 1A only. Neither methoxyflurane anesthesia of the rats before sampling nor washing of the cells with phosphate buffered saline affected the mitotic index. Cultures treated with [³H]thymidine showed the lymphocytes entering into DNA synthesis after approximately 24 h. The time at which BUdR (5-bromo-2′ deoxyuridine) was added, i.e. 0 vs. 24 h incubation, had minimal effect on the mitotic index of cultures harvested at 48 h. However, when harvest was extended to 72 h, mitotic activity was greater in the cultures treated with BUdR at 24 h. No significant differences in mitotic index and the number of average lymphocyte division were detected in cultures exposed to 0.3 to 0.5 μg/ml BUdR at 24 h and harvested at 72 h. Although SCE frequencies increased in the presence of BUdR, the baseline level of SCEs was estimated to be 5 to 6/cell. Average generation time of the lymphocytes dividing between 48 and 72 h was 16.5 h. Because of its simplicity of culture and the reproducible nature of its in vitro growth kinetics, the Sprague-Dawley rat lymphocyte is a suitable model for cytogenetic investigations.     

Simple method of calculating the average duration of mitosis by using a radiation method

On the mitotic index curve of the cellular population following radiation there is a linear part. The time interval between the initial decrease of the mitotic index and the extrapolation of the straight part of the curve to X-axes is the mean duration of mitosis. The values of this parameter for the culture of fibroblasts obtained by this method are practically identical with those obtained by a method based on determination of the mitotic index and the doubling time of the cell count.     

Inhibiting effect of a hepatoma extract on the mitotic rate of regenerating liver

Aqueous tumor extracts were prepared by the homogenization of a fast-growing, undifferentiated, transplantable malignant murine hepatoma in distilled water. After centrifugation, an aliquot of 0.01 ml of the supernatant g body weight was injected intraperitoneally into partially hepatectomized mice. Control animals were injected with saline. Groups of mice were killed at various times in relation to the hepatectomy. Four h before killing the animals were given Colcemid (1 microgram/g body weight). The number of Colcemid-arrested mitoses in the hepatocytes and in the littoral cells, respectively, were counted in 140 microscopic fields. The extract significantly inhibited the mitotic rate in hepatocytes when the injection was given between 22 h before, and up to 26 h after hepatectomy. In the littoral cells, a slight initial stimulation was followed by a slight but significant inhibition which occurred when the injection was given at hepatectomy or until 18 h after hepatectomy. The effect was not modified by exposing the extracts to temperatures of 47 degrees C for 30 min or 22 degrees C for 24 h, but 10 min of boiling destroyed their inhibitory effect. Lyophilization and storing at -18 degrees C for up to 4 weeks did not modify the effect. The mitosis-inhibiting effect was also measurable when the extract was injected subcutaneously. There was an almost linear dose-response curve. The results are discussed in relation to circadian rhythms, the pattern of liver cell proliferation after hepatectomy, and recent similar reports from the literature. The conclusion is drawn that extracts of a hepatoma contain one or more growth-inhibitory factors significantly active on regenerating liver cells, and less significantly on littoral cells.     

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.     

Preferable mitogenicity of beetle lectin, Allo a, for the blood cell culture of macaques and its influence on apoptosis

Lectins with mitogenic properties on lymphocytes are requisite for chromosome research or cell biology using peripheral blood cells. Though some lectins have been used as mitogens to stimulate lymphocytes in chromosome research of macaques, those reagents have not always been optimum. At this time, an attempt was made to test the mitogenicity of lectin Allo A purified from the beetle in Japanese and Rhesus monkeys. Medium with Allo A produced a significantly higher metaphase frequency and cell proliferation (p=0.022) than that with Con A, which has previously yielded a higher quality. Dose effect experiments with ten monkeys revealed that 10 μg/ml was the optimum dose to obtain higher proliferation in both lectins. Though intact blood samples usually have many apoptotic cells, production of an optimum mitogen can be reduced in cell culture as an antagonistic result of higher proliferation. In this connection as well, Allo A was superior to Con A. Thus, Allo A is probably the most useful lectin found so far for obtaining a higher mitotic index of lymphocytes in Old World monkeys.     

Cytogenetic evaluations in human lymphocytes exposed to methyl acetimidate, a lysine-specific protein crosslinking agent

The cytogenetic effects of methyl acetimidate (MAI), a lysine-specific protein crosslinking reagent, were investigated using human peripheral lymphocytes in culture. Lymphocytes were treated with the chemical either prior to PHA exposure or 2-3 days following mitogenic stimulation and assessed for perturbations in cellular proliferation and induction of SCEs. Severe reductions in the mitotic index (MI) and pronounced decreases in the proportion of metaphases proceeding beyond M(1) were observed following G0 exposure to MAI concentrations of as low as 2 mM; with complete suppression of mitotic activity in all cultures exposed to levels of 3 mM MAI or greater. Concentrations resulting in severe depression in MI caused only moderate increases in SCEs. Cells exposed to less than 10 mM MAI during the late S-G2 stages of the cell cycle and harvested at the first metaphase following treatment exhibited profound mitotic delay, impaired prophase to metaphase transitions and abnormal mitotic configurations. These findings demonstrate that protein-specific crosslinking agents may induce a wide spectrum of adverse cytogenetic outcomes in both cycling and noncycling lymphocytes.     

TGF-β1 stimulates cultured human fibroblasts to proliferate and produce tissue-like fibroplasia: A fibronectin matrix-dependent event

During wound repair, fibroblasts accumulate in the injured area until any defect is filled with stratified layers of cells and matrix. Such fibroplasia also occurs in many fibrotic disorders. Transforming growth factor-β (TGF-β), a promotor of granulation tissue in vivo and extracellular matrix production in vitro, is expressed during the active fibroplasia of wound healing and fibroproliferative diseases. Under usual tissue culture conditions, normal fibroblasts grow to confluence and then cease proliferation. In this study, culture conditions with TGF-β1 have been delineated that promote human fibroblasts to grow in stratified layers mimicking in vivo fibroplasia. When medium supplemented with serum, ascorbate, proline, and TGF-β was added thrice weekly to normal human dermal fibroblasts, the cells proliferated and stratified up to 16 cell layers thick within the culture dish, producing a tissue-like fibroplasia. TGF-β stimulated both DNA synthesis as measured by 1H-thymidine uptake and cell proliferation as measured by a Hoechst dye DNA assay in these postconfluent cultures. The stratification was dependent on fibronectin assembly, as demonstrated by anti-fibronectin antibodies which inhibited both basal and TGF-β-stimulated cell proliferation and stratification. Suppression of collagen matrix assembly in cell layers with β-amino-proprionitrile (BAPN) did not inhibit basal or TGF-β stimulated in vitro fibroplasia. BAPN did not interfere with fibronectin matrix assembly as judged by immunofluorescence microscopy. Thus, in concert with serum factors, TGF-β stimulates postconfluent, fibronectin matrix-dependent, fibroblast growth creating a fibroplasia-like tissue in vitro. J Cell Physiol 170:69–80, 1997 © 1997 Wiley-Liss, Inc.