Establishment of a tetraploid cell line from mouse H-1 (ES) cells highly polyploidized with demecolcine

OBJECTIVE: Establishment of tetraploid ES cells. MATERIALS AND METHODS: Mouse H-1 (ES) cells were polyploidized by demecolcine and released from the drug. RESULTS: A tetraploid cell line (4nH1 cells) was established from mouse H-1 (ES) cells (2nH1 cells) highly polyploidized by treatment with demecolcine. Cell cycle parameters of 4nH1 cells were almost the same as those of 2nH1 cells, suggesting that the rate of DNA synthesis was about twice that of the diploid cells. Mode of chromosome number of 4nH1 cells was 76, about twice that of 2nH1 cells. Cell volume of 4nH1 cells was about twice of that of diploid cells, indicating that 4nH1 cells contained about twice as much total intracellular material as 2nH1 cells. Morphology of the 4nH1 cells was flagstone-like, thus differing from that of the spindle-shaped 2nH1 cells, suggesting that the transformation had occurred during the diploid-tetraploid transition. 4nH1 cells exhibited alkaline phosphatase activity and formed teratocarcinomas, implying that they would be pluripotent. CONCLUSION: A pluripotent tetraploid cell line (4nH1 cells) was established.     

The anticancer drug,cisplatin, increases the naturally occurring cell-mediated lysis of tumor cells

Summary It has been proposed that a component of the antitumor potential of the chemotherapeutic agent, cisplatin, resides in the host's ability to respond to cisplatintreated tumor cells. Here we report that tumor cells that are normally resistant to lysis mediated by naturally occurring cytotoxic cells showed an increased sensitivity to lysis mediated by murine spleen cells or human peripheral blood monocytes and lymphocytes when cisplatin was added at the beginning of the lytic assay. This was shown for the lysis of both murine and human tumor cells. The pretreatment of tumor cells, but not effector cells with cisplatin caused an increase in lysis in the presence of murine spleen cells or human peripheral blood leukocytes, indicating that the effect of cisplatin is to reduce resistance to lysis by these effector cells. The lysis of tumor cells by naturally occurring cytotoxic cells was blocked by antibodies specific for tumor necrosis factor. In addition, the ability of cisplatin to increase lysis was seen with cells that are sensitive to natural cytotoxic cells, but not with cells that are sensitive to natural killer cells. These results suggest that the effector cells that mediate the lysis of these tumor cells in the presence of cisplatin are likely to be natural cytotoxic cells. The ability of cisplatin to increase the lysis of tumor cells by naturally occurring cytotoxic cells indicates that these cells may be a host defense mechanism that contributes to the anticancer potential of cisplatin.     

Identification of subsets of human T cells capable of enhanced transendothelial migration.

A critical step in immunologically mediated inflammation is the migration of T cells between endothelial cells of postcapillary venules and into the tissues. To determine whether specific cells are capable of transendothelial migration, T cells that had migrated through endothelial monolayers were retrieved and analyzed. To accomplish this, human umbilical vein endothelial cells (EC) were cultured to confluence on collagen gels and incubated with human T cells. T cells that were nonadherent to the EC, those that bound to the endothelium, and cells that had migrated through the endothelial monolayer and into the collagen were individually harvested and characterized. After a 4-h incubation with EC, T cells distributed themselves such that 77 +/- 2% were nonadherent, 13 +/- 2% were bound to EC, and 10 +/- 1% had migrated into the collagen. The CD4+ T cells that had migrated into the collagen were predominantly CD29bright/CD45RObright and CD45RA-. CD8+ T cells demonstrated a greater transendothelial migratory capacity than the CD4+ T cells. The migrated CD8+ T cells were mainly CD29bright but CD45RA+. Additional phenotypic analysis of the migrating cells indicated that they contained fewer cells that expressed L-selectin. Moreover the surface expression of CD7 was less dense in the T cells that had migrated than in the nonadherent T cells. Finally the T cells that migrated were not enriched for CD45RBdim T cells. Prolonging the incubation with EC to 36 h increased the number of T cells that migrated but did not alter the predominance of CD29bright T cells in the migrated population. Stimulation of EC with IL-1 or IFN-gamma also increased the number of adherent and migrating T cells, respectively, but did not alter the phenotype of the migrating cells. These results indicate that the capacity for transendothelial migration is an intrinsic ability of certain subpopulations of T cells and is related to their stage of differentiation as identified by their surface phenotype.     

Regulation of myogenic stem cell behaviour by vessel cells: The "ménage à trois" of satellite cells,periendothelial cells and endothelial cells

In skeletal muscle, satellite cells, that are responsible of muscle repair, are localized close to capillaries. Although angiogenesis is known for a long time to be crucial for muscle repair and satellite cell survival, cellular interplays between vessel cells and satellite/myogenic cells have been poorly explored. We analyzed the interrelationships between myogenic cells, endothelial cells, and periendothelial cells that includes smooth muscle cells and endomysial fibroblasts. We found that endothelial cells strongly stimulate myogenic cell growth and, inversely, myogenic cells increase angiogenesis. VEGF plays a essential role in this bidirectional interaction. On the contrary, periendothelial cells promote the return to quiescence of a subset of muscle precursor cells to quiescence that ensures self-renewal of adult muscle stem cells. We have shown that Angiopoietin-1/Tie-2 signalling controls the entry into quiescence. We propose that during muscle regeneration, i.e. while vessels are not stabilized, endothelial cells and myogenic cells interact with each other to promote both myogenesis and angiogenesis, that have been shown to be concomitant processes in several models. On the other hand, once homeostasis of muscle is reached, the proximity of satellite cells and periendothelial cells allows the responsiveness of satellite cells, that bear Tie-2 receptor, to the secretion of Angiopoietin-1 by periendothelial cells, that, in the same time, stabilize vessels by promoting quiescence of endothelial cells.     

Stochastic elimination of cancer cells

Tissues of multicellular organisms consist of stem cells and differentiated cells. Stem cells divide to produce new stem cells or differentiated cells. Differentiated cells divide to produce new differentiated cells. We show that such a tissue design can reduce the rate of fixation of mutations that increase the net proliferation rate of cells. It has, however, no consequence for the rate of fixation of neutral mutations. We calculate the optimum relative abundance of stem cells that minimizes the rate of generating cancer cells. There is a critical fraction of stem cell divisions that is required for a stochastic elimination ('wash out') of cancer cells.     

Lineage of anuran epidermal basal cells and their differentiation potential in relation to metamorphic skin remodeling

The anuran remodels the larval epidermis into the adult one during metamorphosis. Larval and adult epidermal cells of the bullfrog were characterized by determining the presence of huge cytoplasmic keratin bundles and the expression profiles of specific marker genes, namely colalpha1 (collagen alpha1 (I)), rlk (larval keratin) and rak (adult keratin). We identified four types of epidermal basal cells: (i) basal skein cells that have keratin bundles and express colalpha1 and rlk; (ii) rak+-basal skein cells that have keratin bundles and express colalpha1, rlk, and rak; (iii) larval basal cells that express rlk and rak; and (iv) adult basal cells that express rak. These traits suggested that these basal cells are on the same lineage in which basal skein cells are the original progenitor cells that consecutively differentiate into rak+-basal skein cells into larval basal cells, and finally into adult basal cells. To directly verify the differentiation potential of larval basal cells into adult ones, the mono-layered epidermis composed of larval basal cells was cultured in the presence of aldosterone and thyroid hormone. In this culture, larval basal cells differentiated into adult basal cells that reconstituted the adult epidermis. Thus, it was concluded that larval basal cells are the direct progenitor cells of the adult epidermal stem cells.     

Anergy in memory CD4+ T cells is induced by B cells

Induction of tolerance in memory T cells has profound implications in the treatment of autoimmune diseases and transplant rejection. Previously, we reported that the presentation of low densities of agonist peptide/MHC class II complexes induced anergy in memory CD4(+) T cells. In the present study, we address the specific interaction of different types of APCs with memory CD4(+) T cells. A novel ex vivo anergy assay first suggested that B cells induce anergy in memory T cells, and an in vivo cell transfer assay further confirmed those observations. We demonstrated that B cells pulsed with defined doses of Ag anergize memory CD4 cells in vivo. We established that CD11c(+) dendritic cells do not contribute to anergy induction to CD4 memory T cells, because diphtheria toxin receptor-transgenic mice that were conditionally depleted of dendritic cells optimally induced anergy in memory CD4(+) T cells. Moreover, B cell-deficient muMT mice did not induce anergy in memory T cells. We showed that B2 follicular B cells are the specific subpopulation of B cells that render memory T cells anergic. Furthermore, we present data showing that anergy in this system is mediated by CTLA-4 up-regulation on T cells. This is the first study to demonstrate formally that B cells are the APCs that induce anergy in memory CD4(+) T cells.     

Stem cells for the replacement of inner ear neurons and hair cells

Stem cells in the nervous system have some capacity to restore damaged tissue. Proliferation of stem cells endows them with self-renewal ability and accounts for in vitro formation of neurospheres, clonally derived colonies of floating cells. However, damage to the nervous system is not readily repaired, suggesting that the stem cells do not provide an easily recruited source of cells for regeneration. The vestibular and auditory organs, despite their limited ability to replace damaged cells, appear to contain cells with stem cell properties. These inner ear stem cells, identified by neurosphere formation and by their expression of markers of inner ear progenitors, can differentiate to hair cells and neurons. Differentiated cells obtained from inner ear stem cells expressed sensory neuron markers and, after co-culture with the organ of Corti, grew processes that extended to hair cells. The neurons expressed synaptic vesicle markers at points of contact with hair cells. Exogenous stem cells have also been used for hair cell and neuron replacement. Embryonic stem cells are one potential source of both hair cells and sensory neurons. Neural progenitors made from embryonic stem cells, transplanted into the inner ear of gerbils that had been de-afferented by treatment with a toxin, differentiated into cells that expressed neuronal markers and grew processes both peripherally into the organ of Corti and centrally. The regrowth of these neurons suggests that it may be possible to replace auditory neurons that have degenerated with neurons that restore auditory function by regenerating connections to hair cells.     

In vitro induction of CD25+ CD4+ regulatory T cells by the neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH)

Recently, we have found that the neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) not only suppresses IFN-gamma production, but also induces TGF-beta1 production by activated effector T cells. These alpha-MSH- treated effector T cells function as regulatory T cells in that they suppress IFN-gamma production and hypersensitivity mediated by other effector T cells. Experimental autoimmune uveoretinitis (EAU) was suppressed in its severity and incidence in mice that were injected with primed T cells activated in vitro by APC and antigen in the presence of alpha-MSH. Moreover, it appeared that alpha-MSH had converted a population of effector T cells polarized to mediate hypersensitivity into a population of T cells that now mediated immunoregulation. To characterize these alpha-MSH- treated T cells, primed T cells were TCR-stimulated in the presence of alpha-MSH in vitro and their lymphokine profile was examined. Such effector T cells displayed enhanced levels of TGF-beta1 production and no IFN-gamma or IL-10, with IL-4 levels remaining unchanged in comparison with inactivated T cells. In addition, if soluble TGF-beta receptor II was added to cocultures of alpha-MSH-treated T cells and activated Th1 cells, the alpha-MSH-treated T cells could not suppress IFN-gamma production by the Th1 cells. These results suggest that alpha-MSH induces T cells with a regulatory lymphokine pattern, and that through their production of TGF-beta1 these cells suppress other effector T cells. Examination of the alpha-MSH-treated T cells showed that alpha-MSH did not alter the phosphorylation of CD3 molecules following TCR engagement. Primed T cells express the melanocortin 5 receptor (MC5r), a receptor that is linked to an intracellular signalling pathway shared by other cytokine receptors. Blocking the receptor with antibody prevented alpha-MSH from suppressing IFN-gamma production by the activated regulatory T cells, suggesting that alpha-MSH immunoregulation is through the MC5r on primed T cells. Surface staining and cell sorting of the alpha-MSH- treated primed T cells showed that the regulatory T cells are CD25+ CD4+ T cells. From these results we find that alpha-MSH can mediate the induction of CD25+ CD4+ regulatory T cells. These regulatory T cells require specific antigen for activation, but through non-specific TGF-beta1-mediated mechanisms they can suppress other effector T cells.     

DNA stable pentaploid H1 (ES) cells obtained from an octaploid cell induced from tetraploid cells polyploidized using demecolcine

Pentaploid H1 (ES) cells (5H1 cells) were accidentally obtained through one‐cell cloning of octaploid H1 (ES) cells (8H1 cells) that were established from tetraploid H1 (ES) cells (4H1 cells) polyploidized using demecolcine. The number of chromosomes of 5H1 cells was 100, unlike the 40 of diploid H1 (ES) cells (2H1 cells), 80 of 4H1, and 160 of 8H1 cells. The durations of G₁, S, and G₂/M phases of 5H1 cells were 3, 7, and 6 h, respectively, almost the same as those of 2H1, 4H1, and 8H1 cells. The cell volume of 5H1 cells was half of that of 8H1 cells, suggesting that 5H1 cells were created through abnormal cell divisions of 8H1 cells. The morphology of growing 5H1 cells was a spherical cluster similar to that of 2H1 cells and differing from the flagstone‐like shape of 4H1 and 8H1 cells. Pentaploid solid tumors were formed from 5H1 cells after interperitoneal injection into the mouse abdomen, and they contained endodermal, mesodermal, and ectodermal cells as well as undifferentiated cells, suggesting both that the DNA content of 5H1 cells was retained during tumor formation and that the 5H1 cells were pluripotent. The DNA content of 5H1 cells was stable in long‐term culturing as 2H1 cells, meaning that 5H1 and 2H1 cells shared similarities in DNA structure. The excellent stability of the DNA content of 5H1 cells was explained using a hypothesis for the DNA structure of polyploid cells because the pairing of homologous chromosomes in 5H1 cells is spatially forbidden. J. Cell. Physiol. 223: 369–375, 2010. © 2010 Wiley‐Liss, Inc.     

Reconstitution of the vascular wall in vitro. A novel model to study interactions between endothelial and smooth muscle cells

To study the biology of the endothelium under conditions that mimic the architecture of the vessel wall, endothelial cells were grown on a collagen lattice containing a multilayer of smooth muscle cells. Light and electron microscopy of such cultures revealed a confluent monolayer of flattened endothelial cells. In co-culture, endothelial cells tend to elongate, whereas in the absence of smooth muscle cells, the endothelial cells show the polygonal morphology typical for cultures of endothelial cells grown on polystyrene substrates. As conditioned culture media of endothelial cells contain substances that may both promote or inhibit the growth of smooth muscle cells, the availability of this vessel wall model prompted us to examine to what extent endothelial cells regulate the proliferation of smooth muscle cells when these cells are maintained in co-culture. Here we show that endothelial cells suppress the proliferation of co-existing smooth muscle cells. This finding suggests that under physiological conditions the balance of the action of growth-promoting and growth-inhibiting substances produced by endothelial cells is in favour of the latter.     

Polyploidization of 2nH1 (ES) cells by K-252a and staurosporine

Mouse 2nH1 (ES) cells were examined for polyploidization using K-252a and staurosporine. Though 2nH1 cells were polyploidized by both K-252a and staurosporine, tetraploid cells, 4nH1K cells, were obtained only from cell populations exposed to K-252a. The probability of successful establishment of tetraploid cells was 2/9, suggesting that the highly polyploidized-tetraploid transition might occur infrequently. Cell cycle parameters of 4nH1K cells were almost the same as those of 2nH1 cells, suggesting that the rate of DNA synthesis was about twice that of the diploid cells. The cell volume of 4nH1K cells was about twice of that of diploid cells, indicating that 4nH1K cells contained about twice as much total intracellular material as 2nH1 cells. The morphology of the 4nH1K cells was flagstone-like, thus differing from that of the spindle-shaped 2nH1 cells, suggesting that morphological transformation occurred during the diploid-tetraploid transition. 4nH1K cells exhibited alkaline phosphatase activity and formed teratocarcinomas, implying that they were pluripotent. These characteristics of 4nH1K cells were similar to those of tetraploid 4nH1 cells that have been established through polyploidization by demecolcine, suggesting that 4nH1K and 4nH1 cells are similar irrespective of the different mechanisms of polyploidization.     

CD31 (PECAM-1)-bright cells derived from AC133-positive cells in human peripheral blood as endothelial-precursor cells

To clarify the process of endothelial differentiation, we isolated AC133(+) cells and induced the in vitro differentiation of these cells into endothelial cells. AC133(+) cells efficiently differentiated into endothelial cells when the cells were cultured on fibronectin-coated dishes in the presence of vascular endothelial growth factor. Time-course analysis of the alteration of endothelial markers on cultured AC133(+) cells revealed that the expression of CD31 (PECAM-1) on AC133(+) cells was the earliest marker among all of the tested markers. Based on the hypothesis that CD31 is an early indicator during the endothelial differentiation, we examined the relationship between CD31 expression and the ability to differentiate into endothelial cells in cells derived from AC133(+) cells. CD31-bright cells, which were sorted from cultured AC133(+) cells, differentiated more efficiently into endothelial cells than had CD31-positive or CD31-negative cells, suggesting that CD31-bright cells may be precursor cells for endothelial cells. In the present study, we identified CD31(+) cells derived from cultured AC133(+) cells that are able to differentiate to endothelial cells as precursor cells.     

Human umbilical cord-derived cells can often serve as feeder cells to maintain primate embryonic stem cells in a state capable of producing hematopoietic cells

Clinical application of human embryonic stem (ES) cells will require the establishment of methods for their culture, either in the presence or absence of human-derived feeder cells. We have tested the ability of non-immortalized cultured cells derived from human umbilical cord (HUC cells) to support ES cell culture. A primate ES cell line that had been established and maintained with mouse embryonic fibroblasts was cultured on HUC cells for >3 months (HUC-maintained ES cells). These cells retained their expression of alkaline phosphatase, SSEA-4, Oct-3/4, and to a lesser extent Nanog, but did not express Rex-1. Nevertheless, HUC-maintained ES cells could produce ectoderm-, mesoderm- and endoderm-derived cells in teratomata that they formed in immunodeficient mice. We show that HUC-maintained ES cells could give rise to hematopoietic cells, although this ability of HUC cells varied among HUC cell populations derived from different neonates. HUC cells are promising as human material with which to maintain ES cells in a state that retains their ability to produce mature cells, including hematopoietic cells.     

A role of HLA-DQ molecules of stimulator-adherent cells in the regulation of human autologous mixed lymphocyte reaction

In this study, we have found that treatment of stimulator autologous adherent cells with anti-HLA-DQ mAb resulted in markedly enhanced proliferative response of T cells in human autologous mixed lymphocyte reaction system wherein T cells were cultured with autologous adherent cells at near ratio of adherent cells to T cells in peripheral blood, in which T cells minimally proliferate. However, treatment of stimulator-adherent cells with anti-HLA class I, anti-DR and anti-DP mAb had no effect on the proliferative response of T cells under the condition. It was further observed that CD4-enriched cells could significantly proliferate in the presence of autologous adherent cells either untreated or treated with anti-DQ mAb, although treatment of adherent cells with anti-DR mAb blocked proliferative response of CD4-enriched cells. Moreover, the proliferative response of CD4-enriched cells was suppressed by addition of CD8-enriched cells in the presence of untreated adherent cells, whereas the proliferative response of CD4-enriched cells could not be suppressed by CD8-enriched cells when the adherent cells in the culture were treated with anti-DQ mAb. These observations suggest that CD8 T cells are required for suppression of proliferative response of CD4 T cells to HLA-DR molecules on autologous stimulator-adherent cells, and that HLA-DQ molecules on the surface of adherent cells play an important role in the induction of suppression by CD8 T cells.     

Nontransformed cells can normalize gap junctional communication with transformed cells

We demonstrate that the Src kinase can augment gap junctional communication between cells derived from homozygous null Cx43 knockout mice. The total conductance between Src transformed cells was nearly twice that of nontransformed cells. In addition, the unitary conductance of the majority of single channel events between transformed cells was about 35% greater than that of nontransformed cells. Analysis showed that both nontransformed and transformed cells expressed at least two populations of channels, suggesting that Src increased junctional conductance by up-regulating one population and/or by increasing the unitary conductance of another population of channels. Interestingly, the conductance displayed by heterologous pairs of transformed and nontransformed cells resembled that of nontransformed cells. The majority of single channel events between heterologous pairs shifted back to lower conductances that were exhibited by nontransformed cells. Thus, nontransformed cells can effectively "normalize" the conductance of gap junction channels expressed by adjacent tumor cells.     

Sex reversal of genetic females (XX) induced by the transplantation of XY somatic cells in the medaka,Oryzias latipes

In order to investigate the function of gonadal somatic cells in the sex differentiation of germ cells, we produced chimera fish containing both male (XY) and female (XX) cells by means of cell transplantation between blastula embryos in the medaka, Oryzias latipes. Sexually mature chimera fish were obtained from all combinations of recipient and donor genotypes. Most chimeras developed according to the genetic sex of the recipients, whose cells are thought to be dominant in the gonads of chimeras. However, among XX/XY (recipient/donor) chimeras, we obtained three males that differentiated into the donor's sex. Genotyping of their progeny and of strain-specific DNA fragments in their testes showed that, although two of them produced progeny from only XX spermatogenic cells, their testes all contained XY cells. That is, in the two XX/XY chimeras, germ cells consisted of XX cells but testicular somatic cells contained both XX and XY cells, suggesting that the XY somatic cells induced sex reversal of the XX germ cells and the XX somatic cells. The histological examination of developing gonads of XX/XY chimera fry showed that XY donor cells affect the early sex differentiation of germ cells. These results suggest that XY somatic cells start to differentiate into male cells depending on their sex chromosome composition, and that, in the environment produced by XY somatic cells in the medaka, germ cells differentiate into male cells regardless of their sex chromosome composition.     

Pluripotent stem cells from the adult mouse inner ear

In mammals, the permanence of acquired hearing loss is mostly due to the incapacity of the cochlea to replace lost mechanoreceptor cells, or hair cells. In contrast, damaged vestibular organs can generate new hair cells, albeit in limited numbers. Here we show that the adult utricular sensory epithelium contains cells that display the characteristic features of stem cells. These inner ear stem cells have the capacity for self-renewal, and form spheres that express marker genes of the developing inner ear and the nervous system. Inner ear stem cells are pluripotent and can give rise to a variety of cell types in vitro and in vivo, including cells representative of ectodermal, endodermal and mesodermal lineages. Our observation that these stem cells are capable of differentiating into hair cell-like cells implies a possible use of such cells for the replacement of lost inner-ear sensory cells.