Culture of murine mastocytes: heterogeneity of the ultrastructural aspects

In cultures of normal mouse hematopoietic cells containing mast cell growth factor develop cells with many features of mast cells. These cells seem heterogeneous with respect to size, cell surface, granules maturity and morphology of nucleus using transmission and scanning electron microscopy. Alcian blue-safranin staining shows that most of the proteoglycan synthesized by cultured mast cells is weakly sulfated and non heparin-mucopolysaccharides. These results support the view that cultured mast cells resemble to mucosal mast cells, and are clearly different from serosal mast cells.     

Presence of mast cell precursors in fetal liver of mice

When liver cells obtained from 13- to 18-day embryos of beige (Chediak-Higashi syndrome) mice were transplanted into irradiated normal adult mice, tissue mast cells with giant granules showing beige mouse origin developed in the normal recipient mice. Mast cell precursors seem to have developed earlier in the liver of embryos than mast cells themselves since no mast cells were detectable in any tissues of 13- and 14-day embryos. This result suggests that tissue mast cells originate from hematopoietic tissues not only in adult mice but also in mouse embryos.     

Noninduced single-stranded breaks in DNA in murine F9 teratocarcinoma cells

Our previous study demonstrated the high incidence of non-induced DNA single strand breaks (SSB) in preimplantation mouse embryo genom (Patkin et al., 1994). F9 mouse teratocarcinoma cell line is an in vitro model for early embryonal differentiation, since F9 cells remind in many respects the inner cell mass cells of mouse blastocyst and are capable of differentiation under retinoic acid (RA) and dibutyryl cAMP (db-cAMP) treatment. Using gap filling reaction of F9 metaphase chromosomes and single-cell DNA electrophoresis, we have observed multiple SSB in undifferentiated F9 cells as well as in F9 cells at the early steps of RA-induced differentiation (days of RA treatment), but not in terminally differentiated F9 cells and in mouse embryonal fibroblasts. Rad51 nuclear protein that binds specifically single stranded DNA is highly expressed in all cells of undifferentiated F9 population and is not expressed in terminally differentiated F9 population. Multiple SSB could lead to enhanced rate of sister chromatid exchanges (SCE) in F9 cells. In undifferentiated F9 population the level of SCE was 9.6 +/- 0.44 per metaphase, that was not higher than in NIH 3T3 cell line. However, RA treatment for 48 h led to rising the SCE level up to 16.68 +/- 0.72 followed by its decrease to the initial rate by 72 h of RA treatment. Since the enhanced level of SSB in undifferentiated F9 cells and in mouse blastocyst does not normally lead to chromosomal instability, we consider SSB to be a natural consequence of fast-going DNA replication in these cells.     

Impaired NK cell development in an IFN-gamma transgenic mouse: aberrantly expressed IFN-gamma enhances hematopoietic stem cell apoptosis and affects NK cell differentiation

Aberrant expression of IFN-gamma has been demonstrated to cause a wide variety of alterations in cell function and development. Previously we reported that constitutive expression of IFN-gamma in bone marrow (BM) and thymus results in a total absence of B cells and a substantial decrease in the number of hematopoietic progenitor cells. In this study, we demonstrate a severe deficiency of NK1.1(+)CD3(-) cells in this transgenic mouse model. Compared with normal control littermates, we found a pronounced reduction of NK cells in IFN-gamma transgenic mouse spleen and liver despite maintenance of normal function. In addition, we observed a reduced number of BM cells in the IFN-gamma transgenic mouse despite normal expression of hematopoietic growth factors in the BM. Interestingly, these cells were less responsive to stem cell factor (SCF) despite c-kit expression on hematopoietic stem cells (HSCs). We observed that addition of exogenous IFN-gamma inhibited proliferation of HSCs and differentiation of NK precursors from HSCs in normal mice in response to SCF, IL-7, fms-like tyrosine kinase 3 ligand, and IL-15. Furthermore, we found that HSCs express the IFN-gammaRalpha subunit and undergo apoptosis in response to exogenous IFN-gamma. Thus, we have demonstrated the occurrence of a severe deficiency of NK cells and lower numbers of BM cells in an IFN-gamma transgenic mouse model. Furthermore, because exogenous IFN-gamma affects the responsiveness to hematopoietic growth factors such as SCF in vitro, our results indicate that chronic expression of IFN-gamma in vivo leads to widespread immune system defects, including alterations in NK cell differentiation.     

Retinoid X receptor (RXR) agonist-induced activation of dominant-negative RXR-retinoic acid receptor alpha403 heterodimers is developmentally regulated during myeloid differentiation

The multiple biologic activities of retinoic acid (RA) are mediated through RAR and retinoid X receptor (RXR) nuclear receptors that interact with specific DNA target sequences as heterodimers (RXR-RAR) or homodimers (RXR-RXR). RA receptor activation appears critical to regulating important aspects of hematopoiesis, since transducing a COOH-terminally truncated RARalpha exhibiting dominant-negative activity (RARalpha403) into normal mouse bone marrow generates hematopoietic growth factor-dependent cell lines frozen at the multipotent progenitor (EML) or committed promyelocyte (MPRO) stages. Nevertheless, relatively high, pharmacological concentrations of RA (1 to 10 microM) overcome these differentiation blocks and induce terminal granulocytic differentiation of the MPRO promyelocytes while potentiating interleukin-3 (IL-3)-induced commitment of EML cells to the granulocyte/monocyte lineage. In the present study, we utilized RXR- and RAR-specific agonists and antagonists to determine how RA overcomes the dominant-negative activity of the truncated RARalpha in these different myeloid developmental stages. Unexpectedly, we observed that an RXR-specific, rather than an RAR-specific, agonist induces terminal granulocytic differentiation of MPRO promyelocytes, and this differentiation is associated with activation of DNA response elements corresponding to RAR-RXR heterodimers rather than RXR-RXR homodimers. This RXR agonist activity is blocked by RAR-specific antagonists, suggesting extensive cross-talk between the partners of the RXR-RARalpha403 heterodimer. In contrast, in the more immature, multipotent EML cells we observed that this RXR-specific agonist is inactive either in potentiating IL-3-mediated commitment of EML cells to the granulocyte lineage or in transactivating RAR-RXR response elements. RA-triggered GALdbd-RARalpha hybrid activity in these cells indicates that the multipotent EML cells harbor substantial nuclear hormone receptor coactivator activity. However, the histone deacetylase (HDAC) inhibitor trichostatin A readily activates an RXR-RAR reporter construct in the multipotent EML cells but not in the committed MPRO promyelocytes, indicating that differences in HDAC-containing repressor complexes in these two closely related but distinct hematopoietic lineages might account for the differential activation of the RXR-RARalpha403 heterodimers that we observed at these different stages of myeloid development.     

Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells

Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vitro translation proved that the exosome mRNAs were functional. Quality control RNA analysis of total RNA derived from exosomes also revealed presence of small RNAs, including microRNAs. The RNA from mast cell exosomes is transferable to other mouse and human mast cells. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred exosomal mRNA can be translated after entering another cell. In summary, we show that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location. We propose that this RNA is called "exosomal shuttle RNA" (esRNA).     

Regulatory mechanisms of mast cell differentiation

Mast cells are a progeny of the multipotential hematopoietic stem cell. Most of progenies of the stem cell complete their differentiation within the bone marrow, but precursors of mast cells leave the bone marrow, migrate in blood, and invade into tissues. After the invasion, precursors proliferate and differentiate into mast cells. An appreciable proportion of mast cells retain proliferative potential after differentiation, and even after degranulation, some mast cells can proliferate and recover the original morphology. Proliferation of mast cells are regulated by both T cell-derived factors (i.e., IL-3 and IL-4) and fibroblast-derived factor(s). Mice of either W/Wv or Sl/Sld genotype lack mast cells, but mast cells do develop when bone marrow cells of W/Wv or Sl/Sld mice were cultured in the presence of T cell-derived factors. Mast cells derived from W/Wv mice cannot respond fibroblast-derived factor(s) and fibroblasts derived from Sl/Sld mice cannot support mast cells of normal mouse origin. Phenotypes of mast cells are determined by the environment in which the mast cells differentiated. However, when mast cells are transplanted into a new environment which is different from the original one, the mast cells acquire the phenotype which are dependent on the second environment.     

Control of alkaline phosphatase activity in C3H10T1/2 cells: role of retinoic acid and cell density.

The enzyme alkaline phosphatase (AP) has been shown to be lost or inappropriately expressed during carcinogenesis in some tissues. Because retinoic acid (RA) appears to play a role in the normal regulation of the enzyme (RA up-regulates AP in a variety of cell types) we have suggested that altered AP expression in some cancers may be caused by a defect in the ability of the cells to respond normally to retinoid. We have begun to use the chemically transformable mouse embryo fibroblast cell, C3H10T1/2, to investigate this possibility. In this initial study we characterized AP regulation in normal C3H10T1/2 cells and show that: (1) 10(-7) M RA increases AP activity within 3-4 h in serum-free medium; (2) serum inhibits short-term induction (0-8 h) in a concentration-dependent manner (10% serum causes complete inhibition); (3) during long-term RA exposure (24 h and 48 h), induction can be detected in serum-containing medium; (4) AP induction is dose related at RA concentrations from 10(-10) M to 10(-6) M in serum-free medium; (5) 10(-5) M RA is ineffective at inducing AP in serum-free medium during 8 h but is the most effective concentration in serum-containing medium during 24 h and 48 h exposures; (6) AP inducibility by RA requires near-confluent cell densities; and (7) when cultures become confluent, cells become constitutive for AP and no longer require RA for enzyme expression. The effects of serum and cell density on AP inducibility by RA and implications of the RA up-regulation of AP for teratogenesis are discussed.     

A cloned MCGF cDNA encodes a multilineage hematopoietic growth factor: multiple activities of interleukin 3

We recently isolated from a mouse T cell cDNA library a full-length clone that encodes a mast cell growth factor (1). On the basis of sequence homologies, this cloned factor must be similar if not identical to purified interleukin 3 (IL 3) (2, 3). Here, we report the first biologic characterization of the cloned gene product expressed in COS-7 monkey cells. Our results establish that a single molecular species promotes the growth and differentiation of a wide spectrum of hematopoietic cell types, including multipotential stem cells and various committed progenitor cells. The relationship of cloned IL 3 to other colony-stimulating factors is discussed.     

Identification of a novel population of human cord blood cells with hema-topoietic and chondrocytic potential

With the exception of mature erythrocytes, cells within the human hematopoietic system are characterized by the cell surface expression of the pan-leukocyte receptor CD45. Here, we identify a novel subset among mononuclear cord blood cells depleted of lineage commitment markers (Lin-) that are devoid of CD45 expression. Surprisingly, functional examination of Lin-CD45- ceils also lacking cell surface CD34 revealed they were capable of multipotential hematopoietic progenitor capacity. Co-culture with mouse embryonic limb bud cells demonstrated that Lin^-CD45^-CD34^- cells were capable of contributing to cartilage nodules and differentiating into human chondrocytes. BMP-4, a mesodermal factor known to promote chondrogenesis, significantly augmented Lin^-CD45^-CD34^-differentiation into chondrocytes. Moreover, unlike CD34~ human hematopoietic stem cells, Lin^-CD45^-CD34^- cells were unable to proliferate or survive in liquid cultures, whereas single Lin^-CD45^-CD34^- cells were able to chimerize the inner cell mass (1CM) of murine blastocysts and proliferate in this embryonic environment. Our study identifies a novel population of Lin-CD45-CD34^- cells capable of commitment into both hematopoietic and chondrocytic lineages, suggesting that human cord blood may provide a more ubiquitous source of tissue with broader developmental potential than previously appreciated.     

Multiple apoptotic defects in hematopoietic cells from mice lacking lipocalin 24p3

The lipocalin mouse 24p3 has been implicated in diverse physiological processes, including apoptosis, iron trafficking, development and innate immunity. Studies from our laboratory as well as others demonstrated the proapoptotic activity of 24p3 in a variety of cultured models. However, a general role for the lipocalin 24p3 in the hematopoietic system has not been tested in vivo. To study the role of 24p3, we derived 24p3 null mice and back-crossed them onto C57BL/6 and 129/SVE backgrounds. Homozygous 24p3(-/-) mice developed a progressive accumulation of lymphoid, myeloid, and erythroid cells, which was not due to enhanced hematopoiesis because competitive repopulation and recovery from myelosuppression were the same as for wild type. Instead, apoptotic defects were unique to many mature hematopoietic cell types, including neutrophils, cytokine-dependent mast cells, thymocytes, and erythroid cells. Thymocytes isolated from 24p3 null mice also displayed resistance to apoptosis-induced by dexamethasone. Bim response to various apoptotic stimuli was attenuated in 24p3(-/-) cells, thus explaining their resistance to the ensuing cell death. The results of these studies, in conjunction with those of previous studies, reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Interestingly, these functions are limited to relatively mature blood cell compartments.     

Serglycin proteoglycan promotes apoptotic versus necrotic cell death in mast cells

The mechanisms that govern whether a cell dies by apoptosis or necrosis are not fully understood. Here we show that serglycin, a secretory granule proteoglycan of hematopoietic cells, can have a major impact on this decision. Wild type and serglycin(-/-) mast cells were equally sensitive to a range of cell death-inducing regimens. However, whereas wild type mast cells underwent apoptotic cell death, serglycin(-/-) cells died predominantly by necrosis. Investigations of the underlying mechanism revealed that cell death was accompanied by leakage of secretory granule compounds into the cytosol and that the necrotic phenotype of serglycin(-/-) mast cells was linked to defective degradation of poly(ADP-ribose) polymerase-1. Cells lacking mouse mast cell protease 6, a major serglycin-associated protease, exhibited similar defects in apoptosis as observed in serglycin(-/-) cells, indicating that the pro-apoptotic function of serglycin is due to downstream effects of proteases that are complex-bound to serglycin. Together, these findings implicate serglycin in promoting apoptotic versus necrotic cell death.     

Retinoic acid metabolism links the periodical differentiation of germ cells with the cycle of Sertoli cells in mouse seminiferous epithelium

Homeostasis of tissues relies on the regulated differentiation of stem cells. In the epithelium of mouse seminiferous tubules, the differentiation process from undifferentiated spermatogonia (A(undiff)), which harbor the stem cell functions, to sperm occurs in a periodical manner, known as the "seminiferous epithelial cycle". To identify the mechanism underlying this periodic differentiation, we investigated the roles of Sertoli cells (the somatic supporting cells) and retinoic acid (RA) in the seminiferous epithelial cycle. Sertoli cells cyclically change their functions in a coordinated manner with germ cell differentiation and support the entire process of spermatogenesis. RA is known to play essential roles in this periodic differentiation, but its precise mode of action and its regulation remains largely obscure. We showed that an experimental increase in RA signaling was capable of both inducing A(undiff) differentiation and resetting the Sertoli cell cycle to the appropriate stage. However, these actions of exogenous RA signaling on A(undiff) and Sertoli cells were strongly interfered by the differentiating germ cells of intimate location. Based on the expression of RA metabolism-related genes among multiple cell types - including germ and Sertoli cells - and their regulation by RA signaling, we propose here that differentiating germ cells play a primary role in modulating the local RA metabolism, which results in the timed differentiation of A(undiff) and the appropriate cycling of Sertoli cells. Similar regulation by differentiating progeny through the modulation of local environment could also be involved in other stem cell systems.     

Interleukin 3-dependent hematopoietic progenitor cell lines

Several biological phenotypes of growth factor-dependent cell lines have been described in recent years, including those with T lymphocyte, neutrophil granulocyte, basophil/mast cell, B lymphocyte, and multipotential stem cell properties. The growth factors for each cell lineage are a subject of intense study. Continuous mouse bone marrow cultures infected with RNA type C viruses (retroviruses) produce nonadherent hematopoietic cells over a longer duration than control cultures. Marrow cultures derived from strains with spontaneously induced ecotropic endogenous retrovirus demonstrate a greater longevity than those from strains with no replicating virus. Cultures infected with murine leukemia virus also generate a greater number, compared with controls, of cloned permanent suspension cell lines dependent for growth on a 41,000-dalton glycoprotein (interleukin 3 [IL 3]). Some are multipotential with capacity for differentiation to erythroid, neutrophil, eosinophil, and basophil/mast cell types. Other cloned IL 3-dependent cell lines are committed to a single pathway. Studies with Friend spleen focus-forming virus indicate that the first effect in the marrow culture is mediated through a subset of adherent hematopoietic stem cells. Bone marrow culture-derived IL 3-dependent cell lines provide a model with which to study the role of viral genes in the control of differentiation and self-renewal capacity of hematopoietic stem cells.     

Fibroblast growth factor-10 promotes cardiomyocyte differentiation from embryonic and induced pluripotent stem cells

Background

The fibroblast growth factor (FGF) family is essential to normal heart development. Yet, its contribution to cardiomyocyte differentiation from stem cells has not been systemically studied. In this study, we examined the mechanisms and characters of cardiomyocyte differentiation from FGF family protein treated embryonic stem (ES) cells and induced pluripotent stem (iPS) cells.

Methodology/Principal Findings

We used mouse ES cells stably transfected with a cardiac-specific α-myosin heavy chain (αMHC) promoter-driven enhanced green fluorescent protein (EGFP) and mouse iPS cells to investigate cardiomyocyte differentiation. During cardiomyocyte differentiation from mouse ES cells, FGF-3, -8, -10, -11, -13 and -15 showed an expression pattern similar to the mesodermal marker Brachyury and the cardiovascular progenitor marker Flk-1. Among them, FGF-10 induced cardiomyocyte differentiation in a time- and concentration-dependent manner. FGF-10 neutralizing antibody, small molecule FGF receptor antagonist PD173074 and FGF-10 and FGF receptor-2 short hairpin RNAs inhibited cardiomyocyte differentiation. FGF-10 also increased mouse iPS cell differentiation into cardiomyocyte lineage, and this effect was abolished by FGF-10 neutralizing antibody or PD173074. Following Gene Ontology analysis, microarray data indicated that genes involved in cardiac development were upregulated after FGF-10 treatment. In vivo, intramyocardial co-administration of FGF-10 and ES cells demonstrated that FGF-10 also promoted cardiomyocyte differentiation.

Conclusion/Significance

FGF-10 induced cardiomyocyte differentiation from ES cells and iPS cells, which may have potential for translation into clinical applications.     

Establishment of mouse embryonic stem cell-derived erythroid progenitor cell lines able to produce functional red blood cells

Background

The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If erythroid cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. To evaluate the feasibility of establishing useful erythroid cell lines, we attempted to establish such cell lines from mouse embryonic stem (ES) cells.

Methodology/Principal Findings

We developed a robust method to obtain differentiated cell lines following the induction of hematopoietic differentiation of mouse ES cells and established five independent hematopoietic cell lines using the method. Three of these lines exhibited characteristics of erythroid cells. Although their precise characteristics varied, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia. In addition, we did not observe formation of any tumors following transplantation of these cells.

Conclusion/Significance

To the best of our knowledge, this is the first report to show the feasibility of establishing erythroid cell lines able to produce mature RBCs. Considering the number of human ES cell lines that have been established so far, the intensive testing of a number of these lines for erythroid potential may allow the establishment of human erythroid cell lines similar to the mouse erythroid cell lines described here. In addition, our results strongly suggest the possibility of establishing useful cell lines committed to specific lineages other than hematopoietic progenitors from human ES cells.     

TGF-beta and retinoic acid: regulators of growth and modifiers of differentiation in human epidermal cells.

In the epidermis of skin, a fine balance exists between proliferating progenitor cells and terminally differentiating cells. We examined the effects of TGF-beta s and retinoic acid (RA) on controlling this balance in normal and malignant human epidermal keratinocytes cultured under conditions where most morphological and biochemical features of epidermis in vivo are retained. Our results revealed marked and pleiotropic effects of both TGF-beta and RA on keratinocytes. In contrast to retinoids, TGF-beta s acted on mitotically active basal cells to retard cell proliferation. Although withdrawal from the cell cycle is a necessary prerequisite for commitment to terminal differentiation, TGF-beta s inhibited normal keratinization in suprabasal cells and promoted the type of differentiation commonly associated with wound-healing and epidermal hyperproliferation. The actions of TGF-beta s and RA on normal keratinization were synergistic, whereas those on abnormal differentiation associated with hyperproliferation were antagonistic. These observations underscore the notion that environmental changes can act separately on proliferating and differentiating cells within the population. Under the conditions used here, the action of TGF-beta s on human keratinocytes was dominant over RA, and TGF-beta s did not seem to be induced as a consequence of RA treatment. This finding is consistent with the fact that RA accelerated, rather than inhibited, proliferation in raft cultures. Collectively, our data suggest that the effects of both factors on epidermal growth and differentiation are multifaceted and the extent to which their action is coupled in keratinocytes may vary under different conditions and/or in different species.     

Retinoic acid induces a specific membrane glycoprotein in human epithelial cell lines

Retinoic acid (RA) inhibits growth, increases the cytokeratin content, and alters the cytoskeleton of the human cervical cell line NHIK 3025. Using RA-treated NHIK 3025 cells as immunogen we prepared murine monoclonal antibodies (IgG1) which recognized an RA-induced cell-surface antigen which could not be detected in untreated NHIK 3025 cells. Analysis of the Triton soluble proteins by SDS-gel electrophoresis and immunoblotting revealed that the cell-surface antigen is a 140-kDa glycoprotein (gp140). gp140 was also shown to be induced by RA in HeLa S3 cells and constitutively expressed in the human trophoblast cell line BeWo. gp140 was also detected in other human epithelial cell lines, but not in human hematopoietic cells. Expression of gp140 was induced in HeLa S3 cells by nanomolar concentrations of RA, and in NHIK 3025 cells by micromolar amounts (1-10 microM). The glycoprotein was detectable 3-6 h following exposure to RA and its expression was reversible upon removal of RA from the medium. Our results indicate that gp140 is a newly identified RA-inducible epithelial membrane glycoprotein which may represent a phenotypic differentiation marker for epithelial cells.     

Effect of retinoic acid on the proliferation and tumorigenicity of mouse mastocytoma cells

Retinoic acid (RA) inhibited the in vitro growth of the mouse mast cell tumor line P₈₁₅ in a dose- and time-dependent manner. The inhibition was accompanied by an increase in the amount of neutral intracellular mucopolysaccharides. Study of cell cycle kinetics showed that exposure to retinoic acid led to a slowing-down of the cell-cycle progression possibly related to a more differentiated cell population disclosed by microscopy with a lower proliferative capacity. In vivo, delays in both tumor appearance and mouse mortality were observed after injecting RA into mice bearing mastocytomas. These results suggest that RA could be of interest in the treatment of human malignant systemic mastocytosis with proliferation of immature mast cells.