Murine Müller cells are progenitor cells for neuronal cells and fibrous tissue cells

Mammalian Müller cells have been reported to possess retinal progenitor cell properties and generate new neurons after injury. This study investigates murine Müller cells under in vitro conditions for their capability of dedifferentiation into retinal progenitor cells. Müller cells were isolated from mouse retina, and proliferating cells were expanded in serum-containing medium. For dedifferentiation, the cultured cells were transferred to serum-replacement medium (SRM) at different points in time after their isolation. Interestingly, early cell passages produced fibrous tissue in which extracellular matrix proteins and connective tissue markers were differentially expressed. In contrast, aged Müller cell cultures formed neurospheres in SRM that are characteristic for neuronal progenitor cells. These neurospheres differentiated into neuron-like cells after cultivation on laminin/ornithine cell culture substrate. Here, we report for the first time that murine Müller cells can be progenitors for both, fibrous tissue cells and neuronal cells, depending on the age of the cell culture.     

Metastatic tumor cells – genotypes and phenotypes

Background

Metastasis is the primary cause of mortality in cancer patients. Therefore, elucidating the genetics and epigenetics of metastatic tumor cells and the mechanisms by which tumor cells acquire metastatic properties constitute significant challenges in cancer research.

Objective

To summarize the current understandings of the specific genotype and phenotype of the metastatic tumor cells.

Method and Result

In-depth genetic analysis of tumor cells, especially with advances in the next-generation sequencing, have revealed insights of the genotypes of metastatic tumor cells. Also, studies have shown that the cancer stem cell (CSC) and epithelial to mesenchymal transition (EMT) phenotypes are associated with the metastatic cascade.

Conclusion

In this review, we will discuss recent advances in the field by focusing on the genomic instability and phenotypic dynamics of metastatic tumor cells.

     

Glucocorticosteroids modify Langerhans cells to produce TGF-β and expand regulatory T cells

Although glucocorticosteroids (GCSs) have been used for many decades in transplantation and (auto)inflammatory diseases, the exact mechanisms responsible for their immunosuppressive properties are not fully understood. The purpose of this study was to characterize the effects of oral GCSs on the cutaneous immune response. We analyzed, by immunofluorescence staining and quantitative RT-PCR, residual skin biopsy material from a clinical study in which we had used oral GCS as positive control for determining the effects of candidate anti-inflammatory compounds on epicutaneous patch tests of Ni-allergic patients. Expectedly, oral GCS treatment led to a reduction of clinical symptoms and infiltrating leukocytes. Notably, we observed increased numbers of dermal FOXP3(+)CD25(+) T cells and epidermal Langerhans cells (LCs) that were associated with upregulated mRNA expression of TGF-β in lesions of GCS-treated Ni-allergic patients. To investigate this phenomenon further, we exposed purified LCs to GCS. They exhibited, in contrast to GCS-nonexposed LCs, 1) a more immature phenotype, 2) higher intracellular amounts of TGF-β, and 3) increased receptor activator for NF-κB expression, conditions that reportedly favor the expansion of regulatory T cells (Tregs). Indeed, we observed an enhancement of functionally suppressive FOXP3(+) T cells when CD3(+) cells were incubated with GCS-pretreated LCs. The expansion of Tregs was inhibited by TGF-β blockage alone, and their suppressive activity was neutralized by a combination of anti-TGF-β and anti-IL-10 Abs. Our data show that systemically applied GCSs endow LCs with Treg-promoting properties and thus shed new light on the mechanisms of GCS-mediated immunosuppression.     

Developmental potency of mouse primitive ectoderm cells,embryonic ectoderm cells,embryonic ectoderm cells and primordial germ cells after blastocyst injection

Developmental potency of primitive and embryonic ectoderm cells from 4.50-day to 6.25-day post-coitum (p.c.) mouse embryos and primordial germ cells from 12.50-day p.c.male genital ridges of fetal mice were studied by direct introducing them into 3.50-day p.c.blastocysts.Sixteen (61.5) overt chimaeras out of 26(50%) offsprings were obtained after transfer of 52 blastocysts injected with 4.50-day primitive ectoderm cells;four (16.0%) overt chimaeras were obtained out of 25 (51.0%) offsprings with 4.75-day primitive ectoderm cells from 49 transferred blastocysts.However,no overt chimaera was obtained with either 5.25-day or 6.25-day embryonic ectoderm cells or 12.50-day male primordial germ cells.GPI analysis of mid-gestation conceptuses developed from injected blastocysts showedthat 5.25-day embryonic ectoderm cells could only contributed to yolk sac of conceptus.Results suggested that implantation acts as a trigger for the determination of primitive ectoderm cells,and their developmental potency becomes limited within a short period of time in normal development.     

Of fat, β cells, and diabetes

The molecular mechanisms linking diet, obesity, and type 2 diabetes are still poorly understood. In a recent paper, Ohtsubo et?al. (2011) show that high lipid levels induce nuclear exclusion of Foxa2 and HNF1α in β cells, leading to impaired expression and glycosylation of proteins controlling glucose-stimulated insulin secretion.     

Neural stem cells, tumour stem cells and brain tumours: dangerous relationships?

Neural stem cells (NSC) have been implicated not only in brain development and neurogenesis but also in tumourigenesis. Brain tumour stem cells (BTSC) have been isolated from several paediatric or adult human brain tumours, however their origin is still disputed. This review discusses the normal role of NSC in the adult mammalian brain and their anatomical location. It compares the molecular characteristics and the biological behaviour of NSC/BTSC, and describes the molecular pathways involved in controlling self-renewal and maintenance of adult NSC/BTSC and brain tumour development. It also assesses the current hypotheses about the origin of BTSC and the clinical consequences.     

Electrochemical immunoassay on expression of integrin β1 on tumor cells and drug-resistant tumor cells

An electrochemical indirect competitive immunoassay protocol as a promising cytosensing strategy was developed to detect integrin β1 expression on human breast cancer MCF-7 cells and adriamycin-resistant human breast cancer MCF-7 (MCF-7/ADR) cells and quantify the cell number. Integrin α5β1 was adsorbed on the gold-nanoparticle modified glassy carbon electrode to bind integrin β1 monoclonal antibody (anti-CD29 mAb). A sandwich structure was then formed using nanocomposites which consisted of horseradish peroxidase (HRP) labeled anti-antibody and gold nanoparticles. HRP bound on the electrode surface could cause an amperometric response of the hydroquinone-H(2)O(2) system. The assembly of the sandwich structure was inhibited by tumor cells to give decreased enzyme-catalytic signals due to the capture of anti-CD29 mAb by integrin β1 on cell membranes. Under optimal conditions the relative current change (S) was proportional to the cell concentration from 1.6×10(3) to 2.0×10(6)cellsmL(-1) with a detection limit of 700cellsmL(-1). Integrin β1 expression in MCF-7/ADR cells was found to be significantly higher than that in MCF-7 cells, indicating the increased adhesion ability of MCF-7/ADR cells.     

Cultured retinal neuronal cells and Müller cells both show net production of lactate

Glucose has long been considered the substrate for energy metabolism in the retina. Recently, an alternative hypothesis (metabolic coupling) suggested that mitochondria in retinal neurons utilize preferentially the lactate produced specifically by Müller cells, the principal glial cell in the retina. These two views of retinal metabolism were examined using confluent cultures of photoreceptor cells, Müller cells, ganglion cells, and retinal pigment epithelial cells incubated in modified Dulbecco's minimal essential medium containing glucose or glucose and lactate. The photoreceptor and ganglion cells represented neural elements, and the Müller and pigment epithelial cells represented non-neural cells. The purpose of the present experiments was two-fold: (1) to determine whether lactate is a metabolic product or substrate in retinal cells, and (2) to examine the evidence that supports the two views of retinal energy metabolism. Measurements were made of lactic acid production, cellular ATP levels, and cellular morphology over 4 h. Results showed that all cell types incubated with 5 mM glucose produced lactate aerobically and anaerobically at linear rates, the anaerobic rate being 2-3-fold higher (Pasteur effect). Cells incubated with both 5 mM glucose and 10 mM lactate produced lactate aerobically and anaerobically at rates similar to those found when cells were incubated with glucose alone. Anaerobic ATP content in the cells was maintained at greater than 50% of the control, aerobic value, and cellular morphology was well preserved under all conditions. The results show that the cultured retinal cells produce lactate, even in the presence of a high starting ambient concentration of lactate. Thus, the net direction of the lactic dehydrogenase reaction is toward lactate formation rather than lactate utilization. It is concluded that retinal cells use glucose, and not glial derived lactate, as their major substrate.     

IL-6 and IFN-α from dsRNA-stimulated dendritic cells control expansion of regulatory T cells

Foxp3⁺CD4⁺ regulatory T cells (Treg) control not only autoimmunity but also the effective immune response against RNA virus infections, which produces virus-derived double-stranded RNA (dsRNA). To induce effective anti-viral immunity, it is a key issue to learn how Treg respond to dsRNA in vitro and in vivo. We here showed that synthetic dsRNA, polyI:C, caused peripheral expansion of functional Treg in a TICAM-1- and IL-6-dependent manner in vivo. PolyI:C did not expand Treg directly, but promoted the expansion of naturally occurring Treg indirectly through IL-6 produced from dendritic cells (DCs). In addition, the expansion of Treg by IL-6 was inhibited by IFN-α from polyI:C-stimulated DCs. These data suggest that the balance of IL-6 and IFN-α in the region of RNA virus infection may determine the number of peripheral Treg, which affects the effective immune responses against viruses.     

Crosstalk between glial and glioblastoma cells triggers the “go-or-grow” phenotype of tumor cells

Background

Glioblastoma (GBM), the most malignant primary brain tumor, leads to poor and unpredictable clinical outcomes. Recent studies showed the tumor microenvironment has a critical role in regulating tumor growth by establishing a complex network of interactions with tumor cells. In this context, we investigated how GBM cells modulate resident glial cells, particularly their paracrine activity, and how this modulation can influence back on the malignant phenotype of GBM cells.

Methods

Conditioned media (CM) of primary mouse glial cultures unexposed (unprimed) or exposed (primed) to the secretome of GL261 GBM cells were analyzed by proteomic analysis. Additionally, these CM were used in GBM cells to evaluate their impact in glioma cell viability, migration capacity and activation of tumor-related intracellular pathways.

Results

The proteomic analysis revealed that the pre-exposure of glial cells to CM from GBM cells led to the upregulation of several proteins related to inflammatory response, cell adhesion and extracellular structure organization within the secretome of primed glial cells. At the functional levels, CM derived from unprimed glial cells favored an increase in GBM cell migration capacity, while CM from primed glial cells promoted cells viability. These effects on GBM cells were accompanied by activation of particular intracellular cancer-related pathways, mainly the MAPK/ERK pathway, which is a known regulator of cell proliferation.

Conclusions

Together, our results suggest that glial cells can impact on the pathophysiology of GBM tumors, and that the secretome of GBM cells is able to modulate the secretome of neighboring glial cells, in a way that regulates the “go-or-grow” phenotypic switch of GBM cells.

     

Identification and characterization of small cells in the adult pancreas: potential progenitor cells?

In this report we describe the identification of a novel cell type in human and canine pancreas using tissue culture techniques. These cells, representing less than 1% of total islet cells, are of a small size (7-10 microm) and highly quiescent. They display a fairly immature morphology, which is characterized by a weakly developed protein synthesis machinery, a few mitochondria and a small number of neuroendocrine granules. These cells, which we have termed "small cells," are usually organized into small clusters, which can be identified within the islets of predominantly small size. They can also be collected as separate structures from preparations of freshly isolated islets. Immunohistochemically, small cells are positive for PDX-1, synaptophysin, insulin, glucagon, somatostatin, pancreatic polypeptide, alpha-fetaprotein and Bcl-2 and negative for cytokeratin 19 and nestin. Insulin secretion studies demonstrated that these cells secrete insulin in a glucose-responsive fashion, although do not respond to secretagogues such as IBMX and arginine as do mature beta cells. Although this study does not provide evidence of the proliferative and differentiation potential of small cells, their immature morphology, along with a small size and quiescence, let us hypothesize that these cells may serve as progenitors contributing to the islet growth.     

Bone marrow as a source of stem cells and germ cells? Perspectives for transplantation

Recent publications have suggested the existence of germ stem cells in the mouse at postnatal stages. The mechanism of de novo oocyte formation is proposed to involve a contribution from the bone marrow to the germ cell pool, via the bloodstream. Critical examination of the data underpinning these contentious claims is under way from a reproductive biology perspective but little has been said about the nature of this elusive bone marrow population with germ cell potential. Furthermore, whereas the prospect of marrow-derived germ cells may appear propitious for fertility applications, its wider impact on transplantation medicine remains to be considered. This paper examines the evidence leading to the current debate and considers the implications of such findings for the field of bone marrow transplantation. The author is indebted to the Anne McLaren Fellowship Scheme of the University of Nottingham and to the Alzheimer’s Society for their support.     

Neurite outgrowth of mature retinal ganglion cells and PC12 cells requires activity of CK1δ and CK1ε

Mature retinal ganglion cells (RGCs) do not normally regenerate severed axons after optic nerve injury and show only little neurite outgrowth in culture. However, RGCs can be transformed into an active regenerative state after lens injury (LI) enabling these neurons to regrow axons in vitro and in vivo. In the current study we investigated the role of CK1δ and CK1ε activity in neurite outgrowth of LI stimulated RGCs and nerve growth factor (NGF) stimulated PC12 cells, respectively. In both cell types CK1δ and ε were localized in granular particles aligned at microtubules in neurites and growth cones. Although LI treatment did not measurably affect the expression of CK1δ and ε, it significantly elevated the specific kinase activity in the retina. Similarly, CK1δ/ε specific kinase activity was also elevated in NGF treated PC12 cells compared with untreated controls. Neurite extension in PC12 cells was associated with a change in the activity of CK1δ C-terminal targeting kinases, suggesting that activity of these kinases might be necessary for neurite outgrowth. Pharmacological inactivation of CK1δ and ε markedly compromised neurite outgrowth of both, PC12 cells and LI stimulated RGCs in a concentration dependent manner. These data provide evidence for a so far unknown, but essential role of CK1 isoforms in neurite growth.