Embryonic stem cells express multiple Eph-subfamily receptor tyrosine kinases.

Eph and its homologues form the largest subfamily of receptor tyrosine kinases. Normal expression patterns of this subfamily indicate roles in differentiation and development, whereas their overexpression has been linked to oncogenesis. This study investigated the potential role of Eph-related molecules during very early embryonic development by examining their expression in embryonic stem (ES) cells and embryoid bodies differentiated from ES cells in vitro. By use of a strategy based on reverse transcriptase-mediated PCR, nine clones containing Eph-subfamily sequence were isolated from ES cells. Of these, eight were almost identical to one of four previously identified molecules (Sek, Nuk, Eck, and Mek4). However, one clone contained sequence from a novel Eph-subfamily member, which was termed embryonic stem-cell kinase or Esk. Northern analysis showed expression of Esk in ES cells, embryoid bodies, day 12 mouse embryos, and some tissues of the adult animal. Levels of expression were similar in ES cells and embryoid bodies. By comparison, Mek4 showed no significant transcription in the ES cell cultures by Northern analysis, whereas Eck displayed stronger signals in ES cells than in the embryoid bodies. These results suggest that Eph-subfamily molecules may play roles during the earliest phases of embryogenesis. Furthermore, the relative importance of different members of this subfamily appears to change as development proceeds.

Enhanced somatic mutation rates induced in stem cells of mice by low chronic exposure to ethylnitrosourea.

We have found that the somatic mutation rate at the Dlb-1 locus increases exponentially during low daily exposure to ethylnitrosourea over 4 months. This effect, enhanced mutagenesis, was not observed at a lacI transgene in the same tissue, although the two loci respond very similarly to acute doses. Since both mutations are neutral, the mutant frequency was expected to increase linearly with time in response to a constant mutagenic exposure, as it did for lacI. Enhanced mutagenesis does not result from an overall sensitization of the animals, since mice that had first been treated with a low daily dose for 90 days and then challenged with a large acute dose were not sensitized to the acute dose. Nor was the increased mutant frequency due to selection, since animals that were treated for 90 days and then left untreated for up to 60 days showed little change from the 90-day frequency. The effect is substantial: about 8 times as many Dlb-1 mutants were induced between 90 and 120 days as in the first 30 days. This resulted in a reverse dose rate effect such that 90 mg/kg induced more mutants when delivered at 1 mg/kg per day than at 3 mg/kg per day. We postulate that enhanced mutagenesis arises from increased stem cell proliferation and the preferential repair of transcribed genes. Enhanced mutagenesis may be important for risk evaluation, as the results show that chronic exposures can be more mutagenic than acute ones and raise the possibility of synergism between chemicals at low doses.

Targeted disruption of vinculin genes in F9 and embryonic stem cells changes cell morphology, adhesion, and locomotion.

Vinculin, a major constituent of focal adhesions and zonula adherens junctions, is thought to be involved in linking the microfilaments to areas of cell-substrate and cell-cell contacts. To test the role of vinculin in cell adhesion and motility, we used homologous recombination to generate F9 embryonal carcinoma and embryonic stem cell clones homozygous for a disrupted vinculin gene. When compared to wild-type cells, vinculin-mutant cells displayed a rounder morphology and a reduced ability to adhere and spread on plastic or fibronectin. Decreased adhesion of the mutant cells was associated with a reduction in lamellipodial extensions, as observed by time-lapse video microscopy. The locomotive activities of control F9 and the vinculin-null cells were compared in two assays. Loss of vinculin resulted in a 2.4-fold increase in cell motility. These results demonstrate an important role for vinculin in determining cell shape, adhesion, surface protrusive activity, and cell locomotion.

Mouse model of human beta zero thalassemia: targeted deletion of the mouse beta maj- and beta min-globin genes in embryonic stem cells.

beta zero-Thalassemia is an inherited disorder characterized by the absence of beta-globin polypeptides derived from the affected allele. The molecular basis for this deficiency is a mutation of the adult beta-globin structural gene or cis regulatory elements that control beta-globin gene expression. A mouse model of this disease would enable the testing of therapeutic regimens designed to correct the defect. Here we report a 16-kb deletion that includes both adult beta-like globin genes, beta maj and beta min, in mouse embryonic stem cells. Heterozygous animals derived from the targeted cells are severely anemic with dramatically reduced hemoglobin levels, abnormal red cell morphology, splenomegaly, and markedly increased reticulocyte counts. Homozygous animals die in utero; however, heterozygous mice are fertile and transmit the deleted allele to progeny. The anemic phenotype is completely rescued in progeny derived from mating beta zero-thalassemic animals with transgenic mice expressing high levels of human hemoglobin A. The beta zero-thalassemic mice can be used to test genetic therapies for beta zero-thalassemia and can be bred with transgenic mice expressing high levels of human hemoglobin HbS to produce an improved mouse model of sickle cell disease.

Modification of rhodamine staining allows identification of hematopoietic stem cells with preferential short-term or long-term bone marrow-repopulating ability.

We have developed a modified rhodamine (Rho) staining procedure to study uptake and efflux in murine hematopoietic stem cells. Distinct populations of Rho++ (bright), Rho+ (dull), and Rho- (negative) cells could be discriminated. Sorted Rho- cells were subjected to a second Rho staining procedure with the P-glycoprotein blocking agent verapamil (VP). Most cells became Rho positive [Rho-/Rho(VP)+ cells] and some remained Rho negative [Rho-/Rho(VP)- cells]. These cell fractions were characterized by their marrow-repopulating ability in a syngeneic, sex-mismatch transplantation model. Short-term repopulating ability was determined by recipient survival for at least 6 weeks after lethal irradiation and transplantation--i.e., radioprotection. Long-term repopulating ability at 6 months after transplantation was measured by fluorescence in situ hybridization with a Y-chromosome-specific probe, by graft function and recipient survival. Marrow-repopulating cells were mainly present in the small Rho- cell fraction. Transplantation of 30 Rho- cells resulted in 50% radioprotection and > 80% donor repopulation in marrow, spleen, and thymus 6 months after transplantation. Cotransplantation of cells from both fractions in individual mice directly showed that within this Rho- cell fraction, the Rho-/Rho(VP)+ cells exhibited mainly short-term and the Rho-/Rho(VP)- cells exhibited mainly long-term repopulating ability. Our results indicate that hematopoietic stem cells have relatively high P-glycoprotein expression and that the cells responsible for long-term repopulating ability can be separated from cells exhibiting short-term repopulating ability, probably by a reduced mitochondrial Rho-binding capacity.

Long-term repopulation of hematolymphoid cells with only a few hemopoietic stem cells in mice.

A PCR-based assay has been devised for the detection and semiquantitation of cells originating from a few donor hematopoietic stem cells (HSCs) in a background of recipient cells. Upon sequencing a segment of murine Y chromosome contained in the plasmid pY2, oligonucleotide primers were designed for specific amplification of the Y chromosome-restricted segment. The HSCs were isolated from the bone marrow of mice on day 4 following a single i.v. injection of 5-fluorouracil and were readily distinguished from other bone marrow elements by the characteristics of low density, absence of lineage-specific surface markers, lack of expression of transferrin receptor, and a high expression of major histocompatibility complex class I antigen. Injection of as few as four such HSCs was shown to produce donor-derived cells (including lymphoid cells) for at least 8 months after transplantation into syngeneic female recipients. Retransplantation, employing 10(6) bone marrow cells from the initial recipients, also yielded clear evidence of repopulation with detectable levels of male donor cells. On statistical grounds, it is clear that long-term repopulation in vivo may result from even a single HSC having the characteristics defined herein.

Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit, GATA-2, p45 NF-E2, and c-myb and low levels or no mRNA for c-fms and the receptors for granulocyte colony-stimulating factor and interleukins 5 and 7.

Pluripotent hematopoietic stem cells (PHSCs) were highly enriched from mouse bone marrow by counterflow centrifugal elutriation, lineage subtraction, and fluorescence-activated cell sorting based on high c-kit receptor expression (c-kitBR). We used reverse transcriptase polymerase chain reaction to assay the c-kitBR subset and the subsets expressing low (c-kitDULL) and no (c-kitNEG) c-kit receptor for expression of mRNA encoding hematopoietic growth factor receptors and transcription factors. The c-kitBR cells had approximately 3.5-fold more c-kit mRNA than unfractionated bone marrow cells. The c-kitDULL cells had 47-58% of the c-kit mRNA found in c-kitBR cells and the c-kitNEG cells had 4-9% of the c-kit mRNA present in c-kitBR cells. By comparing mRNA levels in c-kitBR cells (enriched for PHSCs) with those of unfractionated bone marrow, we demonstrated that c-kitBR cells contained low or undetectable levels of mRNA for c-fms, granulocyte colony-stimulating factor receptor, interleukin 5 receptor (IL-5R), and IL-7R. These same cells had moderate levels of mRNA for erythropoietin receptor, IL-3R subunits IL-3R alpha (SUT-1), AIC-2A, and AIC-2B, IL-6R and its partner gp-130, and the transcription factor GATA-1 and high levels of mRNA for transcription factors GATA-2, p45 NF-E2, and c-myb. We conclude from these findings that PHSCs are programmed to interact with stem cell factor, IL-3, and IL-6 but not with granulocyte or macrophage colony-stimulating factor. These findings also indicate that GATA-2, p45 NF-E2, and c-myb activities may be involved in PHSC maintenance or proliferation.

High incidence of XXY and XYY males among the offspring of female chimeras from embryonic stem cells.

Injecting male embryonic stem cells into the blastocoel of female embryos occasionally produces female chimeras capable of transmitting the embryonic stem cell genome. In our experiments several embryonic stem cell-derived male offspring from female chimeras were observed to be infertile. Karyotypic analysis of these infertile animals revealed aneuploidy. We examined the karyotypes of an additional 14 offspring not selected for infertility (3 females and 11 males) that had received the embryonic stem cell genome from 5 transmitting female chimeras. The 3 females and 5 of the males had normal karyotypes. Six of the males exhibited nonmosaic aneuploidy, which included four XXY karyotypes, one XYY karyotype, and an X,i(Y) karyotype. The high incidence of XXY and XYY males supports previous evidence for aberrant pairing and segregation of X and Y chromosomes when they are present in oocytes.

Application of Molecular Modelling studies to the search of novel approaches for neuroprotection and stem cells applications

Il lavoro di ricerca presentato in questa tesi di dottorato riguarda l'applicazione di studi di modellistica molecolare per l'individuazione di nuovi approcci farmacologici nel campo della neuroprotezione e del controllo della proliferazione di cellule staminali. Durante il mio dottorato di ricerca, mi sono concentrata sullo studio del sistema degli endocannabinoidi come target per lo sviluppo di nuovi trattamenti neuroprotettivi. In particolare, la mia ricerca ha avuto come obiettivo la modulazione dei livelli di 2-arachidonilglicerolo e arachidonil-etanolamide tramite l'inibizione degli enzimi MGL (monoglyceride lipase) e FAAH (fatty acid amide hydrolase). Il mio progetto di ricerca comprende anche studi di modellistica molecolare per l'individuazione di piccole molecole in grado di inibire il complesso proteina-proteina YAP-TEAD. Tale complesso, coinvolto nei sistemi di regolazione della proliferazione cellulare, rappresenta un target di cruciale importanza nel controllo della proliferazione e differenziazione di cellule staminali e, al tempo stesso, nel controllo dell'espansione tumorale

Yes-associated protein (YAP) is a negative regulator of chondrogenesis in mesenchymal stem cells

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Acknowledgements The authors would like to thank Dr Marius Sudol for the hYAP plasmids (obtained through Addgene), Dr Pete Zammit for the pMSCV-IRES-eGFP plasmid, Dr Robert Judson for subcloning the hYAP cDNAs into the pMSCV-IRES-eGFP plasmid, Dr Lynda Erskine for the provision of mouse embryo samples, and Professor Jimmy Hutchison and the Orthopaedics Department at the Aberdeen Royal Infirmary for the provision of human tissue samples. The authors are also grateful to Denise Tosh and Susan Clark for excellent technical support. This work was funded by Arthritis Research UK (grant 19429).

Single-cell derived clones from human adipose stem cells present different immunomodulatory properties

Human adipose mesenchymal stem cells are a heterogeneous population, where cell cultures derived from single cell-expanded clones present varying degrees of differential plasticity. This work focuses on the immunomodulatory/anti-inflammatory properties of these cells. To this end, 5 single cell clones were isolated (generally called 1.X and 3.X) from 2 volunteers. Regarding the expression level of the lineage-characteristic surface antigens, clones 1.10 and 1.22 expressed the lowest amounts, while clones 3.10 and 3.5 expressed more CD105 than the rest and clone 1.7 expressed higher amounts of CD73 and CD44. Regarding cytokine secretion, all clones were capable of spontaneously releasing high levels of IL-6 and low to moderate levels of IL-8. These differences can be explained in part by the distinct methylation profile exhibited by the clones. Furthermore and after lipopolysaccharide stimulation, clone 3.X produced the highest amounts of pro-inflammatory cytokines such as IL-1β, while clones 1.10 and 1.22 highly expressed IL-4 and IL-5. In co-culture experiments, clones 1.X are altogether more potent inhibitors than clones 3.X for proliferation of total, CD3+T, CD4+T and CD8+T lymphocytes and NK cells. The results of this work indicates that adipose stem cell population is heterogeneous in cytokine production profile, and that isolation, characterization and selection of the appropriate cell clone is a more exact method for the possible treatment of different patients or pathologies.

Phagocytosis of Photoreceptor Outer Segments by Transplanted Human Neural Stem Cells as a Neuroprotective Mechanism in Retinal Degeneration

Purpose. Transplantation of human central nervous system stem cells (HuCNS-SC) into the subretinal space of Royal College of Surgeons (RCS) rats preserves photoreceptors and visual function. To explore possible mechanism(s) of action underlying this neuroprotective effect, we performed a detailed morphologic and ultrastructure analysis of HuCNS-SC transplanted retinas. Methods. The HuCNS-SC were transplanted into the subretinal space of RCS rats. Histologic examination of the transplanted retinas was performed by light and electron microscopy. Areas of the retina adjacent to HuCNS-SC graft (treated regions) were analyzed and compared to control sections obtained from the same retina, but distant from the transplant site (untreated regions). Results. The HuCNS-SC were detected as a layer of STEM 121 immunopositive cells in the subretinal space. In treated regions, preserved photoreceptor nuclei, as well as inner and outer segments were identified readily. In contrast, classic signs of degeneration were observed in the untreated regions. Interestingly, detailed ultrastructure analysis revealed a striking preservation of the photoreceptor–bipolar–horizontal cell synaptic contacts in the outer plexiform layer (OPL) of treated areas, in stark contrast with untreated areas. Finally, the presence of phagosomes and vesicles exhibiting the lamellar structure of outer segments also was detected within the cytosol of HuCNS-SC, indicating that these cells have phagocytic capacity in vivo. Conclusions. This study reveals the novel finding that preservation of specialized synaptic contacts between photoreceptors and second order neurons, as well as phagocytosis of photoreceptor outer segments, are potential mechanism(s) of HuCNS-SC transplantation, mediating functional rescue in retinal degeneration.

Mouse mesenchymal stem cells inhibit high endothelial cell activation and lymphocyte homing to lymph nodes by releasing TIMP-1.

Mesenchymal stem cells (MSC) represent a promising therapeutic approach in many diseases in view of their potent immunomodulatory properties, which are only partially understood. Here, we show that the endothelium is a specific and key target of MSC during immunity and inflammation. In mice, MSC inhibit activation and proliferation of endothelial cells in remote inflamed lymph nodes (LNs), affect elongation and arborization of high endothelial venules (HEVs) and inhibit T-cell homing. The proteomic analysis of the MSC secretome identified the tissue inhibitor of metalloproteinase-1 (TIMP-1) as a potential effector molecule responsible for the anti-angiogenic properties of MSC. Both in vitro and in vivo, TIMP-1 activity is responsible for the anti-angiogenic effects of MSC, and increasing TIMP-1 concentrations delivered by an Adeno Associated Virus (AAV) vector recapitulates the effects of MSC transplantation on draining LNs. Thus, this study discovers a new and highly efficient general mechanism through which MSC tune down immunity and inflammation, identifies TIMP-1 as a novel biomarker of MSC-based therapy and opens the gate to new therapeutic approaches of inflammatory diseases.

Uniting the nation? Disability, stem cells, and the Australian media

In this article, the 2002 Australian debate regarding embryonic stem cells is examined. This shows the importance of an analysis of the media to understanding how disability is constructed in discourses of nationhood and biotechnology. Media representation of disability-for instance, signifying disability as catastrophe-is seen as crucial in securing access to a variety of biotechnologies, such as embryonic stem cells. Analysis of such media moments shows a structure of privileged and excluded voices in debates regarding disability and biotechnology. The diversity of voices in the Australian community regarding disability is not represented in a range of media, nor are people with disability quoted as experts on disability. A recognition of the media's construction of disability must be matched by a commitment to disability as part of a truly civil society. It is only in this way that we will have biotechnologies, and diverse cultural and media representations that meet the requirements of the international disability rights movement motto of 'nothing about us without us', recently emphasized in the Disabled Peoples' International Europe's 2000 statement on biotechnology.

Scaffolds, stem cells, and tissue engineering: A potent combination!

Stem cells, either from embryonic or adult sources, have demonstrated the potential to differentiate into a wide range of tissues depending on culture conditions. This makes them prime candidates for use in tissue engineering applications. Current technology allows us to process biocompatible and biodegradable polymers into three-dimensional (3D) configurations, either as solid porous scaffolds or hydrogels, with controlled macro and/or micro spatial geometry and surface chemistry. Such control provides us with the ability to present highly controlled microenvironments to a chosen cell type. However, the precise microenvironments required for optimal expansion and/or differentiation of stem cells are only now being elucidated, and hence the controlled use of stem cells in tissue engineering remains a very young field. We present here a brief review of the current literature detailing interactions between stem cells and 3D scaffolds of varying morphology and chemical properties, concluding with remaining challenges for those interested in tissue engineering using tailored scaffolds and stem cells.