USP7 inhibition inhibits proliferation and induces megakaryocytic differentiation in MDS cells by upregulating gelsolin.

Myelodysplastic syndrome (MDS), a largely incurable hematological malignancy, is driven by complex genetic and epigenetic alterations from an aberrant clone of hematopoietic stem/progenitor cells (HSPCs). Ubiquitin-specific protease 7 (USP7) has been demonstrated to have an important oncogenic role in the development of several cancer types, but its role in MDS is unknown. Here, we demonstrate that USP7 expression is elevated in MDS cell lines and patient samples. The USP7-selective small-molecule inhibitors P5091 and P22077 inhibited cell proliferation and induced megakaryocytic differentiation in both cell lines and primary cells. Furthermore, pharmacological inhibition of USP7 markedly suppressed the growth of MDS cell lines in xenograft mouse models. To explore the mechanisms underlying the observed phenotypic changes, we employed RNA-seq to compare the differences in genes after USP7 inhibitor treatment and found that gelsolin (GSN) expression was increased significantly after USP7 inhibitor treatment. Furthermore, knockdown of GSN attenuated the proliferation inhibition, apoptosis induction and megakaryocyte differentiation induced by USP7 inhibitors in MDS cells. Collectively, our findings identify previously unknown roles of USP7 and suggest that the USP7/GSN axis may be a potential therapeutic target in MDS.

Murine xenograft bioreactors for human immunopeptidome discovery.

The study of peptides presented by MHC class I and class II molecules is limited by the need for relatively large cell numbers, especially when studying post-translationally modified or otherwise rare peptide species. To overcome this problem, we pose the hypothesis that human cells grown as xenografts in immunodeficient mice should produce equivalent immunopeptidomes as cultured cells. Comparing human cell lines grown either in vitro or as murine xenografts, we show that the immunopeptidome is substantially preserved. Numerous features are shared across both sample types, including peptides and proteins featured, length distributions, and HLA-binding motifs. Peptides well-represented in both groups were from more abundant proteins, or those with stronger predicted HLA binding affinities. Samples grown in vivo also recapitulated a similar phospho-immunopeptidome, with common sequences being those found at high copy number on the cell surface. These data indicate that xenografts are indeed a viable methodology for the production of cells for immunopeptidomic discovery.

Development of a Microforce Sensor and Its Array Platform for Robotic Cell Microinjection Force Measurement.

Robot-assisted cell microinjection, which is precise and can enable a high throughput, is attracting interest from researchers. Conventional probe-type cell microforce sensors have some real-time injection force measurement limitations, which prevent their integration in a cell microinjection robot. In this paper, a novel supported-beam based cell micro-force sensor with a piezoelectric polyvinylidine fluoride film used as the sensing element is described, which was designed to solve the real-time force-sensing problem during a robotic microinjection manipulation, and theoretical mechanical and electrical models of the sensor function are derived. Furthermore, an array based cell-holding device with a trapezoidal microstructure is micro-fabricated, which serves to improve the force sensing speed and cell manipulation rates. Tests confirmed that the sensor showed good repeatability and a linearity of 1.82%. Finally, robot-assisted zebrafish embryo microinjection experiments were conducted. These results demonstrated the effectiveness of the sensor working with the robotic cell manipulation system. Moreover, the sensing structure, theoretical model, and fabrication method established in this study are not scale dependent. Smaller cells, e.g., mouse oocytes, could also be manipulated with this approach.

In vitro and in vivo differentiation of induced pluripotent stem cells into male germ cells.

The introduction of induced pluripotent stem cell (iPSC) lines has been a breakthrough in the field of stem cell research. However, the extent of pluripotency among those cell lines tends to be variable due to their different epigenetic signatures. Mouse iPS cell line 4.1 has been established via retroviral transfer of human transcription factors Oct4, Sox2, Klf4, and c-Myc; the germline competence of this line has not been determined. In the present study, we induced the differentiation of miPS-4.1 cells into male germ cells, in vivo and in vitro. In the in vitro model, the behavior of miPS-4.1 cells was identical to that of differentiating mouse embryonic stem cells (ESCs). We obtained primordial germ cell-like cells (PGC-LC) that were positive for alkaline phosphatase (AP) activity. In continuous culture, these cells expressed pluripotent marker Oct4 and male germline markers C-kit and MVH. For our in vivo model, miPS-4.1 cells were co-transplanted with neonatal testicular cell suspension. We observed ectopically reconstituted seminiferous tubule structures, in which the miPS-4.1 cells were homing and developing. In conclusion, we successfully induced the differentiation of miPS-4.1 cells into male germ cells, albeit their epigenetic characteristics. Our study provides a system to examine the mechanisms of male germ cell development and might help to supply an effective treatment for male infertility in the future.

In vivo GABA release and kinetics of transgene loss in a GABAergic cell line after long-term transplantation into the rat brain.

Ex vivo gene therapy uses modified cells to deliver substances into the brain. Cell line M213-2O CL-4 expresses human glutamate decarboxylase (hGAD(67)) by means of an Epstein-Barr virus-based plasmid. This cell line releases GABA in response to depolarizing stimuli in vitro, and after brain transplantation it modulates seizures in animal models. It is unclear if the functional effects observed can be attributed to GABA release by the grafted cells and if GABA release, in turn, is related to the kinetics of transgene permanence or loss under long-term transplantation conditions. To address these issues, two experiments were performed. The first one evaluated GABA levels in the vicinity of an intranigral transplant by microdialysis followed by high performance liquid chromatography (HPLC) quantification. GABA levels and GAD activity were higher in rats with 8-week-old transplants than in control animals, but this effect was lost in rats with 12-week-old transplants. The second experiment evaluated the number of copies of the plasmid containing the hGAD(67) (GAD1) transgene by real-time PCR after transplantation into the hippocampus at the same times. A time-dependent loss of the plasmid in the transplants was observed. The mechanism of plasmid loss was explored in vitro by analyzing the effects of DNA methylation and the absence of selection pressure. The results suggest that the loss of plasmid copies from transplants under long-term conditions may be related to methylation of plasmid regions involved in its nuclear retention. Taking these data together, we propose that the reported long-term functional effects of transplants of cell line M213-2O CL-4 may not be attributed exclusively to increased GABA release in the area of the graft, but that a paracrine-like action of GABA may lead to the remodeling of neural circuits in the host.

A novel and simple hollow-fiber assay for in vivo evaluation of nonpeptidyl thrombopoietin receptor agonists.

Preclinical in vivo assessment of the pharmacologic activity of nonpeptidyl thrombopoietin receptor (TPOR) agonists is very difficult because of the high species specificity of such agonists. In this study, we have developed a novel and simple in vivo hollow-fiber assay to preclinically evaluate TPOR agonists. The 32D-mpl cell line was generated by stable transfection of human TPOR into 32D lymphoblast cells and shown to be a specific model for nonpeptide TPOR agonists in vitro. Stably transfected 32D-mpl cells were then sealed in hollow fibers and implanted into nude mice. Cells in hollow fibers specifically responded to TPOR agonists, including thrombopoietin and eltrombopag, a nonpeptide small-molecule TPOR agonist, but not to granulocyte colony-stimulating factor or erythropoietin. Oral administration of eltrombopag stimulated 32D-mpl cell proliferation, prevented 32D-mpl cell apoptosis, and stimulated the phosphorylation of cellular signaling transducers and activators of transcription in a TPOR- and dose-dependent manner. These results indicate that the hollow-fiber assay is a specific and efficient model for rapidly evaluating the in vivo activity of small-molecule TPOR agonists.

Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease.

Parkinson disease (PD) involves the selective loss of midbrain dopamine (mDA) neurons and is a possible target disease for stem cell-based therapy. Human induced pluripotent stem cells (hiPSCs) are a potentially unlimited source of patient-specific cells for transplantation. However, it is critical to evaluate the safety of hiPSCs generated by different reprogramming methods. Here, we compared multiple hiPSC lines derived by virus- and protein-based reprogramming to human ES cells (hESCs). Neuronal precursor cells (NPCs) and dopamine (DA) neurons delivered from lentivirus-based hiPSCs exhibited residual expression of exogenous reprogramming genes, but those cells derived from retrovirus- and protein-based hiPSCs did not. Furthermore, NPCs derived from virus-based hiPSCs exhibited early senescence and apoptotic cell death during passaging, which was preceded by abrupt induction of p53. In contrast, NPCs derived from hESCs and protein-based hiPSCs were highly expandable without senescence. DA neurons derived from protein-based hiPSCs exhibited gene expression, physiological, and electrophysiological properties similar to those of mDA neurons. Transplantation of these cells into rats with striatal lesions, a model of PD, significantly rescued motor deficits. These data support the clinical potential of protein-based hiPSCs for personalized cell therapy of PD.

Establishment of a murine epidermal cell line suitable for in vitro and in vivo skin modelling.

BACKGROUND: Skin diseases are a major health problem. Some of the most severe conditions involve genetic disorders, including cancer. Several of these human diseases have been modelled in genetically modified mice, thus becoming a highly valuable preclinical tool for the treatment of these pathologies. However, development of three-dimensional models of skin using keratinocytes from normal and/or genetically modified mice has been hindered by the difficulty to subculture murine epidermal keratinocytes. METHODS: We have generated a murine epidermal cell line by serially passaging keratinocytes isolated from the back skin of adult mice. We have termed this cell line COCA. Cell culture is done in fully defined media and does not require feeder cells or any other coating methods. RESULTS: COCA retained its capacity to differentiate and stratify in response to increased calcium concentration in the cell culture medium for more than 75 passages. These cells, including late passage, can form epidermis-like structures in three-dimensional in vitro models with a well-preserved pattern of proliferation and differentiation. Furthermore, these cells form epidermis in grafting assays in vivo, and do not develop tumorigenic ability. CONCLUSIONS: We propose that COCA constitutes a good experimental system for in vitro and in vivo skin modelling. Also, cell lines from genetically modified mice of interest in skin biology could be established using the method we have developed. COCA keratinocytes would be a suitable control, within a similar background, when studying the biological implications of these alterations.

Long-term delivery of nerve growth factor by encapsulated cell biodelivery in the Göttingen minipig basal forebrain.

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

Subarachnoid transplantation of immortalized galanin-overexpressing astrocytes attenuates chronic neuropathic pain.

Treatment of chronic neuropathic pain resulted from peripheral nerve injury is one of the most difficult problems in modern clinical practice. The use of cell lines as biologic "minipumps" to chronically deliver anti-nociceptive molecules into the pain-processing centers of spinal cord is a newly developing technique for the treatment of pain. Moreover, spinal administration of exogenous galanin (GAL) is a useful target for the treatment of chronic pain after nerve injury. Because of better histocompatibility, lower immunogenicity and reproducibility, immortalized astrocytes (IAST) have been served as a promising cellular vehicle to deliver therapeutic molecules into CNS. In this study, the rat IAST was transfected with rat preprogalanin cDNA and the galanin-synthesizing and secreting cell line, IAST/GAL, was isolated. After cells were transplanted into the subarachnoid space of rats with chronic neuropathic pain induced by spared nerve injury (SNI) of sciatic nerve, their analgesic potential was evaluated by behavioral tests. The results showed that IAST/GAL transfected with preprogalanin gene could express and secrete significantly higher level of GAL protein in vitro and in vivo as compared with control cells. In addition, the pain-related behaviors, thermal hyperalgesia and mechanical allodynia were significantly alleviated during the 1-7 weeks after grafts of IAST/GAL cells, which could be reversed by galanin receptor antagonist M35 temporarily. Taken together, these data suggest that subarachnoid transplant of immortalized galanin-overexpressing astrocytes near the pain-processing centers was able to reverse the development of chronic neuropathic pain, which offers an adjunct approach to currently used therapies for the pain management.

[Does 2009 mark a revival of embryonic stem cells?]. / ¿Resucitan al inicio del 2009 las células troncales procedentes de embriones?

The legal possibility of using federal funds to work with embryonic cells and destroy embryos started on March 2009 in the USA. It has nothing to do with regenerative therapies. It is directed to create banks with human cells, banks directed by a few scientists involved in biotechnology enterprises connected with centers of in vitro reproduction. They pursue the use of ad hoc human embryos for biomedical research. The idea of using cell lines derived from embryos is quite spread, and even the idea of obtaining new lines of this type to validate reprogrammed somatic pluripotential cells, the so called iPS cell (induced pluripotent stem), without destroying embryos or using ovules. This type of cells is indeed of great value in medical research and it is opening new possibilities in Cell Therapy. Recent data are analyzed and considerations are advanced encouraging rational alternatives to eliminate embryonic cells in the evaluation of iPS cells.

Production of offspring from a germline stem cell line derived from neonatal ovaries.

The idea that females of most mammalian species have lost the capacity for oocyte production at birth has been challenged recently by the finding that juvenile and adult mouse ovaries possess mitotically active germ cells. However, the existence of female germline stem cells (FGSCs) in postnatal mammalian ovaries still remains a controversial issue among reproductive biologists and stem cell researchers. We have now established a neonatal mouse FGSC line, with normal karyotype and high telomerase activity, by immunomagnetic isolation and culture for more than 15 months. FGSCs from adult mice were isolated and cultured for more than 6 months. These FGSCs were infected with GFP virus and transplanted into ovaries of infertile mice. Transplanted cells underwent oogenesis and the mice produced offspring that had the GFP transgene. These findings contribute to basic research into oogenesis and stem cell self-renewal and open up new possibilities for use of FGSCs in biotechnology and medicine.

Basophil effector function and homeostasis during helminth infection.

Basophils are effector cells of the innate immune system that are associated with allergic inflammation and infections with helminth parasites. However, their development and in vivo functions are largely unknown. Here, we characterize basophil development, turnover, tissue localization, and effector function during infection with the helminth Nippostrongylus brasiliensis. Our results demonstrate that under homeostatic conditions basophils have a lifespan of about 60 hours. N brasiliensis-induced basophilia is caused by increased de novo production of basophils in the bone marrow. Basophils were found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine, and in the lung parenchyma. Activated basophils promoted systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung, and contributed to efficient worm expulsion, demonstrating that basophils play a crucial role as effector cells in type 2 immune responses.

NT-501: an ophthalmic implant of polymer-encapsulated ciliary neurotrophic factor-producing cells.

Neurotech Pharmaceuticals Inc is developing NT-501, an implantable polymeric device containing a genetically modified cell line that secretes ciliary neurotrophic factor, for the potential treatment of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Phase III clinical trials for RP and a phase II clinical trial for dry AMD are ongoing. A phase I clinical trial showed that NT-501 treatment was well tolerated with variable, but positive improvements in visual acuity.

Transfer of a human chromosomal vector from a hamster cell line to a mouse embryonic stem cell line.

Two transchromosomic mouse embryonic stem (ES) sublines (ESMClox1.5 and ESMClox2.1) containing a human minichromosome (MC) were established from a sample of hybrid colonies isolated in fusion experiments between a normal diploid mouse ES line and a Chinese hamster ovary line carrying the MC. DNA cytometric and chromosome analyses of ESMClox1.5 and ESMClox2.1 indicated a mouse chromosome complement with a heteroploid constitution in a subtetraploid range; the karyotypes showed various degrees of polysomy for different chromosomes. A single copy of the MC was found in the majority of cells in all the isolated hybrid colonies and was stably maintained in the established sublines for more than 100 cell generations either with or without the selective agent. No significant differences from the ES parental cells were observed in growth characteristics of the transchromosomic ES sublines. ESMClox1.5 cells were unable to grow in soft agar; when cultured in hanging drops, they formed embryoid bodies, and when inoculated in nude mice, they produced teratomas. They were able to express the early development markers Oct4 and Nanog, as demonstrated by reverse transcription-polymerase chain reaction assay. All these features are in common with the ES parental line. Further research using the transchromosomic ES sublines described here may allow gene expression studies on transferred human minichromosomes and could shed light on the relationships among ploidy, pluripotency, cell transformation, and tumorigenesis. Disclosure of potential conflicts of interest is found at the end of this article.

A novel culture technique for human embryonic stem cells using porous membranes.

We have developed a novel culture technique for human embryonic stem cells (hESCs) using a porous membrane with feeder cells. The feeder cells were seeded and attached to the bottom of a porous membrane and, subsequently, hESCs were cultured on the top of the membrane. This porous membrane technique (PMT) allowed hESCs to be successfully cultured and to be effectively and efficiently separated from the feeder cell layer without enzyme treatment. hESCs being cultured by PMT were observed to interact with feeder cells through pores of membrane, where the interaction was dependent on the pore size of the membrane used. It was also revealed that the number of attached hESC colonies depended on the concentration of feeder cells on the bottom of the membrane. On the other hand, hESC colonies did not attach to porous membrane, as feeder cells were in the presence of culture dish, not the porous membrane. The hESCs cultured on porous membranes not only exhibited expression of several undifferentiated markers and a normal karyotype, but they also formed teratomas consisting of three germ layers in in vivo study. Compared with the mechanical isolation technique conventionally used, PMT significantly decreased mouse vimentin gene expression in cultured hESCs. Thus, a PMT for hESC culture would be a useful tool to exclude enzyme treatment and to reduce contamination from feeder cells simultaneously. Disclosure of potential conflicts of interest is found at the end of this article.

Lyn is an important component of the signal transduction pathway specific to FLT3/ITD and can be a therapeutic target in the treatment of AML with FLT3/ITD.

Fms-like tyrosine kinase 3 (FLT3) is expressed in hematopoietic progenitor cells. An internal tandem duplication (ITD) of FLT3 (FLT3/ITD) is the most frequent mutation in human adult acute myeloid leukemia (AML). FLT3/ITD contributes to the constitutive activation of FLT3 itself and its downstream signal components, mitogen-activated protein kinase and signal transducers and activators of transcription 5 (STAT5), and enables interleukin (IL)-3-dependent cell lines to grow autonomously. In the present study, we showed the specific association of FLT3/ITD with Lyn, which led to the phosphorylation of Lyn in vivo. We also demonstrated that FLT3/ITD receptors displayed a higher affinity to bind to Lyn than wild-type FLT3 receptors in vitro and that this affinity was relative to the intensity of tyrosil phosphorylation of the receptor. Both treatment with small interfering RNA (siRNA) targeting Lyn and the Src family kinase inhibitor PP2 suppressed the IL-3-independent growth of FLT3/ITD-expressing 32D cells (FLT3/ITD-32D), reducing the constitutive phosphorylation of Lyn and STAT5. PP2 treatment of mice transplanted with FLT3/ITD-32D cells blocked the onset of tumors and decreased the size of established tumors. These results demonstrate that Lyn is an important component of the signal transduction pathway specific to FLT3/ITD and can be a therapeutic target in the treatment of AML with FLT3/ITD.