Transmembrane Protein TMEM230, Regulator of Glial Cell Vascular Mimicry and Endothelial Cell Angiogenesis in High-Grade Heterogeneous Infiltrating Gliomas and Glioblastoma.

High-grade gliomas (HGGs) and glioblastoma multiforme (GBM) are characterized by a heterogeneous and aggressive population of tissue-infiltrating cells that promote both destructive tissue remodeling and aberrant vascularization of the brain. The formation of defective and permeable blood vessels and microchannels and destructive tissue remodeling prevent efficient vascular delivery of pharmacological agents to tumor cells and are the significant reason why therapeutic chemotherapy and immunotherapy intervention are primarily ineffective. Vessel-forming endothelial cells and microchannel-forming glial cells that recapitulate vascular mimicry have both infiltration and destructive remodeling tissue capacities. The transmembrane protein TMEM230 (C20orf30) is a master regulator of infiltration, sprouting of endothelial cells, and microchannel formation of glial and phagocytic cells. A high level of TMEM230 expression was identified in patients with HGG, GBM, and U87-MG cells. In this study, we identified candidate genes and molecular pathways that support that aberrantly elevated levels of TMEM230 play an important role in regulating genes associated with the initial stages of cell infiltration and blood vessel and microchannel (also referred to as tumor microtubule) formation in the progression from low-grade to high-grade gliomas. As TMEM230 regulates infiltration, vascularization, and tissue destruction capacities of diverse cell types in the brain, TMEM230 is a promising cancer target for heterogeneous HGG tumors.

Genetic modulation of the HTR2A gene reduces anxiety-related behavior in mice.

The expanding field of precision gene editing using CRISPR/Cas9 has demonstrated its potential as a transformative technology in the treatment of various diseases. However, whether this genome-editing tool could be used to modify neural circuits in the central nervous system (CNS), which are implicated in complex behavioral traits, remains uncertain. In this study, we demonstrate the feasibility of noninvasive, intranasal delivery of adeno-associated virus serotype 9 (AAV9) vectors containing CRISPR/Cas9 cargo within the CNS resulting in modification of the HTR2A receptor gene. In vitro, exposure to primary mouse cortical neurons to AAV9 vectors targeting the HT2RA gene led to a concentration-dependent decrease in spontaneous electrical activity following multielectrode array (MEA) analysis. In vivo, at 5 weeks postintranasal delivery in mice, analysis of brain samples revealed single base pair deletions and nonsense mutations, leading to an 8.46-fold reduction in mRNA expression and a corresponding 68% decrease in the 5HT-2A receptor staining. Our findings also demonstrate a significant decrease in anxiety-like behavior in treated mice. This study constitutes the first successful demonstration of a noninvasive CRISPR/Cas9 delivery platform, capable of bypassing the blood-brain barrier and enabling modulation of neuronal 5HT-2A receptor pathways. The results of this study targeting the HTR2A gene provide a foundation for the development of innovative therapeutic strategies for a broad range of neurological disorders, including anxiety, depression, attentional deficits, and cognitive dysfunction.

Transmembrane Protein TMEM230, a Target of Glioblastoma Therapy.

Glioblastomas (GBM) are the most aggressive tumors originating in the brain. Histopathologic features include circuitous, disorganized, and highly permeable blood vessels with intermittent blood flow. These features contribute to the inability to direct therapeutic agents to tumor cells. Known targets for anti-angiogenic therapies provide minimal or no effect in overall survival of 12-15 months following diagnosis. Identification of novel targets therefore remains an important goal for effective treatment of highly vascularized tumors such as GBM. We previously demonstrated in zebrafish that a balanced level of expression of the transmembrane protein TMEM230/C20ORF30 was required to maintain normal blood vessel structural integrity and promote proper vessel network formation. To investigate whether TMEM230 has a role in the pathogenesis of GBM, we analyzed its prognostic value in patient tumor gene expression datasets and performed cell functional analysis. TMEM230 was found necessary for growth of U87-MG cells, a model of human GBM. Downregulation of TMEM230 resulted in loss of U87 migration, substratum adhesion, and re-passaging capacity. Conditioned media from U87 expressing endogenous TMEM230 induced sprouting and tubule-like structure formation of HUVECs. Moreover, TMEM230 promoted vascular mimicry-like behavior of U87 cells. Gene expression analysis of 702 patients identified that TMEM230 expression levels distinguished high from low grade gliomas. Transcriptomic analysis of patients with gliomas revealed molecular pathways consistent with properties observed in U87 cell assays. Within low grade gliomas, elevated TMEM230 expression levels correlated with reduced overall survival independent from tumor subtype. Highest level of TMEM230 correlated with glioblastoma and ATP-dependent microtubule kinesin motor activity, providing a direction for future therapeutic intervention. Our studies support that TMEM230 has both glial tumor and endothelial cell intracellular and extracellular functions. Elevated levels of TMEM230 promote glial tumor cell migration, extracellular scaffold remodeling, and hypervascularization and abnormal formation of blood vessels. Downregulation of TMEM230 expression may inhibit both low grade glioma and glioblastoma tumor progression and promote normalization of abnormally formed blood vessels. TMEM230 therefore is both a promising anticancer and antiangiogenic therapeutic target for inhibiting GBM tumor cells and tumor-driven angiogenesis.

Zebrafish Tmem230a cooperates with the Delta/Notch signaling pathway to modulate endothelial cell number in angiogenic vessels.

During embryonic development, new arteries, and veins form from preexisting vessels in response to specific angiogenic signals. Angiogenic signaling is complex since not all endothelial cells exposed to angiogenic signals respond equally. Some cells will be selected to become tip cells and acquire migration and proliferation capacity necessary for vessel growth while others, the stalk cells become trailer cells that stay connected with pre-existing vessels and act as a linkage to new forming vessels. Additionally, stalk and tip cells have the capacity to interchange their roles. Stalk and tip cellular responses are mediated in part by the interactions of components of the Delta/Notch and Vegf signaling pathways. We have identified in zebrafish, that the transmembrane protein Tmem230a is a novel regulator of angiogenesis by its capacity to regulate the number of the endothelial cells in intersegmental vessels by co-operating with the Delta/Notch signaling pathway. Modulation of Tmem230a expression by itself is sufficient to rescue improper number of endothelial cells induced by aberrant expression or inhibition of the activity of genes associated with the Dll4/Notch pathway in zebrafish. Therefore, Tmem230a may have a modulatory role in vessel-network formation and growth. As the Tmem230 sequence is conserved in human, Tmem230 may represent a promising novel target for drug discovery and for disease therapy and regenerative medicine in promoting or restricting angiogenesis.

RNA-Generated and Gene-Edited Induced Pluripotent Stem Cells for Disease Modeling and Therapy.

Cellular reprogramming by epigenomic remodeling of chromatin holds great promise in the field of human regenerative medicine. As an example, human-induced Pluripotent Stem Cells (iPSCs) obtained by reprograming of patient somatic cells are sufficiently similar to embryonic stem cells (ESCs) and can generate all cell types of the human body. Clinical use of iPSCs is dependent on methods that do not utilize genome altering transgenic technologies that are potentially unsafe and ethically unacceptable. Transient delivery of exogenous RNA into cells provides a safer reprogramming system to transgenic approaches that rely on exogenous DNA or viral vectors. RNA reprogramming may prove to be more suitable for clinical applications and provide stable starting cell lines for gene-editing, isolation, and characterization of patient iPSC lines. The introduction and rapid evolution of CRISPR/Cas9 gene-editing systems has provided a readily accessible research tool to perform functional human genetic experiments. Similar to RNA reprogramming, transient delivery of mRNA encoding Cas9 in combination with guide RNA sequences to target specific points in the genome eliminates the risk of potential integration of Cas9 plasmid constructs. We present optimized RNA-based laboratory procedure for making and editing iPSCs. In the near-term these two powerful technologies are being harnessed to dissect mechanisms of human development and disease in vitro, supporting both basic, and translational research. J. Cell. Physiol. 232: 1262-1269, 2017. © 2016 Wiley Periodicals, Inc.

FGF2 and EGF Are Required for Self-Renewal and Organoid Formation of Canine Normal and Tumor Breast Stem Cells.

Recent studies suggest that human tumors are generated from cancer cells with stem cell (SC) properties. Spontaneously occurring cancers in dogs contain a diversity of cells that like for human tumors suggest that certain canine tumors are also generated from cancer stem cells (CSCs). CSCs, like normal SCs, have the capacity for self-renewal as mammospheres in suspension cultures. To understand how cells with SC properties contribute to canine mammary gland tumor development and progression, comparative analysis between normal SCs and CSCs, obtained from the same individual, is essential. We have utilized the property of sphere formation to develop culture conditions for propagating stem/progenitor cells from canine normal and tumor tissue. We show that cells from dissociated mammospheres retain sphere reformation capacity for several serial passages and have the capacity to generate organoid structures ex situ. Utilizing various culture conditions for passaging SCs and CSCs, fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) were found to positively or negatively regulate mammosphere regeneration, organoid formation, and multi-lineage differentiation potential. The response of FGF2 and EGF on SCs and CSCs was different, with increased FGF2 and EGF self-renewal promoted in SCs and repressed in CSCs. Our protocol for propagating SCs from normal and tumor canine breast tissue will provide new opportunities in comparative mammary gland stem cell analysis between species and anticancer treatment and therapies for dogs. J. Cell. Biochem. 118: 570-584, 2017. © 2016 Wiley Periodicals, Inc.

Endogenous retrovirus insertion in the KIT oncogene determines white and white spotting in domestic cats.

The Dominant White locus (W) in the domestic cat demonstrates pleiotropic effects exhibiting complete penetrance for absence of coat pigmentation and incomplete penetrance for deafness and iris hypopigmentation. We performed linkage analysis using a pedigree segregating White to identify KIT (Chr. B1) as the feline W locus. Segregation and sequence analysis of the KIT gene in two pedigrees (P1 and P2) revealed the remarkable retrotransposition and evolution of a feline endogenous retrovirus (FERV1) as responsible for two distinct phenotypes of the W locus, Dominant White, and white spotting. A full-length (7125 bp) FERV1 element is associated with white spotting, whereas a FERV1 long terminal repeat (LTR) is associated with all Dominant White individuals. For purposes of statistical analysis, the alternatives of wild-type sequence, FERV1 element, and LTR-only define a triallelic marker. Taking into account pedigree relationships, deafness is genetically linked and associated with this marker; estimated P values for association are in the range of 0.007 to 0.10. The retrotransposition interrupts a DNAase I hypersensitive site in KIT intron 1 that is highly conserved across mammals and was previously demonstrated to regulate temporal and tissue-specific expression of KIT in murine hematopoietic and melanocytic cells. A large-population genetic survey of cats (n = 270), representing 30 cat breeds, supports our findings and demonstrates statistical significance of the FERV1 LTR and full-length element with Dominant White/blue iris (P < 0.0001) and white spotting (P < 0.0001), respectively.

Assessment of proficiency and competency in laboratory animal biomethodologies.

Personnel working with laboratory animals are required by laws and guidelines to be trained and qualified to perform biomethodologic procedures. The assessment of competency and proficiency is a vital component of a laboratory animal training program, because this process confirms that the trainees have met the learning objectives for a particular procedure. The approach toward qualification assessment differs between organizations because laws and guidelines do not outline how the assessment should be performed or which methods and tools should be used. Assessment of clinical and surgical medicine has received considerable attention over the last few decades and has progressed from simple subjective methods to well-defined and objective methods of assessing competency. Although biomethodology competency and proficiency assessment is discussed in the literature, a standard and objective assessment method has not yet been developed. The development and implementation of an objective and standardized biomethodologic assessment program can serve as a tool to improve standards, ensure consistent training, and decrease research variables yet ensure animal welfare. Here we review the definition and goals of training and assessment, review assessment methods, and propose a method to develop a standard and objective assessment program for the laboratory animal science field, particularly training departments and IACUC.

Analysis of induced pluripotent stem cells from a BRCA1 mutant family.

Understanding BRCA1 mutant cancers is hampered by difficulties in obtaining primary cells from patients. We therefore generated and characterized 24 induced pluripotent stem cell (iPSC) lines from fibroblasts of eight individuals from a BRCA1 5382insC mutant family. All BRCA1 5382insC heterozygous fibroblasts, iPSCs, and teratomas maintained equivalent expression of both wild-type and mutant BRCA1 transcripts. Although no difference in differentiation capacity was observed between BRCA1 wild-type and mutant iPSCs, there was elevated protein kinase C-theta (PKC-theta) in BRCA1 mutant iPSCs. Cancer cell lines with BRCA1 mutations and hormone-receptor-negative breast cancers also displayed elevated PKC-theta. Genome sequencing of the 24 iPSC lines showed a similar frequency of reprogramming-associated de novo mutations in BRCA1 mutant and wild-type iPSCs. These data indicate that iPSC lines can be derived from BRCA1 mutant fibroblasts to study the effects of the mutation on gene expression and genome stability.

Rodent laparoscopy: refinement for rodent drug studies and model development, and monitoring of neoplastic, inflammatory and metabolic diseases.

The refinement of surgical techniques represents a key opportunity to improve the welfare of laboratory rodents, while meeting legal and ethical obligations. Current methods used for monitoring intra-abdominal disease progression in rodents usually involve euthanasia at various time-points for end of study, one-time individual tissue collections. Most rodent organ tumour models are developed by the introduction of tumour cells via laparotomy or via ultrasound-guided indirect visualization. Ischaemic rodent models are often generated using laparotomies. This approach requires a high number of rodents, and in some instances introduces high degrees of morbidity and mortality, thereby increasing study variability and expense. Most importantly, most laparotomies do not promote the highest level of rodent welfare. Recent improvements in laparoscopic equipment and techniques have enabled the adaptation of laparoscopy for rodent procedures. Laparoscopy, which is considered the gold standard for many human abdominal procedures, allows for serial biopsy collections from the same animal, results in decreased pain and tissue trauma as well as quicker postsurgical recovery, and preserves immune function in comparison to the same procedures performed by laparotomy. Laparoscopy improves rodent welfare, decreases inter-animal variability, thereby reducing the number of required animals, allows for the replacement of larger species, decreases expense and improves data yield. This review article compares rodent laparotomy and laparoscopic surgical methods, and describes the utilization of laparoscopy for the development of cancer models and assessment of disease progression to improve data collection and animal welfare. In addition, currently available rodent laparoscopic equipment and instrumentation are presented.

Development and implementation of multimedia content for an electronic learning course on rodent surgery.

The development of new rodent models of human disease and advances in surgical equipment and technologies have increased the demand for expertise in rodent surgery. Because of the limited availability of rodent surgical training courses, electronic (e-) learning is presented as an alternative to in-person education and as a means to hone the expertise of current surgeons in biomedical research, similar to e-learning applications for human surgery training. Translating this model to the biomedical research field provides participants with an opportunity to train themselves on rodent surgical techniques prior to operating on live models. An e-learning rodent surgery course was incorporated into a training class of undergraduate (n = 39) and graduate (n = 12) laboratory animal students, and a portion of the course was presented to laboratory animal professionals (n = 15). The effectiveness of the method was evaluated using written examination and postcourse surveys. The exam data demonstrated that the e-learning course transferred knowledge comparable to a lecture course on surgery that was presented in-person. Students responded favorably to videos, step-by-step photographs of surgical procedures, and the ready accessibility of the course. Critiques included the need to improve video resolution and quality of the voice-overs. These results support the continued development and implementation of electronic rodent surgical technique courses for use in laboratory animal and biomedical research communities.

Development of electronic learning courses for surgical training of animal research personnel.

The animal research community comprises members from a wide variety of backgrounds, some of whom must learn basic surgical skills. Though demand for animal research personnel who have surgical skills is increasing, surgical training opportunities are becoming more scarce. Electronic learning or e-learning platforms can be used as an adjunct to hands-on surgical training. Course developers can adapt these e-learning courses to fit the needs of participants who have varying levels of expertise. The authors outline the steps involved in developing an effective e-learning surgical course. They also describe how to use various equipment and software products to help implement e-learning courses. Though the authors focus on developing surgical courses, course developers could apply the general steps outlined by the authors when developing any e-learning course.

An introduction to electronic learning and its use to address challenges in surgical training.

The animal research community faces a shortage of surgical training opportunities along with an increasing demand for expertise in surgical techniques. One possible means of overcoming this challenge is the use of computer-based or electronic learning (e-learning) to disseminate material to a broad range of animal users. E-learning platforms can take many different forms, ranging from simple text documents that are posted online to complex virtual courses that incorporate dynamic video or audio content and in which students and instructors can interact in real time. The authors present an overview of e-learning and discuss its potential benefits as a supplement to hands-on rodent surgical training. They also discuss a few basic considerations in developing and implementing electronic courses.

A domestic cat X chromosome linkage map and the sex-linked orange locus: mapping of orange, multiple origins and epistasis over nonagouti.

A comprehensive genetic linkage map of the domestic cat X chromosome was generated with the goal of localizing the genomic position of the classic X-linked orange (O) locus. Microsatellite markers with an average spacing of 3 Mb were selected from sequence traces of the cat 1.9x whole genome sequence (WGS), including the pseudoautosomal region 1 (PAR1). Extreme variation in recombination rates (centimorgans per megabase) was observed along the X chromosome, ranging from a virtual absence of recombination events in a region estimated to be >30 Mb to recombination frequencies of 15.7 cM/Mb in a segment estimated to be <0.3 Mb. This detailed linkage map was applied to position the X-linked orange gene, placing this locus on the q arm of the X chromosome, as opposed to a previously reported location on the p arm. Fine mapping placed the locus between markers at positions 106 and 116.8 Mb in the current 1.9x-coverage sequence assembly of the cat genome. Haplotype analysis revealed potential recombination events that could reduce the size of the candidate region to 3.5 Mb and suggested multiple origins for the orange phenotype in the domestic cat. Furthermore, epistasis of orange over nonagouti was demonstrated at the genetic level.

Four independent mutations in the feline fibroblast growth factor 5 gene determine the long-haired phenotype in domestic cats.

To determine the genetic regulation of "hair length" in the domestic cat, a whole-genome scan was performed in a multigenerational pedigree in which the "long-haired" phenotype was segregating. The 2 markers that demonstrated the greatest linkage to the long-haired trait (log of the odds > or = 6) flanked an estimated 10-Mb region on cat chromosome B1 containing the Fibroblast Growth Factor 5 (FGF5) gene, a candidate gene implicated in regulating hair follicle growth cycle in other species. Sequence analyses of FGF5 in 26 cat breeds and 2 pedigrees of nonbreed cats revealed 4 separate mutations predicted to disrupt the biological activity of the FGF5 protein. Pedigree analyses demonstrated that different combinations of paired mutant FGF5 alleles segregated with the long-haired phenotype in an autosomal recessive manner. Association analyses of more than 380 genotyped breed and nonbreed cats were consistent with mutations in the FGF5 gene causing the long-haired phenotype in an autosomal recessive manner. In combination, these genomic approaches demonstrated that FGF5 is the major genetic determinant of hair length in the domestic cat.

Oocytes.

Embryonic stem cells (ESCs), derivatives of totipotential cells of early mammalian embryos, have proven to be one of the most powerful tools for studying developmental and stem cell biology. When injected into embryos, ESCs can contribute to tissues derived from all three germ layers and to the germ line. Prior studies have successfully shown that ESCs can recapitulate features of embryonic development by spontaneously forming somatic lineages in culture. More recent studies using differentiating monolayer cultures and embryoid bodies have shown that mouse ESCs can also form germ cells that are capable of undergoing meiosis and forming both male and female gametes. This chapter provides detailed instruction on how to differentiate ESCs in monolayer cultures to derive germ cells and oocyte-like structures and presents standard methodologies for detecting expression of key genetic pathways required for primordial germ cell (PGC) development and oogenesis in vivo. While the full potential of these ESC-derived germ cells and oocyte-like structures remains to be demonstrated, this assay provides a new approach to studying reproductive developmental biology in vitro.

HIF-2alpha regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth.

The division, differentiation, and function of stem cells and multipotent progenitors are influenced by complex signals in the microenvironment, including oxygen availability. Using a genetic "knock-in" strategy, we demonstrate that targeted replacement of the oxygen-regulated transcription factor HIF-1alpha with HIF-2alpha results in expanded expression of HIF-2alpha-specific target genes including Oct-4, a transcription factor essential for maintaining stem cell pluripotency. We show that HIF-2alpha, but not HIF-1alpha, binds to the Oct-4 promoter and induces Oct-4 expression and transcriptional activity, thereby contributing to impaired development in homozygous Hif-2alpha KI/KI embryos, defective hematopoietic stem cell differentiation in embryoid bodies, and large embryonic stem cell (ES)-derived tumors characterized by altered cellular differentiation. Furthermore, loss of HIF-2alpha severely reduces the number of embryonic primordial germ cells, which require Oct-4 expression for survival and/or maintenance. These results identify Oct-4 as a HIF-2alpha-specific target gene and indicate that HIF-2alpha can regulate stem cell function and/or differentiation through activation of Oct-4, which in turn contributes to HIF-2alpha's tumor promoting activity.

Generating oocytes and sperm from embryonic stem cells.

Embryonic stem (ES) cells, derivatives of cells of early mammalian embryos, have turned out to be one of the most powerful tools in developmental and stem cell biology. When injected into embryos, ES cells can contribute to tissues derived from all three germ layers and to the germline. Amazingly, ES cells in culture are able to recapitulate features of embryonic development spontaneously. In addition to previous successes in deriving somatic cell types, recent studies have shown that both mouse and human ES cells can also give rise to primordial germ cells (PGCs) in culture. These mouse germ cells appear to be capable of undergoing meiosis and forming both male and female gametes. Although the full function of these ES-derived germ cells and gametes remains to be demonstrated, these findings open the door for undertaking new types of reproductive studies and novel approaches in regenerative medicine.

Oct4 is required for primordial germ cell survival.

Previous studies have shown that Oct4 has an essential role in maintaining pluripotency of cells of the inner cell mass (ICM) and embryonic stem cells. However, Oct4 null homozygous embryos die around the time of implantation, thus precluding further analysis of gene function during development. We have used the conditional Cre/loxP gene targeting strategy to assess Oct4 function in primordial germ cells (PGCs). Loss of Oct4 function leads to apoptosis of PGCs rather than to differentiation into a trophectodermal lineage, as has been described for Oct4-deficient ICM cells. These new results suggest a previously unknown function of Oct4 in maintaining viability of mammalian germline.