GIPR gene expression in testis is mouse specific and can impact male mouse fertility.

BACKGROUND: Glucose-dependent insulinotropic polypeptide receptor (Gipr) gene expression has been reported in mouse spermatids and Gipr knockout male mice have previously been reported to have decreased in vitro fertilization, although the role of Gipr signaling in male mouse fertility is not well understood. OBJECTIVES: The purposes of these studies were to determine the role of glucose-dependent insulinotropic polypeptide receptor in male fertility using Gipr knockout mice and anti-glucose-dependent insulinotropic polypeptide receptor antibody-treated wild-type mice and to determine if the expression of Gipr in mouse testes is similar in non-human and human primates. METHODS AND MATERIALS: Adiponectin promoter-driven Gipr knockout male mice (GiprAdipo-/- ) were assessed for in vitro and in vivo fertility, sperm parameters, and testicular histology. CD1 male mice were administered an anti-glucose-dependent insulinotropic polypeptide receptor antibody (muGIPR-Ab) prior to and during mating for assessment of in vivo fertility and sperm parameters. Expression of Gipr/GIPR mRNA in the mouse, cynomolgus monkey, and human testes was assessed by in situ hybridization methods using species-specific probes. RESULTS: GiprAdipo-/- male mice are infertile in vitro and in vivo, despite normal testis morphology, sperm counts, and sperm motility. In contrast, administration of muGIPR-Ab to CD1 male mice did not impact fertility. While Gipr mRNA expression is detectable in the mouse testes, GIPR mRNA expression is not detectable in monkey or human testes. DISCUSSION: The infertility of GiprAdipo-/- male mice correlated with the lack of Gipr expression in the testis and/or adipocyte tissue. However, as administration of muGIPR-Ab did not impact the fertility of adult male mice, it is possible that the observations in genetically deficient male mice are related to Gipr deficiency during development. CONCLUSION: Our data support a role for Gipr expression in the mouse testis during the development of sperm fertilization potential, but based on gene expression data, a similar role for glucose-dependent insulinotropic polypeptide receptor in non-human primate or human male fertility is unlikely.

Identification and Optimization of a Minor Allele-Specific siRNA to Prevent PNPLA3 I148M-Driven Nonalcoholic Fatty Liver Disease.

Human genome wide association studies confirm the association of the rs738409 single nucleotide polymorphism (SNP) in the gene encoding protein patatin like phospholipase domain containing 3 (PNPLA3) with nonalcoholic fatty liver disease (NAFLD); the presence of the resulting mutant PNPLA3 I148M protein is a driver of nonalcoholic steatohepatitis (NASH). While Pnpla3-deficient mice do not display an adverse phenotype, the safety of knocking down endogenous wild type PNPLA3 in humans remains unknown. To expand the scope of a potential targeted NAFLD therapeutic to both homozygous and heterozygous PNPLA3 rs738409 populations, we sought to identify a minor allele-specific small interfering RNA (siRNA). Limiting our search to SNP-spanning triggers, a series of chemically modified siRNA were tested in vitro for activity and selectivity toward PNPLA3 rs738409 mRNA. Conjugation of the siRNA to a triantennary N-acetylgalactosamine (GalNAc) ligand enabled in vivo screening using adeno-associated virus to overexpress human PNPLA3I148M versus human PNPLA3I148I in mouse livers. Structure-activity relationship optimization yielded potent and minor allele-specific compounds that achieved high levels of mRNA and protein knockdown of human PNPLA3I148M but not PNPLA3I148I. Testing of the minor allele-specific siRNA in PNPLA3I148M-expressing mice fed a NASH-inducing diet prevented PNPLA3I148M-driven disease phenotypes, thus demonstrating the potential of a precision medicine approach to treating NAFLD.

Self-Assembled, Biodegradable Magnetic Resonance Imaging Agents: Organic Radical-Functionalized Diblock Copolymers.

We report the design, synthesis, and evaluation of biodegradable amphiphilic poly(ethylene glycol)-b-polycarbonate-based diblock copolymers containing pendant persistent organic radicals (e.g., PROXYL). These paramagnetic radical-functionalized polymers self-assemble into micellar nanoparticles in aqueous media, which preferentially accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect. Through T1 relaxation NMR studies, as well as magnetic resonance imaging (MRI) studies on mice, we show that these nanomaterials are effective as metal-free, biodegradable MRI contrast agents. We also demonstrate anticancer drugs can be readily loaded into the nanoparticles, conferring therapeutic delivery properties in addition to their imaging properties making these materials potential theranostic agents in the treatment of cancer.

High-Resolution Projection Microstereolithography for Patterning of Neovasculature.

To gain a quantitative understanding of the way cells sense, process, and respond to dynamic environmental signals in real-time requires developing in vitro model systems that accurately replicate the 3D structure and function of native tissue. A high-resolution projection stereolithography apparatus (µSLA) capable of multimaterial and grayscale 3D patterning of cells and biomaterials at <5 µm resolution is presented. Murine cells (fibroblasts, myoblasts, endothelial, and bone marrow stromal cells) encapsulated within photosensitive hydrogels using the µSLA remain viable up to two weeks after fabrication. Harnessing the high-resolution fabrication capabilities of this machine, sub-millimeter scale angiogenic cell-encapsulating patches designed to promote targeted growth of neovasculature are printed, as assessed in vitro via enzyme-linked immunosorbent assay (ELISA) and in ovo via a chick chorioallantoic membrane assay (CAM). This application establishes the µSLA as an enabling technology that is widely adaptable to any application that requires high-resolution patterning of cells and cells signals. By providing an efficient and robust method of engineering microscale tissues with encapsulated cells, this apparatus has a range of applications including fundamental studies of extracellular matrix interactions, high throughput drug testing of physiologically relevant substitutes for native tissue, and programmable tissue engineering for applications in regenerative medicine.

Hydrophilic packaging of iron oxide nanoclusters for highly sensitive imaging.

Superparamagnetic iron oxide nanoparticles (SPIONs) are used as imaging probes to provide contrast in magnetic resonance images. Successful use of SPIONs in targeted applications greatly depends on their ability to generate contrast, even at low levels of accumulation, in the tissue of interest. In the present study, we report that SPION nanoclusters packaged to a controlled size by a hyperbranched polyglycerol (HPG) can target tissue defects and have a high relaxivity of 719 mM(-1) s(-1), which was close to their theoretical maximal limit. The resulting nanoclusters were able to identify regions of defective vasculature in an ischemic murine hindlimb using MRI with iron doses that were 5-10 fold lower than those typically used in preclinical studies. Such high relaxivity was attributed to the molecular architecture of HPG, which mimics that of the water retentive polysaccharide, glycogen. The results of this study will be broadly useful in sensitive imaging applications.

Matrix stiffness-modulated proliferation and secretory function of the airway smooth muscle cells.

Multiple pulmonary conditions are characterized by an abnormal misbalance between various tissue components, for example, an increase in the fibrous connective tissue and loss/increase in extracellular matrix proteins (ECM). Such tissue remodeling may adversely impact physiological function of airway smooth muscle cells (ASMCs) responsible for contraction of airways and release of a variety of bioactive molecules. However, few efforts have been made to understand the potentially significant impact of tissue remodeling on ASMCs. Therefore, this study reports how ASMCs respond to a change in mechanical stiffness of a matrix, to which ASMCs adhere because mechanical stiffness of the remodeled airways is often different from the physiological stiffness. Accordingly, using atomic force microscopy (AFM) measurements, we found that the elastic modulus of the mouse bronchus has an arithmetic mean of 23.1 ± 14 kPa (SD) (median 18.6 kPa). By culturing ASMCs on collagen-conjugated polyacrylamide hydrogels with controlled elastic moduli, we found that gels designed to be softer than average airway tissue significantly increased cellular secretion of vascular endothelial growth factor (VEGF). Conversely, gels stiffer than average airways stimulated cell proliferation, while reducing VEGF secretion and agonist-induced calcium responses of ASMCs. These dependencies of cellular activities on elastic modulus of the gel were correlated with changes in the expression of integrin-ß1 and integrin-linked kinase (ILK). Overall, the results of this study demonstrate that changes in matrix mechanics alter cell proliferation, calcium signaling, and proangiogenic functions in ASMCs.

Glacier moraine formation-mimicking colloidal particle assembly in microchanneled, bioactive hydrogel for guided vascular network construction.

This study demonstrates that a new method to align microparticles releasing bioactive molecules in microchannels of a hydrogel allows the guiding of growth direction and spacing of vascular networks.

Flow-mediated stem cell labeling with superparamagnetic iron oxide nanoparticle clusters.

This study presents a strategy to enhance the uptake of superparamagnetic iron oxide nanoparticle (SPIO) clusters by manipulating the cellular mechanical environment. Specifically, stem cells exposed to an orbital flow ingested almost a 2-fold greater amount of SPIO clusters than those cultured statically. Improvements in magnetic resonance (MR) contrast were subsequently achieved for labeled cells in collagen gels and a mouse model. Overall, this strategy will serve to improve the efficiency of cell tracking and therapies.

In situ self-folding assembly of a multi-walled hydrogel tube for uniaxial sustained molecular release.

This study presents a multi-walled poly(ethylene glycol) diacrylate hydrogel tube formed by the simple self-folding of a bi-layered hydrogel patch. The gel tube has the capability to release encapsulated molecules through designated pathways in a sustained manner. Therefore, the gel tube encapsulating the vascular endothelial growth factor significantly increases the vascular densities and vessel diameters at an implantation site.

Leukocyte-mimicking stem cell delivery via in situ coating of cells with a bioactive hyperbranched polyglycerol.

Since stem cells emerged as a new generation of medicine, there are increasing efforts to deliver stem cells to a target tissue via intravascular injection. However, the therapeutic stem cells lack the capacity to detect and adhere to the target tissue. Therefore, this study presents synthesis of a bioactive hyperbranched polyglycerol (HPG) that can noninvasively associate with stem cells and further guide them to target sites, such as inflamed endothelium. The overall process is analogous to the way in which leukocytes are mobilized to the injured endothelium.

Obtaining metaphase spreads from single blastomeres for PGD of chromosomal rearrangements.

It has previously been shown that it is possible to obtain metaphase chromosomes from single blastomeres converted into metaphase in the cytoplasm of a mouse zygote. This method is highly labour intensive and cannot be performed outside the preimplantation genetic diagnosis (PGD) laboratory, so to overcome these limitations, a method was developed for obtaining metaphase spreads from single biopsied blastomeres using different chemicals. The substances tested were calyculin A, caffeine, paclitaxel and colcemid in a total of 496 disaggregated and 234 biopsied blastomeres from day 3 embryos. It was demonstrated that the optimal method involved a combined use of 'selective biopsy' (selection of the biopsied blastomere according to morphological criteria) and exposure to caffeine. This resulted in shortening the mean incubation time of biopsied blastomeres, with a metaphase formation rate of 80%. The method is simple for obtaining metaphases from single blastomeres, and may be implemented in clinical practice of PGD for structural rearrangements.

The magic behind stem cells.

This review article summarizes historical development of stem cell research, presents current knowledge on the plasticity potential of both embryonic and adult stem cells and discusses on the future of stem cell based therapies.

Reprogramming of human somatic cells by embryonic stem cell cytoplast.

Somatic cell nuclear transfer (SCNT) provides the basis for the development of patient-specific stem cell lines. Recent progress in SCNT suggested the presence of reprogramming factors in human embryonic stem (hES) cells, although no method is currently available for replacement of nuclei of hES cells by somatic cell nuclei. An original technique has been developed, involving the fusion of different types of somatic cells with hES cells, which allowed a complete replacement of the nuclei of hES cells by nuclei of somatic cells. The resulting 'cybrids' were demonstrated to have the genotype of the donor somatic cells and 'stemness' of the recipient hES cells. However, the colonies isolated from the resulting fusion contained a mixture of these cybrid cells with the cells with the recipient nuclei, as well as hybrid cells containing both donor and recipient nuclei, so future purification will be necessary before the technique can be considered for future practical application.