Genetic engineered molecular imaging probes for applications in cell therapy: emphasis on MRI approach.

Recent advances in stem cell-based regenerative medicine, cell replacement therapy, and genome editing technologies (i.e. CRISPR-Cas 9) have sparked great interest in in vivo cell monitoring. Molecular imaging promises a unique approach to noninvasively monitor cellular and molecular phenomena, including cell survival, migration, proliferation, and even differentiation at the whole organismal level. Several imaging modalities and strategies have been explored for monitoring cell grafts in vivo. We begin this review with an introduction describing the progress in stem cell technology, with a perspective toward cell replacement therapy. The importance of molecular imaging in reporting and assessing the status of cell grafts and their relation to the local microenvironment is highlighted since the current knowledge gap is one of the major obstacles in clinical translation of stem cell therapy. Based on currently available imaging techniques, we provide a brief discussion on the pros and cons of each imaging modality used for monitoring cell grafts with particular emphasis on magnetic resonance imaging (MRI) and the reporter gene approach. Finally, we conclude with a comprehensive discussion of future directions of applying molecular imaging in regenerative medicine to emphasize further the importance of correlating cell graft conditions and clinical outcomes to advance regenerative medicine.

Cryotolerance of Sperm from Transgenic Rhesus Macaques (Macaca mulatta).

Cryopreservation is an important tool routinely used in preserving sperm for assisted reproductive technologies and for genetic preservation of unique animal models. Here we investigated the viability of fresh and frozen sperm from rhesus macaques on the basis of motility, membrane integrity, and acrosome integrity. Sperm motility was determined by visual evaluation; membrane and acrosome integrity were assessed simultaneously through triple staining with Hoechst 33342, propidium iodide, and fluorescein isothiocyanate-peanut agglutinin. We compared thawed semen that had been cryopreserved by using 2 different media with fresh semen from wildtype (WT) macaques; fresh semen from a model of Huntington disease (HD) with fresh WT semen; and fresh HD with cryopreserved-thawed HD semen. Our new freezing media (TEST EQ) preserved the acrosome better, with less net damage, than did traditional TEST (egg yolk extender containing TES and Tris) media. In addition, the percentage of membrane-damaged cells was similar in fresh HD semen (38.6%±2.9%) and WT semen (35.5%±1.9%). Membrane and acrosomal damage were not different between HD and WT sperm after cryopreservation and subsequent thawing. Furthermore, cryopreservation had similar negative effects on the motility of HD and WT sperm. These data illustrate that semen from a rhesus macaque model of HD is similarly cryotoleratant to that from WT animals.

A two years longitudinal study of a transgenic Huntington disease monkey.

BACKGROUND: A two-year longitudinal study composed of morphometric MRI measures and cognitive behavioral evaluation was performed on a transgenic Huntington's disease (HD) monkey. rHD1, a transgenic HD monkey expressing exon 1 of the human gene encoding huntingtin (HTT) with 29 CAG repeats regulated by a human polyubiquitin C promoter was used together with four age-matched wild-type control monkeys. This is the first study on a primate model of human HD based on longitudinal clinical measurements. RESULTS: Changes in striatal and hippocampal volumes in rHD1 were observed with progressive impairment in motor functions and cognitive decline, including deficits in learning stimulus-reward associations, recognition memory and spatial memory. The results demonstrate a progressive cognitive decline and morphometric changes in the striatum and hippocampus in a transgenic HD monkey. CONCLUSIONS: This is the first study on a primate model of human HD based on longitudinal clinical measurements. While this study is based a single HD monkey, an ongoing longitudinal study with additional HD monkeys will be important for the confirmation of our findings. A nonhuman primate model of HD could complement other animal models of HD to better understand the pathogenesis of HD and future development of diagnostics and therapeutics through longitudinal assessment.

Development of transgenic rats producing human beta-amyloid precursor protein as a model for Alzheimer's disease: transgene and endogenous APP genes are regulated tissue-specifically.

BACKGROUND: Alzheimer's disease (AD) is a devastating neurodegenerative disorder that affects a large and growing number of elderly individuals. In addition to idiopathic disease, AD is also associated with autosomal dominant inheritance, which causes a familial form of AD (FAD). Some instances of FAD have been linked to mutations in the beta-amyloid protein precursor (APP). Although there are numerous mouse AD models available, few rat AD models, which have several advantages over mice, have been generated. RESULTS: Fischer 344 rats expressing human APP driven by the ubiquitin-C promoter were generated via lentiviral vector infection of Fischer 344 zygotes. We generated two separate APP-transgenic rat lines, APP21 and APP31. Serum levels of human amyloid-beta (Abeta)40 were 298 pg/ml for hemizygous and 486 pg/ml for homozygous APP21 animals. Serum Abeta42 levels in APP21 homozygous rats were 135 pg/ml. Immunohistochemistry in brain showed that the human APP transgene was expressed in neurons, but not in glial cells. These findings were consistent with independent examination of enhanced green fluorescent protein (eGFP) in the brains of eGFP-transgenic rats. APP21 and APP31 rats expressed 7.5- and 3-times more APP mRNA, respectively, than did wild-type rats. Northern blots showed that the human APP transgene, driven by the ubiquitin-C promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver. A similar expression pattern was also seen for the endogenous rat APP. The unexpected similarity in the tissue-specific expression patterns of endogenous rat APP and transgenic human APP mRNAs suggests regulatory elements within the cDNA sequence of APP. CONCLUSION: This manuscript describes the generation of APP-transgenic inbred Fischer 344 rats. These are the first human AD model rat lines generated by lentiviral infection. The APP21 rat line expresses high levels of human APP and could be a useful model for AD. Tissue-specific expression in the two transgenic rat lines and in wild-type rats contradicts our current understanding of APP gene regulation. Determination of the elements that are responsible for tissue-specific expression of APP may enable new treatment options for AD.