Experimental approaches to derive CD34+ progenitors from human and nonhuman primate embryonic stem cells

Traditionally, CD34 positive cells are predominantly found in the umbilical cord and bone marrow, thus are considered as hematopoietic progenitors. Increasing evidence has suggested that the CD34+ cells represent a distinct subset of cells with enhanced progenitor activity; CD34 is a general marker of progenitor cells in a variety of cell types. Because the CD34 protein shows expression early on in hematopoietic and vascular-associated tissues, CD34+ cells have enormous potential as cellular agents for research and for clinical cell transplantation. Directed differentiation of embryonic stem cells will give rise to an inexhaustible supply of CD34+ cells, creating an exciting approach for biomedical research and for regenerative medicine. Here, we review the main methods that have been published for the derivation of CD34+ cells from embryonic stem cells; specifically those approaches the human and nonhuman primate stem cells. We summarize current status of this field, compare the methods used, and evaluate the issues in translating the bench science to bedside therapy

A neurodevelopmental perspective to improve innovation in preventive treatment of substance use disorders

Background: Midbrain dopaminergic neurons have been associated with substance use disorders (Blaess & Ang, 2015). Understanding their neurodevelopment during early stages of life is fundamental for innovating preventive care treatments. The animal model Monodelphis domestica has been proposed as an excellent candidate to study neurodevelopmental changes due to the ease of access to see changes in their embryonic development (Mate et al., 1994). The purpose of our study is to inform how brain cells, including and especially dopaminergic neurons, mature by quantifying their number during early development. Additionally, the study aims to compare different midbrain areas and track neurodevelopmental changes across early development. Methods: Monodelphis brains were collected at different developmental times points, brains were sliced, and brain sections processed following standard immunohistochemistry and other staining protocols to visualize different protein markers. ImageJ and Zen software were used to conduct area analysis and neuronal quantification. A modified stereological approach developed by our lab was utilized for precise neuronal quantification. A descriptive analysis was utilized to compare anatomical and neuronal numerical differences across different developmental stages. Inter-rater reliability was utilized to reduce bias during the neuronal quantification process. Results: A preliminary analysis from a previous study (Perez et al., 2021) revealed anatomical differences in area and volume across three different stages, embryonic day 14 (area= 27260.36 μm, m= 381.376, V=81781.0735 um^3), postnatal day 1 (49917.28 μm, m= 404.12, V= 149751.827 μm^3), and postnatal day 6 (81866.66 μm, m=166.016, V= 245599.9853 μm^3). Neuronal and area differences from the stages of postnatal day 21, 30, 8 weeks and 23 weeks will be included once inter-rater reliability is established

Differential Expression of Intestinal Genes in Opossums with High and Low Responses to Dietary Cholesterol

High and low responding opossums (Monodelphis domestica) differ in their plasma very low density lipoprotein and low density lipoprotein (VLDL+LDL) cholesterol concentrations when they consume a high cholesterol diet, which is due in part to absorption of a higher percentage of dietary cholesterol in high responders. We compared the expression of a set of genes that influence cholesterol absorption in high and low responders fed a basal or a high cholesterol and low fat (HCLF) diet. Up-regulation of the ABCG5, ABCG8, and IBABP genes by the HCLF diet in high and low responders may reduce cholesterol absorption to maintain cholesterol homeostasis. Differences in expression of the phospholipase genes (PLA2 and PLB) and phospholipase activity were associated with differences in cholesterol absorption when opossums were fed cholesterol-enriched diets. Higher PLA2 and PLB mRNA levels and higher phospholipase activity may increase cholesterol absorption in high responders by enhancing the release of cholesterol from bile salt micelles for uptake by intestinal cells

A preliminary timeline of the midbrain development in the Monodelphis Domestica animal model

Introduction: The Brazilian short-tailed opossum (Monodelphis Domestica) is an understudied animal model compared to the Mus musculus that has been identified as a perfect candidate to study neurodevelopment (Baggott, L. & Moore, H., 1990). What makes the Monodelphis Domestica a perfect specimen for neurodevelopment is that the embryo develops outside the pouch of the mother providing easy noninvasive access to track changes across different developmental stages (Mate et al., 1994). Objective: The objective of the study is to compare the area and volume in the development of the Monodelphis’s midbrain across three different developmental stages. Our research is beneficial because it facilitates the study of neurodevelopmental mental health disorders and its impact in the brain. Methods: We utilized ImageJ and Zen software to perform the volumetric and area analysis of these stages. To conduct a volumetric analysis a Volume Macro code was used in ImageJ software. The area analysis was completed using Zen software. A descriptive analysis was used to compare the differences in area and volume across the developmental stages

Vertebrate endothelial lipase: comparative studies of an ancient gene and protein in vertebrate evolution

Endothelial lipase (gene: LIPG; enzyme: EL) is one of three members of the triglyceride lipase family that contributes to lipoprotein degradation within the circulation system and plays a major role in HDL metabolism in the body. In this study, in silico methods were used to predict the amino acid sequences, secondary and tertiary structures, and gene locations for LIPG genes and encoded proteins using data from several vertebrate genome projects. LIPG is located on human chromosome 18 and is distinct from other human 'neutral lipase' genes, hepatic lipase (gene: LIPC; enzyme: HL) and lipoprotein lipase (gene: LPL; enzyme: LPL) examined. Vertebrate LIPG genes usually contained 10 coding exons located on the positive strand for most primates, as well as for horse, bovine, opossum, platypus and frog genomes. The rat LIPG gene however contained only 9 coding exons apparently due to the presence of a 'stop' codon' within exon 9. Vertebrate EL protein subunits shared 58-97% sequence identity as compared with 38-45% sequence identities with human HL and LPL. Four previously reported human EL N-glycosylation sites were predominantly conserved among the 10 potential N-glycosylation sites observed for the vertebrate EL sequences examined. Sequence alignments and identities for key EL amino acid residues were observed as well as conservation of predicted secondary and tertiary structures with those previously reported for horse pancreatic lipase (PL) (Bourne et al. 1994). Several potential sites for regulating LIPG gene expression were observed including CpG islands near the LIPG gene promoter and a predicted microRNA binding site near the 3'-untranslated region. Promoter regions containing functional polymorphisms that regulate HDL cholesterol in baboons were conserved among primates but not retained between primates and rodents. Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate LIPG gene subfamily with other neutral triglyceride lipase gene families, LIPC and LPL. It is apparent that the triglyceride lipase ancestral gene for the vertebrate LIPG gene predated the appearance of fish during vertebrate evolution[500 million years ago.Full Tex

A Neural Comparison Between Mus Musculus and Monodelphis Domestica

Mus Musculus is one of the first and widely used animal models in neuroscience. There are many reasons that Mus Musculus is used for research including, short generation length and large litters, but the most important reason is their mammalian brain. Another animal that is gaining interest as an animal model is the Monodelphis Domestica. The Monodelphis Domestica is a marsupial, pups are born underdeveloped and move onto the underbelly of their mothers until they reach a more mature age. One difference is the Monodelphis ventricle size is much larger in the forebrain area. Another difference is the formation of the corpus callosum. In the mouse brain, the corpus callosum forms and fuses before the hippocampus compared to the possum where the corpus callosum is formed more posterior to the formation of the hippocampus. The corpus callosum of the Monodelphis is less prominent than the anterior commissure. In the mouse brain, the majority of the nerve fibers are found in the corpus callosum as opposed to the anterior commissure. The corpus callosum allows communication between both hemispheres of the brain. In the mus musculus, the hippocampus is well defined and begins formation after the formation of the corpus callosum. In the Monodelphis, the hippocampus is not as defined. The mus musculus is a social animal, the more defined hippocampus could be an evolutionary improvement for social interaction. The Monodelphis is a more territorial and isolated species. Looking at the differences between the two, can further knowledge into the behavioral differences

Investigating Monodelphis Domestica as an Alternative to the Mus Musculus as an Animal Model

Background: Mus Musculus is one of the first and one of the most widely used animal models in current neuroscience literature (Phifer-Riley & Nachmann, 2015). However, the research community needs alternatives to rodent models to study the mammalian brain. Research is needed to see if antibodies that target tyrosine hydroxylase, which are well researched in mice, can also be used to study the Monodelphis domestica brain. Methods: Following transcardial perfusions and brain extractions, mouse and opossum brains were processed and stained for tyrosine hydroxylase (and with Nissl). Opossum brains will then be sliced and processed using IHC methods to compare two TH antibodies (EMD Millipore and Pelfreeze). Results: Differences include that the Monodelphis has a much larger ventricle in the forebrain area and the mouse brain corpus callosum forms and fuses before the hippocampus compared to the opossum brain, where these fibers are formed more posterior to the formation of the hippocampus. The corpus callosum of the Monodelphis is also less prominent than the anterior commissure. The results of the different antibodies will be presented at the symposium. Conclusions: Although there are differences between the mouse and the opossum brain, there are also many similarities. Further research is needed to determine what these differences could mean in behavior and cognition. Both EMD Millipore and Pelfreeze make TH antibodies that have been looked at in mice and replicated. More research is needed to determine if the antibodies can be used for other animals, including the Monodelphis

Gamma-aminobutyric acid in the Midbrain: Role in Sensation, Perception, and Neurological Disorders

Background: Altered GABA expression in the midbrain is associated with neurological conditions like epilepsy and Parkinson’s Disease. In the genetically epilepsy prone rat, an increase in GABA neurons in the inferior colliculus increases seizure susceptibility. In contrast, Parkinson’s Disease is associated with altered GABA expression in the substantia nigra. The purpose of this study is to identify patterns between the sensory and motor regions of the midbrain and to quantify a baseline of GABA neurons in these areas. Methods: The Allen Institute’s reference atlas of the mouse and BrainMap.org was used to identify the sensory and motor areas of the mouse’s midbrain. Using the ISH tool in Allen Institute, a prior study on the GAD-1 gene was used to quantify GABA neurons in the midbrain. Midbrain images were obtained from a 56-day old C57BL/6J mouse strain. The total count of GABA neurons in each major sensory and motor areas were recorded. Samples of the Monodelphis domestica midbrain tissues were obtained from the laboratory and used for comparison. Results: The combined motor areas of the midbrain were significantly larger than the combined sensory areas. High density of GABA expression was found in the superior colliculus. Clustering and higher quantity of larger GABA neurons were most prominent in the superior colliculus. The sensory midbrain areas of the Monodelphis were larger in size compared to the mouse. The total count for the superior colliculus was estimated to be 1674 neurons, while the inferior colliculus had a total count of 375 neurons. The total count of GABA neurons in the motor related areas of the midbrain was 3,690. Conclusion: In the Mus musculus, there was a greater density and quantity of larger GABA neurons within the sensory area of the superior colliculus. The total area of the motor areas of the midbrain was significantly larger than the sensory areas in the Mus musculus. When comparing the two midbrains, it appeared that the Monodelphis domestica’s sensory areas of the midbrain were distinctly larger than the mouse. Deviations from our baseline could help indicate whether abnormal GABA expression in the midbrain is linked to neurological conditions

The impact of social isolation on the Neural Pathways of Dopamine Neurons in the Ventral Tegmental Area (VTA) and the Nucelus Accumbens (NAc): Implications for the treatment of depression, anxiety, and drug addiction.

As the literature on the Monodelphis domestica continues to grow, it is important to contribute to the knowledge base regarding neural pathways and their role in social behavior in this species. Previous studies have provided evidence that increased activity in ventral tegmental area (VTA) dopamine neurons were associated with more social activity in mice. It is also known that in traditional rodent models, the Nucleus Accumbens (NAcc) is implicated in interaction reward processes like motivation; however, more research is needed to elucidate the role of the NAcc in social behavior of the M. domestica. The present study was designed to address the knowledge gap regarding the brain and social behavior using M. domestica as an animal model. Using immunohistochemistry, we characterized the expression of Tyrosine Hydroxylase (TH), a marker for dopamine neurons, in both the VTA and the NAc of M. domestica and determined that the pattern of TH expression is similar to what is observed in rodents. Next, the expression of TH in opossums that were exposed to a social stimulus were compared to TH levels in animals that were not exposed to a social stimulus, confirming an effect of isolation on TH immunoreactivity. Social stimuli were provided by housing the opossums in groups of 2-3 same-sex partners or by themselves in their cages. Given that Monodelphis is a model for neurodevelopmental research, this study could serve as the first to look at neurotransmitters that are associated with social behavior in an animal model that is not widely studied. A goal of this presentation is to better inform clinicians about the possible biological basis of social isolation and the negative symptoms associated with it. Using the data that was collected, we can begin to understand the biological markers that are implicated in human psychological disorders and find areas to target with different treatment modalities