Models For Development Of Therapeutics Research Articles

Transcriptional characterization of iPSC-derived microglia as a model for therapeutic development in neurodegeneration

Microglia are the resident immune cells in the brain that play a key role in driving neuroinflammation, a hallmark of neurodegenerative disorders. Inducible microglia-like cells have been developed as an in vitro platform for molecular and therapeutic hypothesis generation and testing. However, there has been no systematic assessment of similarity of these cells to primary human microglia along with their responsiveness to external cues expected of primary cells in the brain. In this study, we performed transcriptional characterization of commercially available human inducible pluripotent stem cell (iPSC)-derived microglia-like (iMGL) cells by bulk and single cell RNA sequencing to assess their similarity with primary human microglia. To evaluate their stimulation responsiveness, iMGL cells were treated with Liver X Receptor (LXR) pathway agonists and their transcriptional responses characterized by bulk and single cell RNA sequencing. Bulk transcriptome analyses demonstrate that iMGL cells have a similar overall expression profile to freshly isolated human primary microglia and express many key microglial transcription factors and functional and disease-associated genes. Notably, at the single-cell level, iMGL cells exhibit distinct transcriptional subpopulations, representing both homeostatic and activated states present in normal and diseased primary microglia. Treatment of iMGL cells with LXR pathway agonists induces robust transcriptional changes in lipid metabolism and cell cycle at the bulk level. At the single cell level, we observe heterogeneity in responses between cell subpopulations in homeostatic and activated states and deconvolute bulk expression changes into their corresponding single cell states. In summary, our results demonstrate that iMGL cells exhibit a complex transcriptional profile and responsiveness, reminiscent of in vivo microglia, and thus represent a promising model system for therapeutic development in neurodegeneration.

Abstract 6043: Chromatin remodeling in patient derived colorectal cancer models

Abstract Patient-derived models of cancer (PDMCs) are becoming the gold standard preclinical models for therapeutic development and precision oncology. However, how tumor cells evolve in PDMC and the implication on therapeutic response remains largely unclear. Herein, we leveraged ATAC-seq to compare the chromatin accessibility landscapes of matched sets of colorectal cancer (CRC) patient-derived organoids (PDOs), patient-derived xenografts (PDXs), PDO-derived PDX (PDOXs), and original patient tumors (PTs), supplemented by mRNA-seq for the transcriptomics information. In the study, nine surgically resected CRC tumor specimens were recruited at Duke. For a PT-PDMCs set, part of the original patient tumor was prepared for sequencing, and the remainders from the same specimens were subjected to derive PDXs and PDOs. Throughout the study, eight PDOs were generated, and three subcutaneous PDXs grew. Besides, we also developed six patient-derived xenografts through organoids. In total, we successfully established 6 matched PT-PDO-xenograft sets, including 3 sets that have both conventional PDXs and PDOXs. The global chromatin accessibility derived from PDMCs and PTs ATAC-seq libraries presented broad congruence (79.6% peaks remained unchanged), suggesting that PDMCs mostly retained their epigenetic identity of patient tumors. Nevertheless, paired differential analysis identified alterations in chromatin accessibility between PTs and PDMCs. We discovered two principal remodeling axes. The first one separates PT from all PDMCs, and the second separate in vitro and in vivo models. Interestingly, PDOXs are much more similar to PDXs than to PDOs, indicating that the PDMCs environment is the driving force for chromatin remodeling. Single cell multiome sequencing (10X Genomics) also agreed that the PDOXs are more similar to PDXs compared to PDOs. Based on our findings, PDOXs may be a good substitution for conventional PDXs. Between in vitro and in vivo models, bivariate genomic footprinting analysis was used to further identify transcription factors (TF) that drive chromatin remodeling differentially. Our data suggested KLF14 and EGR2 have enriched motif-binding footprints in PDOXs from all patient cases. Their silencing enhanced CRC growth, which partially explains why PDXs tend to grow slower than PDOs. Downstream genes of EGR2 and KLF14 have also been analyzed. One of the targets, EPHA4, has been reported it would influence the drug sensitivities. Collectively, patient CRC cells undergo both common and distinct chromatin remodeling in PDOs and PDXs/PDOXs, driven by their respective microenvironments. The datasets allow researchers to look into chromatin accessibility, gene expression, transcriptional factor binding, and pathways that are altered in PDMC compared to original tumors, which will shed light on their predictability for therapeutic profiling. Citation Format: Kun Xiang, Ergang Wang, Qiang Huang, David Hsu, Xiling Shen. Chromatin remodeling in patient derived colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6043.

Abstract 5621: NCG-X mouse: A novel animal model to evaluate preclinical studies of humanized erythroid reconstitution without irradiation

Abstract Hematopoietic stem cell (HSC) transplantation is an adoptive immunotherapy for treating malignant hematopoietic cancers and autoimmune disorders. Preclinical studies of such novel therapies require animal models that enable and sustain human erythrocyte reconstitution. Existing mouse models typically require irradiation to remove mouse HSCs from the bone marrow prior to human immune system reconstitution. Irradiation adversely affects the health of the animal by promoting necrosis and apoptosis of gastrointestinal, neural and muscle tissues, which can lead to wasting, infection, and even death. An irradiation-free mouse model for human erythroid reconstitution is in urgent need. KIT proto-oncogene receptor tyrosine kinase, also known as c-KIT, is an important cell surface marker expressed in both mouse and human HSCs, multipotent progenitors (MPP), and common myeloid progenitors (CMP). KIT signaling plays a key role in cell survival, proliferation, and differentiation. Alterned forms of this protein have been shown to be associated with some types of cancer. Point mutation of Val831Met is known to cause macrocytic anemia. We speculate that mice with this mutation experience spontaneous anemia and inhibited HSC function, which would support human erythroid reconstitution without irradiation. NCG-X mouse was generated based on this hypothesis, building upon the severe immunodeficient NCG generated by GemPharmatech years ago. This new strain of mice are deficient of T/B/NK cells with functional inhibition of HSCs. Here, we report that NCG-X supports human HSC transplantation without radiation, enabling complete avoidance of adverse effects on animal health. When compared with irradiated NCG mice, NCG-X mice are able to not only rebuild the human immune system, but also enable human erythroid cells to reconstruct the bone marrow for a prolonged period of time after engraftment: 16 and 30 weeks post-engraftment have been assayed. Though reduced red blood cells and hemoglobin have been observed, 30 weeks post-engraftment enables consistent support from the start to the finish of the preclinical phase. In conclusion, NCG-X provides an improved and preclinical relevant mouse model for therapeutic development of leukemia, thalassemia, other types of blood diseases and autoimmune diseases. Citation Format: Tingting Gu, Cunxiang Ju, Hongyan Sun, Xiang Gao, MingKun Zhang, Weiwei Yu, Mengting Wang, Shuai Li, Shuai Li, Huiyi Wang, Jing Zhao. NCG-X mouse: A novel animal model to evaluate preclinical studies of humanized erythroid reconstitution without irradiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5621.

Knockout of NMDA Receptors in Parvalbumin Interneurons Recreates Autism‐Like Phenotypes

Autism is a disabling neurodevelopmental disorder characterized by social deficits, language impairment, and repetitive behaviors with few effective treatments. New evidence suggests that autism has reliable electrophysiological endophenotypes and that these measures may be caused by n-methyl-d-aspartic acid receptor (NMDAR) disruption on parvalbumin (PV)-containing interneurons. These findings could be used to create new translational biomarkers. Recent developments have allowed for cell-type selective knockout of NMDARs in order to examine the perturbations caused by disrupting specific circuits. This study examines several electrophysiological and behavioral measures disrupted in autism using a PV-selective reduction in NMDA R1 subunit. Mouse electroencephalograph (EEG) was recorded in response to auditory stimuli. Event-related potential (ERP) component amplitude and latency analysis, social testing, and premating ultrasonic vocalizations (USVs) recordings were performed. Correlations were examined between the ERP latency and behavioral measures. The N1 ERP latency was delayed, sociability was reduced, and mating USVs were impaired in PV-selective NMDA Receptor 1 Knockout (NR1 KO) as compared with wild-type mice. There was a significant correlation between N1 latency and sociability but not between N1 latency and premating USV power or T-maze performance. The increases in N1 latency, impaired sociability, and reduced vocalizations in PV-selective NR1 KO mice mimic similar changes found in autism. Electrophysiological changes correlate to reduced sociability, indicating that the local circuit mechanisms controlling N1 latency may be utilized in social function. Therefore, we propose that behavioral and electrophysiological alterations in PV-selective NR1 KO mice may serve as a useful model for therapeutic development in autism. Autism Res 2013, 6: 69-77. © 2013 International Society for Autism Research, Wiley Periodicals, Inc.

Characterization of the neuropsychological phenotype of glycine N-methyltransferase −/− mice and evaluation of its responses to clozapine and sarcosine treatments

Glycine N-methyltransferase (GNMT) affects cellular methylation capacity through regulating the ratio between S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). The product of its enzymatic reaction—sarcosine has antipsychotic effect in patients with schizophrenia. In this study, through RT-PCR and immunohistochemical staining, we demonstrated that GNMT expressed in various neurons located in the cerebral cortex, hippocampus, substantia nigra and cerebellum. Compared to the wild-type mice, Gnmt−/− mice had significantly lower level of sarcosine in the cerebral cortex. Real-time PCR identified genes involved in the methionine metabolism (Dnmt1 and Dnmt3a), ErbB (Nrg1 and ErbB4) and mTOR (Akt2, S6, S6k1 and S6k2) signaling pathways were dysregulated significantly in the cortex of Gnmt−/− mice. Acoustic startle reflex test demonstrated that Gnmt−/− mice had significantly lower level of prepulse inhibition and the deficit was ameliorated through clozapine or sarcosine treatment. Furthermore, liver-specific-human-GNMT transgenic with Gnmt−/− (Tg-GNMT/Gnmt−/−) mice were used to rule out that the phenotype was due to abnormal liver function. In summary, the neuropsychological abnormalities found in Gnmt−/− mice may represent an endophenotype of schizophrenia. GNMT plays an important role in maintaining normal physiological function of brain and Tg-GNMT/Gnmt−/− mice are useful models for development of therapeutics for patients with schizophrenia.

Cloning and Expression of the Rhesus Monkey Oxytocin Receptor

The oxytocin receptor belongs to the family of G-protein-coupled receptors (GPCRs) characterized by seven transmembrane spanning domains and memediates numerous neurotransmitter and hormonal functions. The cloning of this receptor was initiated to validate the use of the rhesus monkey (Macaca mulatta) as a viable animal model for therapeutic development of oxytocin receptor antagonists by ruling out potential species variations that are sometimes present among GPCRs. The rhesus monkey oxytocin receptor was cloned by the polymerase chain reaction (PCR) and expressed transiently in 293/EBNA cells. The cDNA encodes a protein of 389 amino acids and is highly homologous to that from other species, especially the human receptor which exhibits 97% identity to the rhesus protein. The cloned receptor shows a very similar pharmacological profile to the human oxytocin receptor for a variety of agonists and antagonists from various structural classes. These results substantiate the validity of the rhesus monkey as a useful model for the evaluation of human therapeutics.