modeling-mitochondrial-disease-using-brain-organoids-focus-on

Modeling Mitochondrial Disease Using Brain Organoids: A Focus on Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes

Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (MELAS) are mitochondrial disorders most commonly caused by a m.3243A&#62;G ...

Keio University School of Medicine

Kurume University School of Medicine

The synaptic protein α-synuclein has been identified as a major component of Lewy bodies, a pathological hallmark of Parkinson's disease (PD). Prior to the formation of Lewy bodies, mislocalization and aggregation of the α-synuclein in brain tissue is frequently observed in various neurodegenerative diseases. Aberrant accumulation and localization of α-synuclein are also observed in the aging human brain, for which reason aging is regarded as a risk factor for neurodegenerative disease. To investigate changes in α-synuclein properties in the aging brain, we compared α-synuclein immunoreactivity in brain tissue of young (2-years-old) and middle-aged (6-years-old) common marmoset (Callithrix jacchus). Our analyses revealed marked changes in α-synuclein immunoreactivity in the olfactory bulb of common marmosets of these age cohorts. Perikaryal α-synuclein aggregations were formed in the olfactory bulb in middle-aged animals. We also observed signals of α-synuclein accumulation in hippocampus in this cohort; however, unlike in the olfactory bulb, hippocampal α-synuclein signals were localized in the synaptic terminals. We did not observe either of these features in younger marmosets, which suggest that aging may play a role in these phenomena. Our results using common marmoset brain corresponded with the observation that the α-synuclein aggregations were first occurred from olfactory bulb in human normal aged and PD brain. Therefore, common marmoset is expected as useful model for α-synuclein pathology.

α-Synuclein aggregation in the olfactory bulb of middle-aged common marmoset.

The common marmoset (marmoset; Callithrix jacchus) harbors various desired features as a non-human primate (NHP) model for neuroscience research. Recently, efforts have been made to induce neural cells in vitro from marmoset pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), which are characterized by their capacity to differentiate into all cell types from the three germ layers. Successful generation of marmoset neural cells is not only invaluable for understanding neural development and for modeling neurodegenerative and psychiatric disorders, but is also necessary for the phenotypic screening of genetically-modified marmosets. However, differences in the differentiation propensity among PSC lines hamper the applicability and the reproducibility of differentiation methods. To overcome this limitation, we evaluated the efficacy of small molecules for neural differentiation of marmoset ESCs (cjESCs) and iPSCs using multiple differentiation methods. By immunochemical and transcriptomic analyses, we confirmed that our methods using the small molecules are efficient for various differentiation protocols by either enhancing the yield of a mixture of neural cells including both neurons and glial cells, or a pure population of neurons. Collectively, our findings optimized in vitro neural differentiation methods for marmoset PSCs, which would ultimately help enhance the utility of the animal model in neuroscience.

Evaluating the efficacy of small molecules for neural differentiation of common marmoset ESCs and iPSCs.

We previously reported the efficient targeted introduction of transgenes into the genomic DNA of the common marmoset (Callithrix jacchus) using CRISPR-Cas9. In this study, we generated a marmoset embryonic stem cell (ESC) line that ubiquitously expresses the tamoxifen-inducible Cre-driver ERT2CreERT2. We validated the pluripotency of the ESC line and also successfully demonstrated the temporal control of the Cre-driver in a tamoxifen-dependent manner in the ESCs. This ESC line, named ActiCre-B1, will be a valuable resource for in vitro investigation of phenotypes related to embryonic lethality by targeted knockout of functionally important genes.

Generation and validation of a common marmoset embryonic stem cell line ActiCre-B1 that ubiquitously expresses a tamoxifen-inducible Cre-driver.

Studying the non-human primate (NHP) brain is required for the translation of rodent research to humans, but remains a challenge for molecular, cellular, and circuit-level analyses in the NHP brain due to the lack of in vitro NHP brain system. Here, we report an in vitro NHP cerebral model using marmoset (Callithrix jacchus) embryonic stem cell-derived cerebral assembloids (CAs) that recapitulate inhibitory neuron migration and cortical network activity. Cortical organoids (COs) and ganglionic eminence organoids (GEOs) were induced from cjESCs and fused to generate CAs. GEO cells expressing the inhibitory neuron marker LHX6 migrated toward the cortical side of CAs. COs developed their spontaneous neural activity from a synchronized pattern to an unsynchronized pattern as COs matured. CAs containing excitatory and inhibitory neurons showed mature neural activity with an unsynchronized pattern. The CAs represent a powerful in vitro model for studying excitatory and inhibitory neuron interactions, cortical dynamics, and their dysfunction. The marmoset assembloid system will provide an in vitro platform for the NHP neurobiology and facilitate translation into humans in neuroscience research, regenerative medicine, and drug discovery.

Modeling the marmoset brain using embryonic stem cell-derived cerebral assembloids.

Genome editing technology is widely used in the field of laboratory animal science for the production of genetic disease models and the analysis of gene function. One of the major technical problems in genome editing is the low efficiency of precise knock-in by homologous recombination compared to simple knockout via non-homologous end joining. Many studies have focused on this issue, and various solutions have been proposed; however, they have yet to be fully resolved. In this study, we established a system that can easily determine the genotype at the mouse (Mus musculus) Tyr gene locus for genome editing both in vitro and in vivo. In this genome editing system, by designing the Cas9 cleavage site and donor template, wild-type, knockout, and knock-in genotypes can be distinguished by restriction fragment length polymorphisms of PCR products. Moreover, the introduction of the H420R mutation in tyrosinase allows the determination of knock-in mice with specific coat color patterns. Using this system, we evaluated the effects of small-molecule compounds on the efficiency of genome editing in mouse embryos. Consequently, we successfully identified a small-molecule compound that improves knock-in efficiency in genome editing in mouse embryos. Thus, this genome editing system is suitable for screening compounds that can improve knock-in efficiency.

Article	Year
α-Synuclein aggregation in the olfactory bulb of middle-aged common marmoset. Neuroscience research\| PubMed ID: 26643383	2016
Evaluating the efficacy of small molecules for neural differentiation of common marmoset ESCs and iPSCs. Neuroscience research\| PubMed ID: 31586586
Generation and validation of a common marmoset embryonic stem cell line ActiCre-B1 that ubiquitously expresses a tamoxifen-inducible Cre-driver. Stem cell research\| PubMed ID: 33453576
Modeling the marmoset brain using embryonic stem cell-derived cerebral assembloids. Biochemical and biophysical research communications\| PubMed ID: 37002985	2023
An efficient evaluation system for factors affecting the genome editing efficiency in mouse. Experimental animals\| PubMed ID: 37407493	2023

Seiji Shiozawa

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