The common marmoset monkey (Callithrix jacchus; Cj) is an advantageous nonhuman primate species for modeling age-related disorders, including Parkinson's disease, due to their shorter life span compared to macaques. Cj-derived induced pluripotent stem cells (Cj-iPSCs) from somatic cells are needed for in vitro disease modeling and testing regenerative medicine approaches. Here we report the development of a novel Cj-iPSC line derived from adult marmoset fibroblasts. The Cj-iPSCs showed potent pluripotency properties, including the development of mesodermal lineages in tumors after injection to immunocompromised mice, as well as ectoderm and endoderm lineages after in vitro differentiation regimens, demonstrating differentiated derivatives of all three embryonic layers. In addition, expression of key pluripotency genes (ZFP42, PODXL, DNMT3B, C-MYC, LIN28, KLF4, NANOG, SOX2, and OCT4) was observed. We then tested the neural differentiation capacity and gene expression profiles of Cj-iPSCs and a marmoset embryonic stem cell line (Cj-ESC) after dual-SMAD inhibition. Exposure to CHIR99021 and sonic hedgehog (SHH) for 12 and 16 days, respectively, patterned the cells toward a ventralized midbrain dopaminergic phenotype, confirmed by expression of FOXA2, OTX2, EN-1, and tyrosine hydroxylase. These results demonstrate that common marmoset stem cells will be able to serve as a platform for investigating regenerative medicine approaches targeting the dopaminergic system.

Leucine-rich repeat kinase 2 (LRRK2) G2019S is a relatively common mutation, associated with 1-3% of Parkinson's disease (PD) cases worldwide. G2019S is hypothesized to increase LRRK2 kinase activity. Dopaminergic neurons derived from induced pluripotent stem cells of PD patients carrying LRRK2 G2019S are reported to have several phenotypes compared to wild type controls, including increased activated caspase-3 and reactive oxygen species (ROS), autophagy dysfunction, and simplification of neurites. The common marmoset is envisioned as a candidate nonhuman primate species for comprehensive modeling of genetic mutations. Here, we report our successful use of CRISPR/Cas9 with repair template-mediated homology directed repair to introduce the LRRK2 G2019S mutation, as well as a truncation of the LRRK2 kinase domain, into marmoset embryonic and induced pluripotent stem cells. We found that, similar to humans, marmoset LRRK2 G2019S resulted in elevated kinase activity. Phenotypic evaluation after dopaminergic differentiation demonstrated LRRK2 G2019S-mediated increased intracellular ROS, decreased neuronal viability, and reduced neurite complexity. Importantly, these phenotypes were not observed in clones with LRRK2 truncation. These results demonstrate the feasibility of inducing monogenic mutations in common marmosets and support the use of this species for generating a novel genetic-based model of PD that expresses physiological levels of LRRK2 G2019S.Leucine-rich repeat kinase 2 (LRRK2) G2019S is a relatively common cause of Parkinson's disease (PD) 1 . Located in the kinase domain of LRRK2, G2019S is hypothesized to slow transition of the kinase from active to inactive forms, thus effectively increasing Vmax of the enzyme by ~2 fold. This increased kinase activity has been identified as the basis of many PD-associated dysregulated intracellular mechanisms 2-7 . Relative to other common PD-associated gene mutations (e.g. SNCA, Parkin, PINK1, GBA), LRRK2 G2019S has a relatively high penetrance 1,8,9 . The prevalence, penetrance, and sequence conservation of this mutation makes it an excellent target for genetic modeling of PD in a nonhuman species.Because of its genetic similarity to the human genome and shorter lifespan compared to rhesus monkeys 10 , the common marmoset (Callithrix jacchus) has emerged as a candidate nonhuman primate species for modeling age-related disorders, including PD. Genetic approaches for modeling PD (or other diseases) in monkeys has been achieved by introducing the mutant gene using viral vector technologies, via direct intracerebral delivery or microinjection delivery in the oocyte 11,12 . In both cases, the mutant protein is over-expressed at supraphysiological levels. In addition, intracerebral delivery requires brain surgery, and its effects are limited to the target region, which contradicts the current understanding of neurodegeneration as a multisystem disease.Cj-iPSC parental wild type and LRRK2 G2019S clones were fixed and immunostained for the dopaminergic and neuronal markers...

Induced pluripotent stem cell (iPSC)-derived neurons represent an opportunity for cell replacement strategies for neurodegenerative disorders such as Parkinson’s disease (PD). Improvement in cell graft targeting, distribution, and density can be key for disease modification. We have previously developed a trajectory guide system for real-time intraoperative magnetic resonance imaging (RT-IMRI) delivery of infusates, such as viral vector suspensions for gene therapy strategies. Intracerebral delivery of iPSC-derived neurons presents different challenges than viral vectors, including limited cell survival if cells are kept at room temperature for prolonged periods of time, precipitation and aggregation of cells in the cannula, and obstruction during injection, which must be solved for successful application of this delivery approach. To develop procedures suitable for RT-IMRI cell delivery, we first performed in vitro studies to tailor the delivery hardware (e.g., cannula) and defined a range of parameters to be applied (e.g., maximal time span allowable between cell loading in the system and intracerebral injection) to ensure cell survival. Then we performed an in vivo study to evaluate the feasibility of applying the system to nonhuman primates. Our results demonstrate that the RT-IMRI delivery system provides valuable guidance, monitoring, and visualization during intracerebral cell delivery that are compatible with cell survival.

α-Synuclein (α-syn) is a small presynaptic protein distributed ubiquitously in the central and peripheral nervous system. In normal conditions, α-syn is found in soluble form, while in Parkinson’s disease (PD) it may phosphorylate, aggregate, and combine with other proteins to form Lewy bodies. The purpose of this study was to evaluate, in nonhuman primates, whether α-syn expression is affected by age and neurotoxin challenge. Young adult ( n = 5, 5–10 years old) and aged ( n = 4, 23–25 years old) rhesus monkeys received a single unilateral carotid artery injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Three months post-MPTP the animals were necropsied by transcardiac perfusion, and their brains extracted and processed with immunohistochemical methods. Quantification of tyrosine hydroxylase (TH)-positive substantia nigra (SN) neurons showed a significant 80–89% decrease in the side ipsilateral to MPTP administration in young and old animals. Optical density of TH- immunoreactivity (-ir) in the caudate and putamen presented a 60–70% loss compared with the contralateral side. α-Syn-ir was present in both ipsi- and contra- lateral MPTP-treated nigra, caudate, and putamen, mostly in fibers; its intracellular distribution was not affected by age. Comparison of α-syn-ir between MPTP-treated young and aged monkeys revealed significantly higher optical density for both the ipsi- and contralateral caudate and SN in the aged animals. TH and α-syn immunofluorescence confirmed the loss of nigral TH-ir dopaminergic neurons in the MPTP-treated side of intoxicated animals, but bilateral α-syn expression. Colabeling of GAD67 and α-syn immunofluorescence showed that α-syn expression was present mainly in GABAergic fibers. Our results demonstrate that, 3 months post unilateral intracarotid artery infusion of MPTP, α-syn expression in the SN is largely present in GABAergic fibers, regardless of age. Bilateral increase of α-syn expression in SN fibers of aged, compared with young rhesus monkeys, suggests that α-syn-ir may increase with age, but not after neurotoxin-induced dopaminergic nigral cell loss.

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