Reference Transcriptome for Deriving Marmoset Induced Pluripotent Stem Cells.

Limited access to primary tissue from various nonhuman primate (NHP) species represents a significant unmet need that hampers progress in understanding unique cellular diversity and gene regulation of specific tissues and organs in stem cell translational research. Most comparative biology studies have been limited to using postmortem tissue usually frozen specimens with limited utility for research. The generation of induced pluripotent stem cell (iPSC) lines from somatic cells, such as adult skin or blood cells, offers an alternative to invasive and ethically controversial interventions for acquiring tissue. Pluripotent iPSCs have virtually an unlimited capacity to proliferate and differentiate into all cell types of the body. We are generating high-quality validated NHP iPSC lines to offer to scientific community and facilitate their research programs. We use the non-integrative episomal vector system to generate iPSCs from NHP skin biopsies. In this chapter we describe the validation of NHP iPSC lines by confirming pluripotency and their propensity to differentiate into all three germ layers ectoderm, mesoderm, and endoderm according to established standards and measurable limits for a set of marker genes incorporated into a scorecard.

Standards for Deriving Nonhuman Primate-Induced Pluripotent Stem Cells, Neural Stem Cells and Dopaminergic Lineage.

Humans and nonhuman primates (NHP) are similar in behavior and in physiology, specifically the structure, function, and complexity of the immune system. Thus, NHP models are desirable for pathophysiology and pharmacology/toxicology studies. Furthermore, NHP-derived induced pluripotent stem cells (iPSCs) may enable transformative developmental, translational, or evolutionary studies in a field of inquiry currently hampered by the limited availability of research specimens. NHP-iPSCs may address specific questions that can be studied back and forth between in vitro cellular assays and in vivo experimentations, an investigational process that in most cases cannot be performed on humans because of safety and ethical issues. The use of NHP model systems and cell specific in vitro models is evolving with iPSC-based three-dimensional (3D) cell culture systems and organoids, which may offer reliable in vitro models and reduce the number of animals used in experimental research. IPSCs have the potential to give rise to defined cell types of any organ of the body. However, standards for deriving defined and validated NHP iPSCs are missing. Standards for deriving high-quality iPSC cell lines promote rigorous and replicable scientific research and likewise, validated cell lines reduce variability and discrepancies in results between laboratories. We have derived and validated NHP iPSC lines by confirming their pluripotency and propensity to differentiate into all three germ layers (ectoderm, mesoderm, and endoderm) according to standards and measurable limits for a set of marker genes. The iPSC lines were characterized for their potential to generate neural stem cells and to differentiate into dopaminergic neurons. These iPSC lines are available to the scientific community. NHP-iPSCs fulfill a unique niche in comparative genomics to understand gene regulatory principles underlying emergence of human traits, in infectious disease pathogenesis, in vaccine development, and in immunological barriers in regenerative medicine.

Magnetic Resonance Imaging-Guided Delivery of Neural Stem Cells into the Basal Ganglia of Nonhuman Primates Reveals a Pulsatile Mode of Cell Dispersion.

Optimal stem cell delivery procedures are critical to the success of the cell therapy approach. Variables such as flow rate, suspension solution, needle diameter, cell density, and tissue mechanics affect tissue penetration, backflow along the needle, and the dispersion and survival of injected cells during delivery. Most cell transplantation centers engaged in human clinical trials use custom-designed cannula needles, syringes, or catheters, sometimes precluding the use of magnetic resonance imaging (MRI)-guided delivery to target tissue. As a result, stem cell therapies may be hampered because more than 80% of grafted cells do not survive the delivery-for example, to the heart, liver/pancreas, and brain-which translates to poor patient outcomes. We developed a minimally invasive interventional MRI (iMRI) approach for intraoperatively imaging neural stem cell (NSC) delivery procedures. We used NSCs prelabeled with a contrast agent and real-time magnetic resonance imaging to guide the injection cannula to the target and to track the delivery of the cells into the putamen of baboons. We provide evidence that cell injection into the brain parenchyma follows a novel pulsatile mode of cellular discharge from the delivery catheter despite a constant infusion flow rate. The rate of cell infusion significantly affects the dispersion and viability of grafted cells. We report on our investigational use of a frameless navigation system for image-guided NSC transplantation using a straight cannula. Through submillimeter accuracy and real-time imaging, iMRI approaches may improve the safety and efficacy of neural cell transplantation therapies. Stem Cells Translational Medicine 2017;6:877-885.

Methylene blue modulates functional connectivity in the human brain.

Methylene blue USP (MB) is a FDA-grandfathered drug used in clinics to treat methemoglobinemia, carbon monoxide poisoning and cyanide poisoning that has been shown to increase fMRI evoked blood oxygenation level dependent (BOLD) response in rodents. Low dose MB also has memory enhancing effect in rodents and humans. However, the neural correlates of the effects of MB in the human brain are unknown. We tested the hypothesis that a single low oral dose of MB modulates the functional connectivity of neural networks in healthy adults. Task-based and task-free fMRI were performed before and one hour after MB or placebo administration utilizing a randomized, double-blinded, placebo-controlled design. MB administration was associated with a reduction in cerebral blood flow in a task-related network during a visuomotor task, and with stronger resting-state functional connectivity in multiple regions linking perception and memory functions. These findings demonstrate for the first time that low-dose MB can modulate task-related and resting-state neural networks in the human brain. These neuroimaging findings support further investigations in healthy and disease populations.