Optimization of Differentiation of Nonhuman Primate Pluripotent Cells Using a Combinatorial Approach.

The directed differentiation of pluripotent stem cells to a desired lineage often involves complex and lengthy protocols. In order to study the requirements for differentiation in a systematic way, we present here methodology for an iterative approach using combinations of small molecules and biological factors. The factors are used in a cyclical process in which the best combination of factors and concentrations is selected in one round of testing, followed by a modification of the combination and subsequent rounds. While this may produce the desired differentiation in the cell population under study, it is also possible that other strategies may be needed to optimize the differentiation process. These strategies are described in this chapter.

The Route by Which Intranasally Delivered Stem Cells Enter the Central Nervous System.

Intranasal administration is a promising route of delivery of stem cells to the central nervous system (CNS). Reports on this mode of stem cell delivery have not yet focused on the route across the cribriform plate by which cells move from the nasal cavity into the CNS. In the current experiments, human mesenchymal stem cells (MSCs) were isolated from Wharton's jelly of umbilical cords and were labeled with extremely bright quantum dots (QDs) in order to track the cells efficiently. At 2 h after intranasal delivery in immunodeficient mice, the labeled cells were found under the olfactory epithelium, crossing the cribriform plate adjacent to the fila olfactoria, and associated with the meninges of the olfactory bulb. At all times, the cells were separate from actual nerve tracts; this location is consistent with them being in the subarachnoid space (SAS) and its extensions through the cribriform plate into the nasal mucosa. In their location under the olfactory epithelium, they appear to be within an expansion of a potential space adjacent to the turbinate bone periosteum. Therefore, intranasally administered stem cells appear to cross the olfactory epithelium, enter a space adjacent to the periosteum of the turbinate bones, and then enter the SAS via its extensions adjacent to the fila olfactoria as they cross the cribriform plate. These observations should enhance understanding of the mode by which stem cells can reach the CNS from the nasal cavity and may guide future experiments on making intranasal delivery of stem cells efficient and reproducible.

Induced Pluripotent Stem Cells from Nonhuman Primates.

Induced pluripotent stem cells from nonhuman primates (NHPs) have unique roles in cell biology and regenerative medicine. Because of the relatedness of NHPs to humans, NHP iPS cells can serve as a source of differentiated derivatives that can be used to address important questions in the comparative biology of primates. Additionally, when used as a source of cells for regenerative medicine, NHP iPS cells serve an invaluable role in translational experiments in cell therapy. Reprogramming of NHP somatic cells requires the same conditions as previously established for human cells. However, throughout the process, a variety of modifications to the human cell protocols must be made to accommodate significant species differences.

Marmoset induced pluripotent stem cells: Robust neural differentiation following pretreatment with dimethyl sulfoxide.

The marmoset is an important nonhuman primate model for regenerative medicine. For experimental autologous cell therapy based on induced pluripotent (iPS) cells in the marmoset, cells must be able to undergo robust and reliable directed differentiation that will not require customization for each specific iPS cell clone. When marmoset iPS cells were aggregated in a hanging drop format for 3 days, followed by exposure to dual SMAD inhibitors and retinoic acid in monolayer culture for 3 days, we found substantial variability in the response of different iPS cell clones. However, when clones were pretreated with 0.05-2% dimethyl sulfoxide (DMSO) for 24 hours, all clones showed a very similar maximal response to the directed differentiation scheme. Peak responses were observed at 0.5% DMSO in two clones and at 1% DMSO in a third clone. When patterns of gene expression were examined by microarray analysis, hierarchical clustering showed very similar responses in all 3 clones when they were pretreated with optimal DMSO concentrations. The change in phenotype following exposure to DMSO and the 6 day hanging drop/monolayer treatment was confirmed by immunocytochemistry. Analysis of DNA content in DMSO-exposed cells indicated that it is unlikely that DMSO acts by causing cells to exit from the cell cycle. This approach should be generally valuable in the directed neural differentiation of pluripotent cells for experimental cell therapy.

Directed neural differentiation of induced pluripotent stem cells from non-human primates.

Induced pluripotent stem cells (iPS cells) are important for the future development of regenerative medicine involving autologous cell therapy. Before autologous cell therapy can be applied to human patients, suitable animal models must be developed, and in this context non-human primate models are critical. We previously characterized several lines of marmoset iPS cells derived from newborn skin fibroblasts. In the present studies, we explored methods for the directed differentiation of marmoset iPS cells in the neuroectodermal lineage. In this process we used an iterative process in which combinations of small molecules and protein factors were tested for their effects on mRNA levels of genes that are markers for the neuroectodermal lineage. This iterative process identified combinations of chemicals/factors that substantially improved the degree of marker gene expression over the initially tested combinations. This approach should be generally valuable in the directed differentiation of pluripotent cells for experimental cell therapy.

Patient-specific induced pluripotent stem cells in neurological disease modeling: the importance of nonhuman primate models.

The development of the technology for derivation of induced pluripotent stem (iPS) cells from human patients and animal models has opened up new pathways to the better understanding of many human diseases, and has created new opportunities for therapeutic approaches. Here, we consider one important neurological disease, Parkinson's, the development of relevant neural cell lines for studying this disease, and the animal models that are available for testing the survival and function of the cells, following transplantation into the central nervous system. Rapid progress has been made recently in the application of protocols for neuroectoderm differentiation and neural patterning of pluripotent stem cells. These developments have resulted in the ability to produce large numbers of dopaminergic neurons with midbrain characteristics for further study. These cells have been shown to be functional in both rodent and nonhuman primate (NHP) models of Parkinson's disease. Patient-specific iPS cells and derived dopaminergic neurons have been developed, in particular from patients with genetic causes of Parkinson's disease. For complete modeling of the disease, it is proposed that the introduction of genetic changes into NHP iPS cells, followed by studying the phenotype of the genetic change in cells transplanted into the NHP as host animal, will yield new insights into disease processes not possible with rodent models alone.