Induced pluripotent stem cells (iPS cells) have properties similar to those of embryonic stem cells, but they can be derived from any type of somatic cell, such as a skin fibroblast. iPS cells can provide access to specialized cells, such as neurons, in species that otherwise would be unavailable for biomedical research. Among nonhuman primates, the chimpanzee occupies a unique position for comparative biology. Here we propose to use iPS cells from three primates: the chimpanzee, marmoset, and human. Few studies have yet taken advantage of the unique properties of iPS cells for comparative studies of mammalian aging. Our hypothesis is that differentiated cells (motor neurons) derived from three primate species of very different longevities will exhibit differential resistance to physiological stresses, and that pharmacological interventions in key pathways involved in stress resistance will reveal the extent to which mechanisms of stress resistance differ among neurons of these three primate species.
Specific Aim 1 : To validate conditions for efficient and robust derivation of motor neurons from chimpanzee, marmoset, and human iPS cells. We will derive motor neurons from these three primate species using a three-phase protocol (induction of neuroectoderm, neural patterning, and motor neuron development/maturation), by adapting protocols that have been used for efficient differentiation in human pluripotent cells. We hypothesize that motor neurons from all species will be capable of forming typical neuromuscular junctions (NMJs) when co-cultured with a skeletal muscle cell line.
Specific Aim 2 : To assess whether stress resistance in motor neurons of three primate species varies proportionally to the different longevities of these three species. We will use elevated glucose and elevated oxygen as in vitro conditions that mimic the long-term stresses that neurons may experience in vivo. We hypothesize that robustness of neurons under conditions of stress will vary in proportion to the differing life spans of the primate species. We further hypothesize that quality control mechanisms in mitochondria, at the molecular and organellar levels, will be a major determinant of stress resistance.
Specific Aim 3. To assess the potential for pharmacological interventions in key pathways to ameliorate the effects of stresses in motor neurons of the three primate species. While stress resistance is complex, there are three key pathways: NF-?B, p38 MAP kinase, and cell thiol metabolism, that have been implicated in the adverse effects of oxidative stress and hyperglycemia on neural function, or in cellular protective mechanisms. Using drugs that have been well established to act on these three pathways, we will assess whether these interventions influence the adverse effects of stresses in cultured motor neurons. These experiments will allow, for the first time, direct comparisons of stress resistance in isolated neurons from different species and will provide new insights into the mechanisms by which species differ in longevity.
The molecular mechanisms that underly the large differences in life span among mammalian species are largely unknown, even among closely related primates. Within nonhuman primates, the chimpanzee is unique in its relatedness to humans;however, studying chimpanzee cells presents major difficulties. We will generate motor neurons from induced pluripotent stem cells from chimpanzees, marmosets, and humans, and will study their stress resistance in vitro, in order to understand species differences in longevity.