The long-term objectives of this research are to identify safe and effective anesthetics to be used for human stem cell transplantation and to define the effect anesthetics have on stem cells in vivo. To achieve this goal we will identify the effect of several common anesthetic drugs on stem cells in culture and in animals. Specifically, we will determine whether anesthetics change the rate of growth of stem cells or limit the type of cell they may eventually become.
Statement of Benefit to California:
Human embryonic stem cell based therapies will likely be attempted for multiple diseases in many different organ systems in the next few decades. Stem cell transplant in humans and experimentation in animal models will require sedation or complete general anesthesia for many therapies. Very little research has been done on the role that common anesthetics may play in the biology of human stem cells, and how such anesthetics may affect the function or differentiation of these cells once transplanted. Choosing the correct anesthetic may impact the success or failure of early animal and human clinical trials. This proposal focuses specifically on neural stem cells which have been proposed as a potential treatment for many different pathologic states including Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, stroke, spinal cord injury and traumatic brain injury. Some stem cell transplants will be performed under general anesthesia and some will be performed in individuals likely to undergo multiple or long duration anesthetics around the time of their injury and potential transplant (i.e. traumatic brain injury, spinal cord injury, and neonatal stroke) leading to more anesthetic exposure after the transplant. Understanding the role of anesthetics in stem cell biology is imperative and will provide the basis for developing appropriate anesthetic techniques for stem cell based clinical applications.
SYNOPSIS: This proposal will look at five commonly used anesthetics that act on GABA and NMDA receptors as to their potential effects on neural stem cells (NSCs) derived from hESCs. Aim 1: Dose response and time course of these anesthetics will be studied on NSCs in vitro to determine potential growth inhibitory or toxic effects. Aim 2: Fate choice decisions of these cells exposed in ways derived from Aim 1 in culture will be assayed using immunophenotypic analyses; and, Aim 3: in vivo analysis of rat hippocampal NSCs, using BrdU proliferation, immunolabeling and unbiased stereology/confocal microscopy, will follow anesthetic administration as gleaned from Aims 1 and 2. SIGNIFICANCE AND INNOVATION: This application will examine the effect of anesthetics on hESC proliferation and differentiation. Because anesthetic agents will be needed for transplantation of stem cells, or more differentiated cells derived stem cell, this is an important issue. The idea of understanding the effects of different, commonly used animal and human anesthetics on the growth and differentiation of stem cells is innovative and important. We know little about this subject and it can have profound effects on cell growth and survival in future stem cell therapeutic approaches. That said, the choice of hESCs and particular aspects of experimental design laid out here have serious flaws that warrant the PI to rethink his choice of models. STRENGTHS: There is a need to understand something the field takes rather for granted – using any anesthetic you feel like when isolating or grafting stem cells. There is no question that these drugs should have effects on proliferation, survival, and differentiation of stem cells, and there is a pressing need to characterize these effects. The PI has a dedicated interest in discovering effects of well-used anesthetic compounds on neural stem cell behaviors in vitro and in vivo. The applicants already have preliminary evidence that anesthetic agents can affect growth and differentiation of hippocampal neural stem cells. WEAKNESSES:The PI and his collaborators have little or no experience or expertise with human NSCs, and none with hESCs. The rational for choosing to work on unapproved hESC lines for these studies is extremely weak. Most if not all of the important issues raised by the PI regarding stem cell exposures to anesthetic agents could and probably should be worked out on adult NSCs and if hESCs are needed, approved lines certainly would be fine. Aims 1 and 2 are related because both involve studying hESC-derived NSCs in vitro following exposure to different types and dosages of well-used anesthetics; in contrast Aim 3 will study a completely different issue by looking at the effects of these drugs on adult neural stem cells in the rat hippocampus. It is not clear how Aim 3 is different from ongoing research that is already funded in the applicant’s laboratory. The ability to compare effects of anesthetics on hESCs and hippocampal NSCs is an advantage, but there is little justification for CIRM support of work on non-hESCs that is already funded by another entity. The applicants note that many anesthetic agents can act on GABA or NMDA receptors. They seem to assume that actions on these receptors will provide the explanation for any effects the anesthetics may have on hESCs. The proposal would be much stronger if it included explicit tests using highly selective GABA or NMDA receptor antagonists to see whether blockade of these receptors is sufficient to explain the action of anesthetics or whether additional mechanisms are involved. The PI is encouraged to continue to test and publish some of his hypotheses on the progenitor cell populations documented in Figures 2-6 as proof of principle before going on to hESCs (which are not easy to work with and really not justified to answer most of the proposed questions). One reviewer suggested removing Aim 3 and substituting experiments to address which specific targets of anesthetic agents are required for any effects that are observed on hESCs. DISCUSSION: There was no discussion following the reviewers' comments