Stroke is a medical condition for which we do not have adequate medical treatments. The purpose of this project is to examine the factors that influence the success of using human stem cells to repair injury caused by a stroke (brain ischemia). Because so much remains to be known before stem cell therapies can be used in stroke patients, we propose studies which will create a model of stroke in the laboratory. The model involves slices of the rat hippocampus (a brain region important to learning and memory and very sensitive to stroke damage). A stroke-like insult can be produced in this explanted tissue and cell damage, repair and function of synapses in the tissue can be subsequently followed for long periods of time. The way in which we will examine the effect of human embryonic stem cells is by transplanting them ("seeding") onto the brain tissue in culture. The effects of the stem cells on repairing injury will then be followed over time. Some of the basic questions we will examine include how the specific location in the hippocampus affects the benefit of stem cell application, how the maturity of the stem cells controls neuroprotection, and if we can use new drugs being developed by a biotechnology company (NeuroNascent, Inc.) to improve the benefits of the stem cell therapy. These basic studies are needed to help guide future studies in animals, and eventually, in patients.
Statement of Benefit to California:
Stroke is a medical condition for which we have limited medical treatments. Repairing the brain following a stroke through the use of human embryonic stem cells offers the potential for improved stroke recovery. This work described in this proposal will address basic questions relating to the best ways to use stem cells to improve stroke recovery. Ultimately, the work will contribute to the health of the citizens of California by fostering the development of new treatments for stroke and related brain diseases.
SYNOPSIS: This application will use hippocampal slice explant cultures to test whether addition of hES cells or hES-derived neural precursor cells can ameliorate the effects of oxygen and glucose deprivation as a mimic of ischemia.
SIGNIFICANCE AND INNOVATION: This proposal is non-forward looking with many old ideas that are neither fundamental, mechanistic, nor foundational for the field. It is not clear how or if this work could/would move the field forward.
STRENGTHS: The applicant already has experience with this basic paradigm using postnatal hippocampal neural stem cells. Provided that growth and differentiation of hES cells can be mastered, the proposed experiments should be attainable. Many of the technical aspects are fine.
WEAKNESSES: This proposal is largely descriptive and "hopeful" that ES cells will simply cure the pathology without guidance/control. The proposal is directed at "proving" a series of things rather than investigating whether a series of ideas or hypotheses or biological results are true. Much of the application is written in a way that seems to presuppose the outcome of experiments before they have been performed. For example, from Aim 1: “We will show that hESCs and neuronal progenitor cells derived from hESCs will reduce injury severity following simulated ischemia...” and “We will show that the neuroprotection depends on the specific location of the cell seeding...” If these outcomes are already known, why perform the experiments?
The experiments in Aim 1 involve 2- and 7-day survival times after a suspension of dissociated cells are deposited into the slice. Are the undifferentiated hES cells expected to survive and remain undifferentiated throughout this period? Predifferentiated neural precursors derived from hES cells may do well in typical slice culture conditions, but undifferentiated hES cells probably will not. If they do survive, it is unlikely that they will remain undifferentiated. The applicants need to consider how the differentiation status of the hES cells can be evaluated. What specifically will the Cellomics Array Scanner measure? How will these measurements be interpreted?
Because tetanus-induced LTP is a cooperative phenomenon, cell loss following ischemia could negatively impact this form of LTP without causing any direct modification in the underlying synaptic physiology. Thus the effect of added stem cells on tetanus-induced LTP would need to be interpreted with extreme caution.
The use of novel neurogenic agents in Aim 3 adds interest to the proposal, but the need for twice a month visits between UCSF and Neuronacent in Maryland seems excessive for “intensive planning and project coordination”.
DISCUSSION: Reviewers recommend that the applicant elaborate on how the differentiation status of seeded ES cells will be determined, with particular regard to undifferentiated ES cells over a 7-day time period. The PI should consider a more extensive evaluation of cell/synaptic physiology that might alleviate concerns about interpretation of LTP experiments. Also, the PI should write the proposal so that it is clear what information is already known and what outcomes remain to be tested by the proposed experiments.