Interaction between transplanted human embryonic stem cell-derived neuronal precursors and endogenous neurogenesis in ischemic brain
Acute and chronic neurodegenerative diseases are common, disabling, and poorly responsive to current treatment. Stroke, the most frequent cause of acute neurodegeneration, has a prevalence of about 5 million and an incidence of approximately 700,000 individuals per year in the United States, where it is the third leading cause of death. Even among those who survive stroke, disability due to limb weakness, gait disorders, language impairment and other deficits is common, and roughly 20% of patients require institutional care at 6 months post-stroke. This long-term disability contributes to the average lifetime cost for stroke care of nearly $150,000 and an annual national cost of over $50 billion. The most recent major advance in treatment, the use of thrombolytic agents to dissolve clots in the acute aftermath of stroke, is limited in impact because it appears to be effective only within about the first 3 hours after onset of symptoms. Clinical manifestations of acute and chronic neurodegenerative diseases, including stroke, result primarily from irreversible cellular (especially neuronal) dysfunction and, eventually, cell death. Based on this experience, it is reasonable to conclude that cell-replacement therapy, technically challenging though it may be, is worth pursuing. In addition to the prospect of more completely restoring brain function, cell-replacement therapy has the further advantage that it might be effective at later stages of a disease. This is an important consideration not only in disorders like stroke, which often evolve too quickly for acute treatment to be instituted, but also in chronic neurodegenerations, where cell loss may already be extensive before the onset of symptoms. At least two approaches to nerve cell replacement are possible. First, the brain itself generates new nerve cells throughout life. Their production is increased following stroke and is associated with migration of these new cells to affected brain regions. Moreover, blocking this process impairs recovery, suggesting that it is important for brain repair. A second approach involves cell transplantation from external sources, including human embryonic stem cells. Advantages of this strategy include the ability to expand and mature cells in vitro, engineer cells, and deliver cells directly to sites of injury. A critical step in assessing the feasibility of this approach in stroke is to understand the interaction between transplanted human embryonic stem cells and the stroke brain. We propose to investigate this interaction, and hypothesize that it may help determine the success of cell replacement in the brain following stroke, and that its modification may help optimize the neurotransplantation therapy.
Statement of Benefit to California:
In November 2002, the California Department of Health Services published a report titled, “Heart Disease and Stroke in California: Surveillance and Prevention”. The report showed that in 1999, 96,208 Californians had been hospitalized for stroke. The majority (54%) were women and almost 25% were under age 65. The average length of stay was 8 days, and the cost averaged $2,555 per day, accounting for total hospital charges of $2.1 billion. Although the age-adjusted rate for death from stroke in California had decreased in the two decades leading up to 1999, stroke still accounted for 18,079 deaths, or 8% of all deaths in the state. Stroke death rates were higher in African-Americans than in other ethnic groups. And while the prevalence of certain stroke risk factors, like hypertension, had declined, others, including diabetes and hypercholesterolemia, were increasing. Californians, like others, have very limited treatment options once stroke occurs. If they are lucky enough to arrive within about three hours post-stroke at one of eighteen primary stroke centers in the state accredited by the Joint Commission on Accreditation of Healthcare Organizations, they may be candidates for thrombolytic therapy, which can dissolve the clot responsible for their symptoms. However, many patients do not arrive at the hospital quickly enough to benefit from this form of therapy, and some of those who do suffer bleeding complications. The fact that patients with stroke usually improve spontaneously for up to about three months after the event suggests that the brain is conducive to at least some degree of repair. If spontaneous improvement is incomplete, it might be possible to intervene to enhance recovery. Although many such interventions may be possible, the fact that cell loss is such a prominent feature of stroke implies that cell replacement therapy could be a useful approach. This proposal explores that possibility, by investigating the interaction between the brain’s own cell-replacement mechanism and the transplantation of new nerve cells derived from human embryonic stem cells.
SYNOPSIS OF PROPOSAL: Cerebral vascular accidents or stroke is the most common neurological injury and impairs a large number of Americans each year because of the devastating consequences of this acute and subsequently chronic neurological injury. Because stroke rapidly kills brain cells, the potential of replacing these cells could be an attractive therapeutic approach. One could argue that therapies might involve either enhancement of adult endogenous progenitor cells or the use of cell transplantation back from exogenous sources. Animal data already suggest that following cerebral infarct there is an increase in neurogenesis and blocking exogenous neurogenesis seems to adversely affect repair recovery from ischemia. In this proposal, the investigators will study the reciprocal interactions between implanted exogenous neuroprogenitor cells (NPCs) produced in vitro from hESCs and neurogenesis in vivo in a rodent stroke model. IMPACT AND SIGNIFICANCE: As mentioned above, stroke is a particularly devastating and common neurologic injury, and the value of stem cells in a stroke setting are potentially complicated and arguably controversial – but certainly worth pursuing by a laboratory experienced in stroke models with appropriate collaborations and experience in utilizing stem cells. There are essentially no pharmaceutical interventions in stroke, so an approach employing a systematic study of stem cells in these models is certainly warranted. Overall the methods proposed in the study are not particularly innovative or original, nor is the idea of using stem cells in these models. Nevertheless, good information will be obtained from the systematic evaluation of both the biology of human stem cells in this rat injury model and understanding the effects of endogenous neurogenesis of rat cells on the added human stem cells. Stem cells, angiogenic factors, and biodegradable scaffolds all have the potential to facilitate the rebuilding of cortical tissue with functional integrity following stroke. The goal of this proposal is to look at the interactions of grafted hESCs and endogenous neural precursor cells in the subventricular zone of the brain to see if morphogenetic factors affect either population of cells and subsequent cell replacement. Angiogenic factors like VEGF may also support tissue rebuilding, so there is therefore some potential impact and significance from the proposed studies. Improving recovery of neural tissue and function after stroke would have a strong public health impact. QUALITY OF THE RESEARCH PLAN: This is a crisp, well-defined proposal employing essentially existing techniques in the laboratory. That said, the proposed studies do involve a great deal of cell preparation, factor searching, transplantation into stroke cavities with and without trying biodegradable, biopolymer scaffolds to hold and support the cells, along with cell immuno-phenotyping and behavioral analyses of the potentially recovering animals. Including the electrophysiological characterizations of the newly-generated cells in the ischemic-injured brain, all of the proposed studies represent a rather significant commitment of time and effort (most likely beyond that predicted in the proposal). The research plan is well organized and brings together a number of different types of expertise although the collaborations are just starting. Interesting data will be achieved but may be difficult to interpret due to the complexity of the system. STRENGTHS: The idea of examining the effects of exogenous stem cells on endogenous stem cells and vice versa is intriguing and likely to be scientifically worthy. There probably will be a future need to combine exploitation of endogenous neurogenesis, introduction of neurogenic and angiogenic factors, along with primed cells to rebuild the stroke brain. This proposal goes a long way toward predicting some of the cell and molecular strategies that could facilitate this process. Previous pilot studies by the applicant in which they transplanted GFP-expressing mouse E15 NPCs, following the MCAO model proposed here, were very encouraging and showed increased cell survival, migration within the ischemic area, and expression of classical neuronal markers versus that seen in non-ischemic rodent CNS. The stroke model and conditions created here may therefore be rather supportive for the types of injury and grafting studies performed here now using hESC-derived cells. The PI is well-qualified for the proposed research with extensive experience in generating unique animal models, and the use of multiple outcome measures is a strength. WEAKNESSES: In the first objective, it did not appear that the investigators were actually using a control for the effects of human stem cells alone in wild type animals — that is: does engraftment of the human stem cells have any positive or negative effect on endogenous rat progenitor cells? It is not at all clear that the cross talk between human cells and rat stem cells will be applicable to what might be observed ultimately in humans and therefore, what we learn of the biology from rat to human transplantation remains unclear at least in understanding the cross talk between these cells. The investigators hope to eliminate the endogenous stem cells in a rat to see if this has any effect on the grafted human cells. Although this is a somewhat interesting exercise, it seems just that-- an exercise --it doesn’t appear to provide any clear insight into the real human condition and it is not entirely clear what will be learned from this experiment. The investigators presume that there is some critical interaction between the engrafted human cells and endogenous rat stem cells; however, the population of endogenous cells is quite small and any factors they may be releasing may be quite low relative to those released by the activated endogenous microglial and astroglial populations. The investigator does not seem to take into account these other, overwhelming cellular populations. The quantification of these effects is a little unclear; the investigators are supposed to use stereological methods although it’s not yet clear how reliable quantification of endogenous cells, versus the larger number of cells being added in through an injection, will be actually followed. In the last aim, the investigators hope to use angiogenic factors such as VEGF to enhance neural progenitor cell restoration of tissue function. However, the investigators already have positive data on VEGF alone--- and it’s certainly possible that these two will act in a synergistic or additive way; the timing of these experiments would also likely be critical. Often progenitor cells migrate in tissue by crawling down existing blood vessels; one might imagine it might be necessary to apply endogenous factors in advance of the stem cells. For example, will migration of stem cells occur much faster with or without the presence of VEGF? Overall this is a relatively interesting proposal and certainly the methods, skills, collaborations are in place to carry out these experiments – whether the interpretation of the planned experiments will be useful for human use remains unclear. As the PI points out, methods to ablate SVZ neurogenesis “may lack specificity and affect other cell types or functions, or may affect a non-representative subpopulation of NPCs”. The PI’s choice of follow up studies using surgical, knockout or gene knockdown of genes still may not improve specificity. Very open-ended studies are proposed as backups or follow-ups that do not help the cause, e.g. it is pointed out that the biopolymer scaffolds might negatively affect endogenous neurogenesis via bringing in inflammatory responses or physically acting as a barrier to cell infiltration. The potential testing of other synthetic (e.g., PEO, PVA) and naturally occurring (e.g., collagen, alginate, chitosan, hyaluronate) substances make this part of the proposal itself potentially very time consuming, along with the need to engineer NPCs to overexpress VEGF or other angiogenic factors (e.g. FGF-2, angiopoietins). The combinatorics of variables is a bit daunting. Overall, the biomaterials component is weak and some statements are incorrect. Many of the other statements related to the materials are also very vague. As stated, published works in the field have demonstrated significant improvement in cell transplantation and tissue formation when biomaterials are implanted in conjunction with cells in the brain. The grafted hES cells documented in Fig 4 look at bit undifferentiated, and tissue integrity looks peculiar. These transplant studies will most likely require a great deal of attention to make sure the cells are exposed to the best differentiation conditions; this alone could take most of the attention of this proposal. All of the growth factors released from many different cells in the ischemic cortex themselves are so complex and interactive as to make studies of exogenously introduced factors potentially very complex and difficult to understand. There are already many studies like the ones proposed here being done using ES and adult neural stem and progenitor cells. Another significant challenge in this proposal is isolating the influences of the NPCs and ESCs. There are so many confounding factors in the in vivo environment with other cell types in addition to the NPCs that will change when they are ablated that preliminary in vitro experiments to avoid these confounding factors may help to validate observations found in vivo. For example, a three dimensional system can be used in vitro where cells can be co-cultured and cell-cell interactions can be isolated. From a more simple perspective, the standard transwell system can be used. DISCUSSION: This proposal is from a well-known PI studying stroke who plans to enter the hESC field. Applying ESC to the treatment of stroke is interesting and much could be gained from these studies. Also, it is an interesting idea to study the influence of transplanted cells on endogenous progenitors (humans vs. rats). The PI wants to go both ways in this study, but the methods are not well-worked out. It is not clear to the reviewers what will be learned from this study as designed. In the end, how will any of these results can be translated to a therapeutic setting? It appears that the applicant may be underestimating the considerable challenge that successful VEGF delivery poses. There was unanimous agreement that the weakest part of the grant was the biomaterials section. The PI has established a new collaboration with a biomaterials researcher, and it seems that this new collaborator did not read through the grant because there were incorrect statements made regarding the chemicals, materials, etc.