Damage to the human brain or spinal cord in the form of spinal cord injury, head trauma, stroke, Alzheimer’s disease, Parkinson’s disease, Lou Gehrig’s disease (ALS), and other forms of neurotrauma affects many thousands of Californians every year. Since our current human patient treatment regimes are unable to reverse the damage done in these types of brain and spinal cord injury, a great hope is that human embryonic stem cell (hESC) technology will be developed to replace dead brain or spinal cord cells called neurons. The ultimate goal here would be to be able to implant hESC s into the brain or spinal cord and to guide their development to mature and functional neurons. Before this lofty goal can be realized we need to know how the cellular environment around the hESC implantation can affect the development and maturation of these cells. Inflammation occurs in and around neurotrauma and at neural implantation and this is characterized by the increased presence of a variety of proteins that are inflammatory modulators that are called cytokines. The cytokine in control of the continued progression of inflammation after or during injury is tumor necrosis factor alpha (TNF). A specific class of proteins on the surface of hESCs called TNF receptors can bind TNF and thus can potentially signal to control the activities and development of these hESCs. Thus, increased availability of TNF caused by post traumatic injury, disease, or hESC implantation to the central nervous system suggests that this protein could potentially influence stem cell fate if TNF receptor signaling affects hESC fate determination. In fact, recently published studies suggest that TNF shifts neuronal precursor cells (a type of adult stem cell) from neuron to non-neuronal fates. But other studies have shown that TNF’s actions have good as well as bad consequences for the successful shift from implanted stem cells to mature neurons; TNF increases the survival of the implanted cells but it also shifts the fate of the stem cells to non-neuronal cell types. This proposal seeks to dissect apart the diverse developmental mechanisms initiated by TNF in hESCs and determine which of these mechanisms are good and which are bad for their shift to mature neurons. Our data will be vital to the success of future hESC studies aimed at replacing human brain and spinal cord neurons killed or damaged by trauma or disease . We predict that TNF utilizes separate cellular mechanisms for the increase in survival of hESCs and for the guidance to their ultimate mature cell type. If the data supports our hypothesis, the implications for future potential hESC treatment are profound. It will imply that precise and specialized control over local inflammation and TNFs actions around the site of implantation will be vital to the success of hESC treatment for damaged or diseased brain or spinal cord tissue in human patients.
Statement of Benefit to California:
Damage to the human brain or spinal cord in the form of spinal cord injury, head trauma, stroke, Alzheimer’s disease (AD), Parkinson’s disease, Lou Gehrig’s disease (ALS), or other forms of neurotrauma afflicts a highly significant proportion of Californians. The number of AD patients is just a fraction of this total, yet the Alzheimer’s Foundation estimates the current number affected in California is over half a million. Since our current human patient treatment regimes are unable to reverse the damage done in these types of brain and spinal cord injury, a great hope is that human embryonic stem cell (hESC) technology will be developed to replace dead brain or spinal cord cells called neurons. The ultimate goal here would be to be able to implant hESC s into the brain or spinal cord and to guide their development to mature and functional neurons. Currently this is not possible, partially due to our lack of knowledge about how to properly encourage hESCs to develop into functional brain and spinal cord cells in the adult nervous system. This proposal seeks basic and vital information about inflammation around the implantation site and how this affects the development of hESCs towards functional brain and spinal cord cells. The emotional cost to patients suffering from the above mentioned neurotrauma is obviously tremendous. The financial cost to California from just one of these maladies, Alzheimer’s Disease (AD), represents just a fraction of the total societal cost of all the diseases which this research has the potential to affect. Still, this figure for the cost of AD in California is staggering. A 2001 publication in the Journal of Public Health Policy estimates the societal cost to California for the treatment and care of the state’s patients with AD were $ 22.4 billion in the year 2000. The authors predicted that the cost will soar to $68.1 billion in 2040. With these incomprehensibly large estimates before us it is important to realize that any improvement in current treatments resulting from this line of research will carry a very significant emotional and financial benefit for individual neurotrauma patients, their families, and the state of California.
SYNOPSIS: This proposal seeks to explore the role/s that the TNF-alpha may play on neural stem cells and human embryonic stem cell (hESC) differentiation to a neural phenotype, and the survival of these cells as it relates to injury of the CNS. Using an in vitro system in which neural stem cells and hESCs are added to rat hippocampal neurons, the applicant will dissect the TNF-alpha signaling pathways that may be involved in survival and differentiation. Using antibodies that target specific subsets of the signaling from TNFR1 to TNFR2, the PI will test some of the intracellular signaling pathways downstream of the two TNF receptors. This might give additional information about mechanisms of hESC neural differentiation or might point out pathways that other receptor activations may converge upon. SIGNIFICANCE AND INNOVATION: The role that inflammation and its complex components play on cell survival and differentiation in the CNS is coming under great scrutiny as it is realized that many previously recognized proinflammatory molecules such as TNF-alpha might also have positive effects on repair. The effects of TNFalpha on hESC differentiation along neural lineages have not been studied. Thus, this proposal is significant in that it could identify parts of the TNF signaling cascade that may be involved in stem cell survival and differentiation. This in turn could be important in identifying therapeutic targets in the injured CNS and where stem cell therapy might be applied. STRENGTHS: This is a significant and important area of research in spinal cord injury and stem cell therapy. This proposal from a well qualified investigator with a first rate environment and collaborators is well written and clear, and the PI has extensive experience examining the effects of TNF-alpha on neuronal differentiation and survival. The PI will use a well-developed model system derived from rat hippocampus. WEAKNESSES: hESCs will be labeled and their differentiation (or lack thereof) will be followed in cultures. Although beginning with a culture system has advantages, it cannot reproduce the far more complex environment of a brain, especially a diseased brain, if one wants to think about neuronal differentiation from implanted ESCs as a therapy. Thus, it is impossible to predict whether ESCs will behave in the same way in vivo. The addition of TNFs to the cultures is a direct way to investigate effects on ES cells, but it will not tell the PI if the actions are direct, or indirect (i.e., through other cell types in the cultures). Still, the experiments may give some interesting data on TNF-alpha signaling in ES cells in a neural environment. An important missing point in the Research Design is note of any prior differentiation of hESCs to embryoid bodies and neural stem cells. Presumably without the appropriate culture conditions to generate such induction in their co-culture paradigm, neurons will not develop. DISCUSSION: This is a well developed model in the rat hippocampus, but the comparison here is rat to human, and it would be better to compare human to human. The researchers seem to be looking at a partially rebuilt rat hippocampus, so the rationale is not great. While the in-vitro system is well characterized in the PI's lab, it is not clear that hESCs will differentiate into neuronal lineages in their co-culture system. Since the PI is adding TNF to a culture with neurons, astrocytes, and ESCs, they won't be able to say whether the effect is direct or indirect. How will they determine the effects caused specifically by TNF? Also, why do co-culture as opposed to adding astrocytes? One reviewer questioned whether this work would build on the field because the applicant is not taking advantage of directed differentiation protocols. The PI seems to be trying to tease cells into differentiating when directed protocols already exist.