Suppression of rejection of embryonic stem cells and their derivatives. Human embryonic stem cells (hESC) hold great promise in the treatment of human disease since they can give rise to cells that are lost during disease. In particular, we have the capability to induce the differentiation of hESC into oligodendrocytes and neurons, which may be useful in treatment of a variety of neurological diseases. A significant problem may arise in the long-term survival of hESC-derived oligodendrocytes or neurons, however, due to rejection mediated by cytotoxic T cells (CTL) of the host immune system. Medicinal suppression of the immune system is useful, but has attendant adverse side-effects. In contrast, viruses escape CTL mediated immunity by down-regulating the expression of HLA-A and B cell surface molecules on infected cells. We will mimic this strategy using siRNA, a strategy for which the 2006 Nobel prize in Physiology or Medicine was awarded. HLA-A and B negative and control cells will be introduced into rats subjected to thoracic spinal cord contusion injury, a model that has been used extensively by our collaborator. Survival of experimental and control oligodendrocytes and neurons will be assayed by immunohistology of tissue derived from the site of injury and human cell engraftment. Our approach may be useful in prolonging the survival of hESC-derived cells of all lineages and for all uses, since nearly all cells express MHC-I glycoproteins. Our work is particularly relevant to treatment of spinal cord injury, multiple sclerosis and Parkinson’s disease as well as other diseases where loss of neurons or oligodendrocytes causes adverse effects.
Statement of Benefit to California:
Our work will directly benefit the citizens of California since all clinical uses of of human embryonic stem cells (hESC) will benefit from increased survival of hESC and their derivatives. In particular we will modify hESC-derived brain cells (oligodendrocytes and neurons) to survive longer in patients. These cells will be useful in treating spinal cord injury, multiple sclerosis and Parkinson's disease, which together affect 125,000 to 250,000 people in the state of California.
SYNOPSIS: This proposal focuses on the role of MHC-1 and cytotoxic T cells (CTLs) on the survival of transplanted hESCs. hESCs have been reported to express MHC-1 and differentiation leads to increased expression. In specific aim I, the PI will test whether transduction of a lentiviral shRNA FG12 vector expressing siRNA will repress HLA-A and B molecules present on oligodendrocytes and neurons derived from H7 hESCs. The cells will be assayed for MHC-1 expression by FACS or quantitative RT-PCR. In specific aim II, the PI will assay the survival of HLA-A and B hypomorphic vs. control oligodendrocytes and neurons transplanted into the spinal cord of 10 rats/groups subjected to spinal cord contusion. The assays will make use of imaging and histology. SIGNIFICANCE AND INNOVATION: The transplantation of hESCs in the treatment of neurological diseases may lead to rejection. Therefore, it is important to clarify this risk and devise methods to avoid immune-mediated rejection. This proposal aims to use siRNA to decrease MHC-1 expression and therefore avoid a CTL response. The ability to alter Class 1 expression in human ES cell-derived cells by siRNA-mediated methods would be a potentially important advance in improving transplantation of these cells, though the studies proposed here are more limited in their scope. The proposed approach may avoid the need for other pharmacological methods to avoid rejection, such as treatment with cyclosporine-A. The use of siRNAs to lower the MHC of hESCs in order to avoid rejection is a novel direction, making this is a highly innovative proposal, though the significance is more modest. STRENGTHS: Dr. Camerini is a well-established investigator with expertise in immunology, retrovirus and lentivirus, especially HIV. The ability to adopt the results found using HIV to decrease Class 1 expression and subsequently decrease immunogenicity to T-cells but not to NK cells could be quite an important advance in identifying ways to prevent rejection in successfully transplanting hESCs. The collaboration with Dr. Keirstead, who is a leader in developing neurons and other cells derived from human ES cells for spinal cord injury, adds to the quality of the investigators in this work. The experiment with the spinal cord injury model seems well thought out; the groups of injured rats to be studied seems to be a good model, though more details of how these animals will be assessed could be provided. WEAKNESSES: In general, the strategy to decrease Class 1 expression is sound, but some important details and issues are not provided or discussed in this proposal raising a number of questions. For example, how will the PI differentiate the hESCs into oligodendrocytes and neurons? How will the PI test the extent of differentiation and how pure will these cells be? There is no evidence that the methods described in Aim 1 will be effective in knockdown of the HLA molecules, nor does the PI does state what HLA haplotypes are expressed in the H7 cells that are being studied. This should be known and more specific anti-Class 1 siRNAs could be proposed to be tested. In addition, the applicants will use a FG12 expression vector, produced by their collaborator Qin. However, they state that they do not have a sequence for FG12 and have not been able to design appropriate vectors to be used for these studies. These details should be worked out before submitting the proposal. In general, much of this work seems that it will be done by Dr. Keirstead and not necessarily by the PI. There are no identified post-docs or technicians to complete these studies as all the personnel are “to be determined”. The investigator should provide better evidence of the ability to complete this study as described. It is unclear why this group does not propose knocking down Class 1 in the undifferentiated ES cells, which may be easier to genetically modify than the mature oligodendrocytes and neurons derived from the ES cells. This should at least be proposed as an alternative method. Finally, a recent study in the literature found a low immunogenicity of human neural precursor cells under certain culture conditions despite an increase in expression of MHC-1, perhaps because of a low expression of co-stimulatory molecules (J. Neuroimmunol 161:1-11, 2005). Other studies have found no upregulation of MHC-1 (e.g., Stem Cells 22:1101-1110, 2004). This needs some further discussion. DISCUSSION: There was no discussion further following the reviewers' comments.