Characterization of Immune Responses in Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00163
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Human embryonic stem cells (hESC) provide a promising future therapeutic approach for replacement of diseased or damaged tissue. This proposal addresses the development of therapeutic strategies that can specifically aid two different neurological disorders. Multiple sclerosis (MS) is a chronic condition which affects the brain and spinal cord. In patients with this condition, the myelin sheaths that cover nerve fibers are erroneously destroyed by the body’s own immune system. In spinal cord injuries, the resulting inflammation causes severe damage to myelin-producing cells. The goal of this proposal is to determine if stem cells, at various phases of differentiation to myelin-producing cells, have characteristics which could not only replenish the lost myelin, but also confer a healing microenvironment. The focus will be on two beneficial immune-based processes. First specific aim will determine molecular signaling pathways involved in innate immune responses in hESC and establish if these processes provide a microenvironment conducive to tissue regeneration. Innate-immune responses have been shown to underlie tissue repair gene expression by dying cells. Our preliminary experiments have identified that components of this pathway are present in human embryonic stem cells (hESC), particularly at the later phases of differentiation to mature myelin-producing cells. Levels of factors involved in this process will be evaluated in hESC. To further establish the potential of the hESC to create a regenerative microenvironment for damaged cells, inserts of stressed and dying cells will be co-cultured with hESC. Cell viability, with or without co-exposure to hESC, will be determined using a commercially available cell cytotoxicity assay. The hypothesis to be tested is that hESC can provide a restorative microenvironment for stressed or dying cells and this involves up-regulation of innate immune responses. In the second specific aim, we will evaluate whether hESC can down regulate harmful inflammatory immune responses by a process used by fetal cells to escape the maternal immune response. This process is called ‘immune tolerance. To determine if hESC have the ability to provide an immune privileged microenvironment (similar to the fetus) components of this response will be determined in hESC. The hypothesis to be tested is that hESC can subvert destructive immune responses by processes similar to those that underlie immune tolerance. The long term objective of these studies is to design oligodendrocyte populations derived from hESC that provide an ideal microenvironment for promotion of cell survival, regeneration, and immune tolerance. These can eventually be used therapeutically in chronic demyelinating diseases such as MS or acute traumatic spinal cord injuries with minimal chances of host rejection.
Statement of Benefit to California: 
The proposed research will benefit California and its citizens in several ways. First, the results may aid in the design of effective therapies based on hESC that can benefit patients with multiple sclerosis or spinal cord injury. For the citizens of California who are suffering from these disorders, the development of these stem cell-based therapies will alleviate some of the pain and hardships associated with their condition. Second, successful development of stem-cell based therapies will lighten the economic burden for the state of California, which provides health care services to its citizens. Third, the results gained from the studies proposed may lead to intellectual property rights which can be used as a foundation for creating new biotechnology companies. This will not only create jobs but will also contribute to the economic growth of the state of California.
Progress Report: 
  • Our CIRM SEED grant proposal was to study the pathways of programmed cell death (cell suicide) in human embryonic stem cells. This is a critical area for several reasons: for example, when we transplant stem cells, we need to know how to keep them from dying so that they can be functional. On the other hand, we also need to know how to induce programmed cell death in stem cells, since it is becoming more and more clear that cancers may be propagated by stem cell populations. For these and many other reasons, it is important to know what pathways of programmed cell death are available to stem cells.
  • There are at least five major forms of programmed cell death: apoptosis (the best described pathway), autophagic cell death, PARP-mediated cell death, paraptosis, and calcium-mediated programmed cell death. Each of these programmed cell death pathways is activated by different stimuli and stresses. In the proposed research, we aimed to determine which of the five major forms of programmed cell death occur in human embryonic stem cells (hESCsP). Furthermore, we evaluated how the repertoire of PCD pathways changes when hESCs differentiate into neurons.
  • We first compiled a list of 322 genes whose activity contributes to these various forms of programmed cell death. Of these 322 genes, 311 were found to be represented on the assay system we used. 153 of these genes were measured with a very high detection confidence (0.95 or greater). We performed a special analysis (unsupervised two-way hierarchical cluster analysis) of these genes and represented the expression profiles in a heat-map. Within this group of genes, we chose to focus our attention first on Bcl-2 family members (both pro-apoptotic and anti-apoptotic) because we found transcripts of these gene families to be some of the most differentially expressed within the 43 samples analyzed. We also focused on this gene family because it is a critical family for the control of programmed cell death.
  • We then quantified all members of the Bcl-2 family amongst hESCs and differentiated cells, working under the hypothesis that overly abundant Bcl-2 family member transcripts in hESCs would point toward apoptotic and/or anti-apoptotic signaling cascades that are especially active in hESCs. We were encouraged when we found that the expression of some Bcl-2 family member genes changed dramatically (some up and others down) when hESCs were differentiated to other cell types.
  • We found that apoptosis is readily activated in hESCs, and, surprisingly, that a subset of p53-induced Bcl-2 family genes (e.g., Noxa and Puma) is highly constitutively expressed in hESCs (in comparison to multiple non-stem-cell primary cells). Whereas the pro-apoptotic genes Noxa and Puma are typically expressed only in response to DNA damage and p53 activity, hESCs constitutively express high levels of Noxa and Puma. This finding suggests that embryonic stem cells might be hyper-sensitive to sources of DNA damage like ultraviolet rays and X-irradiation, compared to other cell types, and furthermore, that p53-independent mechanisms of death induced by DNA damage might be operative in hESCs. However, not all p53-induced genes are up-regulated in these cells, since p21 is not up-regulated. These findings raise the important possibility that cultured hESCs may undergo DNA damage despite appropriate culture conditions, which would be a critical issue for hESC growth for transplantation. Another possibility is that p53, the “guardian of the genome”, is indeed protecting hESCs from DNA damage, in part by having a low threshold to activate programmed cell death, but without activating senescence (since p21 was not found to be up-regulated). Thus p53 may, in hESCs, mediate hypersensitivity to DNA damage, as a mechanism to keep the genomes of hESCs “pristine” for long-term functionality. We are performing follow-up studies to determine the mechanism and implications of the striking constitutive up-regulation of this subset of p53 target genes.
  • We are grateful to CIRM for supporting this SEED grant, especially since it has allowed us to identify novel aspects of programmed cell death and the underlying molecules, and to identify a potentially important novel aspect of human embryonic stem cells that may prove to be important in the consideration of transplantation of these cells and their differentiated derivatives.

© 2013 California Institute for Regenerative Medicine