Genome Replacement in Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
ICOC Funds Committed: 
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Embryonic stem cells have tremendous potential value for the treatment of many diseases and injuries, but like other transplants their use is likely to be limited because the immune system of the recipient will probably reject the transplant unless there is a good genetic match between donor and recipient. The ideal solution to this problem would be to replace the genetic material of stem cells with genetic material from the prospective patient, and to use the genetically modified stem cells in therapy. Work on experimental animals has shown that it is possible to do this by implanting a nucleus from one of the patient’s body cells into an oocyte from which the nucleus is removed. The oocyte is then allowed to grow into an embryo, and stem cells are derived from the embryo when it reached the appropriate stage. This method suffers from several drawbacks, the most significant being the need for oocyte donors, the technical difficulty of performing the nuclear replacement, and ethical objections to the production of embryos which are later to be destroyed for the benefit of the patient. We have devised an alternative strategy in which the nuclei of existing stem cell lines will be replaced by nuclei from cells of the prospective patient, by physically fusing together the stem cell with a body cell from the patient under condition preventing the survival of the stem cell nucleus. The resulting cell lines, with the genetic material from the patient in stem cell cytoplasm, will then be grown up and should be suitable for use in cellular therapy. In the proposed research we will compare several methods for incapacitating or removing the stem cell nucleus from the fused cells, and we will rigorously test the resulting cell lines for the correct genetic makeup as well as the retention of developmental properties characteristic of stem cells.
Statement of Benefit to California: 
California has taken the lead in the nation in providing support for stem cell research and especially in support for developing the use of human embryonic stem cells in cellular therapy for many diseases and disorders. Many laboratories are investigating methods to control the differentiation of these cells, so that they will no longer be likely to produce tumors in the patient, and they will be more suited to regenerative therapy in specific organs. But the full potential of these cells cannot be met until a solution is found to the problem of immune rejection by the patient. Claims made outside the U.S. for success in generation of stem cell lines that are genetically tailored to the patient turned out to be fraudulent, so the field still awaits a solution to this problem. The conventional approach to generating patient-specific stem cell lines would be to transplant a somatic cell nucleus into an oocyte obtained from a donor, then allow the oocyte to develop into an embryo and recover stem cells from that embryo. However, this has several drawbacks as stated elsewhere in this proposal. California could continue to lead the nation in embryonic stem cell research and in developing the use of these cells in cellular therapy if a solution cold be found to the problem of immune rejection. Here we propose a novel but feasible method of generating patient-specific embryonic stem cells from existing stem cell lines, avoiding the problems of obtaining egg donors, and of generating embryos for later destruction. If this method could be developed in California it would allow the state to continue the momentum that has built up in stem cell research, and to continue to build upon it by moving to the next stage where these cells are actually used in the clinic to treat some of the most devastating injuries and diseases affecting our people.
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