Year 2

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.