Enhanced Telomere Rejuvenation in Pluripotent Cells Reprogrammed via Nuclear Transfer Relative to Induced Pluripotent Stem Cells

Abstract

Although somatic cell nuclear transfer (SCNT) and induction of pluripotency (to form iPSCs) are both recognized reprogramming methods, there has been relatively little comparative analysis of the resulting pluripotent cells. Here, we examine the capacity of these two reprogramming approaches to rejuvenate telomeres using late-generation telomerase-deficient (Terc(-/-)) mice that exhibit telomere dysfunction and premature aging. We found that embryonic stem cells established from Terc(-/-) SCNT embryos (Terc(-/-) ntESCs) have greater differentiation potential and self-renewal capacity than Terc(-/-) iPSCs. Remarkably, SCNT results in extensive telomere lengthening in cloned embryos and improved telomere capping function in the established Terc(-/-) ntESCs. In addition, mitochondrial function is severely impaired in Terc(-/-) iPSCs and their differentiated derivatives but significantly improved in Terc(-/-) ntESCs. Thus, our results suggest that SCNT-mediated reprogramming mitigates telomere dysfunction and mitochondrial defects to a greater extent than iPSC-based reprogramming. Understanding the basis of this differential could help optimize reprogramming strategies.

The role of telomeres and telomerase reverse transcriptase isoforms in pluripotency induction and maintenance

“…42 Similarly, long term self-renewal and teratoma formation in murine and porcine iPSCs with short telomeres are generally impaired. 31,43 In addition, PSCs with short telomeres in a variety of species display mitochondrial dysfunction, 44 impaired neuroectodermal differentiation, 44 uncapping events, and reduction in H3K27me3 distal to telomeres including global hypomethylation which contributes to reactivation of pluripotency genes, 45 and possibly reprogramming transgenes. 31,33,46 Partially reprogrammed iPSCs often show little TERT activation and short telomeres.…”

“…33,51 mESCs and miPSCs also do not exhibit the in vitro plateau in telomere length that is characteristic of hESCs/hiPSCs. 52 While NT-mESCs generally have completed telomere lengthening and full reprogramming by the blastocyst stage, 44 miPSCs may take up to 30 passages to achieve mESC lengths 53 and only do so sporadically. 32 Control of telomere length is an intricate process involving many factors.…”

“…Prior to differentiation NANOG is also expressed at high levels in mTERC ¡/¡ mESCs with short telomeres. 44 Additionally, Pucci et al found that ESRRB, a KLF4 substitute during reprogramming 79 and a direct target of NANOG, 80 is also strongly upregulated. 45 Previous studies have demonstrated p53 binding to the promoters of NANOG and ESRRB 81 and its ability to either enhance or repress the pluripotency genes.…”

“…For example, removal of the canonical function of mTERT by mTERC knockout leads to mitochondrial dysfunction after iPSC and SCNT reprogramming of cells with short telomeres. 44 However, this mitochondrial dysfunction is likely a p53 mediated effect due to the poorly maintained telomeres. The donor cells came from late-generation Terc¡/¡ mice (hence short telomeres to begin with) and were allowed to grow/differentiate under telomerase-activity-deficient conditions, which may have led to mitochondrial damage.…”

“…The donor cells came from late-generation Terc¡/¡ mice (hence short telomeres to begin with) and were allowed to grow/differentiate under telomerase-activity-deficient conditions, which may have led to mitochondrial damage. 44 As there is evidence that most mitochondrial localized TERT may not carry TERC at all and instead act as a reverse transcriptase for mitochondrial RNA 103 it would be informative to additionally knockdown mTERT 44 to look for decreased ROS scavenging. To add to the complexity, TERT is capable of binding alternative RNA's including the RNA component of mitochondrial RNA processing endoribonuclease (RMRP), with which it can form dsRNA that is processed into siRNA directed against RMRP.…”