Telomere Reprogramming and Maintenance in Porcine iPS Cells

Abstract

Telomere reprogramming and silencing of exogenous genes have been demonstrated in mouse and human induced pluripotent stem cells (iPS cells). Pigs have the potential to provide xenotransplant for humans, and to model and test human diseases. We investigated the telomere length and maintenance in porcine iPS cells generated and cultured under various conditions. Telomere lengths vary among different porcine iPS cell lines, some with telomere elongation and maintenance, and others telomere shortening. Porcine iPS cells with sufficient telomere length maintenance show the ability to differentiate in vivo by teratoma formation test. IPS cells with short or dysfunctional telomeres exhibit reduced ability to form teratomas. Moreover, insufficient telomerase and incomplete telomere reprogramming and/or maintenance link to sustained activation of exogenous genes in porcine iPS cells. In contrast, porcine iPS cells with reduced expression of exogenous genes or partial exogene silencing exhibit insufficient activation of endogenous pluripotent genes and telomerase genes, accompanied by telomere shortening with increasing passages. Moreover, telomere doublets, telomere sister chromatid exchanges and t-circles that presumably are involved in telomere lengthening by recombination also are found in porcine iPS cells. These data suggest that both telomerase-dependent and telomerase-independent mechanisms are involved in telomere reprogramming during induction and passages of porcine iPS cells, but these are insufficient, resulting in increased telomere damage and shortening, and chromosomal instability. Active exogenes might compensate for insufficient activation of endogenous genes and incomplete telomere reprogramming and maintenance of porcine iPS cells. Further understanding of telomere reprogramming and maintenance may help improve the quality of porcine iPS cells.

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

“…27,28 Similarly, human and murine induced pluripotent stem cells (iPSCs) are capable of carrying out re-elongation of telomeres in at least some clones, although the telomere length and activity characteristics appear to be clonally unique. [28][29][30][31][32][33] Conversely, the process of differentiation triggers down-regulation of telomerase and initiates telomeric attrition. 34,35 Following the extended cellular division required to form adult tissues, terminally differentiated cell types typically exhibit low or undetectable levels of telomerase activity and shortened telomeres.…”

“…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.…”

“…46 This disparity is likely due to species differences ( Table 1) that repress ALT pathways in humans, but not murine or porcine cells. 31,46 Consequently, although heterozygous mTERC¡/C miPSCs and to a lesser extent mTERC¡/¡ miPSCs are capable of maintaining pluripotency and telomere length in mice, possibly due to the activation of the ALT pathway, 33 hTERT¡/C hiPSCs show poor telomere elongation, and DKC1 (Dyskerin -a telomerase complex component) hiPSC mutants (TERC deficient) do not elongate telomeres. 42 …”

“…61 Moreover, upregulation of pluripotency genes is strongly connected with expression of TERT, the overexpression of which enhances reprogramming efficiency. 62,63 For example, porcine iPSCs can only maintain telomeres with continued expression of exogenous reprogramming factors to maintain TERT levels, 31 and partially reprogrammed mouse embryonic fibroblasts (MEFs), and porcine embryonic fibroblasts (PEFs) show little TERT promoter activation and poor telomere elongation. However, while low telomerase activity is correlated with partial reprogramming, high levels of TERT alone does not induce a pluripotent state, 48 nor does pluripotency strictly require high levels of TERT.…”

Oocyte-associated transcription factors in reprogramming after somatic cell nuclear transfer: a review

“…Studies using extracts from GV stage oocytes or MIIoocytes have shown that oocytes at different stages had distinctive and unique mechanisms for somatic cell reprogramming, which differed from reprogramming techniques used to produce induced pluripotent stem cells with defined transcription factors [66][67][68]. It has been shown that treatment with MII extract could induce at least partial reprogramming in somatic cells [69].…”