Telomeres and telomerase in Induced Pluripotent stem cells – not what we thought

An important new research article appeared yesterday, in the online edition of Future Medicine: Spontaneous reversal of the developmental aging of normal human cells following transcriptional reprogramming. The study is important because it contradicts an important earlier assumption about induced pluripotent stem cells (iPSCs).  It contradicts the assumption that reverting a cell to iPSC status restores the expression of telomerase and telomere length to equivalent embryonic stem cell length. 

(Note: hiPSCs stands for human iPSCs, hESCs stands for human embryonic stem cells)

In my February 2010 blog post IPSCs, telomerase, and closing the loop in the stem cell supply chain I related “The 2009 study Telomeres Acquire Embryonic Stem Cell Characteristics in Induced Pluripotent Stem Cells was important in that it showed that reversion of cells to iPSC status fully restores telomerase activity to iPSCs, equivalent to that in embryonic stem cells (ESCs). “We show here that telomeres are elongated in iPS cells compared to the parental differentiated cells both when using four (Oct3/4, Sox2, Klf4, cMyc) or three (Oct3/4, Sox2, Klf4) reprogramming factors and both from young and aged individuals.  We demonstrate genetically that, during reprogramming, telomere elongation is usually mediated by telomerase and that iPS telomeres acquire the epigenetic marks of ES cells, including a low density of trimethylated histones H3K9 and H4K20 and increased abundance of telomere transcripts. Finally, reprogramming efficiency of cells derived from increasing generations of telomerase-deficient mice shows a dramatic decrease in iPS cell efficiency, a defect that is restored by telomerase reintroduction.”  The study related to mouse cells. Further, the 2009 publication Balancing Out the Ends during iPSC Nuclear Reprogramming discusses how “telomere length maintenance and long-term proliferative capacity of iPSCs is dependent on telomerase,” and concludes “Although a number of hurdles must still be cleared before iPS-based cell therapy becomes practical, the results (cited above) suggest that reprogramming of telomerase and telomeres may not be one them.” Finally, this 2009 study concluded “While these results reveal some heterogeneity in the reprogramming process with respect to telomere length, human somatic cells reprogrammed to pluripotency generally displayed elongated telomeres that suggest that they will not age prematurely when isolated from subjects of essentially any age. While these results reveal some heterogeneity in the reprogramming process with respect to telomere length, human somatic cells reprogrammed to pluripotency generally displayed elongated telomeres that suggest that they will not age prematurely when isolated from subjects of essentially any age.”  All of this seems now to be largely thrown out of the window.

The new study, based on carefully contrived experimental procedures, concludes the opposite is true for human iPSC cells: they generally end up with shorter telomeres than hESCs, telomeres of lengths commensurate with those of the adult cells which were originally reverted to make the iPSCs . “Aim: To determine whether transcriptional reprogramming is capable of reversing the developmental aging of normal human somatic cells to an embryonic state. Materials & methods: An isogenic system was utilized to facilitate an accurate assessment of the reprogramming of telomere restriction fragment (TRF) length of aged differentiated cells to that of the human embryonic stem (hES) cell line from which they were originally derived. An hES-derived mortal clonal cell strain EN13 was reprogrammed by SOX2, OCT4 and KLF4. The six resulting induced pluripotent stem (iPS) cell lines were surveyed for telomere length, telomerase activity and telomere-related gene expression. In addition, we measured all these parameters in widely-used hES and iPS cell lines and compared the results to those obtained in the six new isogenic iPS cell lines. — Results: We observed variable but relatively long TRF lengths in three widely studied hES cell lines (16.09–21.1 kb) but markedly shorter TRF lengths (6.4–12.6 kb) in five similarly widely studied iPS cell lines. Transcriptome analysis comparing these hES and iPS cell lines showed modest variation in a small subset of genes implicated in telomere length regulation. However, iPS cell lines consistently showed reduced levels of telomerase activity compared with hES cell lines.” 

The new study says restoring a cell to pluripotency and providing the cell with youth are two quite different things and the former does not necessarily imply the latter.  Pluripotency of an iPSC implies that, like an embryonic stem cell, the iPSC can differentiate into any somatic cell type.  Absence of youth in this case means that the restored iPSC cell could probably not lead to many generations of descendents like an embryonic stem cell could,  because its telomere lengths are short like those in old cells.   In fact, iPSC cells can be near senescent. 

As related in the new study report “– recently, the focus of reprogramming research has shifted to defined transcriptional methods, that is, the exogenous expression of transcription factors critical to germline gene expression such as MYC, KLF4, OCT4 and SOX2 [29], or LIN28, NANOG, OCT4 and SOX2 [30]. When introduced into somatic cells, varied combinations of these genes or complementing small molecules are capable of altering the differentiated state of somatic cells, leading to induced pluripotent stem (iPS) cells similar to hES cells. This facile, cost-effective and ethically nonproblematic means of potentially manufacturing a host of transplantable patient-specific cells has led to numerous studies of iPS cell pluripotency.  However, there is little research on the effects of transcriptional reprogramming on cellular aging, in particular on telomere length regulation.”   

In other words, the iPSC researchers and the telomerase researchers were paying insufficient attention to each other.  Telomerase expression and iPSC stem cells are each central to a theory of aging treated in my treatise, Telomere Shortening and Damage and Stem Cell Supply Chain Breakdown.  I have reviewed hundreds of papers related to these topics and my impression is that not paying attention to what the other guys are researching is more the general rule than the exception.  

The researchers in the new study used cells from a single genetic source to sort through what could be a very confusing situation.  “ — In the case of human cells, there are contradictory reports as to the aged status of reprogrammed cells [32–34]. This confusion may be due, in part, to the genetic variability in the subtelomeric ‘X’ region of telomere restriction fragments (TRFs) [35,36], often complicating comparisons of TRF length in differing genotypes. “We therefore undertook an analysis of telomere dynamics during transcriptional reprogramming in an isogenic background of the hES-derived clonal embryonic progenitor cell line EN13, such that TRF length can be measured and compared with both the starting somatic cells (EN13) and the normal hES cells with embryonic TRF length from which EN13 was obtained.” 

“We observed variable but relatively long TRF lengths in hES cell lines of 16.1–21.1 kb, but markedly shorter TRF lengths ranging from 6.4 to 12.6 kb in all of the iPS cell lines studied.  In serially passaged iPS cell lines (iPS[IMR90]‑1, iPS[IMR90]‑4 and iPS[foreskin]‑1), TRF length progressively shortened during propagation.” — iPS cell lines consistently showed reduced levels of telomerase activity compared with hES cell lines.” On the other hand, the researchers discovered that one of six cloned lines of iPSC cells they generated expressed telomerase and that in that particular cell line telomeres got longer in successive cell generations until they got to be comparable to the lengths in hES cells. “In order to verify these results in an isogenic background, we generated six iPS cell clones from the hES‑derived cell line EN13. These iPS cell clones showed initial telomere lengths comparable to the parental EN13 cells, had telomerase activity, expressed embryonic stem cell markers and had a telomere-related transcriptome similar to hES cells.  Subsequent culture of five out of six lines generally showed telomere shortening to lengths similar to that observed in the widely distributed iPS lines. However, the clone EH3, with relatively high levels of telomerase activity, progressively increased TRF length over 60 days of serial culture back to that of the parental hES cell line. Conclusion: Prematurely aged (shortened) telomeres appears to be a common feature of iPS cells created by current pluripotency protocols. However, the spontaneous appearance of lines that express sufficient telomerase activity to extend telomere length may allow the reversal of developmental aging in human cells for use in regenerative medicine.” It is interesting that for mouse iPSCs the same thing is observed to happen.  The iPSCs start out with short telomeres like the mature cells that were reverted had, but they express telomerase and grow longer telomeres in succeeding cell-division generations.   See the 2009 publication Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells . 

The 2010 publication Embryonic Stem Cells/Induced Pluripotent Stem Cells points out what can be important differences between hESCs and hiPSCs, probably because of the fact that the hiPSCs were, in fact, old cells with short telomeres.  “we demonstrate here that hiPSCs are capable of generating hemangioblasts/blast cells (BCs), endothelial cells and hematopoietic cells with phenotypic and morphological characteristics similar to those derived from hESCs, but with a dramatic decreased efficiency. Furthermore, in distinct contrast to the hESC derivatives, functional differences were observed in BCs derived from hiPSCs, including significantly increased apoptosis, severely limited growth and expansion capability, as well as a substantially decreased hematopoietic colony forming capability. After further differentiation into erythroid cells, >1000-fold difference in expansion capability was observed in hiPSC-BCs versus hESC-BCs.” 

What I get out of this is: 

·        Reversion of cells to hiPSC status in many cases results in cells that do not express telomerase and have short telomeres.  They are very different in these respects from hESCs.  Such cells may be approaching senescence despite their pluripotency, cannot reliably be depended on for such tasks as organ renewal, and cannot be used to close the loop in the stem cell supply chain(ref)(ref). 

·        A strain of iPSCs could express telomerase in which case the telomeres in succeeding generations could get longer.  How to reliably produce such strains remains unclear.  Telomerase can be exogenously applied in the process of generating iPSCs to provide them with longer initial telomeres but, without inherent capability to express telomerase, such iPSCs are apt to be of limited use. 

·        It appears that much more is to be learned before iPSCs with long replicative life spans can be reliably produced and used for regenerative purposes.  We need black-belt iPSC and telomerase researchers to put their heads together to figure out what can be done.