Abstract:Ectopic expression of defined pluripotence-associated Factors reprograms adult somatic cells back to an Embryonic Stem Cell-like state. During this process the phenoytpic characteristics of Aging are also reverted. Cells can even be directly reprogrammed from one cell type into another. The challenge is to reprogram old cells into young cells without the need of inducing the pluripotent state or changing the differentiation state.
Introduction
Cellular Reprogramming
Direct Reprogramming
miRNAs Modulate iPSC Generation
Random Methylation and Convergence are Driving Forces for Reprogramming
Reprogramming Restores Telomere Elongation
Longevity Promoting Components Enhance iPSC Generation
Sirt1 Promotes Reprogramming
DHEA Could Improve Reprogramming
Polyamines Might Enhance Reprogramming
Reprogramming of Aged and Senescent Cells
Is it really necessary to reprogram cells back to an embryonic state (i.e. induced Pluripotent Stem Cells; iPSCs) and trying to cure age-related diseases? This approach is like curing the symptoms rather than the root. If we succeed curing just one disease, the cured individuals will probably die later by another of the dozen disease which are all just a function of increasing age. Would it not be sufficient and more logical to reprogram the cells and thus the organism back to the young state? Ideally we could induce such changes in vivo without invasive artificial replacement (Regenerative Medicine) or engineering. We have to understand the nature of Aging (evolution) as well as the cause of Aging, its mechanisms and how to effectively reverse it (Interventions). There are Factors causal for age-related changes and other Factors cable to reverse such changes. These need to be identified as their loss-of-function and gain-of-function would revert aged cells to iYC (induced-Young Cells) and aged organisms to iYO (induced-Young Organisms).
Why our teeth are decaying over time? Because it is developmental programmed. During juvenile we are capable of replacing our tooth one time? The missing capability of replacing teeth in the adulthood is not a Cancer protection or other antagonist pleiotropic effect it is because we are programmed to expire fitness disadvantage compared to the next generation over time in order to speed up evolution. It would be theoretical possible to induce this juvenile process in adulthood again to replace our teeth when required.
Age-related changes have to be investigating and tracked back their causes. These paths will convey on the same effectors. E.g. Autophagy declines, why?; Inflammation increases, why?; NAD metabolism decreases, why? Gene expression changes, why?, because of alteration in chromatin structure, why?, already initiated during juvenile growth and even early development, why?
Which age-related changes on molecular Signatures is common to all Aging organisms? Comparing and contrasting different longevity promoting interventions, such as caloric, methionine restriction, genetic manipulation should also convey on those effectors. Such an approach was already initiated in fruit fly and identified upregulation of Takeout; a juvenile hormone clock output gene.
Somatic cell nuclear transfer (SCNT) [Briggs & Kings 1952; Elsdale et al. 1960; Wilmut et al. 1997] resets the developmental potential of somatic nucleus back to totipotency from which the whole development can be recapitulated. The resulting new borne clone is genetically identical to the somatic cell transferred into the recipient oocyte. The clone does not inherit the age of the donor even if the donor was already old [Mizutani et al. 2008; Wakayama et al. 2010]. In such the age-associated changes present in an aged cells can be reversed. SCNT indicates that mechanisms exists that rejuvenate cells.
Adult somatic cells can be reprogrammed to an embryonic-like state by ectopic expression of defined Factors (OCT4, SOX2, KLF4, c-MYC). The generation of this so called Induced Pluripotent Stem Cells (IPSCs), which are remarkable similar to embryonic stem cells (ESCs) can be further improved by additional Factors (NANOG and LIN28) to increase the efficiency and completeness of Reprogramming. LIN28 overexpression, similar to inhibition of the p53/p21CIP1 pathway, increased cell division rate, resulting in accelerated kinetics of iPSC production [Hanna et al., 2009; Yu et al., 2009].
Reprogramming can be achieved by with just protein extracts from Embryonic Stem Cells [21175196].
iPSC derived from mouse can give rise to newborn clones via tetraploid complementation [Kang et al. 2011]. As the age is effectively reset it is a proof of concept that mechanisms exist to rejuvenate somatic cells [Surani & McLaren 2006].
Direct reprogramming is the transdifferentation from one cell type into another.
The conventional reprogramming towards pluripotency through overexpression of Oct4, Sox2, Klf4 and c-Myc can be shortcut and directed towards cardiogenesis in a fast and efficient fashion. Mouse embryonic fibroblasts were directly reprogrammed to spontaneously contracting patches of differentiated cardiomyocytes [Efe et al. 2011].
Somatic cell Reprogramming towards a pluripotent state alters specific gene expression patterns. Transferring OCT4, SOX, KLF4 and c-MYC into human fibroblasts for 4 days generates partial-iPSCs which do not form tumors in vivo and clearly display the potential to differentiate into endothelial cell in response to defined media and culture conditions [22869753].
Using a unique combination of growth Factors while ensuring appropriate culturing conditions reprograms differentiated somatic cells into Neuronal Stem Cells. Brn4 converts skin cells into Neuronal Stem Cells and is even more effective if cells are stimulated by Growth Factors and exposed to just the right environmental conditions. The cells gradually lose their molecular memory that they were once skin cells and become indistinguishable from Stem Cells normally found in Tissue [http://www.mpg.de/5548755/stem_cells_skin_cells].
Transient Oct4 overexpression enables the direct conversion of connectivity Tissue cells into Neural Stem Cells [http://www.sciencedaily.com/releases/2012/03/120322131506.htm].
Blocking SMAD signaling pathway by Small molecule-based inhibition of glycogen synthase kinase 3 beta (GSK3-β) increased the conversions efficiency from skin cells to neurons by several times, yielding up to > 200% and neuronal purities up to >80% [Ladewig et al., 2012].
microRNA modulate target genes Tissue-specifically and therefore represent potential Reprogramming barriers. Depleting miR-21 and miR-29a enhances the Reprogramming efficiency. p53 and ERK1/2 signaling are regulated by miR-21 and miR-29a and function in Reprogramming. c-Myc enhances Reprogramming partly by repressing miRNAs enriched in differentiated cells, such as miR-21 and miR-29a [21693621; http://rnajournal.cshlp.org/content/17/8/1451.long].
Somatic Reprogramming is inefficient and and can be enhanced by p53-deficiecny. miR-34 is also barrier for somatic cell Reprogramming. miR-34 microRNAs and in particular miR-34a displays p53-dependent induction during Reprogramming. miR-34a and p21 cooperatively regulate somatic Reprogramming downstream of p53. In contrast to p53 deficiency, deficiency in miR-34a increases Reprogramming efficiency and kinetics without compromising self-renewal or differentiation. Reprogramming suppression of miR-34a is due, at least in part, to repression of pluripotence genes, including Nanog, Sox2 and Mycn. Similar miR-34b and c repress Reprogramming and all miR-34a/b/c act cooperatively in this process [22020437].
miR-199a-3p is upregulated by p53 at the post-transcriptional level. Induction of miR-199a-3p decreases Reprogramming efficiency, whereas miR-199a-3p inhibition enhances it. miR-199a-3p overexpression inhibits cell proliferation by imposing G1 cell cycle arrest, while miR-199a-3p inhibition results in a pronounced increase in cell proliferation. Enhancement of Reprogramming is almost completely reversed with replacement of miR-1993a. miR-199a-3p inhibition partially rescues iPSC generation impaired by p53 [22553189].
In mammals there are at least two developmental periods - in germ cells and in preimplantation embryos - in which methylation patterns are reprogrammed genome-wide [Reik et al., 2001].
Reprogramming into induced Pluripotent Stem Cells (iPSC) consists of stochastic de novo methylation and selection / fixation of methylation in an environment suitable for Embryonic Stem Cells (ESCs). Continuous passaging of iPSCs diminished the differences between iPSCs and ESCs, implying that iPSCs lose the characteristics inherited form the parent cells and adapt to very closely resemble ESCs over time [http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1002085].
iPSC generated animals are healthy and have no shortened Lifespan [http://www.welt.de/gesundheit/article13482555/Leberkrankheit-mit-umprogrammierten-Zellen-geheilt.html]
Taken skin cells from an elderly patient with advanced heart failure and Reprogramming them creates healthy and young beating cells which are the equivalent to the stage of his heart cells when he was just born. Grafted into rats these cells established connections with cells in the host Tissue [22621821].
Telomere Length is a reliable marker of Cellular Aging and is maintain by the terminal polymerase Telomerase which is composed of the reverse transcriptase protein (TERT) and its integral RNA component (TERC) as well as other proteins such as dyskerin [22110834].
Reprogramming with three Factors (OCT4, SOX2 and KLF4) is capable of reversing not only the developmental state but apparently also the actually cellular age. Although not every iPSC line has the same proliferation potential and Telomere Length of Embryonic Stem Cells, some iPSC cells express sufficient telomerase activity to extend telomere length [20230312; http://www.businesswire.com/news/home/20110412005815/en/biotime-presents-data-fabs-2011-restoration-cell; http://www.futuremedicine.com/doi/abs/10.2217/rme.10.21].
Reverting skin cells from premature aged individuals with defective Telomerase back to a more embryonic state appears to overcome the molecular defects of these cells. Reprogramming lengthened their Telomeres. The Reprogramming process activated Telomerase. Dyskeratosis congenital is a disorder of Telomere maintenance, individuals often have defects in one of the three components of Telomerase, suffer from degeneration in multiple Tissues and produce a range of abnormalities, including in the skin, blood and gastrointestinal tract. TERC upregulation is a feature of of the pluripotent state. Several Telomerase components are targeted by pluripotency-associated Transcription Factors [http://www.nature.com/nature/journal/v464/n7286/full/nature08792.html].
Reprogramming not only resets the Telomere Length, but also restores the proliferation potential of aged human cells back to that of cells obtained from young Tissues.
Inhibition of mTOR by rapamycin or PP242 or insulin/IGF1 signaling enhances efficiency of Reprogramming to iPSCs. These small molecules also improved longevity of Drosophila melanogaster. Other longevity-promoting components including two sirtuin activators (resveratrol and fisetin), an Autophagy inducer (spermidine), a PI3K (phosphoinsoitide-3-kinase) inhibitor (LY294002) and antioxidant (curciumin) but not AMPK activator (metformin) improved Reprogramming efficiency. Thus, controllers of somatic cell Reprogramming and Organismal Lifespan share common regulatory pathways [Chen et al., 2011].
mTOR regulates Senescence and Autophagy during Cellular Reprogramming. Forced expression of Stemness Factors, is a stressful process that activates Apoptosis and Cellular Senescence. mTOR inhibitors and Autophagy activators (e.g. PP242, rapamycin and resveratrol) improve the speed and efficiency of iPSC generation [22052357].
Sirt1 enhances iPSC generation, in part, through deacetylation of p53, inhibition of p21 and enhancement of Nanog. SIRT1 knockdown decreases while resveratrol increases Reprogramming efficiency. Both treatments are associated with altered acetylated p53 and its downstream Nanog but not p21 expression. The effects of reservatrol and SIRT1 knockdown are most pronounced during initiation phase of Reprogramming. Sirt1 overexpression stimulate the formation of colonies with induced Nanog and reduced p21 expression. miR-34a is a regulator of SIRT1. While its inhibitor increases, its mimics reduces iPSC formation. tenovin-6, a small molecule that activate p53 through SIRT1 inhibition, suppresses Reprogramming.
DHEA (Dehydroepiandrosterone) is an abundant steroid, produced at high levels of in the adrenal cells and persists at the Tissues. DHEA exhibit anti-carncenogic effect. TRP53 apoptotic pathway negative regulates cell growth and self-renewal. Withdraw of DHEA increases TRP53 levels, while DHEA decreases Apoptosis. DHEA could improve the Reprogramming efficiency due to a decrease in p53-mediated Apoptosis and increased cells viability. High levels of DHEA could induce demethylation in some specific CpG sites that could help the Reprogramming process. DNA demethylation through DNA methyltranserase inhibitory reaction regulates gene expression in reprogrammed cells [Shoae-Hassani et al., 2011].
Precise regulation of polyamine levels is critical for Embryonic Stem Cell self-renewal. High levels of Amd1 are essential for maintenance of the ESC state and self-renewal. Increasing Amd1 levels led to delayed ESC differentiation. Downregulation of Amd1 is necessary for differentiation of ESCs into neural precursor cells. Amd1 is translationally regulated by miRNA [http://www.eurekalert.org/pub_releases/2012-02/afst-asm022912.php].
Cells from old [Dimos et al. 2008] or even very old donors (aged over 100, centenarians) were successfully rejuvenated [http://www.sciencedaily.com/releases/2011/11/111103120605.htm].
Somatic mitochondria within human iPSCs revert to an immature ESC-like state with respect to organelle morphology and distribution, expression of Nuclear Factors involved in mitochondrial biogenesis, content of mitochdonrial DNA, intracellular ATP level, oxidative damage, and lactate generation. Upon differentiation, mitochondria with iPSCs and ESCs exhibit analogous maturation and an-aerobic-to-aerobic metabolic modifications [Prigione et al., 2010].
iPSCs from an aged individual (84-years) were able to differentiate into neurons, re-establish Telomerase activity, and reconfigure mitochondrial ultra-structure and functionality to a ESC-like state. Aged iPSCs exhibited high sensitivity to drug-induced Apoptosis and low levels of oxidative stress and DNA damage, in a similar fashion as iPSCs derived from young donors and ESCs. Thus, Cellular Reprogramming is capable of reversing Aging-related features in somatic cells [Prigione et al., 2011].
The agiogenic activities of iPSCs derived from very young (mouse embryonic) or old (21 months) mice are essential the same [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0039562].
iPSCs generated from senescent and centenarian cells have rest Telomere size, gene expression Profiles, oxidative stress and mitochondrial metabolism, and are indistinguishable from human ESCs as well as able to redifferentiate into fully rejuvenated cells.
One week after infection the Senescence Associated Heterochromatin Focis (SAHFs) disappeared in senescent cells from 74 years old individuals and restored proliferation after 18-20 days, similar to cells from 74 years old individuals without additional passaging into Senescence.
iPSCs generated from senescent and centenarian cells did not retain marks of Cellular Aging. During Reprogramming there is a global lengthening of Telomeres. Telomere Length was increased in iPSCs derived from senescent cells after Reprogramming, like Telomeres from centenarians. Reprogrammed senescent cells acquired the capacity to progress into terminal differentiation, forming organized organ-like structures characteristic of the three embryonic lineages in teratoma.
Gene expression Profiles of proliferative and senescent aged fibroblasts clustered together, indicating that they share a general common Aging signature.
Six-factor Reprogramming was more efficient for Telomere function resetting.
From senescent or aged cells derived iPSCs had an increased membrane potential to a level similar to that in hhuman ESCs, indicating that Reprogramming of the altered mitochondrial activity from old and senescent cells to a human ESC-like metabolism.
Fibroblasts derived from senescent or aged iPSCs did not enter prematurely into Senescence, but proliferated at a rate equivalent to young proliferative fibroblasts. The number of publication divisions to reach replicative senescent arrest even increased after Reprogramming through the pluripotent state. Global gene expression Profiles of redifferentiated fibroblasts form old and senescent derived iPSCs actually cluster with young proliferative embryonic fibroblasts derived from Embryonic Stem Cells, whereas their parental counterparts share a common Aging signature.
When redifferentiated back into fibroblasts, reprogrammed cells have rejuvenated extended Lifespans and characteristics of young proliferative embryonic fibroblasts, thus, these cells have been completely rid of their former aged cellular phenotype. This proves the reversibility of major aspects of Cellular Aging physiology and indicates the perceived importance of epigenetic modifications in Aging and provides a new paradigm for cell rejuvenation.
Mammalian cells can be reprogrammed after incubation in germinal vesicle (GV) of the frog oocyte [Halley-Stott et al. 2010]. GV oocyte represents an environment where direct Reprogramming of gene expression can take place without cell division and any new cell types being generated [Jullien et al. 2011]. One of the Factors that restrict Reprogramming efficiency is mH2A [Pasque et al. 2011], which level increase during both in vitro and in vivo Aging [Kreiling et al. 2011].
Chen, T., Shen, L., Yu, J., Wan, H., Guo, A., Chen, J., Long, Y., Zhao, J., and Pei, G. (2011). Rapamycin and other longevity-promoting compounds enhance the generation of mouse induced pluripotent stem cells. Aging cell.
Prigione, A., Fauler, B., Lurz, R., Lehrach, H., and Adjaye, J. (2010). The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells. Stem Cells 28, 721-733.
Prigione, A., Hossini, A.M., Lichtner, B., Serin, A., Fauler, B., Megges, M., Lurz, R., Lehrach, H., Makrantonaki, E., Zouboulis, C.C., et al. (2011). Mitochondrial-associated cell death mechanisms are reset to an embryonic-like state in aged donor-derived iPS cells harboring chromosomal aberrations. PloS one 6, e27352.
Reik, W., Dean, W., and Walter, J. (2001). Epigenetic reprogramming in mammalian development. Science 293, 1089-1093.
Shoae-Hassani, A., Sharif, S., and Verdi, J. (2011). A neurosteroid, DHEA, could improves somatic cell reprogramming. Cell Biol Int.
Briggs, R. & King, T.J. (1952) Transplantation of living nuclei from blastula cells into enucleated frogs’ eggs. Proc. Natl Acad. Sci. USA 38, 455–463.
Elsdale, T.R., Gurdon, J.B. & Fischberg, M. (1960) A description of the technique for nuclear transplantation in Xenopus laevis. J. Embryol. Exp. Morphol. 8, 437–444.
Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J. & Campbell, K.H. (1997) Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–813.
Mizutani, E., Ono, T., Li, C., Maki-Suetsugu, R. & Wakayama, T. (2008) Propagation of senescent mice using nuclear transfer embryonic stem cell lines. Genesis 46, 478–483.
Wakayama, S., Mizutani, E. & Wakayama, T. (2010) Production of cloned mice from somatic cells, ES cells, and frozen bodies. Methods Enzymol. 476, 151–169.
Dimos, J.T., Rodolfa, K.T., Niakan, K.K., Weisenthal, L.M., Mitsumoto, H., Chung, W., Croft, G.F., Saphier, G., Leibel, R., Goland, R., Wichterle, H., Henderson, C.E. & Eggan, K. (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321, 1218–1221.
Kang, L., Wu, T., Tao, Y., Yuan, Y., He, J., Zhang, Y., Luo, T., Kou, Z. & Gao, S. (2011) Viable mice produced from three-factor induced pluripotent stem (iPS) cells through tetraploid complementation. Cell Res. 21, 546–549.
Surani, M.A. & McLaren, A. (2006) Stem cells: a new route to rejuvenation. Nature 443, 284–285.
Ladewig, J., Mertens, J., Kesavan, J., Doerr, J., Poppe, D., Glaue, F., Herms, S., Wernet, P., Kogler, G., Muller, F.J., et al. (2012). Small molecules enable highly efficient neuronal conversion of human fibroblasts. Nature methods.
Efe, J.A., Hilcove, S., Kim, J., Zhou, H., Ouyang, K., Wang, G., Chen, J. & Ding, S. (2011) Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat. Cell Biol. 13, 215–222.
Kreiling, J.A., Tamamori-Adachi, M., Sexton, A.N., Jeyapalan, J.C., Munoz-Najar, U., Peterson, A.L., Manivannan, J., Rog- ers, E.S., Pchelintsev, N.A., Adams, P.D. & Sedivy, J.M. (2011) Age-associated increase in heterochromatic marks in murine and primate tissues. Aging Cell 10, 292–304
Halley-Stott, R.P., Pasque, V., Astrand, C., Miyamoto, K., Simeoni, I., Jullien, J. & Gurdon, J.B. (2010) Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes - a method for quantitative transcriptional reprogramming. Methods 51, 56–65.
Jullien, J., Pasque, V., Halley-Stott, R.P., Miyamoto, K. & Gurdon, J.B. (2011) Mechanisms of nuclear reprogramming by eggs and oocytes: a deterministic process? Nat. Rev. Mol. Cell Biol. 12, 453–459.
Pasque, V., Gillich, A., Garrett, N. & Gurdon, J.B. (2011) Histone variant macroH2A confers resistance to nuclear reprogramming. EMBO J. 30, 2373–2387.
