Challenge

What molecular mechanisms contribute to lower genomic stability in the induced pluripotent stem cells?

Image credit: Jere Weltner et al., 2018 - https://www.nature.com/articles/s41467-018-05067-x

Shubhankar Kulkarni Nov 10, 2020

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What molecular mechanisms contribute to lower genomic stability in the induced pluripotent stem cells (iPSCs)? How can we overcome them?

There are several techniques available to dedifferentiate cells. However, the iPSCs are more prone to mutagenesis than embryonic stem cells.     Why do you think that is? How can we solve it?

[1]https://cancercommun.biomedcentral.com/articles/10.1186/s40880-018-0313-0#:~:text=Induced%20pluripotent%20stem%20cells%20(iPSCs,in%20iPSCs%20than%20in%20ESCs.

induced pluripotent stem cell genomic stability

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Epigenetics contribute to genomic instability in iPSCs

Jamila Nov 11, 2020

Zhang and colleagues wanted to determine what causes genome instability in iPSCs. They believe that epigenetics could play a part in the genomic instability of iPSCs. The researchers found that irradiated iPSCs had reduced levels of H3K9me3 and phosphorylated ATM compared to the other irradiated cell types.    

Histone modifications are crucial for the repair process. H3K9me3 is a histone mark that facilitates DSB repair processes as it activates ataxia telangiectasia-mutated (ATM) kinase, which recruits other repair factors.     Therefore, losing H3K9me3 would mean that ATM is not activated, which prevents DSB repair and thus contributes to genomic instability.     More research should be conducted to elucidate the exact role of epigenetics in iPSC genome instability.

[1]Zhang, Minjie, et al. "Lower genomic stability of induced pluripotent stem cells reflects increased non-homologous end joining." Cancer Communications 38.1 (2018): 49.

[2]Ayrapetov, Marina K., et al. "DNA double-strand breaks promote methylation of histone H3 on lysine 9 and transient formation of repressive chromatin." Proceedings of the National Academy of Sciences 111.25 (2014): 9169-9174.

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Cell cycle defects contribute to genomic instability

Antonio Carusillo Nov 15, 2020

In more recent works, Single Nucleotide Variants (SNVs) have been indeed investigated and, interestingly enough, only part of the mutations detected in the iPSCs could be accounted to pre-existing mutations present in the samples used to be reprogrammed in the respective iPSCs     . Meaning that a part of the mutations observed is generated de novo. In a new study presented, iPSCs were investigated for their ability to repair the DNA repair, in particular, the correct cell-cycle checkpoints were checked     . Cell-cycle is divided into checkpoints that verify the physiology and the genomic integrity of the cell before allowing it to go through the division step. If genomic instability or DNA damage is detected, the check-point stops the cycle before the cell can duplicate its DNA, thus, preventing the mutation to be inherited by the daughter cells     .
What the researchers found out by cell-cycle analysis is that during the first 2 days, after expressing in the cells the Yamanaka factors ( OCT4, Klf4, SOX2, and c-MYC), the check-point controlling the transition from G1 phase (G stands for Growth and it is the first step in which the cell starts increasing its size before division phase occurs) to S phase (S stands for Synthesis and corresponds to when the DNA is duplicated to be “distributed” to the two daughter cells) is impaired. In particular, what they did observe is that an unexpected overexpression of Cyclin D1 (a well-known protoncogene     ) leads to the down-regulation of the P53 onocosupressor.
P53-mediated regulation is very important and is defined as the “guardian” of genomic stability. When DNA damage is detected, p53 is activated and a cascade of events follows. If the DNA damage is resolved, p53 expression goes back to baseline and everything continues. If not, p53 levels stay high until the cell either stops dividing (as occurs in aging when telomeres shorten till below a certain threshold) or apoptosis is induced     .
Cells lacking a proper p53 response can withstand a high level of mutations which normally will trigger apoptosis, like in cancer cells     . These results in iPSCs suggest an increased resistance of the cells to genomic instability which may enable them to accumulate and retain mutations during the production procedure. Is this similar to what may happen in cancer? The researchers also address this by looking at known “cancer signatures” meaning single nucleotide changes that occur across different cancer types at defined DNA sequences. Such signatures can be identified due to the high amount of data generated by Next-Generation Sequencing. They do identify a cancer-like signature in the samples analyzed, such signature was absent before the reprogramming. This does not mean that the iPSCs are prone to be cancerous but that a similar process to what happens in cancer may go on in these cells.

A very compelling detail is that p53 downregulation is also what happens in tissue regeneration in amphibians     .

The next question will be why p53 levels lower and how this is controlled in the process of reprogramming in iPSCs. Maybe amphibians will provide us with some suggestions?

[1]Sugiura M, Kasama Y, Araki R, Hoki Y, Sunayama M, Uda M, Nakamura M, Ando S, Abe M. Induced pluripotent stem cell generation-associated point mutations arise during the initial stages of the conversion of these cells. Stem Cell Reports. 2014 Jan 2;2(1):52-63. doi: 10.1016/j.stemcr.2013.11.006. PMID: 24511470; PMCID: PMC3916761.

[2]Araki, R., Hoki, Y., Suga, T. et al. Genetic aberrations in iPSCs are introduced by a transient G1/S cell cycle checkpoint deficiency. Nat Commun 11, 197 (2020). https://doi.org/10.1038/s41467-019-13830-x

[3]Carusillo, A.; Mussolino, C. DNA Damage: From Threat to Treatment. Cells 2020, 9, 1665.

[4]Alao JP. The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention. Mol Cancer. 2007;6:24. Published 2007 Apr 2. doi:10.1186/1476-4598-6-24

[5]Lai PB, Chi TY, Chen GG. Different levels of p53 induced either apoptosis or cell cycle arrest in a doxycycline-regulated hepatocellular carcinoma cell line in vitro. Apoptosis. 2007 Feb;12(2):387-93. doi: 10.1007/s10495-006-0571-1. PMID: 17191126.

[6]Charni, M., Aloni-Grinstein, R., Molchadsky, A. et al. p53 on the crossroad between regeneration and cancer. Cell Death Differ 24, 8–14 (2017). https://doi.org/10.1038/cdd.2016.117

[7]Yun MH, Gates PB, Brockes JP. Regulation of p53 is critical for vertebrate limb regeneration. Proc Natl Acad Sci U S A. 2013;110(43):17392-17397. doi:10.1073/pnas.1310519110

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iPSCs have low fidelity of DNA damage repair

Shubhankar Kulkarni Nov 10, 2020

In comparison to embryonic stem cells and embryonic fibroblasts, iPSCs have lower DNA damage repair capacity. Double-stranded DNA breaks can be repaired via homologous recombination with high fidelity, or via non-homologous end-joining with lower fidelity.      iPSCs showed greater non-homologous end-joining DNA repair and less homologous recombination DNA repair. Even mice derived from iPSCs had lower DNA damage repair capacity than embryonic stem cell-derived mice and control mice.     

[1]Friedberg EC. A history of the DNA repair and mutagenesis field: The discovery of base excision repair. DNA Repair (Amst). 2016 Jan;37:A35-9. doi: 10.1016/j.dnarep.2015.12.003. PMID: 26861186.

[2]Zhang, M., Wang, L., An, K. et al. Lower genomic stability of induced pluripotent stem cells reflects increased non-homologous end joining. Cancer Commun 38, 49 (2018). https://doi.org/10.1186/s40880-018-0313-0

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Pre-existing variations in parental somatic cells

Subash Chapagain Nov 10, 2020

When New Generation Sequencing (NGS)  was employed to study the iPSCs, it was revealed that a number of genetic variations shown by these iPSCs were present as pre-existing variations in the parental somatic cells from where they were derived. As a consequence of the cloning process during iPSC generation, these variations were fixed     .

In another whole-genome sequencing study of the mouse iPSCs, 157 different single nucleotide variants (SNVs) were characterized in four iPSC clones established from the same mouse embryonic fibroblasts (MEFs), suggesting a strong link that these SNVs are most likely derived from the parental cells     .

In regard to these kinds of pre-existing variations that are continued to the iPSCs, two possible scenarios have been assumed. First, the variations get randomly captured during the iPSC generation. And second, some of the pre-existing variations can facilitate the reprogramming or proliferation of iPSCs, that can preferentially be propagated by selective advantage     .

[1]Gore A, Li Z, Fung HL, et al. Somatic coding mutations in human induced pluripotent stem cells. Nature. 2011;471(7336):63–67. doi: 10.1038/nature09805.

[2]Young MA, Larson DE, Sun CW, et al. Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells. Cell Stem Cell. 2012;10(5):570–582. doi: 10.1016/j.stem.2012.03.002

[3] Liang G, Zhang Y. Genetic and epigenetic variations in iPSCs: potential causes and implications for application. Cell Stem Cell. 2013;13(2):149–159. doi: 10.1016/j.stem.2013.07.001.

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Shubhankar Kulkarni4 years ago

An example of how some mutations facilitate the reprogramming ofiPSCs:  The trisomy 12 is the most frequently observed chromosomal aberration in human iPSCs. [1] Since the chromosome 12 contains a high number of cell cycle-related genes and the pluripotency-related gene NANOG [2], selective proliferative and reprogramming may benefit from trisomy 12 in pluripotent stem cells.

References:
1. Taapken SM, Nisler BS, Newton MA, Sampsell-Barron TL, Leonhard KA, McIntire EM,
Montgomery KD (2011) Karotypic abnormalities in human induced pluripotent stem cells
and embryonic stem cells. Nat Biotechnol 29(4):313–314. https://doi.org/10.1038/nbt.1835
2. Mayshar Y, Ben-David U, Lavon N, Biancotti JC, Yakir B, Clark AT, Plath K, Lowry WE,
Benvenisty N (2010) Identification and classification of chromosomal aberrations in human
induced pluripotent stem cells. Cell Stem Cell 7(4):521–531. https://doi.org/10.1016/j.
stem.2010.07.017

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Shubhankar Kulkarni4 years ago

Subash ChapagainReally helpful information here! As soon as I read the first two paragraphs, my suggestion to avoid the pre-existing variations was going to be to check the somatic cells before dedifferentiation. Only completely genomically "healthy" cells can be extracted and taken to make iPCs.

But then I read the third paragraph :). If the first scenario is true, it is probably easier to solve. A comparison of the different techniques used for dedifferentiation can tell us which of these leads to fewer genetic variations and that can be the preferred method. If the second scenario is true, it makes the job difficult. We need to find out if any specific variations have the selective advantage or variations, in general, that is, the mutated cells, pose a selective advantage irrespective of the mutation.

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Subash Chapagain4 years ago

Shubhankar Kulkarni indeed. In the second case, we might want to screen for the 'minimal' set of such variations/mutations that offer the selective advantage so as to maintain a threshold that allows for effective genetic stability all the while maintaining the pluripotency as well. I am not sure how feasible , practically speaking, that would be. 

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Reprogramming-induced mutations due to ROS and lack of repair

Shubhankar Kulkarni Nov 25, 2020

A report showed that reprogramming-induced mutations possess signatures resembling oxidative stress (C -to - A transversions). These transversions preferentially occur in heterochromatic lamina-associated domains (LADs) that are located at the nuclear periphery. Heterochromatin formation in LADs prevents the access of DNA repair proteins.                LADs are, therefore, sensitive to reactive oxygen species released from the mitochondria. It was shown that the estrogen-related nuclear receptors (ERRα and γ) and their co-factors are transiently induced at the early stage of reprogramming resulting in a burst of oxidative phosphorylation.      This might lead to reprogramming-induced mutations.

[1]Guelen L, Pagie L, Brasset E, Meuleman W, Faza MB, Talhout W, Eussen BH, de Klein A, Wessels L, de Laat W, van Steensel B (2008) Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature 453(7197):948–951. https://doi. org/10.1038/nature06947

[2]Yoshihara M, Jiang L, Akatsuka S, Suyama M, Toyokuni S (2014) Genome-wide profiling of 8-oxoguanine reveals its association with spatial positioning in nucleus. DNA Res 21(6):603–612. https://doi.org/10.1093/dnares/dsu023

[3]Amouroux R, Campalans A, Epe B, Radicella JP (2010) Oxidative stress triggers the preferential assembly of base excision repair complexes on open chromatin regions. Nucleic Acids Res 38(9):2878–2890. https://doi.org/10.1093/nar/gkp1247

[4]Kida YS, Kawamura T, Wei Z, Sogo T, Jacinto S, Shigeno A, Kushige H, Yoshihara E, Liddle C, Ecker JR, Yu RT, Atkins AR, Downes M, Evans RM (2015) ERRs mediate a metabolic switch required for somatic cell reprogramming to Pluripotency. Cell Stem Cell 16(5):547– 555. https://doi.org/10.1016/j.stem.2015.03.001

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Passages induce mutations

Shubhankar Kulkarni Nov 25, 2020

Cells are cultured during and after reprogramming. Passage-induced mutations occur stochastically. Kuijk et al. showed that passage-induced mutations are characterized by C-to-A transversions linked to oxidative stress.      Physiological oxygen concentration (2%) reduces chromosomal abnormalities in cultured human ESCs compared with room oxygen (21%).      Optimization of the culture conditions may help eliminate these mutations.

[1]Kuijk E, Jager M, van der Roest B, Locati M, van Hoeck A, Korzelius J, Janssen R, Besselink N, Boymans S, van Boxtel R, Cuppen E (2018) Mutational impact of culturing human pluripotent and adult stem cells. bioRxiv. doi:https://doi.org/10.1101/430165

[2]Forsyth NR, Musio A, Vezzoni P, Simpson AH, Noble BS, McWhir J (2006) Physiologic oxygen enhances human embryonic stem cell clonal recovery and reduces chromosomal abnormalities. Cloning Stem Cells 8(1):16–23. https://doi.org/10.1089/clo.2006.8.16

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Solution: The right choice of cells for reprogramming

Shubhankar Kulkarni Nov 25, 2020

Recently, a study showed that ~50% of human iPSCs generated from skin fibroblasts contain mutations due to ultraviolet-induced damage.     

Here are a few recommended choices to avoid/ minimize the mutation load in the iPSCs:
Hematopoietic stem cells can be reprogrammed into iPSCs at high efficiency and contain significantly fewer mutations than skin fibroblasts.     
Evidence suggests that cells from younger donors may be advantageous. Aging is associated with increased DNA damage.     
Another study showed that umbilical cord blood-derived iPSCs had a significantly lower frequency of mutations than fibroblast-derived iPSCs.     

[1]D'Antonio M, Benaglio P, Jakubosky D, Greenwald WW, Matsui H, Donovan MKR, Li H, Smith EN, D'Antonio-Chronowska A, Frazer KA (2018) Insights into the mutational burden of human induced pluripotent stem cells from an integrative multi-omics approach. Cell Rep 24(4):883–894. https://doi.org/10.1016/j.celrep.2018.06.091

[2]Wang K, Guzman AK, Yan Z, Zhang S, Hu MY, Hamaneh MB, Yu YK, Tolu S, Zhang J, Kanavy HE, Ye K, Bartholdy B, Bouhassira EE (2019) Ultra-high-frequency reprogramming of individual long-term hematopoietic stem cells yields low somatic variant induced pluripotent stem cells. Cell Rep 26(10):2580–2592.e2587. https://doi.org/10.1016/j. celrep.2019.02.021

[3]Garinis GA, van der Horst GT, Vijg J, Hoeijmakers JH (2008) DNA damage and ageing: newage ideas for an age-old problem. Nat Cell Biol 10(11):1241–1247. https://doi.org/10.1038/ ncb1108-1241

[4]Su RJ, Yang Y, Neises A, Payne KJ, Wang J, Viswanathan K, Wakeland EK, Fang X, Zhang XB (2013) Few single nucleotide variations in exomes of human cord blood induced pluripotent stem cells. PLoS One 8(4):e59908. https://doi.org/10.1371/journal.pone.0059908

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Solution: Efficient delivery systems for the reprogramming factors

Shubhankar Kulkarni Nov 25, 2020

iPSCs generated using retroviral vectors produce insertional mutations and reactivate transgenes      that might lead to tumorigenesis. To overcome this problem,
integration-free vectors (such as expression plasmids,      Sendai virus vectors,      and episomal plasmid vectors          ) and
several DNA-free reprogramming methods like the ones based on direct delivery of proteins,           mRNA     , or miRNA      can be used.
Also, CRISPR Cas9-based gene activation was used to reprogram human skin fibroblasts into iPSCs.     

[1]Baum C, von Kalle C, Staal FJ, Li Z, Fehse B, Schmidt M, Weerkamp F, Karlsson S, Wagemaker G, Williams DA (2004) Chance or necessity? Insertional mutagenesis in gene therapy and its consequences. Mol Ther 9(1):5–13

[2]Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S (2008) Generation of mouse induced pluripotent stem cells without viral vectors. Science 322(5903):949–953. https://doi. org/10.1126/science.1164270

[3]Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M (2009) Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proceedings of the Japan Academy Series B, Physical and. Biol Sci 85(8):348–362

[4]Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson JA (2009) Human induced pluripotent stem cells free of vector and transgene sequences. Science 324(5928):797–801. https://doi.org/10.1126/science.1172482

[5]Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H, Nakagawa M, Tanabe K, Tezuka K, Shibata T, Kunisada T, Takahashi M, Takahashi J, Saji H, Yamanaka S (2011) A more efficient method to generate integration-free human iPS cells. Nat Methods 8(5):409–412. https://doi.org/10.1038/nmeth.1591

[6]Zhou H, Wu S, Joo JY, Zhu S, Han DW, Lin T, Trauger S, Bien G, Yao S, Zhu Y, Siuzdak G, Scholer HR, Duan L, Ding S (2009) Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4(5):381–384. https://doi.org/10.1016/j.stem.2009.04.005

[7]Kim D, Kim CH, Moon JI, Chung YG, Chang MY, Han BS, Ko S, Yang E, Cha KY, Lanza R, Kim KS (2009) Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4(6):472–476. https://doi.org/10.1016/j. stem.2009.05.005

[8]Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A, Daley GQ, Brack AS, Collins JJ, Cowan C, Schlaeger TM, Rossi DJ (2010) Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7(5):618–630. https://doi.org/10.1016/j. stem.2010.08.012

[9]Miyoshi N, Ishii H, Nagano H, Haraguchi N, Dewi DL, Kano Y, Nishikawa S, Tanemura M, Mimori K, Tanaka F, Saito T, Nishimura J, Takemasa I, Mizushima T, Ikeda M, Yamamoto H, Sekimoto M, Doki Y, Mori M (2011) Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell 8(6):633–638. https://doi.org/10.1016/j. stem.2011.05.001

[10]Weltner J, Balboa D, Katayama S, Bespalov M, Krjutskov K, Jouhilahti EM, Trokovic R, Kere J, Otonkoski T (2018) Human pluripotent reprogramming with CRISPR activators. Nat Commun 9(1):2643. https://doi.org/10.1038/s41467-018-05067-x

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Solution: Right choice of culture conditions

Shubhankar Kulkarni Nov 25, 2020

Passages induce aneuploidies,      point mutations,      and copy number variations (CNVs).      Hence, a high number of passages should be avoided. Techniques that avoid mutations are:
In the CryoPause method, iPSCs are dissociated into single cells and stored as ready-to-use aliquots, which limits the number of passages.     
Mechanical passaging on feeder layers is preferred over enzymatic passaging on a feeder-free substrate since the latter is associated with increased accumulation of genetic aberrations.      
Antioxidants in the culture can reduce the occurrence of mutations (C-to-A transversions) that occur during reprogramming and cell passaging     . Antioxidants reduced CNVs in iPSCs.     

[1]Mayshar Y, Ben-David U, Lavon N, Biancotti JC, Yakir B, Clark AT, Plath K, Lowry WE, Benvenisty N (2010) Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell 7(4):521–531. https://doi.org/10.1016/j. stem.2010.07.017

[2]Gore A, Li Z, Fung HL, Young JE, Agarwal S, Antosiewicz-Bourget J, Canto I, Giorgetti A, Israel MA, Kiskinis E, Lee JH, Loh YH, Manos PD, Montserrat N, Panopoulos AD, Ruiz S, Wilbert ML, Yu J, Kirkness EF, Izpisua Belmonte JC, Rossi DJ, Thomson JA, Eggan K, Daley GQ, Goldstein LS, Zhang K (2011) Somatic coding mutations in human induced pluripotent stem cells. Nature 471(7336):63–67. https://doi.org/10.1038/nature09805

[3]Laurent LC, Ulitsky I, Slavin I, Tran H, Schork A, Morey R, Lynch C, Harness JV, Lee S, Barrero MJ, Ku S, Martynova M, Semechkin R, Galat V, Gottesfeld J, Izpisua Belmonte JC, Murry C, Keirstead HS, Park HS, Schmidt U, Laslett AL, Muller FJ, Nievergelt CM, Shamir R, Loring JF (2011) Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. Cell Stem Cell 8(1):106–118. https://doi.org/10.1016/j.stem.2010.12.003

[4]Wong KG, Ryan SD, Ramnarine K, Rosen SA, Mann SE, Kulick A, De Stanchina E, Muller FJ, Kacmarczyk TJ, Zhang C, Betel D, Tomishima MJ (2017) CryoPause: a new method to immediately initiate experiments after cryopreservation of pluripotent stem cells. Stem Cell Rep 9(1):355–365. https://doi.org/10.1016/j.stemcr.2017.05.010

[5]Garitaonandia I, Amir H, Boscolo FS, Wambua GK, Schultheisz HL, Sabatini K, Morey R, Waltz S, Wang YC, Tran H, Leonardo TR, Nazor K, Slavin I, Lynch C, Li Y, Coleman R, Gallego Romero I, Altun G, Reynolds D, Dalton S, Parast M, Loring JF, Laurent LC (2015) Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions. PLoS One 10(2):e0118307. https://doi.org/10.1371/journal. pone.0118307

[6]Rouhani FJ, Nik-Zainal S, Wuster A, Li Y, Conte N, Koike-Yusa H, Kumasaka N, Vallier L, Yusa K, Bradley A (2016) Mutational history of a human cell lineage from somatic to induced pluripotent stem cells. PLoS Genet 12(4):e1005932. https://doi.org/10.1371/journal. pgen.1005932

[7]Ji J, Sharma V, Qi S, Guarch ME, Zhao P, Luo Z, Fan W, Wang Y, Mbabaali F, Neculai D, Esteban MA, McPherson JD, Batada NN (2014) Antioxidant supplementation reduces genomic aberrations in human induced pluripotent stem cells. Stem Cell Rep 2(1):44–51. https://doi.org/10.1016/j.stemcr.2013.11.004

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Solution: Right choice of reprogramming factors

Shubhankar Kulkarni Nov 25, 2020

Nucleosome remodeling and deacetylation repressor complex Mbd3/NuRD blocks iPSC induction, and Mbd3 depletion increased the efficiency of iPSC reprogramming.     
Oocyte factor Zscan4 increased reprogramming efficiency and improved genomic stability.      Aged iPSCs exhibit excessive glutathione-mediated reactive oxygen species (ROS) scavenging activity, blocking the DNA damage repair and apoptosis and allows cells with genomic instability to survive. The pluripotency factor Zscan10 is poorly expressed in A-iPSCs. When Zscan4 was added to the four Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) during reprogramming, ROS–glutathione homeostasis was established and the DNA damage response reinstated genomic stability.     
Increased levels of checkpoint kinase 1 (CHK1) reduced reprogramming-induced replication stress and increases reprogramming efficiency and genomic stability in human iPSCs.      
Chemical reprogramming may reduce the risk of tumorigenesis. Hou et al. used a combination of seven small molecules to generate iPSCs.     
Do comment and mention if you know of any other alternatives.

[1]Rais Y, Zviran A, Geula S, Gafni O, Chomsky E, Viukov S, Mansour AA, Caspi I, Krupalnik V, Zerbib M, Maza I, Mor N, Baran D, Weinberger L, Jaitin DA, Lara-Astiaso D, BlecherGonen R, Shipony Z, Mukamel Z, Hagai T, Gilad S, Amann-Zalcenstein D, Tanay A, Amit I, Novershtern N, Hanna JH (2013) Deterministic direct reprogramming of somatic cells to pluripotency. Nature 502(7469):65–70. https://doi.org/10.1038/nature12587

[2]Jiang J, Lv W, Ye X, Wang L, Zhang M, Yang H, Okuka M, Zhou C, Zhang X, Liu L, Li J (2013) Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation. Cell Res 23(1):92–106. https://doi.org/10.1038/cr.2012.157

[3]Skamagki M, Correia C, Yeung P, Baslan T, Beck S, Zhang C, Ross CA, Dang L, Liu Z, Giunta S, Chang TP, Wang J, Ananthanarayanan A, Bohndorf M, Bosbach B, Adjaye J, Funabiki H, Kim J, Lowe S, Collins JJ, Lu CW, Li H, Zhao R, Kim K. ZSCAN10 expression corrects the genomic instability of iPSCs from aged donors. Nat Cell Biol. 2017 Sep;19(9):1037-1048. doi: 10.1038/ncb3598. Epub 2017 Aug 28. Erratum in: Nat Cell Biol. 2019 Apr;21(4):531-532. PMID: 28846095; PMCID: PMC5843481.

[4]Ruiz S, Lopez-Contreras AJ, Gabut M, Marion RM, Gutierrez-Martinez P, Bua S, Ramirez O, Olalde I, Rodrigo-Perez S, Li H, Marques-Bonet T, Serrano M, Blasco MA, Batada NN, Fernandez-Capetillo O (2015) Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells. Nat Commun 6:8036. https:// doi.org/10.1038/ncomms9036

[5]Hou P, Li Y, Zhang X, Liu C, Guan J, Li H, Zhao T, Ye J, Yang W, Liu K, Ge J, Xu J, Zhang Q, Zhao Y, Deng H (2013) Pluripotent stem cells induced from mouse somatic cells by smallmolecule compounds. Science 341(6146):651–654. https://doi.org/10.1126/science.1239278

What molecular mechanisms contribute to lower genomic stability in the induced pluripotent stem cells?

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Epigenetics contribute to genomic instability in iPSCs

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Cell cycle defects contribute to genomic instability

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iPSCs have low fidelity of DNA damage repair

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Pre-existing variations in parental somatic cells

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Reprogramming-induced mutations due to ROS and lack of repair

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Passages induce mutations

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Solution: The right choice of cells for reprogramming

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Solution: Efficient delivery systems for the reprogramming factors

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Solution: Right choice of culture conditions

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Solution: Right choice of reprogramming factors

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What molecular mechanisms contribute to lower genomic stability in the induced pluripotent stem cells?

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Necessity

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Creative contributions

Epigenetics contribute to genomic instability in iPSCs

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Cell cycle defects contribute to genomic instability

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iPSCs have low fidelity of DNA damage repair

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Pre-existing variations in parental somatic cells

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Reprogramming-induced mutations due to ROS and lack of repair

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Passages induce mutations

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Solution: The right choice of cells for reprogramming

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Solution: Efficient delivery systems for the reprogramming factors

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Solution: Right choice of culture conditions

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Solution: Right choice of reprogramming factors

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