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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
Shubhankar Kulkarni Nov 10, 2020


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Epigenetics contribute to genomic instability in iPSCs

Jamila Nov 11, 2020

[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.

Cell cycle defects contribute to genomic instability

Antonio Carusillo
Antonio Carusillo Nov 15, 2020

[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

iPSCs have low fidelity of DNA damage repair

Shubhankar Kulkarni
Shubhankar Kulkarni Nov 10, 2020

[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

Pre-existing variations in parental somatic cells

Subash Chapagain
Subash Chapagain Nov 10, 2020

[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.

Shubhankar Kulkarni
Shubhankar Kulkarni3 months 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.

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.
Shubhankar Kulkarni
Shubhankar Kulkarni4 months 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.
Subash Chapagain
Subash Chapagain4 months 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.

Reprogramming-induced mutations due to ROS and lack of repair

Shubhankar Kulkarni
Shubhankar Kulkarni Nov 25, 2020

[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

Passages induce mutations

Shubhankar Kulkarni
Shubhankar Kulkarni Nov 25, 2020

[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

Solution: The right choice of cells for reprogramming

Shubhankar Kulkarni
Shubhankar Kulkarni Nov 25, 2020

[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

Solution: Efficient delivery systems for the reprogramming factors

Shubhankar Kulkarni
Shubhankar Kulkarni Nov 25, 2020

[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

Solution: Right choice of reprogramming factors

Shubhankar Kulkarni
Shubhankar Kulkarni Nov 25, 2020

[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

Solution: Right choice of culture conditions

Shubhankar Kulkarni
Shubhankar Kulkarni Nov 25, 2020

[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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