Rescue of premature aging defects in Cockayne syndrome stem cells by CRISPR/Cas9-mediated gene correction

Abstract

Cockayne syndrome (CS) is a rare autosomal reces-sive inherited disorder characterized by a variety of clinical features, including increased sensitivity to sun-light, progressive neurological abnormalities, and the appearance of premature aging. However, the pathogenesis of CS remains unclear due to the limita-tions of current disease models. Here, we generate integration-free induced pluripotent stem cells (iPSCs) from fibroblasts from a CS patient bearing mutations in CSB/ERCC6 gene and further derive isogenic gene-corrected CS-iPSCs (GC-iPSCs) using the CRISPR/Cas9 system. CS-associated phenotypic defects are recapit-ulated in CS-iPSC-derived mesenchymal stem cells (MSCs) and neural stem cells (NSCs), both of which display increased susceptibility to DNA damage stress. Premature aging defects in CS-MSCs are rescued by the targeted correction of mutant ERCC6. We next map the transcriptomic landscapes in CS-iPSCs and GC-iPSCs and their somatic stem cell derivatives (MSCs and NSCs) in the absence or presence of ultraviolet (UV) and replicative stresses, revealing that defects in DNA repair account for CS pathologies. Moreover, we generate autologous GC-MSCs free of pathogenic mutation under a cGMP (Current Good Manufacturing Practice)-compli- ant condition, which hold potential for use as improved biomaterials for future stem cell replacement therapy for CS. Collectively, our models demonstrate novel disease features and molecular mechanisms and lay a founda- tion for the development of novel therapeutic strategies to treat CS.

Keywords

Cockayne syndrome / CRISPR/Cas9 / gene correction / disease modelling / mesenchymal stem cell / neural stem cell

References

[1] Amaro-Ortiz A, Yan B, D’Orazio JA (2014) Ultraviolet radiation, aging and the skin: prevention of damage by topical cAMP manipula-tion. Molecules 19:6202–6219

[2] Anders S, Pyl PT, Huber W (2015) HTSeq: a Python framework to work with high-throughput sequencing data. Bioinformatics 31:166–169

[3] Andrade LN, Nathanson JL, Yeo GW, Menck CF, Muotri AR (2012) Evidence for premature aging due to oxidative stress in iPSCs from Cockayne syndrome. Hum Mol Genet 21:3825–3834

[4] Andressoo JO, Mitchell JR, de Wit J, Hoogstraten D, Volker M, Toussaint W, Speksnijder E, Beems RB, van Steeg H, Jans J (2006) An Xpd mouse model for the combined xeroderma pigmentosum/Cockayne syndrome exhibiting both cancer pre-disposition and segmental progeria. Cancer Cell 10:121–132

[5] Bae S, Park J, Kim JS (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30:1473–1475

[6] Cadet J, Sage E, Douki T (2005) Ultraviolet radiation-mediated damage to cellular DNA. Mutat Res 571:3–17

[7] Castro-Vinuelas R, Sanjurjo-Rodriguez C, Pineiro-Ramil M, Her-mida-Gomez T, Fuentes-Boquete IM, de Toro-Santos FJ, Blanco-Garcia FJ, Diaz-Prado SM (2018) Induced pluripotent stem cells for cartilage repair: current status and future perspectives. Eur Cell Mater 36:96–109

[8] Ciaffardini F, Nicolai S, Caputo M, Canu G, Paccosi E, Costantino M, Frontini M, Balajee AS, Proietti-De-Santis L (2014) The cockayne syndrome B protein is essential for neuronal differentiation and neuritogenesis. Cell Death Dis 5:e1268

[9] Cleaver JE, Lam ET, Revet I (2009) Disorders of nucleotide excision repair: the genetic and molecular basis of heterogeneity. Nat Rev Genet 10:756–768

[10] Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O (2009) Protocols to detect senescence-associated beta-galac-tosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc 4:1798–1806

[11] Ding Z, Sui L, Ren R, Liu Y, Xu X, Fu L, Bai R, Yuan T, Hao Y, Zhang W (2015) A widely adaptable approach to generate integration-free iPSCs from non-invasively acquired human somatic cells. Protein Cell 6:386–389

[12] Duan S, Yuan G, Liu X, Ren R, Li J, Zhang W, Wu J, Xu X, Fu L, Li Y (2015) PTEN deficiency reprogrammes human neural stem cells towards a glioblastoma stem cell-like phenotype. Nat Commun 6:10068

[13] Friedberg EC (2001) How nucleotide excision repair protects against cancer. Nat Rev Cancer 1:22–33

[14] Friedberg EC (2003) DNA damage and repair. Nature 421:436–440

[15] Fu LN, Xu XL, Ren RT, Wu J, Zhang WQ, Yang JP, Ren XQ, Wang S, Zhao Y, Sun L (2016) Modeling xeroderma pigmentosum associated neurological pathologies with patients-derived iPSCs. Protein Cell 7:210–221

[16] Geng L, Liu Z, Zhang W, Li W, Wu Z, Wang W, Ren R, Su Y, Wang P, Sun L (2018) Chemical screen identifies a geroprotective role of quercetin in premature aging. Protein Cell.

[17] Golpanian S, DiFede DL, Pujol MV, Lowery MH, Levis-Dusseau S, Goldstein BJ, Schulman IH, Longsomboon B, Wolf A, Khan A (2016) Rationale and design of the allogeneiC human mesenchymal stem cells (hMSC) in patients with aging fRAilTy via intraveno US delivery (CRATUS) study: A phase I/II, randomized, blinded and placebo controlled trial to evaluate the safety and potential efficacy of allogeneic human mesenchymal stem cell infusion in patients with aging frailty. Oncotarget 7:11899–11912

[18] Golpanian S, DiFede DL, Khan A, Schulman IH, Landin AM, Tompkins BA, Heldman AW, Miki R, Goldstein BJ, Mushtaq M (2017) Allogeneic human mesenchymal stem cell infusions for aging frailty. J Gerontol A 72:1505–1512

[19] Gorgels TG, van der Pluijm I, Brandt RM, Garinis GA, van Steeg H, van den Aardweg G, Jansen GH, Ruijter JM, Bergen AA, van Norren D (2007) Retinal degeneration and ionizing radiation hypersensitivity in a mouse model for Cockayne syndrome. Mol Cell Biol 27:1433–1441

[20] Hishiya A, Watanabe K (2004) Progeroid syndrome as a model for impaired bone formation in senile osteoporosis. J Bone Miner Metab 22:399–403

[21] Jaarsma D, van der Pluijm I,de Waard MC, Haasdijk ED, Brandt R, Vermeij M, Rijksen Y, Maas A, van Steeg H, Hoeijmakers JH (2011) Age-related neuronal degeneration: complementary roles of nucleotide excision repair and transcription-coupled repair in preventing neuropathology. PLoS Genet 7:e1002405

[22] Karikkineth AC, Scheibye-Knudsen M, Fivenson E, Croteau DL, Bohr VA (2017) Cockayne syndrome: clinical features, model systems and pathways. Ageing Res Rev 33:3–17

[23] Kawamura T, Suzuki J, Wang YV, Menendez S, Morera LB, Raya A, Wahl GM, Izpisua Belmonte JC (2009) Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 460:1140–1144

[24] Kemp MG, Spandau DF, Travers JB (2017) Impact of age and insulin-like growth factor-1 on DNA damage responses in UV-irradiated human skin. Molecules 22:356

[25] Kim D, Langmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12:357–360

[26] Kubben N, Zhang W, Wang L, Voss TC, Yang J, Qu J, Liu GH, Misteli T (2016) Repression of the antioxidant NRF2 pathway in premature aging. Cell 165:1361–1374

[27] Laugel V (2013) Cockayne syndrome: the expanding clinical and mutational spectrum. Mech Ageing Dev 134:161–170

[28] Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25:1754–1760

[29] Li Y, Zhang W, Chang L, Han Y, Sun L, Gong X, Tang H, Liu Z, Deng H, Ye Y (2016) Vitamin C alleviates aging defects in a stem cell model for Werner syndrome. Protein Cell 7:478–488

[30] Ling C, Liu Z, Song M, Zhang W, Wang S, Liu X, Ma S, Sun S, Fu L, Chu Q (2019) Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells. Protein Cell. 10:249–271

[31] Liu GH, Barkho BZ, Ruiz S, Diep D, Qu J, Yang SL, Panopoulos AD, Suzuki K, Kurian L, Walsh C (2011a) Recapitulation of premature ageing with iPSCs from Hutchinson–Gilford progeria syndrome. Nature 472:221–225

[32] Liu GH, Suzuki K, Qu J, Sancho-Martinez I, Yi F, Li M, Kumar S, Nivet E, Kim J, Soligalla RD (2011b) Targeted gene correction of laminopathy-associated LMNA mutations in patient-specific iPSCs. Cell Stem Cell 8:688–694

[33] Liu GH, Qu J, Suzuki K, Nivet E, Li M, Montserrat N, Yi F, Xu X, Ruiz S, Zhang W (2012) Progressive degeneration of human neural stem cells caused by pathogenic LRRK2. Nature 491:603–607

[34] Liu GH, Suzuki K, Li M, Qu J, Montserrat N, Tarantino C, Gu Y, Yi F, Xu X, Zhang W (2014) Modelling Fanconi anemia patho-genesis and therapeutics using integration-free patient-derived iPSCs. Nat Commun 5:4330

[35] Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550

[36] McKay BC, Cabrita MA (2013) Arresting transcription and sentenc-ing the cell: the consequences of blocked transcription. Mech Ageing Dev 134:243–252

[37] Muller LU, Milsom MD, Harris CE, Vyas R, Brumme KM, Parmar K, Moreau LA, Schambach A, Park IH, London WB (2012) Overcoming reprogramming resistance of Fanconi anemia cells. Blood 119:5449–5457

[38] Murai M, Enokido Y, Inamura N, Yoshino M, Nakatsu Y, van der Horst GT, Hoeijmakers JH, Tanaka K, Hatanaka H (2001) Early postnatal ataxia and abnormal cerebellar development in mice lacking Xeroderma pigmentosum Group A and Cockayne syn-drome Group B DNA repair genes. Proc Natl Acad Sci USA 98:13379–13384

[39] Natale V (2011) A comprehensive description of the severity groups in Cockayne syndrome. Am J Med Genet A 155A:1081–1095

[40] Newman JC, Bailey AD, Weiner AM (2006) Cockayne syndrome group B protein (CSB) plays a general role in chromatin maintenance and remodeling. Proc Natl Acad Sci USA 103:9613–9618

[41] Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H, Nakagawa M, Tanabe K, Tezuka K (2011) A more efficient method to generate integration-free human iPS cells. Nat Methods 8:409–412

[42] Orozco L, Soler R, Morera C, Alberca M, Sanchez A, Garcia-Sancho J (2011) Intervertebral disc repair by autologous mesenchymal bone marrow cells: a pilot study. Transplantation 92:822–828

[43] Orozco L, Munar A, Soler R, Alberca M, Soler F, Huguet M, Sentis J, Sanchez A, Garcia-Sancho J (2013) Treatment of knee osteoarthritis with autologous mesenchymal stem cells: a pilot study. Transplantation 95:1535–1541

[44] Orozco L, Munar A, Soler R, Alberca M, Soler F, Huguet M, Sentis J, Sanchez A, Garcia-Sancho J (2014) Treatment of knee osteoarthritis with autologous mesenchymal stem cells: two-year follow-up results. Transplantation 97:e66–e68

[45] Pan H, Guan D, Liu X, Li J, Wang L, Wu J, Zhou J, Zhang W, Ren R, Li Y (2016) SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2. Cell Res 26:190–205

[46] Peters DT, Cowan CA, Musunuru K (2008) Genome editing in human pluripotent stem cells. In: StemBook, Cambridge

[47] Proietti-De-Santis L, Drane P, Egly JM (2006) Cockayne syndrome B protein regulates the transcriptional program after UV irradiation. EMBO J 25:1915–1923

[48] Rockx DA, Mason R, van Hoffen A, Barton MC, Citterio E, Bregman DB, van Zeeland AA, Vrieling H, Mullenders LH (2000) UV-induced inhibition of transcription involves repression of tran-scription initiation and phosphorylation of RNA polymerase II. Proc Natl Acad Sci USA 97:10503–10508

[49] Sacco R, Tamblyn L, Rajakulendran N, Bralha FN, Tropepe V, Laposa RR (2013) Cockayne syndrome b maintains neural precursor function. DNA Repair 12:110–120

[50] Setlow RB, Setlow JK (1962) Evidence that ultraviolet-induced thymine dimers in DNA cause biological damage. Proc Natl Acad Sci USA 48:1250–1257

[51] Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13:2498–2504

[52] Shehata L, Simeonov DR, Raams A, Wolfe L, Vanderver A, Li X, Huang Y, Garner S, Boerkoel CF, Thurm A (2014) ERCC6 dysfunction presenting as progressive neurological decline with brain hypomyelination. Am J Med Genet A 164A:2892–2900

[53] Shimamoto A, Kagawa H, Zensho K, Sera Y, Kazuki Y, Osaki M, Oshimura M, Ishigaki Y, Hamasaki K, Kodama Y (2014) Reprogramming suppresses premature senescence phenotypes of Werner syndrome cells and maintains chromosomal stability over long-term culture. PLoS ONE 9:e112900

[54] Soontararak S, Chow L, Johnson V, Coy J, Wheat W, Regan D, Dow S (2018) Mesenchymal stem cells (MSC) derived from induced pluripotent stem cells (iPSC) equivalent to adipose-derived MSC in promoting intestinal healing and microbiome normalization in mouse inflammatory bowel disease model. Stem Cells Transl Med 7:456–467

[55] Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, Hatanaka F, Yamamoto M, Araoka T, Li Z (2016) In vivo genome editing via CRISPR/Cas9 mediated homology-indepen-dent targeted integration. Nature 540:144–149

[56] Szklarczyk D, Morris JH, Cook H, Kuhn M, Wyder S, Simonovic M, Santos A, Doncheva NT, Roth A, Bork P (2017) The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res 45:D362–D368

[57] Tacutu R, Craig T, Budovsky A, Wuttke D, Lehmann G, Taranukha D, Costa J, Fraifeld VE, de Magalhaes JP (2013) Human ageing genomic resources: integrated databases and tools for the biology and genetics of ageing. Nucleic Acids Res 41:D1027–D1033

[58] Tompkins BA, DiFede DL, Khan A, Landin AM, Schulman IH, Pujol MV, Heldman AW, Miki R, Goldschmidt-Clermont PJ, Goldstein BJ (2017) Allogeneic mesenchymal stem cells ameliorate aging frailty: a phase II randomized, double-blind, placebo-controlled clinical trial. J Gerontol A 72:1513–1522

[59] Tripathi S, Pohl MO, Zhou Y, Rodriguez-Frandsen A, Wang G, Stein DA, Moulton HM, DeJesus P, Che J, Mulder LC (2015) Meta-and orthogonal integration of influenza “OMICs” data defines a role for UBR4 in virus budding. Cell Host Microbe 18:723–735

[60] van der Horst GT, van Steeg H, Berg RJ, van Gool AJ, de Wit J, Weeda G, Morreau H, Beems RB, van Kreijl CF, de Gruijl FR (1997) Defective transcription-coupled repair in Cockayne syn-drome B mice is associated with skin cancer predisposition. Cell 89:425–435

[61] van der Horst GT, Meira L, Gorgels TG, de Wit J, Velasco-Miguel S, Richardson JA, Kamp Y, Vreeswijk MP, Smit B, Bootsma D (2002) UVB radiation-induced cancer predisposition in Cockayne syndrome group A (Csa) mutant mice. DNA Repair 1:143–157

[62] van der Pluijm I, Garinis GA, Brandt RM, Gorgels TG, Wijnhoven SW, Diderich KE, de Wit J, Mitchell JR, van Oostrom C, Beems R (2007) Impaired genome maintenance suppresses the growth hormone–insulin-like growth factor 1 axis in mice with Cockayne syndrome. PLoS Biol 5:e2

[63] Velez-Cruz R, Egly JM (2013) Cockayne syndrome group B (CSB) protein: at the crossroads of transcriptional networks. Mech Ageing Dev 134:234–242

[64] Velez-Cruz R, Zadorin AS, Coin F, Egly JM (2013) Sirt1 suppresses RNA synthesis after UV irradiation in combined xeroderma pigmentosum group D/Cockayne syndrome (XP-D/CS) cells. Proc Natl Acad Sci USA 110:E212–E220

[65] Vessoni AT, Herai RH, Karpiak JV, Leal AM, Trujillo CA, Quinet A, Agnez Lima LF, Menck CF, Muotri AR (2016) Cockayne syndrome-derived neurons display reduced synapse density and altered neural network synchrony. Hum Mol Genet 25:1271–1280

[66] Wang S, Wang X, Wu Y, Han C (2015) IGF-1R signaling is essential for the proliferation of cultured mouse spermatogonial stem cells by promoting the G2/M progression of the cell cycle. Stem Cells Dev 24:471–483

[67] Wang S, Wang X, Ma L, Lin X, Zhang D, Li Z, Wu Y, Zheng C, Feng X, Liao S (2016) Retinoic acid is sufficient for the in vitro induction of mouse spermatocytes. Stem Cell Rep 7:80–94

[68] Wang LX, Yi F, Fu LN, Yang JP, Wang S, Wang ZX, Suzuki K, Sun L, Xu XL, Yu Y (2017) CRISPR/Cas9-mediated targeted gene correction in amyotrophic lateral sclerosis patient iPSCs. Protein Cell 8:365–378

[69] Wang P, Liu Z, Zhang X, Li J, Sun L, Ju Z, Li J, Chan P, Liu GH, Zhang W (2018a) CRISPR/Cas9-mediated gene knockout reveals a guardian role of NF-kappaB/RelA in maintaining the homeostasis of human vascular cells. Protein Cell 9:945–965

[70] Wang S, Hu B, Ding Z, Dang Y, Wu J, Li D, Liu X, Xiao B, Zhang W, Ren R (2018b) ATF6 safeguards organelle homeostasis and cellular aging in human mesenchymal stem cells. Cell Discov 4:2

[71] Wang S, Liu Z, Ye Y, Li B, Liu T, Zhang W, Liu GH, Zhang YA, Qu J, Xu D (2018c) Ectopic hTERT expression facilitates repro-graming of fibroblasts derived from patients with Werner syn-drome as a WS cellular model. Cell Death Dis 9:923

[72] Wu Z, Zhang W, Song M, Wang W, Wei G, Li W, Lei J, Huang Y, Sang Y, Chan P (2018) Differential stem cell aging kinetics in Hutchinson–Gilford progeria syndrome and Werner syndrome. Protein Cell 9:333–350

[73] Yamada A, Masutani C, Hanaoka F (2002) Detection of reduced RNA synthesis in UV-irradiated Cockayne syndrome group B cells using an isolated nuclear system. Biochim Biophys Acta 1592:129–134

[74] Yan P, Li Q, Wang L, Lu P, Suzuki K, Liu Z, Lei J, Li W, He X, Wang S (2019) FOXO3-engineered human ESC-derived vascular cells Promote vascular protection and regeneration. Cell Stem Cell.

[75] Yang J, Li J, Suzuki K, Liu X, Wu J, Zhang W, Ren R, Zhang W, Chan P, Izpisua Belmonte JC (2017) Genetic enhancement in cultured human adult stem cells conferred by a single nucleotide recoding. Cell Res 27:1178–1181

[76] Yu QC, Song W, Wang D, Zeng YA (2016) Identification of blood vascular endothelial stem cells by the expression of protein C receptor. Cell Res 26:1079–1098

[77] Zhang W, Li J, Suzuki K, Qu J, Wang P, Zhou J, Liu X, Ren R, Xu X, Ocampo A (2015) Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging. Science 348:1160–1163

[78] Zhang W, Wan H, Feng G, Qu J, Wang J, Jing Y, Ren R, Liu Z, Zhang L, Chen Z (2018) SIRT6 deficiency results in developmental retardation in cynomolgus monkeys. Nature 560:661–665

[79] Zhang X, Liu Z, Liu X, Wang S, Zhang Y, He X, Sun S, Ma S, Shyh-Chang N, Liu F (2019) Telomere-dependent and telomere-independent roles of RAP1 in regulating human stem cell homeostasis. Protein Cell.

RIGHTS & PERMISSIONS

The Author(s)