Abstract
C9orf72 genetic mutation is the most common genetic cause of ALS/FTD accompanied by abnormal protein insufficiency. Induced pluripotent stem cell (iPSC)-derived two-dimensional (2D) and three-dimensional (3D) cultures are providing new approaches. Therefore, this study established neuronal cell types and generated spinal cord organoids (SCOs) derived from C9orf72 knockdown human iPSCs to model ALS disease and screen the unrevealed phenotype. Wild-type (WT) iPSC lines from three healthy donor fibroblasts were established, and pluripotency and differentiation ability were identified by RT-PCR, immunofluorescence and flow cytometry. After infection by the lentivirus with C9orf72-targeting shRNA, stable C9-knockdown iPSC colonies were selected and differentiated into astrocytes, motor neurons and SCOs. Finally, we analyzed the extracted RNA-seq data of human C9 mutant/knockout iPSC-derived motor neurons and astrocytes from the GEO database and the inflammatory regulation-related genes in function and pathways. The expression of inflammatory factors was measured by qRT-PCR. The results showed that both WT-iPSCs and edited C9-iPSCs maintained a similar ability to differentiate into the three germ layers, astrocytes and motor neurons, forming SCOs in a 3D culture system. The constructed C9-SCOs have features of spinal cord development and multiple neuronal cell types, including sensory neurons, motor neurons, and other neurons. Based on the bioinformatics analysis, proinflammatory factors were confirmed to be upregulated in C9-iPSC-derived 2D cells and 3D cultured SCOs. The above differentiated models exhibited low C9orf72 expression and the pathological characteristics of ALS, especially neuroinflammation.
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The data that support the findings of this study are available from the corresponding author upon reasonable request.
Abbreviations
- ALS :
-
Amyotrophic lateral sclerosis
- C9orf72 :
-
Chromosome 9 open reading frame 72
- WT :
-
Wild type;
- HiPSCs :
-
Human induced pluripotent stem cells
- SOX2 :
-
SRY-box transcription factor 2
- OCT4 :
-
Octamer-binding transcription factor 4
- SSEA4 :
-
Stage-specific embryonic antigen 4
- NSCs :
-
Neural stem cells
- GFAP :
-
Glial fibrillary acidic protein
- CHAT :
-
Choline acetyltransferase
- FOXA2 :
-
Forkhead box A2
- IL :
-
Interleukin
- TNFα :
-
Tumour necrosis factor alpha-like
- TGFβ :
-
Transforming growth factor beta
- VEGF :
-
Vascular endothelial growth factor
- IFNγ :
-
Interferon γ
- Cos :
-
Cerebral organoids
- SCOs :
-
Spinal cord organoids
- 3D :
-
Three-dimensional
- GDNF :
-
Glial-derived neurotrophic factor
- BDNF :
-
Brain-derived neurotrophic factor
References
Genge A, Chio A (2023) The future of ALS diagnosis and staging: where do we go from here? Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration 24(3–4):165–174. https://doi.org/10.1080/21678421.2022.2150555
Punjani, R, Larson, TC, Wagner, L, Davis, B, Horton, DK, Kaye, W (2022) Survival and epidemiology of amyotrophic lateral sclerosis (ALS) cases in the chicago and detroit metropolitan cohort: incident cases 2009–2011 and survival through 2018. Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 1–9. https://doi.org/10.1080/21678421.2022.2121167.
Renton AE, Majounie E, Waite A, Simon-Sanchez J, Rollinson S, Gibbs JR, Schymick JC, Laaksovirta H, van Swieten JC, Myllykangas L et al (2011) A hexanucleotide repeat expansion in c9orf72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72(2):257–268. https://doi.org/10.1016/j.neuron.2011.09.010
Dejesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ, Nicholson AM, Finch NA, Flynn H, Adamson J et al (2011) Expanded ggggcc hexanucleotide repeat in noncoding region of c9orf72 causes chromosome 9p-linked FTD and ALS. Neuron 72(2):245–256. https://doi.org/10.1016/j.neuron.2011.09.011
Kaivola K, Pirinen M, Laaksovirta H, Jansson L, Rautila O, Launes J, Hokkanen L, Lahti J, Eriksson JG, Strandberg TE et al (2023) C9orf72 hexanucleotide repeat allele tagging snps: associations with ALS risk and longevity. Front Genet 14:1087098. https://doi.org/10.3389/fgene.2023.1087098
Mackenzie IR, Arzberger T, Kremmer E, Troost D, Lorenzl S, Mori K, Weng SM, Haass C, Kretzschmar HA, Edbauer D et al (2013) Dipeptide repeat protein pathology in c9orf72 mutation cases: clinico-pathological correlations. Acta Neuropathol 126(6):859–879. https://doi.org/10.1007/s00401-013-1181-y
Therrien M, Rouleau GA, Dion PA, Parker JA (2013) Deletion of c9orf72 results in motor neuron degeneration and stress sensitivity in c. Elegans Plos One 8(12):e83450. https://doi.org/10.1371/journal.pone.0083450
Haeusler AR, Donnelly CJ, Periz G, Simko EA, Shaw PG, Kim MS, Maragakis NJ, Troncoso JC, Pandey A, Sattler R et al (2014) C9orf72 nucleotide repeat structures initiate molecular cascades of disease. Nature 507(7491):195–200. https://doi.org/10.1038/nature13124
Zhang K, Donnelly CJ, Haeusler AR, Grima JC, Machamer JB, Steinwald P, Daley EL, Miller SJ, Cunningham KM, Vidensky S et al (2015) The c9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature 525(7567):56–61. https://doi.org/10.1038/nature14973
Ciura S, Lattante S, Le Ber I, Latouche M, Tostivint H, Brice A, Kabashi E (2013) Loss of function of c9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis. Ann Neurol 74(2):180–187. https://doi.org/10.1002/ana.23946
Jiang J, Zhu Q, Gendron TF, Saberi S, Mcalonis-Downes M, Seelman A, Stauffer JE, Jafar-Nejad P, Drenner K, Schulte D et al (2016) Gain of toxicity from ALS/FTD-linked repeat expansions in c9orf72 is alleviated by antisense oligonucleotides targeting ggggcc-containing rnas. Neuron 90(3):535–550. https://doi.org/10.1016/j.neuron.2016.04.006
Lancaster MA, Renner M, Martin CA, Wenzel D, Bicknell LS, Hurles ME, Homfray T, Penninger JM, Jackson AP, Knoblich JA (2013) Cerebral organoids model human brain development and microcephaly. Nature 501(7467):373–379. https://doi.org/10.1038/nature12517
Lancaster MA, Knoblich JA (2014) Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc 9(10):2329–2340. https://doi.org/10.1038/nprot.2014.158
Muzio L, Consalez GG (2013) Modeling human brain development with cerebral organoids. Stem Cell Res Ther 4(6):154. https://doi.org/10.1186/scrt384
Zhang W, Jiang J, Xu Z, Yan H, Tang B, Liu C, Chen C, Meng Q (2023) Microglia-containing human brain organoids for the study of brain development and pathology. Mol Psychiatry 28(1):96–107. https://doi.org/10.1038/s41380-022-01892-1
Adlakha YK (2023) Human 3d brain organoids: steering the demolecularization of brain and neurological diseases. Cell Death Discovery 9(1):221. https://doi.org/10.1038/s41420-023-01523-w
Trujillo CA, Gao R, Negraes PD, Gu J, Buchanan J, Preissl S, Wang A, Wu W, Haddad GG, Chaim IA et al (2019) Complex oscillatory waves emerging from cortical organoids model early human brain network development. Cell Stem Cell 25(4):558–569. https://doi.org/10.1016/j.stem.2019.08.002
Tamaki Y, Ross JP, Alipour P, Castonguay CÉ, Li B, Catoire H, Rochefort D, Urushitani M, Takahashi R, Sonnen JA et al (2023) Spinal cord extracts of amyotrophic lateral sclerosis spread tdp-43 pathology in cerebral organoids. PLoS Genet 19(2):e1010606. https://doi.org/10.1371/journal.pgen.1010606
Szebényi K, Wenger L, Sun Y, Dunn A, Limegrover CA, Gibbons GM, Conci E, Paulsen O, Mierau SB, Balmus G et al (2021) Human ALS/FTD brain organoid slice cultures display distinct early astrocyte and targetable neuronal pathology. Nat Neurosci 24(11):1542–1554. https://doi.org/10.1038/s41593-021-00923-4
Pereira JD, Dubreuil DM, Devlin AC, Held A, Sapir Y, Berezovski E, Hawrot J, Dorfman K, Chander V, Wainger BJ (2021) Human sensorimotor organoids derived from healthy and amyotrophic lateral sclerosis stem cells form neuromuscular junctions. Nat Commun 12(1):4744. https://doi.org/10.1038/s41467-021-24776-4
O’Rourke JG, Bogdanik L, Yanez A, Lall D, Wolf AJ, Muhammad AK, Ho R, Carmona S, Vit JP, Zarrow J et al (2016) C9orf72 is required for proper macrophage and microglial function in mice. Science 351(6279):1324–1329. https://doi.org/10.1126/science.aaf1064
Atanasio A, Decman V, White D, Ramos M, Ikiz B, Lee HC, Siao CJ, Brydges S, Larosa E, Bai Y et al (2016) C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice. Sci Rep 6:23204. https://doi.org/10.1038/srep23204
Li CY, Yang TM, Ou RW, Wei QQ, Shang HF (2021) Genome-wide genetic links between amyotrophic lateral sclerosis and autoimmune diseases. BMC Med 19(1):27. https://doi.org/10.1186/s12916-021-01903-y
Burberry A, Suzuki N, Wang JY, Moccia R, Mordes DA, Stewart MH, Suzuki-Uematsu S, Ghosh S, Singh A, Merkle FT et al (2016) Loss-of-function mutations in the c9orf72 mouse ortholog cause fatal autoimmune disease. Sci Transl Med 8(347):347r–393r. https://doi.org/10.1126/scitranslmed.aaf6038
Joshi AU, Minhas PS, Liddelow SA, Haileselassie B, Andreasson KI, Dorn GN, Mochly-Rosen D (2019) Fragmented mitochondria released from microglia trigger a1 astrocytic response and propagate inflammatory neurodegeneration. Nat Neurosci 22(10):1635–1648. https://doi.org/10.1038/s41593-019-0486-0
Varcianna A, Myszczynska MA, Castelli LM, O’Neill B, Kim Y, Talbot J, Nyberg S, Nyamali I, Heath PR, Stopford MJ et al (2019) Micro-RNAs secreted through astrocyte-derived extracellular vesicles cause neuronal network degeneration in c9orf72 ALS. EBioMedicine 40:626–635. https://doi.org/10.1016/j.ebiom.2018.11.067
Shi Y, Lin S, Staats KA, Li Y, Chang WH, Hung ST, Hendricks E, Linares GR, Wang Y, Son EY et al (2018) Haploinsufficiency leads to neurodegeneration in c9orf72 ALS/FTD human induced motor neurons. Nat Med 24(3):313–325. https://doi.org/10.1038/nm.4490
Birger A, Ben-Dor I, Ottolenghi M, Turetsky T, Gil Y, Sweetat S, Perez L, Belzer V, Casden N, Steiner D et al (2019) Human iPSC-derived astrocytes from ALS patients with mutated c9orf72 show increased oxidative stress and neurotoxicity. EBioMedicine 50:274–289. https://doi.org/10.1016/j.ebiom.2019.11.026
Ziff OJ, Clarke BE, Taha DM, Crerar H, Luscombe NM, Patani R (2022) Meta-analysis of human and mouse ALS astrocytes reveals multi-omic signatures of inflammatory reactive states. Genome Res 32(1):71–84. https://doi.org/10.1101/gr.275939.121
Sommer D, Rajkumar S, Seidel M, Aly A, Ludolph A, Ho R, Boeckers TM, Catanese A (2022) Aging-dependent altered transcriptional programs underlie activity impairments in human c9orf72-mutant motor neurons. Front Mol Neurosci 15:894230. https://doi.org/10.3389/fnmol.2022.894230
Catanese A, Rajkumar S, Sommer D, Freisem D, Wirth A, Aly A, Massa-Lopez D, Olivieri A, Torelli F, Ioannidis V et al (2021) Synaptic disruption and creb-regulated transcription are restored by k(+) channel blockers in ALS. Embo Mol Med 13(7):e13131. https://doi.org/10.15252/emmm.202013131
Dafinca R, Barbagallo P, Farrimond L, Candalija A, Scaber J, Ababneh NA, Sathyaprakash C, Vowles J, Cowley SA, Talbot K (2020) Impairment of mitochondrial calcium buffering links mutations in C9ORF72 and TARDBP in iPS-Derived motor neurons from Patients with ALS/FTD. Stem Cell Reports 14(5):892–908. https://doi.org/10.1016/j.stemcr.2020.03.023
Abo-Rady M, Kalmbach N, Pal A, Schludi C, Janosch A, Richter T, Freitag P, Bickle M, Kahlert AK, Petri S et al (2020) Knocking out c9orf72 exacerbates axonal trafficking defects associated with hexanucleotide repeat expansion and reduces levels of heat shock proteins. Stem Cell Reports 14(3):390–405. https://doi.org/10.1016/j.stemcr.2020.01.010
Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L, Bennett ML, Munch AE, Chung WS, Peterson TC et al (2017) Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541(7638):481–487. https://doi.org/10.1038/nature21029
Balendra R, Isaacs AM (2018) C9orf72-mediated als and ftd: multiple pathways to disease. Nat Rev Neurol 14(9):544–558. https://doi.org/10.1038/s41582-018-0047-2
Koppers M, Blokhuis AM, Westeneng HJ, Terpstra ML, Zundel CA, Vieira DSR, Schellevis RD, Waite AJ, Blake DJ, Veldink JH et al (2015) C9orf72 ablation in mice does not cause motor neuron degeneration or motor deficits. Ann Neurol 78(3):426–438. https://doi.org/10.1002/ana.24453
Dong W, Ma Y, Guan F, Zhang X, Chen W, Zhang L, Zhang L (2021) Ablation of c9orf72 together with excitotoxicity induces ALS in rats. FEBS J 288(5):1712–1723. https://doi.org/10.1111/febs.15501
Boutin ME, Hampton C, Quinn R, Ferrer M, Song MJ (2019) 3d engineering of ocular tissues for disease modeling and drug testing. Adv Exp Med Biol 1186:171–193. https://doi.org/10.1007/978-3-030-2847-8-7
Yuva-Aydemir Y, Almeida S, Gao FB (2018) Insights into c9orf72-related ALS/FTD from drosophila and iPSC models. Trends Neurosci 41(7):457–469. https://doi.org/10.1016/j.tins.2018.04.002
Szebenyi K, Wenger L, Sun Y, Dunn A, Limegrover CA, Gibbons GM, Conci E, Paulsen O, Mierau SB, Balmus G et al (2021) Human ALS/FTD brain organoid slice cultures display distinct early astrocyte and targetable neuronal pathology. Nat Neurosci 24(11):1542–1554. https://doi.org/10.1038/s41593-021-00923-4
Seto Y, Eiraku M (2019) Toward the formation of neural circuits in human brain organoids. Curr Opin Cell Biol 61:86–91. https://doi.org/10.1016/j.ceb.2019.07.010
Qian, X, Song, H, Ming, GL (2019) Brain organoids: advances, applications and challenges. Development 146(8). https://doi.org/10.1242/dev.166074
Tang XY, Wu S, Wang D, Chu C, Hong Y, Tao M, Hu H, Xu M, Guo X, Liu Y (2022) Human organoids in basic research and clinical applications. Signal Transduct Target Ther 7(1):168. https://doi.org/10.1038/s41392-022-01024-9
Kofman S, Mohan N, Sun X, Ibric L, Piermarini E, Qiang L (2022) Human mini brains and spinal cords in a dish: modeling strategies, current challenges, and prospective advances. J Tissue Eng 13:1768603695. https://doi.org/10.1177/20417314221113391
Pinilla G, Kumar A, Floaters MK, Pardo CA, Rothstein J, Ilieva H (2021) Increased synthesis of pro-inflammatory cytokines in c9orf72 patients. Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration 22(7–8):517–527. https://doi.org/10.1080/21678421.2021.1912100
Leng K, Rose I, Kim H, Xia W, Romero-Fernandez W, Rooney B, Koontz M, Li E, Ao Y, Wang S et al (2022) CRISPRi screens in human iPSC-derived astrocytes elucidate regulators of distinct inflammatory reactive states. Nat Neurosci 25(11):1528–1542. https://doi.org/10.1038/s41593-022-01180-9
Kim H, Leng K, Park J, Sorets AG, Kim S, Shostak A, Embalabala RJ, Mlouk K, Katdare KA, Rose I et al (2022) Reactive astrocytes transduce inflammation in a blood-brain barrier model through a tnf-stat3 signaling axis and secretion of alpha 1-antichymotrypsin. Nat Commun 13(1):6581. https://doi.org/10.1038/s41467-022-34412-4
Bottero V, Alrafati F, Santiago JA, Potashkin JA (2021) Transcriptomic and network meta-analysis of frontotemporal dementias. Front Mol Neurosci 14:747798. https://doi.org/10.3389/fnmol.2021.747798
Dickson DW, Baker MC, Jackson JL, Dejesus-Hernandez M, Finch NA, Tian S, Heckman MG, Pottier C, Gendron TF, Murray ME et al (2019) Extensive transcriptomic study emphasizes importance of vesicular transport in c9orf72 expansion carriers. Acta Neuropathol Commun 7(1):150. https://doi.org/10.1186/s40478-019-0797-0
Namboori SC, Thomas P, Ames R, Hawkins S, Garrett LO, Willis C, Rosa A, Stanton LW, Bhinge A (2021) Single-cell transcriptomics identifies master regulators of neurodegeneration in SOD1 ALS iPSC-derived motor neurons. Stem Cell Reports 16(12):3020–3035. https://doi.org/10.1016/j.stemcr.2021.10.010
Shao Q, Liang C, Chang Q, Zhang W, Yang M, Chen JF (2019) C9orf72 deficiency promotes motor deficits of a C9 ALS/FTD mouse model in a dose-dependent manner. Acta Neuropathol Commun 7(1):32. https://doi.org/10.1186/s40478-019-0685-7
Masrori P, Beckers J, Gossye H, Van Damme P (2022) The role of inflammation in neurodegeneration: novel insights into the role of the immune system in c9orf72 hre-mediated ALS/FTD. Mol Neurodegener 17(1):22. https://doi.org/10.1186/s13024-022-00525-z
Guttenplan KA, Weigel MK, Adler DI, Couthouis J, Liddelow SA, Gitler AD, Barres BA (2020) Knockout of reactive astrocyte activating factors slows disease progression in an ALS mouse model. Nat Commun 11(1):3753. https://doi.org/10.1038/s41467-020-17514-9
Renner, H, Grabos, M, Becker, KJ, Kagermeier, TE, Wu, J, Otto, M, Peischard, S, Zeuschner, D, Tsytsyura, Y, Disse, P et al. (2020) A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids. Elife 9. https://doi.org/10.7554/eLife.52904
Zhong S, Zhang S, Fan X, Wu Q, Yan L, Dong J, Zhang H, Li L, Sun L, Pan N et al (2018) A single-cell RNA-seq survey of the developmental landscape of the human prefrontal cortex. Nature 555(7697):524–528. https://doi.org/10.1038/nature25980
Revah O, Gore F, Kelley KW, Andersen J, Sakai N, Chen X, Li MY, Birey F, Yang X, Saw NL et al (2022) Maturation and circuit integration of transplanted human cortical organoids. Nature 610(7931):319–326. https://doi.org/10.1038/s41586-022-05277-w
Acknowledgements
This work was supported by Hebei Medical University, and funded by Natural Science Foundation of China (81801278), China Scholarship Council (201608130015), Natural Science Foundation of Hebei Province (H2019206637), Key Natural Science Foundation of Hebei Province (H2020206557), Overseas researcher Program in Hebei Provincial Department of human resources and social security (C20190509), Natural Science Foundation of Hebei Province (H2015206409), Natural Science Foundation of Hebei Province (H2023206266).
Funding
Funded by Natural Science Foundation of China (81801278), China Scholarship Council (201608130015), Natural Science Foundation of Hebei Province (H2019206637), Key Natural Science Foundation of Hebei Province (H2020206557), Overseas researcher Program in Hebei Provincial Department of human resources and social security (C20190509), Natural Science Foundation of Hebei Province (H2015206409), Natural Science Foundation of Hebei Province (H2023206266).
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JM and HXC conceived and designed the study. RYG conducted the research and investigation process, including performed the experiments and YMC performed the GEO data collection and analysis. JYZ collected the references and extracted and cross-checked the data. RYG and JM wrote the first draft of the manuscript. JM, HXC and RYG revised and discussed the final edition. All authors have read and approved the final manuscript.
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a Hebei Medical University-National University of Ireland Galway Stem Cell Research Center, Hebei Medical University, Shijiazhuang, Hebei Province 050017, China.
b Hebei Research Center for Stem Cell Medical Translational Engineering, Shijiazhuang, Hebei Province 050017, China.
c Human Anatomy Department, Hebei Medical University, Shijiazhuang, Hebei Province 050017, China.
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Guo, R., Chen, Y., Zhang, J. et al. Neural Differentiation and spinal cord organoid generation from induced pluripotent stem cells (iPSCs) for ALS modelling and inflammatory screening. Mol Neurobiol (2023). https://doi.org/10.1007/s12035-023-03836-4
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DOI: https://doi.org/10.1007/s12035-023-03836-4