HBEGF-TNF induce a complex outer retinal pathology with photoreceptor cell extrusion in human organoids

Abstract

Human organoids could facilitate research of complex and currently incurable neuropathologies, such as age-related macular degeneration (AMD) which causes blindness. Here, we establish a human retinal organoid system reproducing several parameters of the human retina, including some within the macula, to model a complex combination of photoreceptor and glial pathologies. We show that combined application of TNF and HBEGF, factors associated with neuropathologies, is sufficient to induce photoreceptor degeneration, glial pathologies, dyslamination, and scar formation: These develop simultaneously and progressively as one complex phenotype. Histologic, transcriptome, live-imaging, and mechanistic studies reveal a previously unknown pathomechanism: Photoreceptor neurodegeneration via cell extrusion. This could be relevant for aging, AMD, and some inherited diseases. Pharmacological inhibitors of the mechanosensor PIEZO1, MAPK, and actomyosin each avert pathogenesis; a PIEZO1 activator induces photoreceptor extrusion. Our model offers mechanistic insights, hypotheses for neuropathologies, and it could be used to develop therapies to prevent vision loss or to regenerate the retina in patients suffering from AMD and other diseases.

The growth factor EPIREGULIN promotes basal progenitor cell proliferation in the developing neocortex

“…# 18265017 ElectroMAX Stbl4 Competent Cells Thermo Fisher Cat. # 11635018 GW 12/13 human fetal brain tissue This study N/A Human: CRTDi004-A iPSC (Völkner et al, 2022 ) https://hpscreg.eu/cell-line/CRTDi004-A Human: HPSI0114i-kolf_2 iPSC Welcome Trust Sanger Institute (HipSci) RRID:CVCL_AE29 Gorilla: GC1 iPSC (Wunderlich et al, 2014 ) N/A Mouse: NSC This paper N/A Mouse: C57BL/6 J Charles River RRID:IMSR; JAX:000664 Mouse: Tubb3 ::GFP (Attardo et al, 2008 ) N/A Recombinant DNA pCAG-GFP (Florio et al, 2015 ) N/A pCas9-JMJC_6B-T2A-EGFP-gRNAs This paper N/A pCAG-Ereg This paper N/A Candidate cis-regulatory elements This paper Table EV 3 Antibodies Goat anti-Sox2 (1:200) R&D Systems AF2018 RRID:AB_355110 Rabbit anti-Ki67 (1:500) Abcam ab15580 RRID:AB_443209 Mouse anti-PCNA (1:500) Millipore CBL407 RRID:AB_93501 Mouse anti-PhVim (1:200) Abcam ab22651 RRID:AB_447222 Rat anti-PH3 (1:500) Abcam ab10543 RRID:AB_2295065 Rat anti-Ctip2 (1:250) Abcam ab18465 RRID:AB_2064130 Rabbit anti-TBR2 (1:250) Abcam ab23345 RRID:AB_778267 Chicken anti-GFP (1:2,000)_ Abcam ab13970 RRID:AB_300798 Rat anti-RFP (1:500) proteintech RRID:AB_2336064 ...…”

“…All experiments involving hiPSCs were performed in accordance with the ethical standards of the institutional and/or national research committee, as well as with the 1964 Helsinki Declaration, and approved by the University Hospital Ethical Review Committee (IRB00001473; IORG0001076; ethical approval number SR-EK-456092021). Human cortical organoids were generated using the previously generated human induced pluripotent stem cell (iPSC) lines CRTDi004-A (Völkner et al, 2022 ) and HPSI0114i-kolf_2 (Welcome Trust Sanger Institute, HipSci), derived from healthy donors. The human iPSC lines were maintained on Matrigel-coated (Corning, 354277) culture dishes in mTeSR1 and passaged using TrypLE Express enzyme (CRTDi004-A) or ReLeSR (HPSI0114i-kolf_2) (Schütze et al, 2023 ).…”

Compartmentalization and synergy of osteoblasts drive bone formation in the regenerating fin

“…Samples were further dissected for embedding into epoxy resin and processed according to a modified protocol for serial block face SEM 67 using osmium tetroxide (OsO 4 ), thiocarbohydrazide (TCH), and again OsO 4 to generate enhanced membrane contrast. 68 , 69 In brief, samples were postfixed overnight in modified Karnovsky fixative (2% glutaraldehyde/2% formaldehyde in 50 mM HEPES, pH 7.4), followed by post-fixation in a 2% aqueous OsO 4 solution containing 1.5% potassium ferrocyanide and 2mM CaCl 2 (30 minutes on ice), washes in water, 1% TCH in water (20 minutes at room temperature), washes in water and a second osmium contrasting step in 2% OsO 4 /water (30 minutes on ice). Samples were washed and en-bloc contrasted with 1% uranyl acetate/water for 2 h on ice, washed in water, and dehydrated in a graded series of ethanol/water mixtures (30%, 50%, 70%, 90%, 96%), followed by 3 changes in pure ethanol on molecular sieve.…”

Pleiotropic physiological functions of Piezo1 in human body and its effect on malignant behavior of tumors

“…This requires a thorough understanding of the specific biological function of Piezo1 and the development of potential pharmacokinetics, or antagonists, with good drug-like properties. In summary, the drug design of Piezo1 faces several challenges and limitations that will require further research and efforts to address ( Völkner et al, 2022 ).…”