“Generation of two Betacellulin CRISPR-Cas9 knockout hiPSC lines to study affected EGF system paradigm in Schizophrenia”

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Hill , Suresh Sundram doi: https://doi.org/10.1101/2025.04.07.647671 Agustin Cota-Coronado a Department of Psychiatry, School of Clinical Sciences, Monash University , Clayton, Victoria, 3168, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: agustincota.cotacoronado{at}monash.edu Murray Manning b Cancer Research Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University , Clayton, VIC, Australia c Functional Genomics Platform, Monash University , Clayton, VIC, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dong-Hyung Kim a Department of Psychiatry, School of Clinical Sciences, Monash University , Clayton, Victoria, 3168, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Joohyung Lee a Department of Psychiatry, School of Clinical Sciences, Monash University , Clayton, Victoria, 3168, Australia d Centre for Endocrinology and Metabolism, Hudson Institute of Medical Research , Clayton, Victoria, 3168, Australia e Department of Anatomy and Developmental Biology, Monash University , Clayton, Victoria, 3168, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Andrew Gibbons a Department of Psychiatry, School of Clinical Sciences, Monash University , Clayton, Victoria, 3168, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Joseph Rosenbluh b Cancer Research Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University , Clayton, VIC, Australia c Functional Genomics Platform, Monash University , Clayton, VIC, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rachel A. Hill a Department of Psychiatry, School of Clinical Sciences, Monash University , Clayton, Victoria, 3168, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Suresh Sundram a Department of Psychiatry, School of Clinical Sciences, Monash University , Clayton, Victoria, 3168, Australia f Mental Health Program, Monash Health , Clayton, Victoria, 3168, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Several members of the epidermal growth factor (EGF) family have been implicated in the biology of schizophrenia ( Ketharanathan et al., 2024 ). The EGF-related ligand, Betacellulin (BTC), plays an important role in the proliferation and differentiation of neural stem cells and our group found markedly reduced BTC levels in patients with schizophrenia. Nevertheless, the interplay of affected BTC and its participation in neural specification and neurodevelopment remains elusive. We generated Knockout (KO) - BTC clones from an existing hiPSC line through CRISPR/Cas9-mediated modification. Furthermore, we validated BTC-KO through genotyping/sequencing, FACS and Western Blot. Finally, we demonstrated trilineage differentiation potential in vitro . 1. Resource Table View this table: View inline View popup 2. Resource utility Epidermal growth factor (EGF) is implicated in the pathogenesis of schizophrenia. We identified low Betacellulin concentrations (EGF-related ligand) in people with schizophrenia. We generated novel cell lines to understand the role of BTC in neurodevelopment and the implications of low or absent BTC in Schizophrenia and related psychiatric disorders. 3. Resource Details Schizophrenia is a psychiatric syndrome that is currently diagnosed solely by its clinical features of positive symptoms such as delusions and hallucinations, negative symptoms such as decreased motivation and anhedonia (reduction of pleasure), and cognitive symptoms such as decreased attention and impaired executive function. It has no laboratory or imaging diagnostics, no defining pathology and no known causes or cure. Symptoms and impacts vary widely between individuals with the disorder. Most current treatments are only effective in targeting the psychotic symptoms of schizophrenia with a substantial proportion of people being refractory to treatment. Given that antenatal, early life and adolescent staged environmental factors and high heritability are associated with this adult-onset disorder, neurodevelopmental processes are strongly implicated. Epidermal growth factor (EGF) has been proposed as an important effector molecule in schizophrenia ( Ketharanathan et al., 2024 ; Mostaid et al., 2017 ; Sotoyama et al., 2013 ). Additionally, betacellulin (BTC), another ligand member of the EGF family that is capable of signalling through EGF’s receptor, induces neural stem cell (NSC) proliferation in vivo and prevents spontaneous differentiation in culture. Furthermore, it has been shown that in vivo infusion in the mouse induced the expansion of neural stem cells and neuroblasts, and promoted neurogenesis in the olfactory bulb and the dentate gyrus ( Gómez-Gaviro et al., 2012 ). Our group recently showed reduced levels of BTC in post-mortem brain tissue from patients with Schizophrenia compared with healthy controls (manuscript in preparation). However, as post-mortem human brain studies do not allow causative determination, we generated these novel resources to understand the role of BTC in early specification and neurodevelopment. We selected the well-characterized hiPSC line KOLF2.1J ( Pantazis et al., 2022 ) and introduced a Cas9-GFP protein through lipofectamine and subsequently two sgRNA directed to the exon 3 in the betacellulin gene. The transfected cells were selected through FACS-sorting for GFP-high cells and performed single-cell deposition. A total of 124 clones were genotyped, of those, 11 clones showed a band in the KO band. Out of those 11 clones, 1 clone did not have a WT band; 2 clones were expanded and sequenced. Subsequently, two clones were genotyped MONPSYi001-A-2-BTC-KO (Hom) was negative for WT PCR, indicating a full KO. Whereas cell line MONPSYi001-A-1-BTC-KO showed a partial deletion of 6 amino acids in the sgRNA1A site, making it a heterozygous clone, compared with the WT line MONPSYi001-A. The colonies exhibited typical pluripotent stem cell morphology ( Fig 1C ) under brightfield light. Then, spontaneously differentiated the 3 cell lines and they all showed robust expression of markers belonging to the three germ layers including ectoderm: Nestin and PAX6 (>95% positives) at Day 10 of neural differentiation, mesoderm: α-Smooth Muscle Actin and Brachyury (>90% positives) and endoderm: SOX17 (>90% positives). Furthermore, cells showed reduced BTC at the protein level and were Mycoplasma negative, validated with nested PCR (Supplementary Figure 1). To our knowledge, these are the first hiPSC lines that can test the low BTC paradigm schizophrenia representing valuable resources for disease modelling and drug screening based on clinical data. Download figure Open in new tab 4. Materials and Methods 4.1 hiPSC culturing and passaging KOLF2.1J human induced pluripotent stem cell line (The Jackson Laboratory, cat # JIPSC1000) and isogenic clones derived from this line were grown in mTSER Plus feeder-free media (STEMCELL, cat # 85850). 10uM Rock inhibitor (STEMCELL, Cat # 72304) was added to mTSER Plus media for 18–24 h after splitting and thawing. 4.2 CRISPR-Cas9 knockouts KOLF2.1J line was transfected with a high-fidelity Cas9-eGFP using Lipofectamine STEM (Thermo Fisher, cat # STEM00001). A Recombinant S. pyogenes Cas9 nuclease, purified from an E. coli strain expressing the nuclease. It holds a nuclear localisation sequence (NLS) and C-terminal 6-His tag at 10 µg/µL. Combined Solution I: 25 UG Cas9-GFP, 3.2µg sgRNA1A, 3.2µg sgRNA2and 500µl OPTI-MEM was used. Solution II: 40µl Lipofectamine STEM and 500µl OPTI-MEM were incubated at RT for 10min, then added to a 2 x T25 flask with ∼70% confluent cells 2 days after transfections the cells were FACS sorted for GFP high cells into 12x 96-well plates (single cell deposition). Bulk cells were collected for gDNA extraction and PCR. 4.3 Genotyping KO PCR products were sequenced to verify the exact KO mutations for the transfected 2 clones. The cell line MONPSYi001-A-2-BTC-KO (Homozygous) was negative for WT PCR, indicating a full KO. Whereas cell line MONPSYi001-A-1-BTC-KO showed a partial deletion of 6 amino acids in the sgRNA1A site, making it a heterozygous clone. 4.3 Western Blot 20 µg of protein was run on either 8 or 12% acrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane using the Trans-Blot Turbo RTA Transfer Kit (Bio-Rad, Cat # 1704274). Primary antibodies raised against BTC (1:500, Invitrogen, Cat # PA5-102636), Cas9 (1:500, Invitrogen, Cat # MA1-201), β-actin (1:1000, Sigma-Aldrich, Cat # A5316), were applied overnight at 4°C and labelled with AlexaFluor488 anti-mouse (1:1000, Invitrogen, Cat # A21202) or AlexaFluor647 anti-rabbit (1:1000, Invitrogen, Cat # A31573) secondary antibodies for 1h at room temperature. PVDF membranes were scanned and imaged using the ChemicDoc MP Imaging System (Bio-Rad). BTC was normalized against the internal control, β-actin, and Cas9 were normalized against the internal control, β-tubulin. Blot density was analyzed using the Fiji-ImageJ v1.54f software with the gel analysis routine method. 4.4 Mycoplasma detection Two independent nested PCR-based protocols (I/II) were performed for detecting Mycoplasma/Acholeplasma species. Two positive controls: +ve1 and +ve2 are shown as Mycoplasma positive samples, giving the expected products at 219-426bp (protocol I) and 165-355bp (protocol II). None of the new cell lines gave a positive band, indicating them as mycoplasma-free. 4.5 Pluripotency assessment iPSC clones were grown on laminin-521 coated Ibidi 8-well plates (cat# fixed with 4% PFA, and stained with pluripotency markers listed in Table 3. Images were taken on a Zeiss 880 confocal microscope. The percentage of cells positive for OCT3/4 and NANOG was calculated from immunofluorescence images using the Fiji software, compared with total DAPI-positive cells. 4.6 Differentiation potential iPSC clones were differentiated into three layers according to STEMdiff™ Trilineage Differentiation Kit (StemCell, Cat# 05230), fixed in 4% PFA, and stained with differentiation markers listed in Table 3. Images were taken on a Nikon C1 confocal microscope at 20X magnification ( Fig 1F ). View this table: View inline View popup Table 1: Characterization and validation View this table: View inline View popup Table 2: Reagents details Footnotes ↵ 1 Dr Cota-Coronado and Professor Sundram jointly supervised this work References ↵ Gómez-Gaviro , M.V. , Scott , C.E. , Sesay , A.K. , Matheu , A. , Booth , S. , Galichet , C. , Lovell-Badge , R. , 2012 . Betacellulin promotes cell proliferation in the neural stem cell niche and stimulates neurogenesis . Proc. Natl. Acad. Sci . 109 , 1317 – 1322 . doi: 10.1073/pnas.1016199109 OpenUrl Abstract / FREE Full Text ↵ Ketharanathan , T. , Pereira , A. , Sundram , S. , 2024 . Gene expression changes in Brodmann’s Area 46 differentiate epidermal growth factor and immune system interactions in schizophrenia and mood disorders . Schizophrenia 10 , 76 . doi: 10.1038/s41537-024-00488-8 OpenUrl CrossRef ↵ Mostaid , M.S. , Lee , T.T. , Chana , G. , Sundram , S. , Shannon Weickert , C. , Pantelis , C. , Everall , I. , Bousman , C. , 2017 . Peripheral Transcription of NRG-ErbB Pathway Genes Are Upregulated in Treatment-Resistant Schizophrenia . Front. Psychiatry 8 , 225 . doi: 10.3389/fpsyt.2017.00225 OpenUrl CrossRef PubMed ↵ Pantazis , C.B. , Yang , A. , Lara , E. , McDonough , J.A. , Blauwendraat , C. , Peng , L. , Oguro , H. , Kanaujiya , J. , Zou , J. , Sebesta , D. , Pratt , G. , Cross , E. , Blockwick , J. , Buxton , P. , Kinner-Bibeau , L. , Medura , C. , Tompkins , C. , Hughes , S. , Santiana , M. , Faghri , F. , Nalls , M.A. , Vitale , D. , Ballard , S. , Qi , Y.A. , Ramos , D.M. , Anderson , K.M. , Stadler , J. , Narayan , P. , Papademetriou , J. , Reilly , L. , Nelson , M.P. , Aggarwal , S. , Rosen , L.U. , Kirwan , P. , Pisupati , V. , Coon , S.L. , Scholz , S.W. , Priebe , T. , Öttl , M. , Dong , J. , Meijer , M. , Janssen , L.J.M. , Lourenco , V.S. , Van Der Kant , R. , Crusius , D. , Paquet , D. , Raulin , A.-C. , Bu , G. , Held , A. , Wainger , B.J. , Gabriele , R.M.C. , Casey , J.M. , Wray , S. , Abu-Bonsrah , D. , Parish , C.L. , Beccari , M.S. , Cleveland , D.W. , Li , E. , Rose , I.V.L. , Kampmann , M. , Calatayud Aristoy , C. , Verstreken , P. , Heinrich , L. , Chen , M.Y. , Schüle , B. , Dou , D. , Holzbaur , E.L.F. , Zanellati , M.C. , Basundra , R. , Deshmukh , M. , Cohen , S. , Khanna , R. , Raman , M. , Nevin , Z.S. , Matia , M. , Van Lent , J. , Timmerman , V. , Conklin , B.R. , Johnson Chase , K. , Zhang , K. , Funes , S. , Bosco , D.A. , Erlebach , L. , Welzer , M. , Kronenberg-Versteeg , D. , Lyu , G. , Arenas , E. , Coccia , E. , Sarrafha , L. , Ahfeldt , T. , Marioni , J.C. , Skarnes , W.C. , Cookson , M.R. , Ward , M.E. , Merkle , F.T. , 2022 . A reference human induced pluripotent stem cell line for large-scale collaborative studies . Cell Stem Cell 29 , 1685 - 1702.e22 . doi: 10.1016/j.stem.2022.11.004 OpenUrl CrossRef PubMed ↵ Sotoyama , H. , Namba , H. , Chiken , S. , Nambu , A. , Nawa , H. , 2013 . Exposure to the cytokine EGF leads to abnormal hyperactivity of pallidal GABA neurons: implications for schizophrenia and its modeling . J. Neurochem . 126 , 518 – 528 . doi: 10.1111/jnc.12223 OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted April 09, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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