{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T02:20:31Z","timestamp":1778638831046,"version":"3.51.4"},"reference-count":70,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2025,11,5]],"date-time":"2025-11-05T00:00:00Z","timestamp":1762300800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2025,11,5]],"date-time":"2025-11-05T00:00:00Z","timestamp":1762300800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"publisher","award":["RS-2025-00518006"],"award-info":[{"award-number":["RS-2025-00518006"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003052","name":"Ministry of Trade, Industry and Energy","doi-asserted-by":"publisher","award":["20023068"],"award-info":[{"award-number":["20023068"]}],"id":[{"id":"10.13039\/501100003052","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100014189","name":"Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry","doi-asserted-by":"publisher","award":["RS-2024-00398561"],"award-info":[{"award-number":["RS-2024-00398561"]}],"id":[{"id":"10.13039\/501100014189","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Stem Cell Res Ther"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>. Porcine pluripotent stem cells hold significant promise as a large-animal model for human neurodevelopment and disease modeling. However, efficient protocols for their directed differentiation into midbrain dopaminergic (mDA) neurons and the generation of 3D midbrain-like organoids remain limited. This study aimed to establish species-optimized conditions for the derivation of functional mDA neurons from porcine embryonic stem cells (pESCs) under both two- and three-dimensional environments.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>. In vitro fertilization (IVF)- and parthenogenetic activation (PA)-derived pESCs were subjected to neural induction using stepwise exposure to dual SMAD inhibition, SHH, CHIR99021, and FGF8. For monolayer culture, adherent monolayers were differentiated on Matrigel- or poly-L-ornithine\/laminin I\/fibronectin-coated surfaces. For 3D culture, porcine midbrain-like organoids (pMLOs) were formed under low-adhesion conditions. Functional and molecular characterization was performed via immunofluorescence, patch-clamp electrophysiology, microelectrode array (MEA) recordings, dopamine ELISA, and RNA sequencing.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>. Porcine embryonic stem cells required higher SHH and GSK3 inhibition thresholds to efficiently induce FOXA2\u207a ventral midbrain progenitors, revealing a species-specific divergence from human protocols. IVF-derived pESCs showed markedly enhanced dopaminergic differentiation and functional maturation compared to PA-derived pESCs. Notably, in 3D culture, neuroepithelial structures emerged as early as day 5, and functionally mature mDA neurons\u2014confirmed by TH expression, dopamine release, and spontaneous synaptic activity\u2014were detected by day 28. This timeline represents an accelerated maturation relative to comparable human mDA differentiation systems, where functional properties typically arise after day 35. Single-cell RNA sequencing further delineated dynamic dopaminergic lineage trajectories and revealed porcine-specific gene expression patterns associated with early mDA identity acquisition.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>. This study presents the first robust platform for generating functionally validated mDA neurons and midbrain-like organoids from porcine stem cells. The findings highlight species-specific signaling dynamics and establish porcine in vitro models as scalable, translational systems for investigating neurodevelopment and disease mechanisms.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s13287-025-04693-9","type":"journal-article","created":{"date-parts":[[2025,11,5]],"date-time":"2025-11-05T12:31:40Z","timestamp":1762345900000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["In vitro derivation of midbrain dopaminergic neurons from porcine embryonic stem cells in multi-dimensional conditions"],"prefix":"10.1186","volume":"16","author":[{"given":"Hyerin","family":"Choi","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dongjin","family":"Oh","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ali","family":"Jawad","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zheng","family":"Haomiao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jaehyung","family":"Ham","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juyoung","family":"Heo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aram","family":"Oh","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Huijin","family":"Jin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jaehyeok","family":"Seo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Byoung Chol","family":"Oh","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sang-Hwan","family":"Hyun","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2025,11,5]]},"reference":[{"key":"4693_CR1","doi-asserted-by":"crossref","unstructured":"Pain B, Baquerre C, Coulpier M. Cerebral organoids and their potential for studies of brain diseases in domestic animals. Vet Res. 2021;52(1):65.","DOI":"10.1186\/s13567-021-00931-z"},{"issue":"2","key":"4693_CR2","doi-asserted-by":"publisher","first-page":"248","DOI":"10.1016\/j.neuron.2015.12.008","volume":"89","author":"JC Silbereis","year":"2016","unstructured":"Silbereis JC, Pochareddy S, Zhu Y, Li MF, Sestan N. The cellular and molecular landscapes of the developing human central nervous system. Neuron. 2016;89(2):248\u201368.","journal-title":"Neuron"},{"key":"4693_CR3","doi-asserted-by":"crossref","unstructured":"Beauchamp A, Yee Y, Darwin BC, Raznahan A, Mars RB, Lerch JP. Whole-brain comparison of rodent and human brains using Spatial transcriptomics. Elife. 2022;11:e79418.","DOI":"10.7554\/eLife.79418"},{"issue":"4","key":"4693_CR4","doi-asserted-by":"publisher","first-page":"683","DOI":"10.1016\/j.neuron.2014.12.060","volume":"85","author":"C Dehay","year":"2015","unstructured":"Dehay C, Kennedy H, Kosik KS. The outer subventricular zone and Primate-Specific cortical complexification. Neuron. 2015;85(4):683\u201394.","journal-title":"Neuron"},{"key":"4693_CR5","doi-asserted-by":"crossref","unstructured":"Zhang RR, Quan HX, Wang YF, Luo FC. Neurogenesis in primates versus rodents and the value of non-human primate models. Natl Sci Rev. 2023;10(11):nwad248.","DOI":"10.1093\/nsr\/nwad248"},{"issue":"2","key":"4693_CR6","doi-asserted-by":"publisher","first-page":"179","DOI":"10.1038\/pr.2011.21","volume":"71","author":"MS Conrad","year":"2012","unstructured":"Conrad MS, Dilger RN, Nickolls A, Johnson RW. Magnetic resonance imaging of the neonatal piglet brain. Pediatr Res. 2012;71(2):179\u201384.","journal-title":"Pediatr Res"},{"key":"4693_CR7","doi-asserted-by":"crossref","unstructured":"Conrad MS, Sutton BP, Dilger RN, Johnson RW. An in vivo Three-Dimensional magnetic resonance Imaging-Based averaged brain collection of the neonatal piglet (Sus scrofa). PLoS ONE. 2014;9(9):e107650.","DOI":"10.1371\/journal.pone.0107650"},{"issue":"4","key":"4693_CR8","doi-asserted-by":"publisher","first-page":"291","DOI":"10.1159\/000339311","volume":"34","author":"MS Conrad","year":"2012","unstructured":"Conrad MS, Dilger RN, Johnson RW. Brain growth of the domestic pig (Sus scrofa) from 2 to 24 weeks of age: A longitudinal MRI study. Dev Neurosci. 2012;34(4):291\u20138.","journal-title":"Dev Neurosci"},{"issue":"1","key":"4693_CR9","doi-asserted-by":"publisher","first-page":"25","DOI":"10.1038\/s41536-021-00135-1","volume":"17","author":"J Thomas","year":"2021","unstructured":"Thomas J, Zimmerlin L, Huo JS, Considine M, Cope L, Zambidis ET. Running the full human developmental clock in interspecies chimeras using alternative human stem cells with expanded embryonic potential. NPJ Regen Med. 2021;17(1):25.","journal-title":"NPJ Regen Med"},{"key":"4693_CR10","doi-asserted-by":"crossref","unstructured":"Wang S, Li T, Zhou TT, Pu LL, Wang HY, Yin XX, Hao XQ, Ren L, Liang ZH. Survival in patients with Parkinson\u2019s disease: a ten-year follow-up study in northern China. Bmc Neurol. 2022;22(1):367.","DOI":"10.1186\/s12883-022-02899-5"},{"issue":"13","key":"4693_CR11","doi-asserted-by":"publisher","first-page":"1615","DOI":"10.1002\/mds.25898","volume":"29","author":"AD Macleod","year":"2014","unstructured":"Macleod AD, Taylor KSM, Counsell CE. Mortality in parkinson\u2019s disease: A systematic review and Meta-analysis. Mov Disord. 2014;29(13):1615\u201322.","journal-title":"Mov Disord"},{"issue":"9","key":"4693_CR12","doi-asserted-by":"publisher","first-page":"492","DOI":"10.1038\/nrneurol.2015.123","volume":"11","author":"RA Barker","year":"2015","unstructured":"Barker RA, Drouin-Ouellet J, Parmar M. Cell-based therapies for Parkinson disease-past insights and future potential. Nat Reviews Neurol. 2015;11(9):492\u2013503.","journal-title":"Nat Reviews Neurol"},{"key":"4693_CR13","unstructured":"Sonne J, Reddy V, Beato MR. Neuroanatomy, substantia Nigra. In: StatPearls. Treasure Island (FL); 2023."},{"issue":"2","key":"4693_CR14","doi-asserted-by":"publisher","first-page":"267","DOI":"10.1002\/path.4654","volume":"238","author":"IE Holm","year":"2016","unstructured":"Holm IE, Alstrup AK, Luo Y. Genetically modified pig models for neurodegenerative disorders. J Pathol. 2016;238(2):267\u201387.","journal-title":"J Pathol"},{"issue":"4\u20135","key":"4693_CR15","doi-asserted-by":"publisher","first-page":"481","DOI":"10.1007\/s00429-009-0217-5","volume":"213","author":"MS Nielsen","year":"2009","unstructured":"Nielsen MS, Sorensen JC, Bjarkam CR. The substantia Nigra Pars compacta of the Gottingen minipig: an anatomical and Stereological study. Brain Struct Funct. 2009;213(4\u20135):481\u20138.","journal-title":"Brain Struct Funct"},{"issue":"2","key":"4693_CR16","doi-asserted-by":"publisher","first-page":"149","DOI":"10.1002\/cne.903220202","volume":"322","author":"K Ostergaard","year":"1992","unstructured":"Ostergaard K, Holm IE, Zimmer J. Tyrosine hydroxylase and acetylcholinesterase in the domestic pig mesencephalon: an immunocytochemical and histochemical study. J Comp Neurol. 1992;322(2):149\u201366.","journal-title":"J Comp Neurol"},{"key":"4693_CR17","doi-asserted-by":"crossref","unstructured":"Wang XL, Cao CW, Huang JJ, Yao J, Hai T, Zheng QT, Wang X, Zhang HY, Qin GS, Cheng JB, et al. One-step generation of triple gene-targeted pigs using CRISPR\/Cas9 system. Sci Rep-Uk. 2016;6:20620.","DOI":"10.1038\/srep20620"},{"issue":"3","key":"4693_CR18","doi-asserted-by":"publisher","first-page":"402","DOI":"10.1093\/nsr\/nwz013","volume":"6","author":"JG Zhao","year":"2019","unstructured":"Zhao JG, Lai LX, Ji WZ, Zhou Q. Genome editing in large animals: current status and future prospects. Natl Sci Rev. 2019;6(3):402\u201320.","journal-title":"Natl Sci Rev"},{"issue":"6","key":"4693_CR19","doi-asserted-by":"publisher","first-page":"1175","DOI":"10.1007\/s00018-014-1744-7","volume":"72","author":"XQ Zhou","year":"2015","unstructured":"Zhou XQ, Xin JG, Fan NN, Zou QJ, Huang J, Ouyang Z, Zhao Y, Zhao BT, Liu ZM, Lai SS, et al. Generation of CRISPR\/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer. Cell Mol Life Sci. 2015;72(6):1175\u201384.","journal-title":"Cell Mol Life Sci"},{"issue":"18","key":"4693_CR20","doi-asserted-by":"publisher","first-page":"3113","DOI":"10.1242\/dev.118570","volume":"142","author":"M Huch","year":"2015","unstructured":"Huch M, Koo BK. Modeling mouse and human development using organoid cultures. Development. 2015;142(18):3113\u201325.","journal-title":"Development"},{"issue":"10","key":"4693_CR21","doi-asserted-by":"publisher","first-page":"2329","DOI":"10.1038\/nprot.2014.158","volume":"9","author":"MA Lancaster","year":"2014","unstructured":"Lancaster MA, Knoblich JA. Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc. 2014;9(10):2329\u201340.","journal-title":"Nat Protoc"},{"issue":"1","key":"4693_CR22","doi-asserted-by":"publisher","first-page":"31","DOI":"10.1083\/jcb.201610056","volume":"216","author":"M Simian","year":"2017","unstructured":"Simian M, Bissell MJ. Organoids: A historical perspective of thinking in three dimensions. J Cell Biol. 2017;216(1):31\u201340.","journal-title":"J Cell Biol"},{"key":"4693_CR23","doi-asserted-by":"crossref","unstructured":"Reis RL. 2nd Consensus conference on definitions on biomaterials science. J Tissue Eng Regen Med. 2020, 14(4):561\u2013562.","DOI":"10.1002\/term.3016"},{"issue":"3","key":"4693_CR24","doi-asserted-by":"publisher","first-page":"246","DOI":"10.1038\/ncb3312","volume":"18","author":"A Fatehullah","year":"2016","unstructured":"Fatehullah A, Tan SH, Barker N. Organoids as an model of human development and disease. Nat Cell Biol. 2016;18(3):246\u201354.","journal-title":"Nat Cell Biol"},{"issue":"3","key":"4693_CR25","doi-asserted-by":"publisher","first-page":"2052","DOI":"10.1111\/jcmm.14111","volume":"23","author":"E Kim","year":"2019","unstructured":"Kim E, Kim M, Hwang SU, Kim J, Lee G, Park YS, Hyun SH. Neural induction of porcine-induced pluripotent stem cells and further differentiation using glioblastoma-cultured medium. J Cell Mol Med. 2019;23(3):2052\u201363.","journal-title":"J Cell Mol Med"},{"issue":"12","key":"4693_CR26","doi-asserted-by":"publisher","first-page":"1880","DOI":"10.1002\/term.3143","volume":"14","author":"LS Machado","year":"2020","unstructured":"Machado LS, Pieri NCG, Botigelli RC, de Castro RVG, de Souza AF, Bridi A, Lima MA, Neto PF, Pess\u00f4a LVD, Martins SMMK, et al. Generation of neural progenitor cells from porcine-induced pluripotent stem cells. J Tissue Eng Regen M. 2020;14(12):1880\u201391.","journal-title":"J Tissue Eng Regen M"},{"issue":"3","key":"4693_CR27","doi-asserted-by":"publisher","first-page":"232","DOI":"10.1007\/s11626-022-00648-z","volume":"58","author":"P Horcharoensuk","year":"2022","unstructured":"Horcharoensuk P, Yang- S, Chakritbudsabong W, Samatiwat P, Pramong R, Rungarunlert S, Rungsiwiwut R. Melatonin attenuates dimethyl sulfoxide- and Zika virus-induced degeneration of Porcine induced neural stem cells. Vitro Cell Dev-An. 2022;58(3):232\u201342.","journal-title":"Vitro Cell Dev-An"},{"key":"4693_CR28","doi-asserted-by":"crossref","unstructured":"Choi H, Oh D, Kim M, Jawad A, Zheng HM, Cai L, Lee J, Kim E, Lee G, Jang H, et al. Establishment of Porcine embryonic stem cells in simplified serum free media and feeder free expansion. Stem Cell Res Ther. 2024;15(1):245.","DOI":"10.1186\/s13287-024-03858-2"},{"issue":"9","key":"4693_CR29","doi-asserted-by":"publisher","first-page":"1962","DOI":"10.1038\/nprot.2017.078","volume":"12","author":"S Nolbrant","year":"2017","unstructured":"Nolbrant S, Heuer A, Parmar M, Kirkeby A. Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation. Nat Protoc. 2017;12(9):1962\u201379.","journal-title":"Nat Protoc"},{"issue":"4","key":"4693_CR30","doi-asserted-by":"publisher","first-page":"357","DOI":"10.1038\/nmeth.3317","volume":"12","author":"D Kim","year":"2015","unstructured":"Kim D, Landmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015;12(4):357\u2013U121.","journal-title":"Nat Methods"},{"issue":"3","key":"4693_CR31","doi-asserted-by":"publisher","first-page":"290","DOI":"10.1038\/nbt.3122","volume":"33","author":"M Pertea","year":"2015","unstructured":"Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol. 2015;33(3):290\u2013.","journal-title":"Nat Biotechnol"},{"issue":"9","key":"4693_CR32","doi-asserted-by":"publisher","first-page":"1650","DOI":"10.1038\/nprot.2016.095","volume":"11","author":"M Pertea","year":"2016","unstructured":"Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL. Transcript-level expression analysis of RNA-seq experiments with HISAT, stringtie and ballgown. Nat Protoc. 2016;11(9):1650\u201367.","journal-title":"Nat Protoc"},{"key":"4693_CR33","doi-asserted-by":"crossref","unstructured":"Love MI, Huber W, Anders S. Moderated Estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550.","DOI":"10.1186\/s13059-014-0550-8"},{"issue":"W1","key":"4693_CR34","doi-asserted-by":"publisher","first-page":"W191","DOI":"10.1093\/nar\/gkz369","volume":"47","author":"U Raudvere","year":"2019","unstructured":"Raudvere U, Kolberg L, Kuzmin I, Arak T, Adler P, Peterson H, Vilo J. G:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res. 2019;47(W1):W191\u20138.","journal-title":"Nucleic Acids Res"},{"issue":"22","key":"4693_CR35","doi-asserted-by":"publisher","first-page":"e2609","DOI":"10.21769\/BioProtoc.2609","volume":"7","author":"X Xu","year":"2017","unstructured":"Xu X, Radulescu CI, Utami KH, Pouladi MA. Obtaining Multi-electrode array recordings from human induced pluripotent stem Cell-Derived neurons. Bio Protoc. 2017;7(22):e2609.","journal-title":"Bio Protoc"},{"issue":"1","key":"4693_CR36","doi-asserted-by":"publisher","first-page":"29","DOI":"10.1016\/j.stem.2016.10.003","volume":"20","author":"N Kee","year":"2017","unstructured":"Kee N, Volakakis N, Kirkeby A, Dahl L, Storvall H, Nolbrant S, Lahti L, Bjorklund AK, Gillberg L, Joodmardi E, et al. Single-Cell analysis reveals a close relationship between differentiating dopamine and subthalamic nucleus neuronal lineages. Cell Stem Cell. 2017;20(1):29\u201340.","journal-title":"Cell Stem Cell"},{"issue":"16","key":"4693_CR37","doi-asserted-by":"publisher","first-page":"3373","DOI":"10.1242\/dev.094565","volume":"140","author":"JV Veenvliet","year":"2013","unstructured":"Veenvliet JV, Dos Santos MT, Kouwenhoven WM, von Oerthel L, Lim JL, van der Linden AJ, Koerkamp MJ, Holstege FC, Smidt MP. Specification of dopaminergic subsets involves interplay of En1 and Pitx3. Development. 2013;140(16):3373\u201384.","journal-title":"Development"},{"issue":"1","key":"4693_CR38","doi-asserted-by":"publisher","first-page":"135","DOI":"10.1016\/j.stem.2016.09.004","volume":"20","author":"A Kirkeby","year":"2017","unstructured":"Kirkeby A, Nolbrant S, Tiklova K, Heuer A, Kee N, Cardoso T, Ottosson DR, Lelos MJ, Rifes P, Dunnett SB, et al. Predictive markers guide differentiation to improve graft outcome in clinical translation of hESC-Based therapy for parkinson\u2019s disease. Cell Stem Cell. 2017;20(1):135\u201348.","journal-title":"Cell Stem Cell"},{"issue":"2","key":"4693_CR39","doi-asserted-by":"publisher","first-page":"343","DOI":"10.1016\/j.stem.2021.01.005","volume":"28","author":"TW Kim","year":"2021","unstructured":"Kim TW, Piao J, Koo SY, Kriks S, Chung SY, Betel D, Socci ND, Choi SJ, Zabierowski S, Dubose BN, et al. Biphasic activation of WNT signaling facilitates the derivation of midbrain dopamine neurons from hESCs for translational use. Cell Stem Cell. 2021;28(2):343\u201355. e345.","journal-title":"Cell Stem Cell"},{"issue":"35","key":"4693_CR40","doi-asserted-by":"publisher","first-page":"12629","DOI":"10.1523\/JNEUROSCI.0684-11.2011","volume":"31","author":"SC Gantz","year":"2011","unstructured":"Gantz SC, Ford CP, Neve KA, Williams JT. Loss of Mecp2 in substantia Nigra dopamine neurons compromises the nigrostriatal pathway. J Neurosci. 2011;31(35):12629\u201337.","journal-title":"J Neurosci"},{"issue":"10","key":"4693_CR41","doi-asserted-by":"publisher","first-page":"3463","DOI":"10.1523\/JNEUROSCI.09-10-03463.1989","volume":"9","author":"AA Grace","year":"1989","unstructured":"Grace AA, Onn SP. Morphology and electrophysiological properties of immunocytochemically identified rat dopamine neurons recorded in vitro. J Neurosci. 1989;9(10):3463\u201381.","journal-title":"J Neurosci"},{"issue":"11","key":"4693_CR42","doi-asserted-by":"publisher","first-page":"4264","DOI":"10.1523\/JNEUROSCI.12-11-04264.1992","volume":"12","author":"S Rayport","year":"1992","unstructured":"Rayport S, Sulzer D, Shi WX, Sawasdikosol S, Monaco J, Batson D, Rajendran G. Identified postnatal mesolimbic dopamine neurons in culture: morphology and electrophysiology. J Neurosci. 1992;12(11):4264\u201380.","journal-title":"J Neurosci"},{"issue":"1","key":"4693_CR43","doi-asserted-by":"publisher","first-page":"337","DOI":"10.1016\/j.stemcr.2022.10.016","volume":"18","author":"K Nishimura","year":"2023","unstructured":"Nishimura K, Yang SZ, Lee KW, Asgr\u00edmsd\u00f3ttir ES, Nikouei K, Paslawski W, Gnodde S, Lyu G, Hu LJ, Salt\u00f3 C, et al. Single-cell transcriptomics reveals correct developmental dynamics and high-quality midbrain cell types by improved hESC differentiation. Stem Cell Rep. 2023;18(1):337\u201353.","journal-title":"Stem Cell Rep"},{"issue":"1","key":"4693_CR44","doi-asserted-by":"publisher","first-page":"4183","DOI":"10.1038\/s41467-020-17876-0","volume":"11","author":"D Agarwal","year":"2020","unstructured":"Agarwal D, Sandor C, Volpato V, Caffrey TM, Monzon-Sandoval J, Bowden R, Alegre-Abarrategui J, Wade-Martins R, Webber C. A single-cell atlas of the human substantia Nigra reveals cell-specific pathways associated with neurological disorders. Nat Commun. 2020;11(1):4183.","journal-title":"Nat Commun"},{"issue":"3\u20134","key":"4693_CR45","doi-asserted-by":"publisher","first-page":"344","DOI":"10.1007\/s12031-017-0942-9","volume":"62","author":"FN Emamzadeh","year":"2017","unstructured":"Emamzadeh FN. Role of apolipoproteins and alpha-Synuclein in parkinson\u2019s disease. J Mol Neurosci. 2017;62(3\u20134):344\u201355.","journal-title":"J Mol Neurosci"},{"key":"4693_CR46","doi-asserted-by":"crossref","unstructured":"Fiorenzano A, Storm P, Sozzi E, Bruzelius A, Corsi S, Kajtez J, Mudannayake J, Nelander J, Mattsson B, \u00c5kerblom M et al. TARGET- seq: Linking single- cell transcriptomics of human dopaminergic neurons with their target specificity. P Natl Acad Sci USA. 2024, 121(47).","DOI":"10.1073\/pnas.2410331121"},{"issue":"3","key":"4693_CR47","doi-asserted-by":"publisher","first-page":"651","DOI":"10.1016\/j.stemcr.2018.07.014","volume":"11","author":"D Gyllborg","year":"2018","unstructured":"Gyllborg D, Ahmed M, Toledo EM, Theofilopoulos S, Yang SZ, Ffrench-Constant C, Arenas E. The matricellular protein R-Spondin 2 promotes midbrain dopaminergic neurogenesis and differentiation. Stem Cell Rep. 2018;11(3):651\u201364.","journal-title":"Stem Cell Rep"},{"issue":"40","key":"4693_CR48","doi-asserted-by":"publisher","first-page":"12542","DOI":"10.1523\/JNEUROSCI.2521-09.2009","volume":"29","author":"SM Kolk","year":"2009","unstructured":"Kolk SM, Gunput RAF, Tran TS, van den Heuvel DMA, Prasad AA, Hellemons AJCGM, Adolfs Y, Ginty DD, Kolodkin AL, Burbach JPH, et al. Semaphorin 3F is a bifunctional guidance cue for dopaminergic axons and controls their Fasciculation, Channeling, rostral Growth, and intracortical targeting. J Neurosci. 2009;29(40):12542\u201357.","journal-title":"J Neurosci"},{"issue":"7","key":"4693_CR49","doi-asserted-by":"publisher","first-page":"2374","DOI":"10.1016\/j.celrep.2020.01.084","volume":"30","author":"C Salesse","year":"2020","unstructured":"Salesse C, Charest J, Doucet-Beaupr\u00e9 H, Castonguay AM, Labrecque S, De Koninck P, L\u00e9vesque M. Opposite control of excitatory and inhibitory synapse formation by Slitrk2 and Slitrk5 on dopamine neurons modulates hyperactivity behavior. Cell Rep. 2020;30(7):2374\u201386.","journal-title":"Cell Rep"},{"issue":"15","key":"4693_CR50","doi-asserted-by":"publisher","first-page":"2917","DOI":"10.1007\/s00018-014-1596-1","volume":"71","author":"G Lupo","year":"2014","unstructured":"Lupo G, Bertacchi M, Carucci N, Augusti-Tocco G, Biagioni S, Cremisi F. From pluripotency to forebrain patterning: an in vitro journey astride embryonic stem cells. Cell Mol Life Sci. 2014;71(15):2917\u201330.","journal-title":"Cell Mol Life Sci"},{"issue":"6","key":"4693_CR51","doi-asserted-by":"publisher","first-page":"703","DOI":"10.1016\/j.celrep.2012.04.009","volume":"1","author":"A Kirkeby","year":"2012","unstructured":"Kirkeby A, Grealish S, Wolf DA, Nelander J, Wood J, Lundblad M, Lindvall O, Parmar M. Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Rep. 2012;1(6):703\u201314.","journal-title":"Cell Rep"},{"issue":"3","key":"4693_CR52","doi-asserted-by":"publisher","first-page":"230","DOI":"10.1038\/nrn1628","volume":"6","author":"M Placzek","year":"2005","unstructured":"Placzek M, Briscoe J. The floor plate: multiple cells, multiple signals. Nat Rev Neurosci. 2005;6(3):230\u201340.","journal-title":"Nat Rev Neurosci"},{"issue":"3","key":"4693_CR53","doi-asserted-by":"publisher","first-page":"155","DOI":"10.1016\/j.tig.2004.01.002","volume":"20","author":"U Strahle","year":"2004","unstructured":"Strahle U, Lam CS, Ertzer R, Rastegar S. Vertebrate floor-plate specification: variations on common themes. Trends Genet. 2004;20(3):155\u201362.","journal-title":"Trends Genet"},{"issue":"1\u20132","key":"4693_CR54","doi-asserted-by":"publisher","first-page":"90","DOI":"10.1016\/j.mod.2010.11.002","volume":"128","author":"YE Mavromatakis","year":"2011","unstructured":"Mavromatakis YE, Lin W, Metzakopian E, Ferri AL, Yan CH, Sasaki H, Whisett J, Ang SL. Foxa1 and Foxa2 positively and negatively regulate Shh signalling to specify ventral midbrain progenitor identity. Mech Dev. 2011;128(1\u20132):90\u2013103.","journal-title":"Mech Dev"},{"issue":"10","key":"4693_CR55","doi-asserted-by":"publisher","first-page":"707","DOI":"10.1007\/s13238-022-00912-8","volume":"13","author":"P Yin","year":"2022","unstructured":"Yin P, Li S, Li XJ, Yang W. New pathogenic insights from large animal models of neurodegenerative diseases. Protein Cell. 2022;13(10):707\u201320.","journal-title":"Protein Cell"},{"issue":"111","key":"4693_CR56","doi-asserted-by":"publisher","first-page":"14393","DOI":"10.1038\/s41598-021-93904-3","volume":"13","author":"ZQ Du","year":"2021","unstructured":"Du ZQ, Liang H, Liu XM, Liu YH, Wang C, Yang CX. Single cell RNA-seq reveals genes vital to in vitro fertilized embryos and Parthenotes in pigs. Sci Rep. 2021;13(111):14393.","journal-title":"Sci Rep"},{"key":"4693_CR57","doi-asserted-by":"crossref","unstructured":"Xu P, He H, Gao Q, Zhou Y, Wu Z, Zhang X, Sun L, Hu G, Guan Q, You Z et al. Human midbrain dopaminergic neuronal differentiation markers predict cell therapy outcomes in a Parkinson\u2019s disease model. J Clin Invest. 2022;132(14):e156768.","DOI":"10.1172\/JCI156768"},{"issue":"4","key":"4693_CR58","doi-asserted-by":"publisher","first-page":"999","DOI":"10.1016\/j.cell.2018.06.021","volume":"174","author":"A Zeisel","year":"2018","unstructured":"Zeisel A, Hochgerner H, Lonnerberg P, Johnsson A, Memic F, van der Zwan J, Haring M, Braun E, Borm LE, La Manno G, et al. Molecular architecture of the mouse nervous system. Cell. 2018;174(4):999\u2013e10141022.","journal-title":"Cell"},{"issue":"1","key":"4693_CR59","doi-asserted-by":"publisher","first-page":"7302","DOI":"10.1038\/s41467-021-27464-5","volume":"12","author":"A Fiorenzano","year":"2021","unstructured":"Fiorenzano A, Sozzi E, Birtele M, Kajtez J, Giacomoni J, Nilsson F, Bruzelius A, Sharma Y, Zhang Y, Mattsson B, et al. Single-cell transcriptomics captures features of human midbrain development and dopamine neuron diversity in brain organoids. Nat Commun. 2021;12(1):7302.","journal-title":"Nat Commun"},{"issue":"2","key":"4693_CR60","doi-asserted-by":"publisher","first-page":"248","DOI":"10.1016\/j.stem.2016.07.005","volume":"19","author":"J Jo","year":"2016","unstructured":"Jo J, Xiao YX, Sun AX, Cukuroglu E, Tran HD, Goke J, Tan ZY, Saw TY, Tan CP, Lokman H, et al. Midbrain-like organoids from human pluripotent stem cells contain functional dopaminergic and Neuromelanin-Producing neurons. Cell Stem Cell. 2016;19(2):248\u201357.","journal-title":"Cell Stem Cell"},{"issue":"6","key":"4693_CR61","doi-asserted-by":"publisher","first-page":"727","DOI":"10.1002\/stem.3163","volume":"38","author":"TH Kwak","year":"2020","unstructured":"Kwak TH, Kang JH, Hali S, Kim J, Kim KP, Park C, Lee JH, Ryu H, Na JE, Jo J, et al. Generation of homogeneous midbrain organoids with in vivo-like cellular composition facilitates neurotoxin-based parkinson\u2019s disease modeling. Stem Cells. 2020;38(6):727\u201340.","journal-title":"Stem Cells"},{"issue":"1","key":"4693_CR62","doi-asserted-by":"publisher","first-page":"7361","DOI":"10.1038\/s41467-023-43141-1","volume":"14","author":"A Pagliaro","year":"2023","unstructured":"Pagliaro A, Finger R, Zoutendijk I, Bunschuh S, Clevers H, Hendriks D, Artegiani B. Temporal morphogen gradient-driven neural induction shapes single expanded neuroepithelium brain organoids with enhanced cortical identity. Nat Commun. 2023;14(1):7361.","journal-title":"Nat Commun"},{"issue":"2","key":"4693_CR63","doi-asserted-by":"publisher","first-page":"566","DOI":"10.1016\/j.cell.2016.09.027","volume":"167","author":"G La Manno","year":"2016","unstructured":"La Manno G, Gyllborg D, Codeluppi S, Nishimura K, Salto C, Zeisel A, Borm LE, Stott SRW, Toledo EM, Villaescusa JC, et al. Molecular diversity of midbrain development in Mouse, Human, and stem cells. Cell. 2016;167(2):566\u2013.","journal-title":"Cell"},{"issue":"3","key":"4693_CR64","doi-asserted-by":"publisher","first-page":"930","DOI":"10.1016\/j.celrep.2014.10.008","volume":"9","author":"JF Poulin","year":"2014","unstructured":"Poulin JF, Zou J, Drouin-Ouellet J, Kim KYA, Cicchetti F, Awatramani RB. Defining midbrain dopaminergic neuron diversity by Single-Cell gene expression profiling. Cell Rep. 2014;9(3):930\u201343.","journal-title":"Cell Rep"},{"issue":"50","key":"4693_CR65","doi-asserted-by":"publisher","first-page":"15923","DOI":"10.1523\/JNEUROSCI.3910-09.2009","volume":"29","author":"B Kadkhodaei","year":"2009","unstructured":"Kadkhodaei B, Ito T, Joodmardi E, Mattsson B, Rouillard C, Carta M, Muramatsu SI, Sumi-Ichinose C, Nomura T, Metzger D, et al. Nurr1 is required for maintenance of maturing and adult midbrain dopamine neurons. J Neurosci. 2009;29(50):15923\u201332.","journal-title":"J Neurosci"},{"key":"4693_CR66","doi-asserted-by":"crossref","unstructured":"Tripathy SJ, Toker L, Li B, Crichlow CL, Tebaykin D, Mancarci BO, Pavlidis P. Transcriptomic correlates of neuron electrophysiological diversity. Plos Comput Biol. 2017;13(10):e1005814.","DOI":"10.1371\/journal.pcbi.1005814"},{"issue":"5","key":"4693_CR67","doi-asserted-by":"publisher","first-page":"1238","DOI":"10.1016\/j.cell.2016.04.032","volume":"165","author":"XY Qian","year":"2016","unstructured":"Qian XY, Nguyen HN, Song MM, Hadiono C, Ogden SC, Hammack C, Yao B, Hamersky GR, Jacob F, Zhong C, et al. Brain-Region-Specific organoids using Mini-bioreactors for modeling ZIKV exposure. Cell. 2016;165(5):1238\u201354.","journal-title":"Cell"},{"key":"4693_CR68","doi-asserted-by":"publisher","first-page":"194","DOI":"10.1016\/j.conb.2019.03.001","volume":"56","author":"P Arlotta","year":"2019","unstructured":"Arlotta P, Pasca S. Cell diversity in the human cerebral cortex: from the embryo to brain organoids. Curr Opin Neurobiol. 2019;56:194\u20138.","journal-title":"Curr Opin Neurobiol"},{"issue":"5","key":"4693_CR69","doi-asserted-by":"publisher","first-page":"2600","DOI":"10.3390\/ijms22052600","volume":"22","author":"SU Hwang","year":"2021","unstructured":"Hwang SU, Eun K, Kim M, Yoon JD, Cai L, Choi H, Oh D, Lee G, Kim H, Kim E, et al. Establishment of 3D Neuro-Organoids derived from pig embryonic Stem-Like cells. Int J Mol Sci. 2021;22(5):2600.","journal-title":"Int J Mol Sci"},{"issue":"4","key":"4693_CR70","doi-asserted-by":"publisher","first-page":"447","DOI":"10.1089\/cell.2009.0078","volume":"12","author":"JR Yang","year":"2010","unstructured":"Yang JR, Liao CH, Pang CY, Huang LL, Lin YT, Chen YL, Shiue YL, Chen LR. Directed differentiation into neural lineages and therapeutic potential of Porcine embryonic stem cells in rat parkinson\u2019s disease model. Cell Reprogram. 2010;12(4):447\u201361.","journal-title":"Cell Reprogram"}],"container-title":["Stem Cell Research &amp; Therapy"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s13287-025-04693-9.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s13287-025-04693-9\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s13287-025-04693-9.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,5]],"date-time":"2025-11-05T13:08:29Z","timestamp":1762348109000},"score":1,"resource":{"primary":{"URL":"https:\/\/stemcellres.biomedcentral.com\/articles\/10.1186\/s13287-025-04693-9"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,5]]},"references-count":70,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["4693"],"URL":"https:\/\/doi.org\/10.1186\/s13287-025-04693-9","relation":{},"ISSN":["1757-6512"],"issn-type":[{"value":"1757-6512","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,5]]},"assertion":[{"value":"6 June 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"22 September 2025","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"5 November 2025","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"This study was conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals from the National Veterinary and Quarantine Services. The study protocol was approved by the Institutional Animal Care and Use Committee at Chungbuk National University on March 15, 2023 under the project name \u201cDevelopment of porcine midbrain organoids by the analysis of midbrain differentiation mechanism using pluripotent stem cells\u201d (Approval Number: CBNUA-2082-23-02). The author used ChatGPT (OpenAI, San Francisco, CA, USA) to improve the language of the manuscript draft.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"617"}}