title: VSX2 and ASCL1 Are Indicators of Neurogenic Competence in Human Retinal Progenitor Cultures.
abstract: Three dimensional (3D) culture techniques are frequently used for CNS tissue modeling and organoid production, including generation of retina-like tissues. A proposed advantage of these 3D systems is their potential to more closely approximate in vivo cellular microenvironments, which could translate into improved manufacture and/or maintenance of neuronal populations. Visual System Homeobox 2 (VSX2) labels all multipotent retinal progenitor cells (RPCs) and is known to play important roles in retinal development. In contrast, the proneural transcription factor Acheate scute-like 1 (ASCL1) is expressed transiently in a subset of RPCs, but is required for the production of most retinal neurons. Therefore, we asked whether the presence of VSX2 and ASCL1 could gauge neurogenic potential in 3D retinal cultures derived from human prenatal tissue or ES cells (hESCs). Short term prenatal 3D retinal cultures displayed multiple characteristics of human RPCs (hRPCs) found in situ, including robust expression of VSX2. Upon initiation of hRPC differentiation, there was a small increase in co-labeling of VSX2+ cells with ASCL1, along with a modest increase in the number of PKCα+ neurons. However, 3D prenatal retinal cultures lost expression of VSX2 and ASCL1 over time while concurrently becoming refractory to neuronal differentiation. Conversely, 3D optic vesicles derived from hESCs (hESC-OVs) maintained a robust VSX2+ hRPC population that could spontaneously co-express ASCL1 and generate photoreceptors and other retinal neurons for an extended period of time. These results show that VSX2 and ASCL1 can serve as markers for neurogenic potential in cultured hRPCs. Furthermore, unlike hESC-OVs, maintenance of 3D structure does not independently convey an advantage in the culture of prenatal hRPCs, further illustrating differences in the survival and differentiation requirements of hRPCs extracted from native tissue vs. those generated entirely in vitro.
authors: Waisman Center, University of Wisconsin, Madison, Wisconsin, United States of America; McPherson Eye Research Institute, University of Wisconsin, Madison, Wisconsin, United States of America.
authors: Lynda S
authors: LS
authors: Wright
authors: Department of Ophthalmology, Lozano Blesa University Hospital, Zaragoza, Spain; Aragones Health Sciences Institute, Zaragoza, Spain.
authors: Isabel
authors: I
authors: Pinilla
authors: Waisman Center, University of Wisconsin, Madison, Wisconsin, United States of America.
authors: Jishnu
authors: J
authors: Saha
authors: Waisman Center, University of Wisconsin, Madison, Wisconsin, United States of America; New England College of Optometry, Boston, Massachusetts, United States of America.
authors: Joshua M
authors: JM
authors: Clermont
authors: Waisman Center, University of Wisconsin, Madison, Wisconsin, United States of America.
authors: Jessica S
authors: JS
authors: Lien
authors: Waisman Center, University of Wisconsin, Madison, Wisconsin, United States of America.
authors: Katarzyna D
authors: KD
authors: Borys
authors: Waisman Center, University of Wisconsin, Madison, Wisconsin, United States of America.
authors: Elizabeth E
authors: EE
authors: Capowski
authors: Waisman Center, University of Wisconsin, Madison, Wisconsin, United States of America; McPherson Eye Research Institute, University of Wisconsin, Madison, Wisconsin, United States of America.
authors: M Joseph
authors: MJ
authors: Phillips
authors: Waisman Center, University of Wisconsin, Madison, Wisconsin, United States of America; McPherson Eye Research Institute, University of Wisconsin, Madison, Wisconsin, United States of America; Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin, United States of America.
authors: David M
authors: DM
authors: Gamm
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology; D018398:Homeodomain Proteins / Q000502:physiology*; D006801:Humans; D021621:Imaging, Three-Dimensional; D058953:Neural Stem Cells / Q000502:physiology*; D055495:Neurogenesis / Q000502:physiology; D016133:Polymerase Chain Reaction; D012160:Retina / Q000166:cytology* / Q000196:embryology / Q000502:physiology; D014157:Transcription Factors / Q000502:physiology*
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D018398:Homeodomain Proteins; D014157:Transcription Factors; C065500:ASCL1 protein, human; C490289:VSX2 protein, human