VSX2 and ASCL1 Are Indicators of Neurogenic Competence in Human Retinal Progenitor Cultures

<div><p>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 <i>in vivo</i> 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 <i>in situ</i>, 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 <i>in vitro</i>.</p></div

Prenatal retinal neurospheres lose VSX2 expression over time in culture.

<p>KI67+ hRPCs in the outer neuroblastic layer of 96 day human prenatal retina co-express the neural stem cell markers (<b>A</b>) NESTIN and (<b>B</b>) SOX2. Short term prenatal retinal neurosphere cultures also contain abundant (<b>C</b>) KI67+/NESTIN+ and (<b>D</b>) KI67+/SOX2+ hRPCs. Nearly all VSX2+ hRPCs in short term prenatal retinal neurosphere cultures co-label with (<b>E</b>) NESTIN and (<b>F</b>) SOX2. Prenatal retinal neurosphere cultures (n = 5) from 79–108 day gestation tissue were sampled at 1 week, 1 month, and 2 months. After 2 months, very little VSX2 immunostaining is detected, although (<b>G</b>) NESTIN and (<b>H</b>) SOX2 remain highly expressed. The percentage of VSX2, KI67, NESTIN, and SOX2 immunopositive cells were quantified (<b>I</b>) in short term cultures and (<b>J</b>) over a 2 month period. Nuclei were visualized with DAPI and cell count data is expressed as % immunopositive cells. Scale bars: 50 μm (panels A,B); 20 μm (panels C-H).</p

VSX2+ hRPCs from 3D hESC-OVs maintain neurogenic competence and continue along a normal developmental trajectory in long term cultures.

<p>VSX2+/ ASCL1+ hRPCs were detected in 3D hESC-OVs at (<b>A</b>) 30 days, (<b>B</b>) 50 days, and (<b>C</b>) 90 days of differentiation. (<b>D</b>) The percentages of ASCL1+ cells that co-labeled with VSX2 were quantified in 30, 50, and 90 day hESC-OVs. (<b>E</b>) By 50 days, many photoreceptor precursor cells identified by RECOVERIN immunoreactivity were present in hESC-OVs, and by 90 days (<b>F,G</b>), RECOVERIN+ cells increased in abundance. (<b>H</b>) Quantification of cells expressing ASCL1 or RECOVERIN was performed at 20, 30, 50, and 90 days of differentiation. Nuclei were visualized with DAPI and cell count data is expressed as % immunopositive cells. ND: nondetectable. <i>Arrows</i> in panels A-C demarcate ASCL1+/VSX2+ cells. Scale bars: 50 μm (panels E,F); 20 μm (panels A-C,); 10 μm (panel G).</p

The Current Status and Future Prospects of KAGRA, the Large-Scale Cryogenic Gravitational Wave Telescope Built in the Kamioka Underground

International audienceKAGRA is a gravitational-wave (GW) detector constructed in Japan with two unique key features: It was constructed underground, and the test-mass mirrors are cooled to cryogenic temperatures. These features are not included in other kilometer-scale detectors but will be adopted in future detectors such as the Einstein Telescope. KAGRA performed its first joint observation run with GEO600 in 2020. In this observation, the sensitivity of KAGRA to GWs was inferior to that of other kilometer-scale detectors such as LIGO and Virgo. However, further upgrades to the detector are ongoing to reach the sensitivity for detecting GWs in the next observation run, which is scheduled for 2022. In this article, the current situation, sensitivity, and future perspectives are reviewed.</jats:p