Overexpression of c-MYC, BMI1 and BCL-XL (collectively referred to as MBX) can confer immortalization potential on human induced pluripotent stem cell (hiPSC)-derived megakaryocyte progenitors, which may serve as seed cells for in vitro platelet production. However, existing construction strategies are limited by the risks of random integration and basal leaky expression. Using CRISPR-Cas9-mediated homology-directed repair (HDR), this study was designed to integrate the trans-regulatory element M2rtTA of the Tet-On system and the MBX sequence driven by the cis-acting response element TRE into the safe harbor loci ROSA26 and AAVS1 of the hiPSC genome, respectively, aiming to construct a doxycycline (DOX)-inducible hiPSC line with the potential to generate immortalized megakaryocyte progenitors.

① Recombinant single guide RNA plasmids targeting the AAVS1 and ROSA26 loci, namely px458-sgAAVS1 and px458-sgROSA26, as well as the donor plasmids pAAVS1-TRE-MBX-EGFP and pROSA26-Hygro-M2rtTA were constructed. ② The CAG-driven trans-regulatory element M2rtTA was integrated into the ROSA26 locus to generate CAG-M2rtTA hiPSCs. ③ Based on the constructed CAG-M2rtTA hiPSCs, the TRE-driven MBX tandem cassette and EGFP reporter gene were integrated into the AAVS1 locus to generate CAG-M2rtTA/TRE-MBX hiPSCs. ④ The cells were divided into an induction group (DOX-ON), a non-induction group (non-DOX) and a wild-type blank control group (WT). Differences in EGFP and MBX expression after DOX induction were detected by fluorescence microscopy and qPCR. ⑤ The 3 groups of cells were subjected to synchronous directed megakaryocytic differentiation. For the DOX-ON group, 2 μg/mL DOX was added to the culture medium on day 12 of differentiation, and this culture condition was maintained until day 30. During this period, the cells were passaged normally, and EGFP expression was observed. On day 18 of differentiation, cell samples were harvested from the 3 groups. Wright-Giemsa staining and flow cytometry (detection of CD41a, CD42b and DNA ploidy) were performed to evaluate the regulatory effect of DOX on the directed differentiation of CAG-M2rtTA/TRE-MBX hiPSC cells into mature megakaryocytes. Meanwhile, part of the DOX-ON cells collected on day 18 were subjected to DOX withdrawal to establish the DOX-OFF group. Following drug withdrawal, these cells were cultured for another 6 d under the same differentiation conditions as the other groups, after which the expression of CD41a and CD42b was detected by flow cytometry. The experiment was performed with 3 biological replicates, and statistical analysis was conducted using the independent-samples t test.

① Sanger sequencing confirmed the successful construction of the targeting plasmids px458-sgAAVS1 and px458-sgROSA26. PCR analysis showed that M2rtTA and MBX-EGFP were precisely integrated into the ROSA26 and AAVS1 loci, respectively, yielding CAG-M2rtTA/TRE-MBX hiPSCs. ② After 48 h of DOX induction, EGFP fluorescence was observed in the DOX-ON group. qPCR showed that the mRNA expression levels of c-MYC, BMI1 and BCL-XL were (14.78±1.28)-fold (P<0.0001), (7.87±0.24)-fold (P<0.0001) and (6.70±0.11)-fold (P=0.0010)-fold higher than those in the WT group, respectively. No marked difference was observed between the non-DOX and WT groups. ③ During the megakaryocytic differentiation, the DOX-ON group maintained sustained EGFP expression and proliferative capacity until day 30 of differentiation and passage 5. On day 18 of differentiation, the DOX-ON group retained megakaryocyte progenitor-like morphology, with a DNA ploidy >4N cell proportion of 5.0%, and CD41a⁺CD42b^- and CD41a⁺CD42b⁺ cell proportions of 48.1% and 10.9%, respectively. The non-DOX group exhibited mature megakaryocyte-like morphology, with a DNA ploidy >4N cell proportion of 17.9%, and CD41a⁺CD42b^- and CD41a⁺CD42b⁺ cell proportions of 22.3% and 40.7%, respectively, showing results consistent with that of the WT group. ④ At 6 d after DOX withdrawal, the proportion of CD41a^-CD42b^- cells in the DOX-OFF group decreased to 19.9%, whereas that of CD41a⁺CD42b⁺ cells increased to 33.5%, suggesting that the cells restored their terminal megakaryocytic differentiation capacity after MBX expression was switched off.

The CAG-M2rtTA/TRE-MBX hiPSC line has been successfully constructed via CRISPR-Cas9-mediated HDR. This cell line can maintain stable proliferation at the megakaryocyte progenitor stage through DOX-induced MBX overexpression.

To systematically identify the key genes regulating the exit from naïve pluripotency in embryonic stem cells（ESCs）in order to provide novel targets and theoretical insights into the mechanisms for pluripotency transition and early cell fate determination.

Nanog-green fluorescent protein（Nanog-GFP）reporter-labeled ESCs were infected with a genome-wide Brie knockout library, and further cultured under leukemia inhibitory factor/serum（LIF/S）conditions for 14 d. Flow cytometry was used to sort Nanog-GFP⁺（naïve-state）and Nanog-GFP^-（primed state）cell populations, followed by genomic DNA extraction and high-throughput sequencing. Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout（MAGeCK）was applied to identify differential genes between GFP^-/Input, GFP⁺/Input, and GFP⁺/GFP^- groups. Metascape and Gene Set Enrichment Analysis（GSEA）were conducted for functional enrichment analysis. Then the obtained candidate genes were employed to construct knockout models, and their roles were assessed through cell morphology observation, Nanog-positive rate detection, colony formation assays, and pluripotency gene expression analysis.

The GFP⁺/Input screening revealed 2921 negatively regulated genes（mainly enriched in basic life processes, such as RNA metabolism and cell cycle）and 1393 positively regulated genes（enriched in the processes of nervous system development, carbohydrate metabolism, and vascular system development）. In the GFP^-/Input screening, 2765 negatively regulated genes（enriched in RNA metabolism, cell cycle, and other fundamental processes）and 1303 positively regulated genes（enriched in neural development, cell survival, and endothelial migration）were identified. The GFP⁺/GFP^- comparison identified 1001 negatively regulated genes [involved in stress response and inhibition of mitogen-activated protein kinase（MAPK）signaling] and 983 positively regulated genes [related to fibroblast growth factor/extracellular signal-regulated kinase（FGF/ERK）signaling pathway and glucose metabolism). These genes, were not only known pluripotency regulators（e. g., Nanog, Nr5a2, Klf2, Klf4）and exit-associated genes（e. g., Gata6, Grb2, Zeb1, Fgfr1）, but also some novel candidates（e. g., Dmrt1, Rxra, Zbtb14 and Tmem41b）. Functional validation showed that transient knockout of Dmrt1, Tmem41b, and Hic2 significantly increased the proportion of Nanog⁺ cells（P<0. 01）, suggesting their role in suppressing ground-state exit. ESCs with stable Dmrt1 knockout exhibited a more naïve-state phenotype, presenting compact, dome-shaped colonies, with increased ratio of undifferentiated colonies（P<0. 01）, up-regulation of ground-state markers（Nanog, Nr5a2, Dppa3, P<0. 01）, and down-regulation of primed-state markers（Fgf5, Lefty1, Dnmt3b, P<0. 01）. Rescue experiments for Dmrt1 expression reversed these above phenotypes.

A candidate gene set regulating exit from naïve pluripotency in ESC is screened out and identified with genome-wide CRISPR. Our findings implicate Dmrt1 plays a critical role in promoting the exit.

To investigate the occurrence of stress depression caused by retinal cells death driven by activation of photosensitive STIM1 calcium channel and the changes in brain inflammation related to the visually-associated brain regions.

A mouse model of retinal injury driven by light was constructed by injecting STIM1 virus into the vitreous body of mouse eyeball. HE staining and depression-related behavior test were performed to assess the severity of retinopathy and incidence of depression. Reverse transcription-PCR（RT-PCR）, Western blotting and immunofluorescence assay were used to analyze the changes of microglial cells, inflammatory factors（IL-6 and MIP-1α）, nerve cell damage marker（S100B）, apoptotic protein（Caspase-3）and myelin basic protein（MBP）in the depressive mice.

①The cell density in each layer of the mouse retina were decreased significantly under the light mode of 3 h/d for 7 consecutive days（P<0.05）. ②In the open field test, the times of entering the central area, moving distance in the central area and total distance of movement were obviously decreased（P<0.05）, the index of sucrose preference of the sucrose preference test was significantly decreased（P<0.01）, and the immobile time in the forced swimming test and the tail suspension test was notably increased（P<0.001）in the mice from the photosensitive group. The positive rate of depression in all the 4 tests（open field test, sucrose preference test, forced swimming test and tail suspension test）was 23.26%. ③The density of IBa-1⁺ cells in the visual cortex and dorsolateral geniculate nucleus was elevated in the depressive mice of the photosensitive group（P<0.05）. The expression of IL-6 and MIP-1α at mRNA level was obviously elevated（P<0.000 1）, as well as the expression of S100B and Caspase-3 at protein level was significantly enhanced（P<0.001）, while the MBP protein in the white matter was clearly weakened（P<0.05）. The intensity of MBP fluorescence signal was evidently reduced in the corpus callosum（cc）area and cingulate gyrus（cg）area in white matter（P<0.000 1）.

A stress depression-like mouse model caused by light-driven retinal injury is successfully constructed. Pathogenesis of depression is closely associated with inflammatory environment and neuronal apoptosis in the vision-related brain regions and demyelinating injury in white matter.