Single-Cell RNA Sequencing of hESC-Derived 3D Retinal Organoids Reveals Novel Genes Regulating RPC Commitment in Early Human Retinogenesis.

The development of the mammalian retina is a complicated process involving the generation of distinct types of neurons from retinal progenitor cells (RPCs) in a spatiotemporal-specific manner. The progression of RPCs during retinogenesis includes RPC proliferation, cell-fate commitment, and specific neuronal differentiation. In this study, by performing single-cell RNA sequencing of cells isolated from human embryonic stem cell (hESC)-derived 3D retinal organoids, we successfully deconstructed the temporal progression of RPCs during early human retinogenesis. We identified two distinctive subtypes of RPCs with unique molecular profiles, namely multipotent RPCs and neurogenic RPCs. We found that genes related to the Notch and Wnt signaling pathways, as well as chromatin remodeling, were dynamically regulated during RPC commitment. Interestingly, our analysis identified that CCND1, a G1-phase cell-cycle regulator, was coexpressed with ASCL1 in a cell-cycle-independent manner. Temporally controlled overexpression of CCND1 in retinal organoids demonstrated a role for CCND1 in promoting early retinal neurogenesis. Together, our results revealed critical pathways and novel genes in early retinogenesis of humans

High-precision, non-invasive anti-microvascular approach via concurrent ultrasound and laser irradiation

Antivascular therapy represents a proven strategy to treat angiogenesis. By applying synchronized ultrasound bursts and nanosecond laser irradiation, we developed a novel, selective, non-invasive, localized antivascular method, termed photo-mediated ultrasound therapy (PUT). PUT takes advantage of the high native optical contrast among biological tissues and can treat microvessels without causing collateral damage to the surrounding tissue. In a chicken yolk sac membrane model, under the same ultrasound parameters (1 MHz at 0.45 MPa and 10 Hz with 10% duty cycle), PUT with 4 mJ/cm2 and 6 mJ/cm2 laser fluence induced 51% (p = 0.001) and 37% (p = 0.018) vessel diameter reductions respectively. With 8 mJ/cm2 laser fluence, PUT would yield vessel disruption (90%, p < 0.01). Selectivity of PUT was demonstrated by utilizing laser wavelengths at 578 nm or 650 nm, where PUT selectively shrank veins or occluded arteries. In a rabbit ear model, PUT induced a 68.5% reduction in blood perfusion after 7 days (p < 0.001) without damaging the surrounding cells. In vitro experiments in human blood suggested that cavitation may play a role in PUT. In conclusion, PUT holds significant promise as a novel non-invasive antivascular method with the capability to precisely target blood vessels.R01AR060350R01CA1867694K12EY022299-4BL2014089

Tyrosine-mutated AAV2-mediated shRNA silencing of PTEN promotes axon regeneration of adult optic nerve.

Activating PI3K/AKT/mTOR signaling pathway via deleting phosphatase and tensin homolog (PTEN) has been confirmed to enhance intrinsic growth capacity of neurons to facilitate the axons regeneration of central nervous system after injury. Considering conditional gene deletion is currently not available in clinical practice, we exploited capsid residue tyrosine 444 to phenylalanine mutated single-stranded adeno-associated virus serotype 2 (AAV2) as a vector delivering short hairpin RNA to silence PTEN to promote retinal ganglion cells (RGCs) survival and axons regeneration in adult rat optic nerve axotomy paradigm. We found that mutant AAV2 displayed higher infection efficiency to RGCs and Müller cells by intravitreal injection, mediated PTEN suppression, resulted in much more RGCs survival and more robust axons regeneration compared with wild type AAV2, due to the different extent of the mTOR complex-1 activation and glutamate aspartate transporter (GLAST) regulation. These results suggest that high efficiency AAV2-mediated PTEN knockdown represents a practicable therapeutic strategy for optic neuropathy

Western-blotting for the expression of GLAST in retina 6 weeks after axotomy.

<p>Compared to intact control, ONA resulted in dramatic down-regulation of GLAST, yet Y444F AAV2-shRNA.PTEN, compared with Wt AAV2-shRNA.PTEN or Wt AAV2-GFP, inhibited the reduction significantly. *<i>P</i> < 0.05, **<i>P</i> < 0.01 in ANOVA followed by Bonferroni’s post-test.</p

PTEN knockdown promoting axons regeneration in optic nerve 6 weeks after axotomy.

<p>Fluorescent images of optic nerve longitudinal sections showed CTB-FITC labeled regenerating axons of rats treated with Y444F AAV2-shRNA.PTEN (A), Wt AAV2-shRNA.PTEN (B), and Wt AAV2-GFP (C) respectively. Quantification of the fluorescence intensity at different distances proximal to and distal to the ONA site showed the significant difference in Y444F AAV2-shRNA.PTEN, Wt AAV2-shRNA.PTEN and Wt AAV2-GFP groups (D). *<i>P</i> < 0.05, **<i>P</i> < 0.01 in ANOVA followed by Bonferroni’s post-test. Scale bar, 100μm. Arrow, ONA site.</p

FluoroGold retrograde labeling evaluating the transduction efficiency of AAV2 vectors 4 weeks after injection.

<p>Retinal whole-mounts displayed FluoroGold-labeled (white) RGCs and gene-transduced (green) RGCs from the same retinal regions of rats intravitreally injected Wt AAV2-shRNA.PTEN-GFP or Y444F AAV2-shRNA.PTEN-GFP vector (A-H). Quantifying the percentage of GFP-positive cells in FluoroGold-labeled cells revealed the transduction capacity of Y444F AAV2-shRNA.PTEN-GFP was significantly stronger than that of Wt AAV2-shRNA.PTEN-GFP (I). ** <i>P</i> < 0.01 as tested by Student’s <i>t</i>-test. Scale bar, 100μm.</p

FluoroGold retrograde labeling of RGCs identifying the complete axotomy.

<p>No RGCs was labeled with FluoroGold in retinal longitudinal sections (A) and whole-mounts (C) of axotomized rats while numerous RGCs were labeled with FluoroGold in retinal longitudinal sections (B)and whole-mounts (D) of intact rats. Scale bar, 100μm.</p

Immunofluorescence displaying RGCs and Müller cells transgene expressing GFP 4 weeks after intravitreal injection.

<p>Merged image showed colocalization of GFP fluorescence and TUJ1 staining in retinal flat-mounts from eyes treated with Y444F AAV2-shRNA.PTEN-GFP (A-C) or Wt AAV2-shRNA.PTEN-GFP (D-F), showed colocalization of GFP fluorescence and GS staining in retinal sections from eyes treated with Y444F AAV2-shRNA.PTEN-GFP (G-I) or Wt AAV2-shRNA.PTEN-GFP (J-L). Scale bar, 50μm.</p

TUJ1 immuno-labeling evaluating RGCs survival 6 weeks after axotomy.

<p>The number of survived RGCs decreased significantly 6 weeks after axotomy. Compared with Wt AAV2-GFP, both Y444F AAV2-shRNA.PTEN and Wt AAV2-shRNA.PTEN significantly prompted RGCs survival, and the pro-survival effect of Y444F AAV2-shRNA.PTEN was stronger than that of Wt AAV2-shRNA.PTEN. **<i>P</i> < 0.01 in Student’s <i>t</i>-test or ANOVA followed by Bonferroni’s post-test. Scale bar, 50μm.</p

Immunofluorescence testing PTEN in retinas 4 weeks after intravitreal injection of AAV2 vectors.

<p>Immunofluorescence of retinal sections showed PTEN expression in retinas following Wt AAV2-GFP (A-C), Y444FA AV2-shRNA.PTEN (D-F), or Wt AAV2-shRNA.PTEN (G-I) injection. Quantification of PTEN expression, measured by ImageJ densitometry method, revealed a significant difference in GCL and INL treated with Wt AAV2-GFP, Wt AAV2-shRNA.PTEN, and Y444F AAV2-shRNA.PTEN respectively (J). **<i>P</i> < 0.01 in ANOVA followed by Bonferroni’s post-test. Scale bar, 100μm.</p