Rapid and efficient generation of a transplantable population of functional retinal ganglion cells from fibroblasts.

Glaucoma and other optic neuropathies lead to progressive and irreversible vision loss by damaging retinal ganglion cells (RGCs) and their axons. Cell replacement therapy is a potential promising treatment. However, current methods to obtain RGCs have inherent limitations, including time-consuming procedures, inefficient yields and complex protocols, which hinder their practical application. Here, we have developed a straightforward, rapid and efficient approach for directly inducing RGCs from mouse embryonic fibroblasts (MEFs) using a combination of triple transcription factors (TFs): ASCL1, BRN3B and PAX6 (ABP). We showed that on the 6th day following ABP induction, neurons with molecular characteristics of RGCs were observed, and more than 60% of induced neurons became iRGCs (induced retinal ganglion cells) in the end. Transplanted iRGCs had the ability to survive and appropriately integrate into the RGC layer of mouse retinal explants and N-methyl-D-aspartic acid (NMDA)-damaged retinas. Moreover, they exhibited electrophysiological properties typical of RGCs, and were able to regrow dendrites and axons and form synaptic connections with host retinal cells. Together, we have established a rapid and efficient approach to acquire functional RGCs for potential cell replacement therapy to treat glaucoma and other optic neuropathies.

Maf1 controls retinal neuron number by both RNA Pol III- and Pol II-dependent mechanisms.

The generation of appropriate numbers and types of neurons is a prerequisite for assembling functional neural circuits. However, the molecular basis regulating retinal neuron number remains poorly understood. Here, we report that inactivation of the RNA polymerase (Pol) III inhibitor gene Maf1 in mice results in decreased retinal thickness and neuron number that cause attenuated electroretinogram (ERG) responses. Its absence causes aberrant differentiation of all retinal neuron types primarily by an RNA Pol II-dependent mechanism while promoting retinal progenitor cell proliferation via both Pol III- and Pol II-dependent mechanisms. Chromatin profiling and transcription assay reveal that Maf1 binds widely to the genome to regulate the expression of a large set of Pol II-transcribed genes involved in retinal cell proliferation, differentiation, and/or survival. Together, our data suggest that Maf1 may control retinal neuron number by a balanced regulation of cell proliferation, differentiation, and death via both Pol III-dependent and Pol II-dependent mechanisms.

Neural cell isolation from adult macaques for high-throughput analyses and neurosphere cultures.

The low number of neural progenitor cells (NPCs) present in the adult and aged primate brains represents a challenge for generating high-yield and viable in vitro cultures of primary brain cells. Here we report a step-by-step approach for the fast and reproducible isolation of high-yield and viable primary brain cells, including mature neurons, immature cells and NPCs, from adult and aged macaques. We describe the anesthesia, transcardial perfusion and brain tissue preparation; the subsequent microdissection of the regions of interest and their enzymatic dissociation, leading to the separation of single cells. The cell isolation steps of our protocol can also be used for routine cell culturing, in particular for NPC expansion and differentiation, suitable for studies of hippocampal neurogenesis in the adult macaque brain. The purified primary brain cells are largely free from myelin debris and erythrocytes, paving the way for multiple downstream applications in vitro and in vivo. When combined with single-cell profiling techniques, this approach allows an unbiased and comprehensive mapping of cell states in the adult and aged macaque brain, which is needed to advance our understanding of human cognitive and neurological diseases. The neural cell isolation protocol requires 4 h and a team of four to six users with expertize in primary brain cell isolation to avoid tissue hypoxia during the time-sensitive steps of the procedure.

Identification of TPBG-Expressing Amacrine Cells in DAT-tdTomato Mouse.

Purpose: Neurons are the bricks of the neuronal system and experimental access to certain neuron subtypes will be of great help to decipher neuronal circuits. Here, we identified trophoblast glycoprotein (TPBG)-expressing GABAergic amacrine cells (ACs) that were selectively labeled in DAT-tdTomato transgenic mice. Methods: Retina and brain sections were prepared for immunostaining with antibodies against various biomarkers. Patch-sequencing was performed to obtain the transcriptomes of tdTomato-positive cells in DAT-tdTomato mice. Whole-cell recordings were conducted to identify responses to light stimulation. Results: Tyrosine hydroxylase immunoreactive cells were colocalized with tdTomato-positive cells in substantia nigra pars compacta, but not in the retina. Transcriptomes collected from tdTomato-positive cells in retinas via Patch-sequencing exhibited the expression of marker genes of ACs (Pax6 and Slc32a1) and marker genes of GABAergic neurons (Gad1, Gad2, and Slc6a1). Immunostaining with antibodies against relevant proteins (GAD67, GAD65, and GABA) also confirmed transcriptomic results. Furthermore, tdTomato-positive cells in retinas selectively expressed Tpbg, a marker gene for distinct clusters molecularly defined, which was proved with TPBG immunoreactivity in fluorescently labeled cells. Finally, tdTomato-positive cells recorded showed ON-OFF responses to light stimulation. Conclusions: Ectopic expression occurs in the retina but not in the substantia nigra pars compacta in the DAT-tdTomato mouse, and fluorescently labeled cells in the retina are TPBG-expressing GABAergic ACs. This type of transgenic mice has been proved as an ideal tool to achieve efficient labeling of a distinct subset of ACs that selectively express Tpbg.

Single-cell transcriptomics of adult macaque hippocampus reveals neural precursor cell populations.

The extent to which neurogenesis occurs in adult primates remains controversial. In this study, using an optimized single-cell RNA sequencing pipeline, we profiled 207,785 cells from the adult macaque hippocampus and identified 34 cell populations comprising all major hippocampal cell types. Analysis of their gene expression, specification trajectories and gene regulatory networks revealed the presence of all key neurogenic precursor cell populations, including a heterogeneous pool of radial glia-like cells (RGLs), intermediate progenitor cells (IPCs) and neuroblasts. We identified HMGB2 as a novel IPC marker. Comparison with mouse single-cell transcriptomic data revealed differences in neurogenic processes between species. We confirmed that neurogenesis is recapitulated in ex vivo neurosphere cultures from adult primates, further supporting the existence of neural precursor cells (NPCs) that are able to proliferate and differentiate. Our large-scale dataset provides a comprehensive adult neurogenesis atlas for primates.

In vivo Regeneration of Ganglion Cells for Vision Restoration in Mammalian Retinas.

Glaucoma and other optic neuropathies affect millions of people worldwide, ultimately causing progressive and irreversible degeneration of retinal ganglion cells (RGCs) and blindness. Previous research into cell replacement therapy of these neurodegenerative diseases has been stalled due to the incapability for grafted RGCs to integrate into the retina and project properly along the long visual pathway. In vivo RGC regeneration would be a promising alternative approach but mammalian retinas lack regenerative capacity. It therefore has long been a great challenge to regenerate functional and properly projecting RGCs for vision restoration in mammals. Here we show that the transcription factors (TFs) Math5 and Brn3b together are able to reprogram mature mouse Müller glia (MG) into RGCs. The reprogrammed RGCs extend long axons that make appropriate intra-retinal and extra-retinal projections through the entire visual pathway to innervate both image-forming and non-image-forming brain targets. They exhibit typical neuronal electrophysiological properties and improve visual responses in RGC loss mouse models. Together, our data provide evidence that mammalian MG can be reprogrammed by defined TFs to achieve in vivo regeneration of functional RGCs as well as a promising new therapeutic approach to restore vision to patients with glaucoma and other optic neuropathies.

Generation of self-organized sensory ganglion organoids and retinal ganglion cells from fibroblasts.

Neural organoids provide a powerful tool for investigating neural development, modeling neural diseases, screening drugs, and developing cell-based therapies. Somatic cells have previously been reprogrammed by transcription factors (TFs) into sensory ganglion (SG) neurons but not SG organoids. We identify a combination of triple TFs Ascl1, Brn3b/3a, and Isl1 (ABI) as an efficient means to reprogram mouse and human fibroblasts into self-organized and networked induced SG (iSG) organoids. The iSG neurons exhibit molecular features, subtype diversity, electrophysiological and calcium response properties, and innervation patterns characteristic of peripheral sensory neurons. Moreover, we have defined retinal ganglion cell (RGC)-specific identifiers to demonstrate the ability for ABI to reprogram induced RGCs (iRGCs) from fibroblasts. Unlike iSG neurons, iRGCs maintain a scattering distribution pattern characteristic of endogenous RGCs. iSG organoids may serve as a model to decipher the pathogenesis of sensorineural diseases and screen effective drugs and a source for cell replacement therapy.

Reprogramming Fibroblasts to Neural Stem Cells by Overexpression of the Transcription Factor Ptf1a.

Neural stem cells (NSCs) have the features of both neural progenitors and stem cells, and show great potentials in translational research and regenerative medicine. Studies on NSCs have been greatly accelerated by the introduction of induced neural stem cells (iNSCs). The iNSCs are usually differentiated from induced pluripotent stem cells (iPSCs) or transdifferentiated from somatic cells such as fibroblasts or glial cells. Here, we describe a detailed protocol to reprogram human and mouse fibroblasts into iNSCs by overexpression of a transcription factor Ptf1a delivered by lentiviruses. The obtained iNSC lines have a strong self-renewal ability and are capable of differentiating into various types of neurons, astrocytes, and oligodendrocytes both in vitro and in vivo. The protocol is quite simple but powerful to produce iNSC lines.

A Novel Mutation p.S93R in CRYBB1 Associated with Dominant Congenital Cataract and Microphthalmia.

Purpose: To identify the pathogenetic mutations in a four-generation Chinese family with dominant congenital cataracts and microphthalmia.Methods: A four-generation Chinese family with dominant congenital cataracts were recruited. Genomic DNAs were collected from their peripheral blood leukocytes and subjected to whole exome sequencing. The genetic mutations were identified by bioinformatic analyses and verified by Sanger sequencing.Results: Whole exome sequencing revealed a c.279C>G point mutation in the CRYBB1 gene which was further verified by Sanger sequencing. The nucleotide replacement results in a novel mutation p.S93R in a conserved residue of ßB1 crystallin which is predicted to disrupt normal ßB1 structure and function.Conclusions: We identified a novel missense mutation p.S93R in CRYBB1 in a Chinese family with autosomal dominant congenital cataracts and microphthalmia. This serine residue is extremely conserved evolutionarily in more than 50 ßγ-crystallins of many species. These data will be very helpful to further understand the structural and functional features of crystallins.

Necessity and Sufficiency of Ldb1 in the Generation, Differentiation and Maintenance of Non-photoreceptor Cell Types During Retinal Development.

During mammalian retinal development, the multipotent progenitors differentiate into all classes of retinal cells under the delicate control of transcriptional factors. The deficiency of a transcription cofactor, the LIM-domain binding protein Ldb1, has been shown to cause proliferation and developmental defects in multiple tissues including cardiovascular, hematopoietic, and nervous systems; however, it remains unclear whether and how it regulates retinal development. By expression profiling, RNA in situ hybridization and immunostaining, here we show that Ldb1 is expressed in the progenitors during early retinal development, but later its expression gradually shifts to non-photoreceptor cell types including bipolar, amacrine, horizontal, ganglion, and Müller glial cells. Retina-specific ablation of Ldb1 in mice resulted in microphthalmia, optic nerve hypoplasia, retinal thinning and detachment, and profound vision impairment as determined by electroretinography. In the mutant retina, there was precocious differentiation of amacrine and horizontal cells, indicating a requirement of Ldb1 in maintaining the retinal progenitor pool. Additionally, all non-photoreceptor cell types were greatly reduced which appeared to be caused by a generation defect and/or retinal degeneration via excessive cell apoptosis. Furthermore, we showed that misexpressed Ldb1 was sufficient to promote the generation of bipolar, amacrine, horizontal, ganglion, and Müller glial cells at the expense of photoreceptors. Together, these results demonstrate that Ldb1 is not only necessary but also sufficient for the development and/or maintenance of non-photoreceptor cell types, and implicate that the pleiotropic functions of Ldb1 during retinal development are context-dependent and determined by its interaction with diverse LIM-HD (LIM-homeodomain) and LMO (LIM domain-only) binding protein partners.

Direct reprogramming of fibroblasts into neural stem cells by single non-neural progenitor transcription factor Ptf1a.

Induced neural stem cells (iNSCs) reprogrammed from somatic cells have great potentials in cell replacement therapies and in vitro modeling of neural diseases. Direct conversion of fibroblasts into iNSCs has been shown to depend on a couple of key neural progenitor transcription factors (TFs), raising the question of whether such direct reprogramming can be achieved by non-neural progenitor TFs. Here we report that the non-neural progenitor TF Ptf1a alone is sufficient to directly reprogram mouse and human fibroblasts into self-renewable iNSCs capable of differentiating into functional neurons, astrocytes and oligodendrocytes, and improving cognitive dysfunction of Alzheimer's disease mouse models when transplanted. The reprogramming activity of Ptf1a depends on its Notch-independent interaction with Rbpj which leads to subsequent activation of expression of TF genes and Notch signaling required for NSC specification, self-renewal, and homeostasis. Together, our data identify a non-canonical and safer approach to establish iNSCs for research and therapeutic purposes.

Lmo4 and Other LIM domain only factors are necessary and sufficient for multiple retinal cell type development.

Understanding the molecular basis by which distinct cell types are specified is a central issue in retinogenesis and retinal disease development. Here we examined the role of LIM domain only 4 (Lmo4) in retinal development using both gain-of-function and loss-of-function approaches. By immunostaining, Lmo4 was found to be expressed in mouse retina from E10.5 to mature stages. Retroviral delivery of Lmo4 into retinal progenitor cells could promote the amacrine, bipolar and Müller cell fates at the expense of photoreceptors. It also inhibited the fate of early-born retinal ganglion cells. Using a dominant-negative form of Lmo4 which suppresses transcriptional activities of all LIM domain only factors, we demonstrated that LIM domain only factors are both necessary and sufficient for promoting amacrine and bipolar cell development, but not for the differentiation of ganglion, horizontal, Müller, or photoreceptor cells. Taken together, our study uncovers multiple roles of Lmo4 during retinal development and demonstrates the importance of LIM domain only factors in ensuring proper retinal cell specification and differentiation. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 900-915, 2016.

Tfap2a and 2b act downstream of Ptf1a to promote amacrine cell differentiation during retinogenesis.

Retinogenesis is a precisely controlled developmental process during which different types of neurons and glial cells are generated under the influence of intrinsic and extrinsic factors. Three transcription factors, Foxn4, RORß1 and their downstream effector Ptf1a, have been shown to be indispensable intrinsic regulators for the differentiation of amacrine and horizontal cells. At present, however, it is unclear how Ptf1a specifies these two cell fates from competent retinal precursors. Here, through combined bioinformatic, molecular and genetic approaches in mouse retinas, we identify the Tfap2a and Tfap2b transcription factors as two major downstream effectors of Ptf1a. RNA-seq and immunolabeling analyses show that the expression of Tfap2a and 2b transcripts and proteins is dramatically downregulated in the Ptf1a null mutant retina. Their overexpression is capable of promoting the differentiation of glycinergic and GABAergic amacrine cells at the expense of photoreceptors much as misexpressed Ptf1a is, whereas their simultaneous knockdown has the opposite effect. Given the demonstrated requirement for Tfap2a and 2b in horizontal cell differentiation, our study thus defines a Foxn4/RORß1-Ptf1a-Tfap2a/2b transcriptional regulatory cascade that underlies the competence, specification and differentiation of amacrine and horizontal cells during retinal development.

[Cloning and functional analysis of Phyllostachys edulis MYB transcription factor PeMYB2].

MYB-type transcription factor is one of the largest families in plants, which plays important roles in accepting stress signals from environment and regulating the expression of stress-tolerant genes. In this paper, using homologous cloning and RACE technology, a MYB-type transcription factor, designated PeMYB2, was cloned from Phyllostachys edulis. The results of bioinformatics showed that PeMYB2 is a typical R2R3-MYB. It contained two tandem repeats in its N-terminus, and a membrane protein DUF3651 in its C-terminus. In addition, phylogenetic analysis indicated that PeMYB2 shared the highest homology with 85.98% to OsMYB18 protein from Oryza sativa spp. Japonica. In addition, a yeast one-hybrid assay showed that PeMYB2 could activate the expression of downstream genes. After PeMYB2 was transformed into Arabidopsis thaliana, seven PeMYB2 transgenic Arabidopsis lines were obtained. Phenotypic analysis of the transgenic and wild-type Arabidopsis showed that over-expression of PeMYB2 caused delayed flower or dwarfism in transgenic Arabidopsis. Under the abiotic stress conditions, such as salt and cold stresses, the over-expression of PeMYB2 in Arabidopsis had higher survival rate than the wild-type Arabidopsis. Expression analysis of saline stress response marker genes in the transgenic and wild-type plants under the salt stress condition showed that PeMYB2 regulated the expression of NXH1, SOS1, RD29A, and COR15A. As the result, PeMYB2 might play an important role in various responses to abiotic stresses in P. edulis.

Photocatalytic degradation of Acid Blue 62 over CuO-SnO2 nanocomposite photocatalyst under simulated sunlight.

The novel CuO-SnO2 nanocomposite oxide photocatalysts were prepared by simple co-precipitation method, and characterized by X-ray diffraction, transmission electron microscopy, N2 adsorption-desorption measurement and UV-Vis diffuse reflectance spectroscopy. The photocatalytic activities of CuO-SnO2, evaluated using the photodegradation of Acid Blue 62 as a probe reaction under the irradiation of Xenon light, were also found to be related to the calcination temperature and the molar ratio of Cu to Sn. The maximum photocatalytic activity of the CuO-SnO2 photocatalyst was observed to be calcined at 500 degrees C for 3 h (the molar ratio of Cu to Sn was 1:1) due to the sample with good crystallization and high surface area. It also showed much higher photocatalytic activity in treatment dye wastewater under simulated sunlight irradiation compared to Degussa P25 TiO2.