Transplanted human cones incorporate into the retina and function in a murine cone degeneration model.

Once human photoreceptors die, they do not regenerate, thus, photoreceptor transplantation has emerged as a potential treatment approach for blinding diseases. Improvements in transplant organization, donor cell maturation, and synaptic connectivity to the host will be critical in advancing this technology for use in clinical practice. Unlike the unstructured grafts of prior cell-suspension transplantations into end-stage degeneration models, we describe the extensive incorporation of induced pluripotent stem cell (iPSC) retinal organoid-derived human photoreceptors into mice with cone dysfunction. This incorporative phenotype was validated in both cone-only as well as pan-photoreceptor transplantations. Rather than forming a glial barrier, Müller cells extended throughout the graft, even forming a series of adherens junctions between mouse and human cells, reminiscent of an outer limiting membrane. Donor-host interaction appeared to promote polarization as well as the development of morphological features critical for light detection, namely the formation of inner and well-stacked outer segments oriented toward the retinal pigment epithelium. Putative synapse formation and graft function were evident at both structural and electrophysiological levels. Overall, these results show that human photoreceptors interacted readily with a partially degenerated retina. Moreover, incorporation into the host retina appeared to be beneficial to graft maturation, polarization, and function.

A comprehensive library of human transcription factors for cell fate engineering.

Human pluripotent stem cells (hPSCs) offer an unprecedented opportunity to model diverse cell types and tissues. To enable systematic exploration of the programming landscape mediated by transcription factors (TFs), we present the Human TFome, a comprehensive library containing 1,564 TF genes and 1,732 TF splice isoforms. By screening the library in three hPSC lines, we discovered 290 TFs, including 241 that were previously unreported, that induce differentiation in 4 days without alteration of external soluble or biomechanical cues. We used four of the hits to program hPSCs into neurons, fibroblasts, oligodendrocytes and vascular endothelial-like cells that have molecular and functional similarity to primary cells. Our cell-autonomous approach enabled parallel programming of hPSCs into multiple cell types simultaneously. We also demonstrated orthogonal programming by including oligodendrocyte-inducible hPSCs with unmodified hPSCs to generate cerebral organoids, which expedited in situ myelination. Large-scale combinatorial screening of the Human TFome will complement other strategies for cell engineering based on developmental biology and computational systems biology.

Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in vivo Genome Editing.

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors.

Optogenetics for neural transplant manipulation and functional analysis.

Transplantation of neural stem cells (NSCs) or NSC-derived neurons into the brain is a promising therapeutic approach to restore neuronal function. Rapid progress in the NSCs research field, particularly due to the exploitation of induced pluripotent stem cells (iPSCs), offers great potential and an unlimited source of stem cell-derived neural grafts. Studying the functional integration of these grafts into host brain tissues and their effects on each other have been boosted by the implementation of optogenetic technologies. Optogenetics provides high spatiotemporal functional manipulations of grafted or host neurons in parallel. This review aims to highlight the impact of optogenetics in neural stem cell transplantations.

MiRNA Regulatory Functions in Photoreceptors.

MicroRNAs (miRNAs) are important regulators of gene expression. These small, non-coding RNAs post-transcriptionally silence messenger RNAs (mRNAs) in a sequence-specific manner. In this way, miRNAs control important regulatory functions, also in the retina. If dysregulated, these molecules are involved in several retinal pathologies. For example, several miRNAs have been linked to essential photoreceptor functions, including light sensitivity, synaptic transmission, and modulation of inflammatory responses. Mechanistic miRNA knockout and knockdown studies further linked their functions to degenerative retinal diseases. Of note, the type and timing of genetic manipulation before, during, or after retinal development, is important when studying specific miRNA knockout effects. Within this review, we focus on miR-124 and the miR-183/96/182 cluster, which have assigned functions in photoreceptors in health and disease. As a single miRNA can regulate hundreds of mRNAs, we will also discuss the experimental validation and manipulation approaches to study complex miRNA/mRNA regulatory networks. Revealing these networks is essential to understand retinal pathologies and to harness miRNAs as precise therapeutic and diagnostic tools to stabilize the photoreceptors' transcriptomes and, thereby, function.

Combined Experimental and System-Level Analyses Reveal the Complex Regulatory Network of miR-124 during Human Neurogenesis.

Non-coding RNAs regulate many biological processes including neurogenesis. The brain-enriched miR-124 has been assigned as a key player of neuronal differentiation via its complex but little understood regulation of thousands of annotated targets. To systematically chart its regulatory functions, we used CRISPR/Cas9 gene editing to disrupt all six miR-124 alleles in human induced pluripotent stem cells. Upon neuronal induction, miR-124-deleted cells underwent neurogenesis and became functional neurons, albeit with altered morphology and neurotransmitter specification. Using RNA-induced-silencing-complex precipitation, we identified 98 high-confidence miR-124 targets, of which some directly led to decreased viability. By performing advanced transcription-factor-network analysis, we identified indirect miR-124 effects on apoptosis, neuronal subtype differentiation, and the regulation of previously uncharacterized zinc finger transcription factors. Our data emphasize the need for combined experimental- and system-level analyses to comprehensively disentangle and reveal miRNA functions, including their involvement in the neurogenesis of diverse neuronal cell types found in the human brain.

On-demand optogenetic activation of human stem-cell-derived neurons.

The widespread application of human stem-cell-derived neurons for functional studies is impeded by complicated differentiation protocols, immaturity, and deficient optogene expression as stem cells frequently lose transgene expression over time. Here we report a simple but precise Cre-loxP-based strategy for generating conditional, and thereby stable, optogenetic human stem-cell lines. These cells can be easily and efficiently differentiated into functional neurons, and optogene expression can be triggered by administering Cre protein to the cultures. This conditional expression system may be applied to stem-cell-derived neurons whenever timed transgene expression could help to overcome silencing at the stem-cell level.

LDB1 overexpression is a negative prognostic factor in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer in western countries and is driven by the Wnt signaling pathway. LIM-domain-binding protein 1 (LDB1) interacts with the Wnt signaling pathway and has been connected to malignant diseases. We therefore aimed to evaluate the role of LDB1 in CRC. RESULTS: Overexpression of LDB1 in CRC is associated with strikingly reduced overall and metastasis free survival in all three independent patient cohorts. The expression of LDB1 positively correlates with genes involved in the Wnt signaling pathway (CTNNB1, AXIN2, MYC and CCND1). Overexpression of LDB1 in CRC cell lines induced Wnt pathway upregulation as well as increased invasivity and proliferation. Upon separate analysis, the role of LDB1 proved to be more prominent in proximal CRC, whereas distal CRC seems to be less influenced by LDB1. MATERIALS AND METHODS: The expression of LDB1 was measured via RT-qPCR in 59 clinical tumor and normal mucosa samples and correlated to clinical end-points. The role of LDB1 was examined in two additional large patient cohorts from publicly available microarray and RNAseq datasets. Functional characterization was done by lentiviral overexpression of LDB1 in CRC cell lines and TOP/FOP, proliferation and scratch assays. CONCLUSIONS: LDB1 has a strong role in CRC progression, confirmed in three large, independent patient cohorts. The in vitro data confirm an influence of LDB1 on the Wnt signaling pathway and tumor cell proliferation. LDB1 seems to have a more prominent role in proximal CRC, which confirms the different biology of proximal and distal CRC.

Biophysical Properties of Optogenetic Tools and Their Application for Vision Restoration Approaches.

Optogenetics is the use of genetically encoded light-activated proteins to manipulate cells in a minimally invasive way using light. The most prominent example is channelrhodopsin-2 (ChR2), which allows the activation of electrically excitable cells via light-dependent depolarization. The combination of ChR2 with hyperpolarizing-light-driven ion pumps such as the Cl(-) pump halorhodopsin (NpHR) enables multimodal remote control of neuronal cells in culture, tissue, and living animals. Very soon, it became obvious that this method offers a chance of gene therapy for many diseases affecting vision. Here, we will give a brief introduction to retinal function and retinal diseases; optogenetic vision restoration strategies will be highlighted. We will discuss the functional and structural properties of rhodopsin-based optogenetic tools and analyze the potential for the application of vision restoration.

Circulating tumor cells exhibit stem cell characteristics in an orthotopic mouse model of colorectal cancer.

The prognosis of colorectal cancer (CRC) is closely linked to the occurrence of distant metastases, which putatively develop from circulating tumor cells (CTCs) shed into circulation by the tumor. As far more CTCs are shed than eventually metastases develop, only a small subfraction of CTCs harbor full tumorigenic potential. The aim of this study was to further characterize CRC-derived CTCs to eventually identify the clinically relevant subfraction of CTCs.We established an orthotopic mouse model of CRC which reliably develops metastases and CTCs. We were able to culture the resulting CTCs in vitro, and demonstrated their tumor-forming capacity when re-injected into mice. The CTCs were then subjected to qPCR expression profiling, revealing downregulation of epithelial and proliferation markers. Genes associated with cell-cell adhesion (claudin-7, CD166) were significantly downregulated, indicating a more metastatic phenotype of CTCs compared to bulk tumor cells derived from hepatic metastases. The stem cell markers DLG7 and BMI1 were significantly upregulated in CTC, indicating a stem cell-like phenotype and increased capacity of tumor formation and self-renewal. In concert with their in vitro and in vivo tumorigenicity, these findings indicate stem cell properties of mouse-derived CTCs.In conclusion, we developed an orthotopic mouse model of CRC recapitulating the process of CRC dissemination. CTCs derived from this model exhibit stem-cell like characteristics and are able to form colonies in vitro and tumors in vivo. Our results provide new insight into the biology of CRC-derived CTCs and may provide new therapeutic targets in the metastatic cascade of CRC.

Thermal activation of catalytic microjets in blood samples using microfluidic chips.

We demonstrate that catalytic microjet engines can out-swim high complex media composed of red blood cells and serum. Despite the challenge presented by the high viscosity of the solution at room temperature, the catalytic microjets can be activated at physiological temperature and, consequently, self-propel in diluted solutions of blood samples. We prove that these microjets self-propel in 10× diluted blood samples using microfluidic chips.

Characterization and manipulation of foamy virus membrane interactions.

Foamy viruses (FVs), a unique type of retroviruses, are characterized by several unusual features in their replication strategy. FVs, common to all non-human primates and several other species, display an extremely broad tropism in vitro. Basically, all mammalian cells and species examined, but also cells of amphibian or bird origin, are permissive to FV glycoprotein (Env)-mediated capsid release into the cytoplasm. The nature of the broadly expressed, and potentially evolutionary conserved, FV entry receptor molecule(s) is poorly characterized. Although recent data indicate that proteoglycans serve as an important factor for FV Env-mediated target cell attachment, additional uncharacterized molecules appear to be essential for the pH-dependent fusion of viral and cellular lipid membranes after endocytic uptake of virions. Furthermore, FVs show a very special assembly strategy. Unlike other retroviruses, the FV capsid precursor protein (Gag) undergoes only very limited proteolytic processing during assembly. This results in an immature morphology of capsids found in released FV virions. In addition, the FV Gag protein appears to lack a functional membrane-targeting signal. As a consequence, FVs utilize a specific interaction between capsid and cognate viral glycoprotein for initiation of thebudding process. Genetic fusion of heterologous targeting domains for plasma but not endosomal membranes to FV Gag enables glycoprotein-independent particle egress. However, this is at the expense of normal capsid morphogenesis and infectivity. The low-level Gag precursor processing and the requirement for a reversible, artificial Gag membrane association for effective pseudotyping of FV capsids by heterologous glycoproteins strongly suggest that FVs require a transient interaction of capsids with cellular membranes for viral replication. Under natural condition, this appears to be achieved by the lack of a membrane-targeting function of the FV Gag protein and the accomplishment of capsid membrane attachment through an unusual specific interaction with the cognate glycoprotein.

A small-molecule-controlled system for efficient pseudotyping of prototype foamy virus vectors.

Foamy virus (FV) vector systems have recently demonstrated their power as efficient gene transfer tools for different target tissues. Unfortunately, FVs cannot be naturally pseudotyped by heterologous viral glycoproteins due to an unusual particle morphogenesis involving a FV Env-dependent particle release process. Therefore, current FV vector systems are constrained to the broad host cell range provided by the cognate viral glycoprotein. We evaluated different approaches for pseudotyping of FV vectors, in which the specific FV Gag-Env interaction, essential for particle egress, is substituted by a small-molecule controlled heterodimerization (HD) system. In one system developed, one HD-domain (HDD) is fused to a membrane-targeting domain (MTD), such as the human immunodeficiency virus (HIV) Gag matrix (MA) subunit, with a second fused to the FV capsid protein. Coexpression of both components with different heterologous viral glycoproteins allowed an efficient, dimerizer-dependent pseudotyping of FV capsids. With this system FV vesicular stomatitis virus glycoprotein (VSV-G) pseudotype titers greater than 1 × 10(6) IU/ml were obtained, at levels comparable to authentic FV vector particles. As a proof-of-principle we demonstrate that Pac2 cells, naturally resistant to FV vectors, become permissive to FV VSV-G pseudotypes. Similar to other retroviral vectors, this FV pseudotyping system now enables adaptation of cell-specific targeting approaches for FVs.

Orthoretroviral-like prototype foamy virus Gag-Pol expression is compatible with viral replication.

BACKGROUND: Foamy viruses (FVs) unlike orthoretroviruses express Pol as a separate precursor protein and not as a Gag-Pol fusion protein. A unique packaging strategy, involving recognition of briding viral RNA by both Pol precursor and Gag as well as potential Gag-Pol protein interactions, ensures Pol particle encapsidation. RESULTS: Several Prototype FV (PFV) Gag-Pol fusion protein constructs were generated to examine whether PFV replication is compatible with an orthoretroviral-like Pol expression. During their analysis, non-particle-associated secreted Pol precursor protein was discovered in extracellular wild type PFV particle preparations of different origin, copurifying in simple virion enrichment protocols. Different analysis methods suggest that extracellular wild type PFV particles contain predominantly mature p85(PR-RT) and p40(IN) Pol subunits. Characterization of various PFV Gag-Pol fusion constructs revealed that PFV Pol expression in an orthoretroviral manner is compatible with PFV replication as long as a proteolytic processing between Gag and Pol proteins is possible. PFV Gag-Pol translation by a HIV-1 like ribosomal frameshift signal resulted in production of replication-competent virions, although cell- and particle-associated Pol levels were reduced in comparison to wild type. In-frame fusion of PFV Gag and Pol ORFs led to increased cellular Pol levels, but particle incorporation was only marginally elevated. Unlike that reported for similar orthoretroviral constructs, a full-length in-frame PFV Gag-Pol fusion construct showed wildtype-like particle release and infectivity characteristics. In contrast, in-frame PFV Gag-Pol fusion with C-terminal Gag ORF truncations or non-removable Gag peptide addition to Pol displayed wildtype particle release, but reduced particle infectivity. PFV Gag-Pol precursor fusion proteins with inactivated protease were highly deficient in regular particle release, although coexpression of p71(Gag) resulted in a significant copackaging of these proteins. CONCLUSIONS: Non-particle associated PFV Pol appears to be naturally released from infected cells by a yet unknown mechanism. The absence of particle-associated Pol precursor suggests its rapid processing upon particle incorporation. Analysis of different PFV Gag-Pol fusion constructs demonstrates that orthoretroviral-like Pol expression is compatible with FV replication in principal as long as fusion protein processing is possible. Furthermore, unlike orthoretroviruses, PFV particle release and infectivity tolerate larger differences in relative cellular Gag/Pol levels.

Analysis of prototype foamy virus particle-host cell interaction with autofluorescent retroviral particles.

BACKGROUND: The foamy virus (FV) replication cycle displays several unique features, which set them apart from orthoretroviruses. First, like other B/D type orthoretroviruses, FV capsids preassemble at the centrosome, but more similar to hepadnaviruses, FV budding is strictly dependent on cognate viral glycoprotein coexpression. Second, the unusually broad host range of FV is thought to be due to use of a very common entry receptor present on host cell plasma membranes, because all cell lines tested in vitro so far are permissive. RESULTS: In order to take advantage of modern fluorescent microscopy techniques to study FV replication, we have created FV Gag proteins bearing a variety of protein tags and evaluated these for their ability to support various steps of FV replication. Addition of even small N-terminal HA-tags to FV Gag severely impaired FV particle release. For example, release was completely abrogated by an N-terminal autofluorescent protein (AFP) fusion, despite apparently normal intracellular capsid assembly. In contrast, C-terminal Gag-tags had only minor effects on particle assembly, egress and particle morphogenesis. The infectivity of C-terminal capsid-tagged FV vector particles was reduced up to 100-fold in comparison to wild type; however, infectivity was rescued by coexpression of wild type Gag and assembly of mixed particles. Specific dose-dependent binding of fluorescent FV particles to target cells was demonstrated in an Env-dependent manner, but not binding to target cell-extracted- or synthetic- lipids. Screening of target cells of various origins resulted in the identification of two cell lines, a human erythroid precursor- and a zebrafish- cell line, resistant to FV Env-mediated FV- and HIV-vector transduction. CONCLUSIONS: We have established functional, autofluorescent foamy viral particles as a valuable new tool to study FV--host cell interactions using modern fluorescent imaging techniques. Furthermore, we succeeded for the first time in identifying two cell lines resistant to Prototype Foamy Virus Env-mediated gene transfer. Interestingly, both cell lines still displayed FV Env-dependent attachment of fluorescent retroviral particles, implying a post-binding block potentially due to lack of putative FV entry cofactors. These cell lines might ultimately lead to the identification of the currently unknown ubiquitous cellular entry receptor(s) of FVs.