Biology subjects

Baranov, P.

Publications and source records attributed to Baranov, P..

17 recordsLinked to original sources

bioRxiv2026

A lifespan single-cell atlas of the human developing hippocampus benchmarks familial Alzheimer's disease brain organoids.

Familial Alzheimer's disease (fAD) is an early-onset form of AD caused by autosomal-dominant variants in APP, PSEN1, or PSEN2, with PSEN1 accounting for most genetically defined cases [1]. The hippocampus is among the earliest and most severely affected brain regions in AD [2,3]. Human induced pluripotent stem cell (iPSC)-derived brain organoids recapitulate key features of early human brain development and provide a tractable model for studying how fAD mutations perturb neurodevelopmental processes [4]. However, their interpretation is complicated by heterogeneous regional identity, variable maturation state, and cell-type composition across protocols [5,6]. Existing single-cell studies of human hippocampus cover prenatal [7] and postnatal [8-10] stages but do not provide a continuous developmental reference. By elevating the atlas approach in utilizing single-cell RNA-sequencing data, we obtain standardized information on the organoid cell class and type composition and maturation states. Here, we constructed the Human Developing Hippocampus Atlas (HuDeHA), an integrated single-cell reference comprising 658,059 cells spanning post-conceptional week 3 to 15.3 years, and used it to benchmark iPSC-derived brain organoids carrying PSEN1 E280A which is associated with fAD in a large Colombian population. Reference-based mapping revealed altered cellular composition in PSEN1 E280A organoids, including reduced radial glia and increased neural crest-derived neurons. These changes were accompanied by cross-lineage transcriptional alterations, including broad upregulation of the ventral patterning factor MEIS2 and reduced expression of the {beta}-binding protein transthyretin (TTR) in choroid-plexus and ependymal-associated populations. Reconstructed neuronal-lineage trajectories showed a shift toward mature states in PSEN1 E280A organoids. Together, these findings establish HuDeHA as a resource for developmental benchmarking of hippocampus-relevant organoid systems and describe cell-lineage-specific developmental changes in PSEN1 E280A organoids that may inform interpretation of early cellular alterations in fAD.

neuroscience↗

bioRxiv2026

Quantitative assessment of cell fate commitment in single-cell transcriptomics using scCS

Cell fate trajectory inference is one of the key downstream methods in single-cell RNA-sequencing data analysis. Multiple existing tools allow studying such and help identify continuous cell fate dynamics, important genes along the trajectory, and reconstruct the transcriptional states a cell passes to its estimated final state. These methods have been essential to study various biological systems, yet cell fate by itself currently presents mostly qualitative analysis, and existing tools do not allow to directly quantify the fate-related parameters, including commitment, transition speed, fate affinity and entropy. Such quantifications may be performed through multiple parameters and result in better understanding and description of cell fates. We present scCS (single-cell Commitment Scoring), a scverse-friendly Python framework for this problem. scCS introduces Discounted Future-Fate Propagation (DFFP), which models the source transition graph as a geometrically stopped random walk that can reach endpoint anchors or stop unresolved, with a user-defined finite expected graph horizon. For each cell, the resulting probabilities are separated into total fate reach, relative affinity among reached fates, entropy-based fate specificity and reach-supported resolved commitment, while Signed Ordering Flux independently quantifies local progression. scCS also provides endpoint-anchor, graph-coverage and horizon-sensitivity diagnostics, an instantaneous local-direction mode, standardized visualizations, gene-level analyses and replicate-aware comparisons across experimental conditions. Applications to pancreatic endocrinogenesis and neural crest-Schwann-cell differentiation illustrate the general framework. scCS converts an explicit biological fate hypothesis into auditable cell-, population- and replicate-level quantities without redefining the source dynamics.

bioinformatics↗

bioRxiv2026

Mitochondrial cytochrome c accumulation accompanies reduced electron flux through complex IV without enhancing cell sensitivity to apoptosis

We show that chronic impairment of mitochondrial respiration is associated with marked accumulation of cytochrome c (Cytc) protein. Using SCO2-deficient HCT116 cells lacking functional cytochrome c oxidase and wild-type cells exposed to sustained hypoxia, we found that substantial mitochondrial Cytc accumulation parallels reduced electron flux through Cytc. SCO2-deficient cells exhibited equally elevated Cytc levels under normoxia (19% O2) and hypoxia (0.1-3% O2). Wild-type cells under sustained hypoxia accumulated Cytc, reaching levels comparable to those in SCO2-deficient cells. This effect was reversible upon reoxygenation. Increased Cytc protein levels were also observed in other cell models, including primary cortical neurons cultured under chronic hypoxia and in cerebral cortex tissue from hypoxia-exposed mice. Cytc accumulation occurred independently of CYCS transcription, mRNA translation, HIF activation, ROS production and changes in mitochondrial network. Pharmacological inhibition of complex III was likewise accompanied by increased Cytc levels, whereas mitochondrial uncoupling had no effect, suggesting that impaired electron transfer rather than membrane depolarisation per se underlies this association. Raman spectroscopy revealed enrichment of reduced Cytc and an increased Cytc-to-cytochrome b ratio in respiration-deficient cells. Further supporting a stabilisation-based mechanism, the fraction of membrane-unbound ferro-Cytc was decreased in SCO2-deficient cells, consistent with moderate cardiolipin enrichment, which is known to enhance retention of Cytc at the inner mitochondrial membrane. Despite elevated mitochondrial Cytc content, SCO2-deficient cells were less susceptible to apoptosis induced by intermittent hypoxia or dichloroacetate. Together, these findings indicate that reduced electron flux through complex IV is associated with Cytc accumulation through increased protein stability and membrane retention without enhancing apoptotic sensitivity.

Cell Biology↗

bioRxiv2025

Mechanism of Ribosome Stalling by the AMD1 C-terminal Tail Arrest Peptide

AMD1 encodes Adenosylmethionine decarboxylase 1 (AMD1), a key enzyme required for polyamine biosynthesis. A subset of ribosomes translating the AMD1 coding sequence (CDS) read through the stop codon and pause at the next in-frame stop codon 384 nucleotides downstream. The resulting C-terminal extension (C-tail) is universally conserved across all vertebrates, implying that its molecular function is critical to their fitness. Despite growing evidence that such cis-acting elements regulate translation of their genes, the molecular mechanism by which the C-tail mediates ribosome stalling remains unclear. Here, we determined the structure of the ribosome nascent chain complex paused by the AMD1 C-tail which traps eukaryotic release factor 1 (eRF1) with the ATP-binding cassette sub-family E member 1 (ABCE1). The nascent chain forms a molecular clamp that positions an arginine finger in the peptidyl-transferase center, occluding the accommodation of the eRF1 GGQ motif thereby hampering translation termination. Analysis of aggregated ribosome profiling data revealed several genes with a pattern of stop codon readthrough followed by ribosome stalling at a specific location, suggesting that regulatory readthrough-stall mechanisms may not be limited to AMD1.

biophysics↗

bioRxiv2025

Human retinal organoid single-cell atlas allows to reconstruct retinal development at high resolution and identify nature restricted transcriptional states in vitro

The vertebrate retina is a highly specialized neural structure crucial for the initial capture and processing of visual information. Perturbations in retinal development contribute significantly to congenital and acquired forms of blindness, underscoring the necessity of elucidating human retinal ontogeny. Human pluripotent stem cell-derived retinal organoids have emerged as a powerful model capable of self-organizing and recapitulating key aspects of retinal morphogenesis, offering valuable insights into retinal development and disease. Despite their utility, the extensive cellular heterogeneity, dynamic state transitions, and existence of unique, the development of this system has not yet been fully resolved, posing significant challenges for interpreting their developmental fidelity and therapeutic applicability. Here, we present the Human Retinal Organoid Cell Atlas (HROCA), a comprehensive single-cell transcriptomic reference spanning retinal organoid development from Day 10 to Day 365, integrating data from 15 studies, 71 batches, and encompassing 458,309 cells. HROCA provides a detailed benchmark for organoid differentiation, identifying known retinal cell classes and novel cellular states at single-cell resolution. By associating HROCA with the human fetal retina atlas (HFR), we offer an in-depth comparison between in vitro and in vivo retinal development, revealing protocol-specific differences, limitations in neuronal maturation, and developmental timing discrepancies. HROCA expands our understanding of retinal developmental plasticity, highlighting unique cell populations such as chimeric, non-canonical, and apatride cells, which do not conform to classical retinal cell types but exist consistently across developmental timelines in vitro. This atlas serves as a crucial resource for understanding retinal development, evaluating organoid models, and guiding future improvements in regenerative medicine strategies.

developmental biology↗

bioRxiv2025

Translation of numerous upstream open reading frames rather than their products is essential for proliferation of human cells of distinct origin

Upstream open reading frames (uORFs) are a widespread class of translated regions (translons) occurring in 5' leaders of mRNAs and serving critical roles in post-transcriptional regulation. However, their specific biological activities in human cells remains to be fully elucidated. Here, we conducted a genome-wide CRISPR-Cas9 loss-of-function screen of 978 uORFs identified with ribosome profiling, across human cell lines of distinct origin (HAP1, A549 and HEK293T). A total of 155 uORFs were identified as being essential for cell proliferation. These uORFs showed a high cell-type specificity, with only a few being universally essential. Subsequent analysis has revealed that the primary reason underlying the uORF essentiality is not encoded micropeptides, but rather cis-regulatory mechanisms. Moreover, uORFs located within short 5' UTRs were disproportionately sensitive to frameshift-inducing indels, which frequently lead to the uORF extension and overlap with the coding region (CDS), resulting in translational repression. Finally, by intersecting regions of essential uORF with ClinVar and dbSNP datasets, we identified naturally occurring variants with the potential to disrupt their function and contribute to disease phenotypes. These findings highlight a pervasive and underappreciated layer of translational control in human cells and establish uORFs as critical cis-regulatory elements with potential relevance to human health.

molecular biology↗

bioRxiv2025

Single-cell transcriptome of retinal myeloid cells in response to transplantation of human neurons reveals reversibility of microglial activation

The host retinal microglia and macrophage activation remains a major challenge for the integration of donor neurons following transplantation. Previously, we and others have shown that it is possible to increase donor retinal ganglion cell (RGC) survival by inhibiting the microglia-RGC interaction with Annexin V or through reprogramming microglia with the soluble Fas ligand. However, the exact mechanisms of the microglia/macrophage activation and their heterogeneity following transplantation remain unknown. To address this question, the donor RGC were differentiated from Brn3b-Tdtomato-Thy1.2 human embryonic stem cells using a 3D protocol, followed by dissociation and RGC purification. RGC were delivered subretinally (1.5x104 viable cells/eye) into 3-6-month-old CX3CR1GFPknock-in mice. Three days after transplantation retinas were dissociated into single-cell suspension and GFP-positive myeloid cells isolated using FACS. Of the sorted cells, up to 10,000 viable cells per sample were used for single-cell RNA library preparation and sequenced using the 10X Genomics Chromium platform. In addition, several retinas were fixed and stained for donor RGC (mCherry) and host microglia/macrophages (Iba1). RNA Velocity was used to reconstruct the myeloid cell population and activation trajectory from scRNAseq data. We observed continuous bi-directional transition of microglia/macrophages from a homeostatic to an activated state. We also observed that the response to the transplant falls into the classic disease-associated-microglia (DAM) activation paradigm with a decrease in expression of the homeostatic gene Tmem119 and an increase in expression of disease-associated genes including Apoe, Lgals3, and Spp1. Our findings show that the host retinal myeloid cell population undergoes activation upon transplantation of stem-cell derived donor RGC, with a molecular profile of the activated cells similar to that of activated myeloid cells associated with neurodegenerative diseases of the brain and the eye. Advanced integrated transcriptomic analysis shows separate activated-to-homeostatic and homeostatic-to-activated trajectories suggesting the reversibility of this process.

neuroscience↗

bioRxiv2025

Optimizing Tissue Clearing Methods for Improved Imaging of Whole-Mount Retinas

Gene and cell therapies are promising approaches for restoring vision in hereditary and advanced optic neuropathies. However, these therapeutic approaches must be accurately evaluated through a combination of methods, including advanced imaging, to reach the clinic. We present a whole-mount tissue-clearing methodology to improve imaging of donor neuron integration in the retina following cell transplantation in mice. Mouse retinas were processed using five different clearing methods, comparing tissue transparency, pigmentation, and immunohistochemical clarity. Among the tested methods, ScaleS consistently outperformed its peers, demonstrating a 46% increase in tissue transparency and an 89% increase in immunohistochemical clarity compared to controls. We developed a modified version of ScaleS, termed ScaleH, by adding polyvinyl alcohol to reduce fluorescence decay and enhance sample stability. ScaleH maintained fluorescence stability over extended periods (32% less decay) and proved compatible with immunolabeling and endogenous fluorescent reporters, enabling improved visualization of transplanted human stem cell-derived retinal neurons in the mouse retina. Moreover, ScaleH also improved optic nerve imaging, demonstrating the potential for broader neurobiological applications. Our clearing workflow supports robust, high-resolution imaging for evaluating the integration of transplanted cells in regenerative studies.

bioengineering↗

bioRxiv2025

Comprehensive analysis of yeast +1 ribosomal frameshifting unveils a novel stimulator affirming two distinct frameshifting mechanisms

Ribosomal frameshifting is an important, albeit rare, mRNA decoding mechanism that generally allows the synthesis of a single protein from two different reading frames. For most +1 frameshifting cases, the mechanism is commonly presumed to involve dissociation of the P-site tRNA from its cognate codon followed by its movement to the +1 codon, setting the new +1 frame for incoming tRNAs. This movement is stabilized by P-site tRNA pairing with the +1 codon. However, in several occurrences in the yeast Saccharomyces cerevisiae, P-site tRNA re-pairing with the +1 codon is impossible. Two alternative hypotheses exist explaining this observation. One model suggests that +1 frameshifting occurs according to a common mechanism involving P-site movement, while its re-pairing with +1 codon is not essential. The alternative model suggests a distinct mechanism in which the A-site tRNA acceptance at the +1 codon occurs in the absence of P-site tRNA movement relative to mRNA. Here we set out to perform a comprehensive comparative analysis of all known +1 ribosomal frameshifting sites in S. cerevisiae. This included a novel case of +1 ribosomal frameshifting that we discovered during this study. It is required for the expression of LLP1 gene encoding dolichol-linked oligosaccharide pyrophosphatase. During the analysis of all frameshifting contexts, we identified a conserved RNA secondary structure located almost immediately upstream of the ABP140 frameshifting site. This structure substantially increases +1 frameshifting efficiency. The RNA stimulators location suggests that mRNA exiting the ribosome forms this structure, creating an mRNA pulling effect, thus favouring positioning of the +1 codon in the P-site. Placing the stimulator upstream of various known frameshifting sites, revealed that its stimulatory action is selective to those frameshifting sites where P-site tRNA re-pairing is possible, reinforcing the idea of two distinct mechanisms of ribosomal frameshifting.

molecular biology↗

bioRxiv2024

Unraveling the developmental heterogeneity within the human retina to reconstruct the continuity of retinal ganglion cell maturation and stage-specific intrinsic and extrinsic factors

Tissue development is a complex spatiotemporal process with multiple interdependent components. Anatomical, histological, sequencing, and evolutional strategies can be used to profile and explain tissue development from different perspectives. The introduction of scRNAseq methods and the computational tools allows to deconvolute developmental heterogeneity and draw a decomposed uniform map. In this manuscript, we decomposed the development of a human retina with a focus on the retinal ganglion cells (RGC). To increase the temporal resolution of retinal cell classes maturation state we assumed the working hypothesis that that maturation of retinal ganglion cells is a continuous, non-discrete process. We have assembled the scRNAseq atlas of human fetal retina from fetal week 8 to week 27 and applied the computational methods to unravel maturation heterogeneity into a uniform maturation track. We align RGC transcriptomes in pseudotime to map RGC developmental fate trajectories against the broader timeline of retinal development. Through this analysis, we identified the continuous maturation track of RGC and described the cell-intrinsic (DEGs, maturation gene profiles, regulons, transcriptional motifs) and -extrinsic profiles (neurotrophic receptors across maturation, cell-cell interactions) of different RGC maturation states. We described the genes involved in the retina and RGC maturation, including de novo RGC maturation drivers. We demonstrate the application of the human fetal retina atlas as a reference tool, allowing automated annotation and universal embedding of scRNAseq data. Altogether, our findings deepen the current knowledge of the retina and RGC maturation by bringing in the maturation dimension for the cell class vs. state analysis. We show how the pseudotime application contributes to developmental-oriented analyses, allowing to order the cells by their maturation state. This approach not only improves the downstream computational analysis but also provides a true maturation track transcriptomics profile.

bioinformatics↗

bioRxiv2024

Programmed ribosomal frameshifting during PLEKHM2 mRNA decoding generates a constitutively active mediator of kinesin-1-dependent lysosome transport

Programmed ribosomal frameshifting is a process where a proportion of ribosomes change their reading frame on an mRNA1, rephasing the ribosome relative to the mRNA. While frameshifting is commonly employed by viruses2, very few phylogenetically conserved examples are known in nuclear encoded genes and some of the evidence is controversial3,4. Here we report a +1 frameshifting event during decoding of the human gene PLEKHM25. This frameshifting occurs at the sequence UCC_UUU_CGG, which is conserved in vertebrates and is similar to an influenza virus sequence that frameshifts with similar efficiency6,7. The new C-terminal domain generated by this frameshift forms an -helix, which relieves PLEKHM2 from autoinhibition and allows it to move to the tips of cells via association with kinesin-1 without requiring activation by ARL8. Reintroducing both the canonically-translated and frameshifted protein are necessary to restore normal contractile function of PLEKHM2-knockout cardiomyocytes, demonstrating the necessity of frameshifting for normal cardiac activity.

molecular biology↗

bioRxiv2024

Modulation of microglia activation after human donor retinal ganglion cell transplantation

Stem cell-derived retinal ganglion cell transplantation therapy offers a promising avenue for restoring vision in patients with significant retinal ganglion cell loss. The major challenge in this therapeutic approach is ensuring the survival of transplanted human donor retinal ganglion cells within the host retina. Here, we demonstrated the pivotal role of host retinal microglia and macrophages in the rejection, acceptance and survival of human donor cells. To identify the potential targets for treatment in microglia-retinal ganglion cell interaction, we assembled a single-cell atlas for mouse retina during development, aging, maturation and across neurodegenerative conditions. To our knowledge, this is the largest integrated atlas containing 1,053,629 cells including 36,080 myeloid cells. The downstream analysis highlighted phagocytosis among other known pathways involved in microglia activation, neuroinflammation and host retinal ganglion cell damage. We hypothesized that the same mechanisms are responsible for donor retinal ganglion cell removal. We showed that it is possible to improve the survival of human donor stem cell-derived retinal ganglion cells through modulation of the host retinal microglia. Pretreatment of human donor retinal ganglion cells with annexin V and/or soluble Fas ligand before transplantation led to a 2.5-fold increase in donor cell survival, including the outgrowth of axons targeting optic nerve head. Detailed analyses of the host mouse retina post-transplantation revealed morphological changes microglia and macrophages typical for activation in neuroinflammation. These findings indicate that soluble Fas ligand and annexin V treatment can be used to improve the success rate of transplantation of neurons within the retina and central nervous system.

cell biology↗

bioRxiv2024

Sustained neurotrophic factor cotreatment enhances donor and host retinal ganglion cell survival in mice

Retinal ganglion cells (RGCs) lack regenerative capacity in mammals, and their degeneration in glaucoma leads to irreversible blindness. Autologous and allogeneic RGC replacement with stem cell-derived neurons has been established as a promising strategy for vision restoration, but it has been limited by relatively low (<1%) survival rates of donor cells in the hostile microenvironment of a diseased retina and optic nerve. Brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) are known to support the survival of neurons, including RGCs in. vitro and in vivo. Here, we aim to improve donor RGC survival by supplementing our in vitro cultures and in transplants with a slow-release formulation of these neuroprotective agents. We show that slow-release BDNF/GDNF significantly enhances RGC differentiation, survival, and function in vitro. Furthermore, we demonstrated that BDNF/GDNF co-treatment improved mouse and human stem cell-derived donor RGC transplantation outcomes by 2.7- and 15-fold in mice, respectively. Lastly, we show that slow-release BDNF/GDNF provides neuroprotective effects on host RGCs, preserving retinal function in a model of optic neuropathy. Altogether, this approach of engineering the retinal microenvironment with slow-release neurotrophic factors significantly enhances both donor and host neuron survival, representing a promising approach for treating glaucoma and other optic neuropathies. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/583961v3_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@62d2eorg.highwire.dtl.DTLVardef@1805635org.highwire.dtl.DTLVardef@9bbea7org.highwire.dtl.DTLVardef@101e2f9_HPS_FORMAT_FIGEXP M_FIG C_FIG Engineering the retinal environment with neurotrophic factors to mimic development improves donor and host neuron survival, outgrowth, and function in an optic neuropathy mouse model.

bioengineering↗

bioRxiv2023

Suppressing DNMT3a Alleviates the Intrinsic Epigenetic Barrier for Optic Nerve Regeneration and Restores Vision in Adult Mice

The limited regenerative potential of the optic nerve in adult mammals presents a major challenge for restoring vision after optic nerve trauma or disease. The mechanisms of this regenerative failure are not fully understood1,2. Here, through small-molecule and genetic screening for epigenetic modulators3, we identify DNA methyltransferase 3a (DNMT3a) as a potent inhibitor of axon regeneration in mouse and human retinal explants. Selective suppression of DNMT3a in retinal ganglion cells (RGCs) by gene targeting or delivery of shRNA leads to robust, full-length regeneration of RGC axons through the optic nerve and restoration of vision in adult mice after nerve crush injury. Genome-wide bisulfite and transcriptome profiling in combination with single nucleus RNA-sequencing of RGCs revealed selective DNA demethylation and reactivation of genetic programs supporting neuronal survival and axonal growth/regeneration by DNMT3a deficiency. This was accompanied by the suppression of gene networks associated with apoptosis and inflammation. Our results identify DNMT3a as the central orchestrator of an RGC-intrinsic mechanism that limits optic nerve regeneration. Suppressing DNMT3a expression in RGCs unlocks the epigenetic switch for optic nerve regeneration and presents a promising therapeutic avenue for effectively reversing vision loss resulted from optic nerve trauma or diseases.

neuroscience↗

bioRxiv2023

Ribosome Decision Graphs for the Representation of Eukaryotic RNA Translation Complexity

The application of ribosome profiling has revealed an unexpected abundance of translation in addition to that responsible for the synthesis of previously annotated protein-coding regions. Multiple short sequences have been found to be translated within single RNA molecules, both within annotated protein-coding and non-coding regions. The biological significance of this translation is a matter of intensive investigation. However, current schematic or annotation-based representations of mRNA translation generally do not account for the apparent multitude of translated regions within the same molecules. They also do not take into account the stochasticity of the process that allows alternative translations of the same RNA molecules by different ribosomes. There is a need for formal representations of mRNA complexity that would enable the analysis of quantitative information on translation and more accurate models for predicting the phenotypic effects of genetic variants affecting translation. To address this, we developed a conceptually novel abstraction that we term Ribosome Decision Graphs (RDGs). RDGs represent translation as multiple ribosome paths through untranslated and translated mRNA segments. We termed the later translons. Non-deterministic events, such as initiation, re-initiation, selenocysteine insertion or ribosomal frameshifting are then represented as branching points. This representation allows for an adequate representation of eukaryotic translation complexity and focuses on locations critical for translation regulation. We show how RDGs can be used for depicting translated regions, analysis of genetic variation and quantitative genome-wide data on translation for characterisation of regulatory modulators of translation.

genomics↗

bioRxiv2022

Non-triplet genetic code in ciliate Euplotes ciliates is a result of neutral evolution

Although several variants of the standard genetic code are known, its triplet character is universal with an exception in ciliates Euplotes, where stop codons at internal mRNA positions specify ribosomal frameshifting. How did Euplotes spp. evolved and maintained such an unusual genetic code remains a mystery. To investigate these questions, we explored the evolution of frameshifting occurrence in Euplotes genes. We sequenced and analyzed several transcriptomes from different Euplotes spp to characterize the gain-and-loss dynamics of frameshift sites. Surprisingly, we found a sharp asymmetry between frameshift gain and frameshift loss events with the former exceeding the latter by about 10 folds. Further analysis of mutation rates in protein-coding and non-coding regions revealed that this asymmetry is expected based on single nucleotide mutation rates and does not require positive selection for frameshifting. We found that the number of frameshift sites in Euplotes spp is increasing and is far from the steady state. The steady equilibrium state is expected in about 0.1 to 1 billion years leading to about a 10 fold increase in the number of frameshift sites in Euplotes genes.

evolutionary biology↗

bioRxiv2022

Introduced chemokine gradients guide transplanted and regenerated retinal neurons toward their natural position in the retina

Ongoing cell replacement studies and clinical trials have demonstrated the need to control donor and newborn cell behavior within their target tissue. Here we present a methodology to guide stem cell-derived and endogenously regenerated neurons by engineering the microenvironment. Being an "approachable part of the brain," the eye provides a unique opportunity to study donor neuron fate, migration, and integration within the central nervous system. Glaucoma and other optic neuropathies lead to the permanent loss of retinal ganglion cells (RGCs) - the neurons in the retina that transfer all visual information from the eye to the brain. Cell transplantation and transdifferentiation strategies have been proposed to restore RGCs, and one of the significant barriers to successful RGC integration into the existing retinal circuitry is cell migration towards their natural position in the retina. Here we describe a framework for identifying, selecting, and applying chemokines to direct cell migration in vivo within the retina. We have performed an in silico analysis of the single-cell transcriptome of the developing human retina and identified six receptor-ligand candidates to guide stem cell-derived or newborn neurons. The lead candidates were then tested in functional in vitro assays for their ability to guide stem cell-derived RGCs. For the in vivo studies, donor and newborn neurons were differentiated in human and mouse retinal organoids or endogenously reprogrammed with proneuronal transcription factors, respectively. An exogenous stromal cell-derived factor-1 (SDF1) gradient led to a 2.7-fold increase in donor RGC migration into the ganglion cell layer and a 3.3-fold increase in the displacement of newborn RGCs out of the inner nuclear layer. Furthermore, by altering the migratory profile of donor RGCs toward multipolar migration, overall migration was improved in mature retinal tissues. Together, these results highlight the ability and importance of engineering the tissue microenvironment and the individual cells for research and clinical applications in gene and cell therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/510158v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@1856140org.highwire.dtl.DTLVardef@1b3ca26org.highwire.dtl.DTLVardef@9c9b34org.highwire.dtl.DTLVardef@19187c0_HPS_FORMAT_FIGEXP M_FIG C_FIG In brief, the "in silico - in vitro - in vivo" funnel holds significant potential for identifying targets to control cellular processes in research and clinical applications. In this report, Soucy et al. describes a framework for identifying, selecting, and applying chemokines to direct retinal ganglion cell migration in vivo within the adult mouse retina.

bioengineering↗