Small-Molecule-Directed Endogenous Regeneration of Visual Function in a Mammalian Retinal Degeneration Model.

Degenerative retinal diseases associated with photoreceptor loss are a leading cause of visual impairment worldwide, with limited treatment options. Phenotypic profiling coupled with medicinal chemistry were used to develop a small molecule with proliferative effects on retinal stem/progenitor cells, as assessed in vitro in a neurosphere assay and in vivo by measuring Msx1-positive ciliary body cell proliferation. The compound was identified as having kinase inhibitory activity and was subjected to cellular pathway analysis in non-retinal human primary cell systems. When tested in a disease-relevant murine model of adult retinal degeneration (MNU-induced retinal degeneration), we observed that four repeat intravitreal injections of the compound improved the thickness of the outer nuclear layer along with the regeneration of the visual function, as measured with ERG, visual acuity, and contrast sensitivity tests. This serves as a proof of concept for the use of a small molecule to promote endogenous regeneration in the eye.

Direct neuronal reprogramming of NDUFS4 patient cells identifies the unfolded protein response as a novel general reprogramming hurdle.

Mitochondria account for essential cellular pathways, from ATP production to nucleotide metabolism, and their deficits lead to neurological disorders and contribute to the onset of age-related diseases. Direct neuronal reprogramming aims at replacing neurons lost in such conditions, but very little is known about the impact of mitochondrial dysfunction on the direct reprogramming of human cells. Here, we explore the effects of mitochondrial dysfunction on the neuronal reprogramming of induced pluripotent stem cell (iPSC)-derived astrocytes carrying mutations in the NDUFS4 gene, important for Complex I and associated with Leigh syndrome. This led to the identification of the unfolded protein response as a major hurdle in the direct neuronal conversion of not only astrocytes and fibroblasts from patients but also control human astrocytes and fibroblasts. Its transient inhibition potently improves reprogramming by influencing the mitochondria-endoplasmic-reticulum-stress-mediated pathways. Taken together, disease modeling using patient cells unraveled novel general hurdles and ways to overcome these in human astrocyte-to-neuron reprogramming.

Human pluripotent stem cell fate trajectories toward lung and hepatocyte progenitors.

In this study, we interrogate molecular mechanisms underlying the specification of lung progenitors from human pluripotent stem cells (hPSCs). We employ single-cell RNA-sequencing with high temporal precision, alongside an optimized differentiation protocol, to elucidate the transcriptional hierarchy of lung specification to chart the associated single-cell trajectories. Our findings indicate that Sonic hedgehog, TGF-ß, and Notch activation are essential within an ISL1/NKX2-1 trajectory, leading to the emergence of lung progenitors during the foregut endoderm phase. Additionally, the induction of HHEX delineates an alternate trajectory at the early definitive endoderm stage, preceding the lung pathway and giving rise to a significant hepatoblast population. Intriguingly, neither KDR+ nor mesendoderm progenitors manifest as intermediate stages in the lung and hepatic lineage development. Our multistep model offers insights into lung organogenesis and provides a foundation for in-depth study of early human lung development and modeling using hPSCs.

Comprehensive chromatin proteomics resolves functional phases of pluripotency and identifies changes in regulatory components.

The establishment of cellular identity is driven by transcriptional and epigenetic regulators of the chromatin proteome - the chromatome. Comprehensive analyses of the chromatome composition and dynamics can therefore greatly improve our understanding of gene regulatory mechanisms. Here, we developed an accurate mass spectrometry (MS)-based proteomic method called Chromatin Aggregation Capture (ChAC) followed by Data-Independent Acquisition (DIA) and analyzed chromatome reorganizations during major phases of pluripotency. This enabled us to generate a comprehensive atlas of proteomes, chromatomes, and chromatin affinities for the ground, formative and primed pluripotency states, and to pinpoint the specific binding and rearrangement of regulatory components. These comprehensive datasets combined with extensive analyses identified phase-specific factors like QSER1 and JADE1/2/3 and provide a detailed foundation for an in-depth understanding of mechanisms that govern the phased progression of pluripotency. The technical advances reported here can be readily applied to other models in development and disease.

Depletion of the RNA-binding protein PURA triggers changes in posttranscriptional gene regulation and loss of P-bodies.

The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular roles and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6 and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs are not affected. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study proposes PURA Syndrome as a new model to study the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders.

Human genetic defects in SRP19 and SRPRA cause severe congenital neutropenia with distinctive proteome changes.

The mechanisms of coordinated changes in proteome composition and their relevance for the differentiation of neutrophil granulocytes are not well studied. Here, we discover 2 novel human genetic defects in signal recognition particle receptor alpha (SRPRA) and SRP19, constituents of the mammalian cotranslational targeting machinery, and characterize their roles in neutrophil granulocyte differentiation. We systematically study the proteome of neutrophil granulocytes from patients with variants in the SRP genes, HAX1, and ELANE, and identify global as well as specific proteome aberrations. Using in vitro differentiation of human induced pluripotent stem cells and in vivo zebrafish models, we study the effects of SRP deficiency on neutrophil granulocyte development. In a heterologous cell-based inducible protein expression system, we validate the effects conferred by SRP dysfunction for selected proteins that we identified in our proteome screen. Thus, SRP-dependent protein processing, intracellular trafficking, and homeostasis are critically important for the differentiation of neutrophil granulocytes.

Intron-encoded cistronic transcripts for minimally invasive monitoring of coding and non-coding RNAs.

Despite their fundamental role in assessing (patho)physiological cell states, conventional gene reporters can follow gene expression but leave scars on the proteins or substantially alter the mature messenger RNA. Multi-time-point measurements of non-coding RNAs are currently impossible without modifying their nucleotide sequence, which can alter their native function, half-life and localization. Thus, we developed the intron-encoded scarless programmable extranuclear cistronic transcript (INSPECT) as a minimally invasive transcriptional reporter embedded within an intron of a gene of interest. Post-transcriptional excision of INSPECT results in the mature endogenous RNA without sequence alterations and an additional engineered transcript that leaves the nucleus by hijacking the nuclear export machinery for subsequent translation into a reporter or effector protein. We showcase its use in monitoring interleukin-2 (IL2) after T cell activation and tracking the transcriptional dynamics of the long non-coding RNA (lncRNA) NEAT1 during CRISPR interference-mediated perturbation. INSPECT is a method for monitoring gene transcription without altering the mature lncRNA or messenger RNA of the target of interest.

Induced pluripotent stem cells and cerebral organoids from the critically endangered Sumatran rhinoceros.

Less than 80 Sumatran rhinos (SR, Dicerorhinus sumatrensis) are left on earth. Habitat loss and limited breeding possibilities are the greatest threats to the species and lead to a continuous population decline. To stop the erosion of genetic diversity, reintroduction of genetic material is indispensable. However, as the propagation rate of captive breeding is far too low, innovative technologies have to be developed. Induced pluripotent stem cells (iPSCs) are a powerful tool to fight extinction. They give rise to each cell within the body including gametes and provide a unique modality to preserve genetic material across time. Additionally, they enable studying species-specific developmental processes. Here, we generate iPSCs from the last male Malaysian SR Kertam, who died in 2019, and characterize them comprehensively. Differentiation in cells of the three germ layers and cerebral organoids demonstrate their high quality and great potential for supporting the rescue of this critically endangered species.

Is hypoimmunogenic stem cell therapy safe in times of pandemics?

The manipulation of human leukocyte antigens (HLAs) and immune modulatory factors in "universal" human pluripotent stem cells (PSCs) holds promise for immunological tolerance without HLA matching. This paradigm raises concerns should "universal" grafts become virally infected. Furthermore, immunological manipulation might functionally impair certain progeny, such as hematopoietic stem cells. We discuss the risks and benefits of hypoimmunogenic PSCs, and the need to further advance HLA matching and autologous strategies.

Naïve-like pluripotency to pave the way for saving the northern white rhinoceros from extinction.

The northern white rhinoceros (NWR) is probably the earth's most endangered mammal. To rescue the functionally extinct species, we aim to employ induced pluripotent stem cells (iPSCs) to generate gametes and subsequently embryos in vitro. To elucidate the regulation of pluripotency and differentiation of NWR PSCs, we generated iPSCs from a deceased NWR female using episomal reprogramming, and observed surprising similarities to human PSCs. NWR iPSCs exhibit a broad differentiation potency into the three germ layers and trophoblast, and acquire a naïve-like state of pluripotency, which is pivotal to differentiate PSCs into primordial germ cells (PGCs). Naïve culturing conditions induced a similar expression profile of pluripotency related genes in NWR iPSCs and human ESCs. Furthermore, naïve-like NWR iPSCs displayed increased expression of naïve and PGC marker genes, and a higher integration propensity into developing mouse embryos. As the conversion process was aided by ectopic BCL2 expression, and we observed integration of reprogramming factors, the NWR iPSCs presented here are unsuitable for gamete production. However, the gained insights into the developmental potential of both primed and naïve-like NWR iPSCs are fundamental for in future PGC-specification in order to rescue the species from extinction using cryopreserved somatic cells.

The ART of bringing extinction to a freeze - History and future of species conservation, exemplified by rhinos.

The ongoing mass extinction of animal species at an unprecedented rate is largely caused by human activities. Progressive habitat destruction and fragmentation is resulting in accelerated loss of biodiversity on a global scale. Over decades, captive breeding programs of non-domestic species were characterized by efforts to optimize species-specific husbandry, to increase studbook-based animal exchange, and to improve enclosure designs. To counter the ongoing dramatic loss of biodiversity, new approaches are warranted. Recently, new ideas, particularly the application of assisted reproduction technologies (ART), have been incorporated into classical zoo breeding programs. These technologies include semen and oocyte collection, artificial insemination, and in-vitro embryo generation. More futuristic ideas of advanced ART (aART) implement recent advances in biotechnology and stem-cell related approaches such as cloning, inner cell mass transfer (ICM), and the stem-cell-associated techniques (SCAT) for the generation of gametes and ultimately embryos of highly endangered species, such as the northern white rhinoceros (Ceratotherium simum cottoni) of which only two female individuals are left. Both, ART and aART greatly depend on and benefit from the rapidly evolving cryopreservation techniques and biobanking not only of genetic, but also of viable cellular materials suitable for the generation of induced pluripotent stem cells (iPSC). The availability of cryopreserved materials bridges gaps in time and space, thereby optimizing the available genetic variability and enhancing the chance to restore viable populations.

Rescue of STAT3 Function in Hyper-IgE Syndrome Using Adenine Base Editing.

STAT3-hyper IgE syndrome (STAT3-HIES) is a primary immunodeficiency presenting with destructive lung disease along with other symptoms. CRISPR-Cas9-mediated adenine base editors (ABEs) have the potential to correct one of the most common STAT3-HIES causing heterozygous STAT3 mutations (c.1144C>T/p.R382W). As a proof-of-concept, we successfully applied ABEs to correct STAT3 p.R382W in patient fibroblasts and induced pluripotent stem cells (iPSCs). Treated primary STAT3-HIES patient fibroblasts showed a correction efficiency of 29% ± 7% without detectable off-target effects evaluated through whole-genome and high-throughput sequencing. Compared with untreated patient fibroblasts, corrected single-cell clones showed functional rescue of STAT3 signaling with significantly increased STAT3 DNA-binding activity and target gene expression of CCL2 and SOCS3. Patient-derived iPSCs were corrected with an efficiency of 30% ± 6% and differentiated to alveolar organoids showing preserved plasticity in treated cells. In conclusion, our results are supportive for ABE-based gene correction as a potential causative treatment of STAT3-HIES.

Chromatin-Associated Membraneless Organelles in Regulation of Cellular Differentiation.

Membrane-free intracellular biocondensates are enclosures of proteins and nucleic acids that form by phase separation. Extensive ensembles of nuclear "membraneless organelles" indicate their involvement in genome regulation. Indeed, nuclear bodies have been linked to regulation of gene expression by formation of condensates made of chromatin and RNA processing factors. Important questions pertain to the involvement of membraneless organelles in determining cell identity through their cell-type-specific composition and function. Paraspeckles provide a prism to these questions because they exhibit striking cell-type-specific patterns and since they are crucial in embryogenesis. Here, we outline known interactions between paraspeckles and chromatin, and postulate how such interactions may be important in regulation of cell fate transitions. Moreover, we propose long non-coding RNAs (lncRNAs) as candidates for similar regulation because many form foci that resemble biocondensates and exhibit dynamic patterns during differentiation. Finally, we outline approaches that could ascertain how chromatin-associated membraneless organelles regulate cellular differentiation.

In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states.

Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals.

Nucleus size and DNA accessibility are linked to the regulation of paraspeckle formation in cellular differentiation.

BACKGROUND: Many long noncoding RNAs (lncRNAs) have been implicated in general and cell type-specific molecular regulation. Here, we asked what underlies the fundamental basis for the seemingly random appearance of nuclear lncRNA condensates in cells, and we sought compounds that can promote the disintegration of lncRNA condensates in vivo. RESULTS: As a basis for comparing lncRNAs and cellular properties among different cell types, we screened lncRNAs in human pluripotent stem cells (hPSCs) that were differentiated to an atlas of cell lineages. We found that paraspeckles, which form by aggregation of the lncRNA NEAT1, are scaled by the size of the nucleus, and that small DNA-binding molecules promote the disintegration of paraspeckles and other lncRNA condensates. Furthermore, we found that paraspeckles regulate the differentiation of hPSCs. CONCLUSIONS: Positive correlation between the size of the nucleus and the number of paraspeckles exist in numerous types of human cells. The tethering and structure of paraspeckles, as well as other lncRNAs, to the genome can be disrupted by small molecules that intercalate in DNA. The structure-function relationship of lncRNAs that regulates stem cell differentiation is likely to be determined by the dynamics of nucleus size and binding site accessibility.

FICD activity and AMPylation remodelling modulate human neurogenesis.

Posttranslational modification (PTM) of proteins represents an important cellular mechanism for controlling diverse functions such as signalling, localisation or protein-protein interactions. AMPylation (also termed adenylylation) has recently been discovered as a prevalent PTM for regulating protein activity. In human cells AMPylation has been exclusively studied with the FICD protein. Here we investigate the role of AMPylation in human neurogenesis by introducing a cell-permeable propargyl adenosine pronucleotide probe to infiltrate cellular AMPylation pathways and report distinct modifications in intact cancer cell lines, human-derived stem cells, neural progenitor cells (NPCs), neurons and cerebral organoids (COs) via LC-MS/MS as well as imaging methods. A total of 162 AMP modified proteins were identified. FICD-dependent AMPylation remodelling accelerates differentiation of neural progenitor cells into mature neurons in COs, demonstrating a so far unknown trigger of human neurogenesis.

BART-Seq: cost-effective massively parallelized targeted sequencing for genomics, transcriptomics, and single-cell analysis.

We describe a highly sensitive, quantitative, and inexpensive technique for targeted sequencing of transcript cohorts or genomic regions from thousands of bulk samples or single cells in parallel. Multiplexing is based on a simple method that produces extensive matrices of diverse DNA barcodes attached to invariant primer sets, which are all pre-selected and optimized in silico. By applying the matrices in a novel workflow named Barcode Assembly foR Targeted Sequencing (BART-Seq), we analyze developmental states of thousands of single human pluripotent stem cells, either in different maintenance media or upon Wnt/ß-catenin pathway activation, which identifies the mechanisms of differentiation induction. Moreover, we apply BART-Seq to the genetic screening of breast cancer patients and identify BRCA mutations with very high precision. The processing of thousands of samples and dynamic range measurements that outperform global transcriptomics techniques makes BART-Seq first targeted sequencing technique suitable for numerous research applications.

Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition.

RNA-binding proteins (RBPs) and long non-coding RNAs (lncRNAs) are key regulators of gene expression, but their joint functions in coordinating cell fate decisions are poorly understood. Here we show that the expression and activity of the RBP TDP-43 and the long isoform of the lncRNA Neat1, the scaffold of the nuclear compartment "paraspeckles," are reciprocal in pluripotent and differentiated cells because of their cross-regulation. In pluripotent cells, TDP-43 represses the formation of paraspeckles by enhancing the polyadenylated short isoform of Neat1. TDP-43 also promotes pluripotency by regulating alternative polyadenylation of transcripts encoding pluripotency factors, including Sox2, which partially protects its 3' UTR from miR-21-mediated degradation. Conversely, paraspeckles sequester TDP-43 and other RBPs from mRNAs and promote exit from pluripotency and embryonic patterning in the mouse. We demonstrate that cross-regulation between TDP-43 and Neat1 is essential for their efficient regulation of a broad network of genes and, therefore, of pluripotency and differentiation.

Pathological ASXL1 Mutations and Protein Variants Impair Neural Crest Development.

The neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 (ASXL1). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1-900) recapitulated the NC phenotypes in vitro and in ovo, raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC.

Altered neuronal migratory trajectories in human cerebral organoids derived from individuals with neuronal heterotopia.

Malformations of the human cortex represent a major cause of disability1. Mouse models with mutations in known causal genes only partially recapitulate the phenotypes and are therefore not unlimitedly suited for understanding the molecular and cellular mechanisms responsible for these conditions2. Here we study periventricular heterotopia (PH) by analyzing cerebral organoids derived from induced pluripotent stem cells (iPSCs) of patients with mutations in the cadherin receptor-ligand pair DCHS1 and FAT4 or from isogenic knockout (KO) lines1,3. Our results show that human cerebral organoids reproduce the cortical heterotopia associated with PH. Mutations in DCHS1 and FAT4 or knockdown of their expression causes changes in the morphology of neural progenitor cells and result in defective neuronal migration dynamics only in a subset of neurons. Single-cell RNA-sequencing (scRNA-seq) data reveal a subpopulation of mutant neurons with dysregulated genes involved in axon guidance, neuronal migration and patterning. We suggest that defective neural progenitor cell (NPC) morphology and an altered navigation system in a subset of neurons underlie this form of PH.