Stage-specific dynamic reorganization of genome topology shapes transcriptional neighborhoods in developing human retinal organoids.

We have generated a high-resolution Hi-C map of developing human retinal organoids to elucidate spatiotemporal dynamics of genomic architecture and its relationship with gene expression patterns. We demonstrate progressive stage-specific alterations in DNA topology and correlate these changes with transcription of cell-type-restricted gene markers during retinal differentiation. Temporal Hi-C reveals a shift toward A compartment for protein-coding genes and B compartment for non-coding RNAs, displaying high and low expression, respectively. Notably, retina-enriched genes are clustered near lost boundaries of topologically associated domains (TADs), and higher-order assemblages (i.e., TAD cliques) localize in active chromatin regions with binding sites for eye-field transcription factors. These genes gain chromatin contacts at their transcription start site as organoid differentiation proceeds. Our study provides a global view of chromatin architecture dynamics associated with diversification of cell types during retinal development and serves as a foundational resource for in-depth functional investigations of retinal developmental traits.

Large-scale phage cultivation for commensal human gut bacteria.

Phages are highly abundant in the human gut, yet most of them remain uncultured. Here, we present a gut phage isolate collection (GPIC) containing 209 phages for 42 commensal human gut bacterial species. Genome analysis of the phages identified 34 undescribed genera. We discovered 22 phages from the Salasmaviridae family that have small genomes (∼10-20 kbp) and infect Gram-positive bacteria. Two phages from a candidate family, Paboviridae, with high prevalence in the human gut were also identified. Infection assays showed that Bacteroides and Parabacteroides phages are specific to a bacterial species, and strains of the same species also exhibit substantial variations in phage susceptibility. A cocktail of 8 phages with a broad host range for Bacteroides fragilis strains effectively reduced their abundance in complex host-derived communities in vitro. Our study expands the diversity of cultured human gut bacterial phages and provides a valuable resource for human microbiome engineering.

Spatial profiling of microbial communities by sequential FISH with error-robust encoding.

Spatial analysis of microbiomes at single cell resolution with high multiplexity and accuracy has remained challenging. Here we present spatial profiling of a microbiome using sequential error-robust fluorescence in situ hybridization (SEER-FISH), a highly multiplexed and accurate imaging method that allows mapping of microbial communities at micron-scale. We show that multiplexity of RNA profiling in microbiomes can be increased significantly by sequential rounds of probe hybridization and dissociation. Combined with error-correction strategies, we demonstrate that SEER-FISH enables accurate taxonomic identification in complex microbial communities. Using microbial communities composed of diverse bacterial taxa isolated from plant rhizospheres, we apply SEER-FISH to quantify the abundance of each taxon and map microbial biogeography on roots. At micron-scale, we identify clustering of microbial cells from multiple species on the rhizoplane. Under treatment of plant metabolites, we find spatial re-organization of microbial colonization along the root and alterations in spatial association among microbial taxa. Taken together, SEER-FISH provides a useful method for profiling the spatial ecology of complex microbial communities in situ.

In vitro modeling and rescue of ciliopathy associated with IQCB1/NPHP5 mutations using patient-derived cells.

Mutations in the IQ calmodulin-binding motif containing B1 (IQCB1)/NPHP5 gene encoding the ciliary protein nephrocystin 5 cause early-onset blinding disease Leber congenital amaurosis (LCA), together with kidney dysfunction in Senior-Løken syndrome. For in vitro disease modeling, we obtained dermal fibroblasts from patients with NPHP5-LCA that were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into retinal pigment epithelium (RPE) and retinal organoids. Patient fibroblasts and RPE demonstrated aberrantly elongated ciliary axonemes. Organoids revealed impaired development of outer segment structures, which are modified primary cilia, and mislocalization of visual pigments to photoreceptor cell soma. All patient-derived cells showed reduced levels of CEP290 protein, a critical cilia transition zone component interacting with NPHP5, providing a plausible mechanism for aberrant ciliary gating and cargo transport. Disease phenotype in NPHP5-LCA retinal organoids could be rescued by adeno-associated virus (AAV)-mediated IQCB1/NPHP5 gene augmentation therapy. Our studies thus establish a human disease model and a path for treatment of NPHP5-LCA.

CRISPR/Cas-Based Genome Editing for Human Gut Commensal Bacteroides Species.

Bacteroides is the most abundant genus in the human gut microbiome and has been increasingly used as model organisms for studying the function and ecology of the gut microbiome. However, genome editing tools for such commensal gut microbes are still lacking. Here we developed a versatile, highly efficient CRISPR/Cas-based genome editing tool that allows markerless gene deletion and insertion in human gut Bacteroides species. We constructed multiple CRISPR/Cas systems in all-in-one Bacteroides-E. coli shuttle plasmids and systematically evaluated the genome editing efficiency in Bacteroides thetaiotaomicron, including the mode of Cas protein expression (constitutive, inducible), different Cas proteins (FnCas12a, SpRY, SpCas9), and sgRNAs. Using the anhydrotetracycline (aTc)-inducible CRISPR/FnCas12a system, we successfully deleted large genomic fragments up to 50 kb to study the function of metabolic gene clusters. Furthermore, we demonstrated that CRISPR/FnCas12a can be broadly applied to engineer multiple human gut Bacteroides species, including Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis, and Bacteroides vulgatus. We envision that CRISPR/Cas-based genome editing tools for Bacteroides will greatly facilitate mechanistic studies of the gut commensal and the development of engineered live biotherapeutics.

Potential roles of mesenchymal stromal cells in islet allo- and xenotransplantation for type 1 diabetes mellitus.

Islet transplantation is poised to play an important role in the treatment of type 1 diabetes mellitus (T1DM). However, there are several challenges limiting its widespread use, including the instant blood-mediated inflammatory reaction, hypoxic/ischemic injury, and the immune response. Mesenchymal stem/stromal cells (MSCs) are known to exert regenerative, immunoregulatory, angiogenic, and metabolic properties. Here, we review recent reports on the application of MSCs in islet allo- and xenotransplantation. We also document the clinical trials that have been undertaken or are currently underway, relating to the co-transplantation of islets and MSCs. Increasing evidence indicates that co-transplantation of MSCs prolongs islet graft survival by locally secreted protective factors that reduce immune reactivity and promote vascularization, cell survival, and regeneration. MSC therapy may be a promising option for islet transplantation in patients with T1DM.

Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids.

Mutations in the photoreceptor transcription factor gene cone-rod homeobox (CRX) lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs48 mutation, we established an in vitro model of CRX-LCA in retinal organoids that showed defective photoreceptor maturation by histology and gene profiling, with diminished expression of visual opsins. Adeno-associated virus (AAV)-mediated CRX gene augmentation therapy partially restored photoreceptor phenotype and expression of phototransduction-related genes as determined by single-cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying K88N mutation revealed the loss of opsin expression as a common phenotype, which was alleviated by AAV-mediated augmentation of CRX. Our studies provide a proof-of-concept for developing gene therapy of dominant CRX-LCA and other CRX retinopathies.

Structure-guided manipulation of the regioselectivity of the cyclosporine A hydroxylase CYP-sb21 from Sebekia benihana.

The cytochrome P450 enzyme CYP-sb21 from the rare actinomycete Sebekia benihana is capable of hydroxylating the immunosuppressive drug molecule cyclosporine A (CsA) primarily at the 4th N-methyl leucine (MeLeu4), giving rise to γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH). This oxidative modification of CsA leads to dramatically reduced immunosuppressive activity while retaining the hair growth-promoting side-effect, thus demonstrating great application potential in both pharmaceutical and cosmetic industries. However, this P450 enzyme also hydroxylates CsA at the unwanted position of the 9th N-methyl leucine (MeLeu9), indicating that the regioselectivity needs to be improved for the development of CsA-4-OH into a commercial hair growth stimulator. Herein, we report the crystal structure of CYP-sb21 in its substrate-free form at 1.85 Å. Together with sequence and 3D structure comparisons, Autodock-based substrate docking, molecular dynamics (MD) simulation, and site-directed mutagenesis, we identified a number of key residues including R294, E264, and M179 that can improve catalytic efficiency or change the regioselectivity of CYP-sb21 towards CsA, setting the stage for better enzymatic preparation of CsA-4-OH. This study also provides new insights into the substrate recognition and binding mechanism of P450 enzymes that accommodate bulky substrates.

A simple and efficient method for generating human retinal organoids.

Purpose: Retinal organoids (ROs) derived from human pluripotent stem cells largely recapitulate key features of in vivo retinal development, thus permitting the study of retinogenesis, disease modeling, and therapeutic development. However, the complexities of current protocols limit the use of this in vitro system in applications requiring large-scale production of organoids. Currently, widely used methods require the isolation of presumed optic vesicle-like structures from adherent cultures by dissection, a labor-intensive and time-consuming step that involves extensive practice and training. Method: We report a simple and efficient method for generating ROs by scraping the entire adherent culture and growing the resulting cell aggregates in a free-floating condition. Results: Within 1 to 7 days following the procedure, emerging morphologically well-defined optic vesicles can be identified and harvested with ease. The transition from two-dimensional (2D) to 3D culture condition favored the formation of ROs from areas devoid of typical optic vesicle-like structures, thus increasing the RO yield. Moreover, ROs generated by this approach were more often associated with the pigment epithelium. Conclusions: This improved, robust, and efficient protocol should facilitate large-scale differentiation of pluripotent stem cells into retinal organoids in support of human disease modeling and therapy development.

Downregulation of Gabarapl1 significantly attenuates antibody binding to porcine aortic endothelial cells.

After hyperacute rejection in pig-to-primate xenotransplantation had been overcome by the introduction of α1,3-galactosyltransferase gene-knockout (GTKO) pigs, acute and chronic antibody-mediated rejection became one of the major barriers to long-term graft survival. This was associated with exposure of non-Gal antigens to the recipient's immune system and indicated that further genetic engineering of the pigs would be necessary. We here report that Gabarapl1, a regulator of tumorigenesis, plays a role in the regulation of immunogenicity of porcine aortic endothelial cells (PAECs). Knockdown of Gabarapl1 in PAECs results in a remarkable reduction in binding of serum antibody from PAEC-immunized monkeys, associated with decreased serum cytotoxicity of pig cells. Expression of swine leukocyte antigens (SLA) II DR was downregulated by Gabarapl1 knockdown. However, suppression of expression of SLA II is associated with less reduction of antibody binding than achieved by Gabarapl1 knockdown, suggesting that other Gabarapl1-regulated xenoantigens may be more important. These findings indicate a hitherto unknown relationship between Gabarapl1 and xenoimmunogenicity, suggesting a potential new strategy to reduce rejection initiated by the presence of non-Gal antigens.

Compartmentalized biosynthesis of mycophenolic acid.

Mycophenolic acid (MPA) from filamentous fungi is the first natural product antibiotic to be isolated and crystallized, and a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. However, some key biosynthetic mechanisms of such an old and important molecule have remained unclear. Here, we elucidate the MPA biosynthetic pathway that features both compartmentalized enzymatic steps and unique cooperation between biosynthetic and ß-oxidation catabolism machineries based on targeted gene inactivation, feeding experiments in heterologous expression hosts, enzyme functional characterization and kinetic analysis, and microscopic observation of protein subcellular localization. Besides identification of the oxygenase MpaB' as the long-sought key enzyme responsible for the oxidative cleavage of the farnesyl side chain, we reveal the intriguing pattern of compartmentalization for the MPA biosynthetic enzymes, including the cytosolic polyketide synthase MpaC' and O-methyltransferase MpaG', the Golgi apparatus-associated prenyltransferase MpaA', the endoplasmic reticulum-bound oxygenase MpaB' and P450-hydrolase fusion enzyme MpaDE', and the peroxisomal acyl-coenzyme A (CoA) hydrolase MpaH'. The whole pathway is elegantly comediated by these compartmentalized enzymes, together with the peroxisomal ß-oxidation machinery. Beyond characterizing the remaining outstanding steps of the MPA biosynthetic steps, our study highlights the importance of considering subcellular contexts and the broader cellular metabolism in natural product biosynthesis.

Circulating pig-specific DNA as a novel biomarker for monitoring xenograft rejection.

Monitoring for immune rejection is crucial for long-term survival of pig xenografts. Circulating DNA is a promising non-invasive biomarker for either organ injury or response to therapy. In this study, circulating pig-specific DNA (cpsDNA) was monitored during xenograft rejection. Potential targets of cpsDNA were selected by in silico analysis, and species specificity of selected primers was confirmed by PCR. Subsequently, cpsDNA as a biomarker was evaluated using a complement-dependent cytotoxicity (CDC) assay in vitro. Then, early diagnosis and response to rapamycin were assessed by an in vivo imaging model of pig-to-mouse cell transplantation. Finally, cpsDNA was monitored in a pig-to-monkey artery patch transplantation model. The results showed that (a) a method of cpsDNA quantitation was established for application in mouse and nonhuman primate models; (b) cpsDNA reflected CDC in vitro; (c) cpsDNA in vivo mirrored xenograft rejection, and correlated with xenograft loss in pig-to-mouse cell transplantation; (d) cpsDNA was significantly reduced when rapamycin was administered; and (e) dynamic cpsDNA was detectable in pig-to-monkey artery patch transplantation. In conclusion, measurement of cpsDNA could prove to be a less invasive, but more specific and sensitive low-cost biomarker enabling monitoring of xenograft rejection and the response to immunosuppressive therapy.

A potential role of TLR2 in xenograft rejection of porcine iliac endothelial cells: An in vitro study.

BACKGROUND: Porcine vascular endothelial cells are a major participant in xenograft rejection. The Toll-like receptor 2 (TLR2) pathway plays an important role in both innate and adaptive immunity. The specific role of TLR2 in the response to a xenograft has not been reported. Whether the TLR2 pathway in pig vascular endothelial cells is involved in acute rejection needs to be investigated, and the mechanism is explored. METHODS: We used a modified antibody-dependent complement-mediated cytotoxicity (ADCC) assay to conduct in vitro experiments. In porcine iliac artery endothelial cells (PIECs), siRNA was used to knock down the expression of TLR2, CXCL8, and CCL2. The effect of human serum or inactivated human serum on the expression of TLR2 was analyzed by real-time PCR and Western blotting, and transwell assays were used to assess the chemotactic efficiency of PIECs on human monocyte-macrophages (THP-1 cells) and human neutrophils. The downstream signaling pathways activated by human serum were detected by Western blotting, and the regulation of proinflammatory chemokines and cytokines by TLR2 signaling was assessed by real-time PCR and ELISA. RESULTS: TLR2 was significantly upregulated in PIECs after exposure to human serum, and porcine proinflammatory chemokines, CXCL8 and CCL2, were induced, at least partially, in a TLR2-dependent pattern; the upregulated chemokines participated in the chemotaxis of human neutrophils and THP-1 cells across the species barrier. CONCLUSIONS: (i) TLR2 is significantly upregulated in PIECs by human serum, (ii) the elevated TLR2 participates in the chemotaxis of inflammatory cells through the secretion of chemokine CCL2 and CXCL8, and (iii) blockade of TLR2 would be beneficial for xenograft survival.

Potential Antigens Involved in Delayed Xenograft Rejection in a Ggta1/Cmah Dko Pig-to-Monkey Model.

When hyperacute rejection is avoided by deletion of Gal expression in the pig, delayed xenograft rejection (DXR) becomes a major immunologic barrier to successful xenotransplantation. This study was to investigate the potential antigens involved in DXR. We isolated primary renal microvascular endothelial cells (RMEC) and aortic endothelial cells (AEC) from a GGTA1/CMAH double-knockout (DKO) pig (and a GGTA1-KO pig) and immunized cynomolgus monkeys with both of these cells. After sensitization, monkey serum antibody binding and cytotoxicity to RMEC was significantly higher than to AEC(p < 0.05), suggesting that RMEC are more immunogenic than AEC. Transcriptome sequencing of GGTA1/CMAH DKO pigs indicated that the expression of 1,500 genes was higher in RMEC than in AEC, while expression of 896 genes was lower. Next, we selected 101 candidate genes expressed only in pig RMEC, but not in pig AEC or in monkey or human RMEC. When these genes were knocked out individually in GGTA1/CMAH DKO RMEC, 32 genes were associated with reduced antibody binding, indicating that these genes might be primary immunologic targets involved in DXR. These genes may be important candidates for deletion in producing pigs against which there is a reduced primate immune response in pig kidney xenograft.

Single-cell analysis reveals lineage segregation in early post-implantation mouse embryos.

The mammalian post-implantation embryo has been extensively investigated at the tissue level. However, to unravel the molecular basis for the cell-fate plasticity and determination, it is essential to study the characteristics of individual cells. In particular, the individual definitive endoderm (DE) cells have not been characterized in vivo Here, we report gene expression patterns in single cells freshly isolated from mouse embryos on days 5.5 and 6.5. Initial transcriptome data from 124 single cells yielded signature genes for the epiblast, visceral endoderm, and extra-embryonic ectoderm and revealed a unique distribution pattern of fibroblast growth factor (FGF) ligands and receptors. Further analysis indicated that early-stage epiblast cells do not segregate into lineages of the major germ layers. Instead, some cells began to diverge from epiblast cells, displaying molecular features of the premesendoderm by expressing higher levels of mesendoderm markers and lower levels of Sox3 transcripts. Analysis of single-cell high-throughput quantitative RT-PCR data from 441 cells identified a late stage of the day 6.5 embryo in which mesoderm and DE cells emerge, with many of them coexpressing Oct4 and Gata6 Analysis of single-cell RNA-sequence data from 112 cells of the late-stage day 6.5 embryos revealed differentially expressed signaling genes and networks of transcription factors that might underlie the segregation of the mesoderm and DE lineages. Moreover, we discovered a subpopulation of mesoderm cells that possess molecular features of the extraembryonic mesoderm. This study provides fundamental insight into the molecular basis for lineage segregation in post-implantation mouse embryos.

PHB Associates with the HIRA Complex to Control an Epigenetic-Metabolic Circuit in Human ESCs.

The chromatin landscape and cellular metabolism both contribute to cell fate determination, but their interplay remains poorly understood. Using genome-wide siRNA screening, we have identified prohibitin (PHB) as an essential factor in self-renewal of human embryonic stem cells (hESCs). Mechanistically, PHB forms protein complexes with HIRA, a histone H3.3 chaperone, and stabilizes the protein levels of HIRA complex components. Like PHB, HIRA is required for hESC self-renewal. PHB and HIRA act together to control global deposition of histone H3.3 and gene expression in hESCs. Of particular note, PHB and HIRA regulate the chromatin architecture at the promoters of isocitrate dehydrogenase genes to promote transcription and, thus, production of α-ketoglutarate, a key metabolite in the regulation of ESC fate. Our study shows that PHB has an unexpected nuclear role in hESCs that is required for self-renewal and that it acts with HIRA in chromatin organization to link epigenetic organization to a metabolic circuit.

Transplantation of rat embryonic stem cell-derived retinal progenitor cells preserves the retinal structure and function in rat retinal degeneration.

INTRODUCTION: Degenerative retinal diseases like age-related macular degeneration (AMD) are the leading cause of blindness. Cell transplantation showed promising therapeutic effect for such diseases, and embryonic stem cell (ESC) is one of the sources of such donor cells. Here, we aimed to generate retinal progenitor cells (RPCs) from rat ESCs (rESCs) and to test their therapeutic effects in rat model. METHODS: The rESCs (DA8-16) were cultured in N2B27 medium with 2i, and differentiated to two types of RPCs following the SFEBq method with modifications. For rESC-RPC1, the cells were switched to adherent culture at D10, while for rESC-RPC2, the suspension culture was maintained to D14. Both RPCs were harvested at D16. Primary RPCs were obtained from P1 SD rats, and some of them were labeled with EGFP by infection with lentivirus. To generate Rax::EGFP knock-in rESC lines, TALENs were engineered to facilitate homologous recombination in rESCs, which were cotransfected with the targeting vector and TALEN vectors. The differentiated cells were analyzed with live image, immunofluorescence staining, flow cytometric analysis, gene expression microarray, etc. RCS rats were used to mimic the degeneration of retina and test the therapeutic effects of subretinally transplanted donor cells. The structure and function of retina were examined. RESULTS: We established two protocols through which two types of rESC-derived RPCs were obtained and both contained committed retina lineage cells and some neural progenitor cells (NPCs). These rESC-derived RPCs survived in the host retinas of RCS rats and protected the retinal structure and function in early stage following the transplantation. However, the glia enriched rESC-RPC1 obtained through early and longer adherent culture only increased the b-wave amplitude at 4 weeks, while the longer suspension culture gave rise to evidently neuronal differentiation in rESC-RPC2 which significantly improved the visual function of RCS rats. CONCLUSIONS: We have successfully differentiated rESCs to glia enriched RPCs and retinal neuron enriched RPCs in vitro. The retinal neuron enriched rESC-RPC2 protected the structure and function of retina in rats with genetic retinal degeneration and could be a candidate cell source for treating some degenerative retinal diseases in human trials.

WNT signaling determines tumorigenicity and function of ESC-derived retinal progenitors.

Tumor formation constitutes a major obstacle to the clinical application of embryonic stem cell-derived (ESC-derived) cells. In an attempt to find major extracellular signaling and intrinsic factors controlling tumorigenicity and therapeutic functionality of transplanted ESC-derived retinal progenitor cells (ESC-RPCs), we evaluated multiple kinds of ESC-RPCs in a mouse retinal degeneration model and conducted genome-wide gene expression profiling. We identified canonical WNT signaling as a critical determinant for the tumorigenicity and therapeutic function of ESC-RPCs. The function of WNT signaling is primarily mediated by TCF7, which directly induces expression of Sox2 and Nestin. Inhibition of WNT signaling, overexpression of dominant-negative Tcf7, and silencing Tcf7, Sox2, or Nestin all resulted in drastically reduced tumor formation and substantially improved retinal integration and visual preservation in mice. These results demonstrate that the WNT signaling cascade plays a critical role in modulating the tumorigenicity and functionality of ESC-derived progenitors.