Improved Protocol for Reproducible Human Cortical Organoids Reveals Early Alterations in Metabolism with MAPT Mutations.

Cerebral cortical-enriched organoids derived from human pluripotent stem cells (hPSCs) are valuable models for studying neurodevelopment, disease mechanisms, and therapeutic development. However, recognized limitations include the high variability of organoids across hPSC donor lines and experimental replicates. We report a 96-slitwell method for efficient, scalable, reproducible cortical organoid production. When hPSCs were cultured with controlled-release FGF2 and an SB431542 concentration appropriate for their TGFBR1 / ALK5 expression level, organoid cortical patterning and reproducibility were significantly improved. Well-patterned organoids included 16 neuronal and glial subtypes by single cell RNA sequencing (scRNA-seq), frequent neural progenitor rosettes and robust BCL11B+ and TBR1+ deep layer cortical neurons at 2 months by immunohistochemistry. In contrast, poorly-patterned organoids contain mesendoderm-related cells, identifiable by negative QC markers including COL1A2 . Using this improved protocol, we demonstrate increased sensitivity to study the impact of different MAPT mutations from patients with frontotemporal dementia (FTD), revealing early changes in key metabolic pathways.

Virucidal N95 Respirator Face Masks via Ultrathin Surface-Grafted Quaternary Ammonium Polymer Coatings.

N95 respirator face masks serve as effective physical barriers against airborne virus transmission, especially in a hospital setting. However, conventional filtration materials, such as nonwoven polypropylene fibers, have no inherent virucidal activity, and thus, the risk of surface contamination increases with wear time. The ability of face masks to protect against infection can be likely improved by incorporating components that deactivate viruses on contact. We present a facile method for covalently attaching antiviral quaternary ammonium polymers to the fiber surfaces of nonwoven polypropylene fabrics that are commonly used as filtration materials in N95 respirators via ultraviolet (UV)-initiated grafting of biocidal agents. Here, C12-quaternized benzophenone is simultaneously polymerized and grafted onto melt-blown or spunbond polypropylene fabric using 254 nm UV light. This grafting method generated ultrathin polymer coatings which imparted a permanent cationic charge without grossly changing fiber morphology or air resistance across the filter. For melt-blown polypropylene, which comprises the active filtration layer of N95 respirator masks, filtration efficiency was negatively impacted from 72.5 to 51.3% for uncoated and coated single-ply samples, respectively. Similarly, directly applying the antiviral polymer to full N95 masks decreased the filtration efficiency from 90.4 to 79.8%. This effect was due to the exposure of melt-blown polypropylene to organic solvents used in the coating process. However, N95-level filtration efficiency could be achieved by wearing coated spunbond polypropylene over an N95 mask or by fabricating N95 masks with coated spunbond as the exterior layer. Coated materials demonstrated broad-spectrum antimicrobial activity against several lipid-enveloped viruses, as well as Staphylococcus aureus and Escherichia coli bacteria. For example, a 4.3-log reduction in infectious MHV-A59 virus and a 3.3-log reduction in infectious SuHV-1 virus after contact with coated filters were observed, although the level of viral deactivation varied significantly depending on the virus strain and protocol for assaying infectivity.

Scalable, effective, and rapid decontamination of SARS-CoV-2 contaminated N95 respirators using germicidal ultraviolet C (UVC) irradiation device.

Particulate respirators such as N95s are an essential component of personal protective equipment (PPE) for front-line workers. This study describes a rapid and effective UVC irradiation system that would facilitate the safe re-use of N95 respirators and provides supporting information for deploying UVC for decontamination of SARS-CoV-2 during the COVID-19 pandemic. To assess the inactivation potential of the proposed UVC germicidal device as a function of time by using 3 M 8211-N95 particulate respirators inoculated with SARS-CoV-2. A germicidal UVC device to deliver tailored UVC dose was developed and test coupons (2.5 cm2) of the 3 M-N95 respirator were inoculated with 106 plaque-forming units (PFU) of SARS-CoV-2 and were UV irradiated. Different exposure times were tested (0-164 s) by fixing the distance between the lamp and the test coupon to 15.2 cm while providing an exposure of at least 5.43 mWcm-2. Primary measure of outcome was titration of infectious virus recovered from virus-inoculated respirator test coupons after UVC exposure. Other measures included the method validation of the irradiation protocol, using lentiviruses (biosafety level-2 agent) and establishment of the germicidal UVC exposure protocol. An average of 4.38 × 103 PFU ml-1 (SD 772.68) was recovered from untreated test coupons while 4.44 × 102 PFU ml-1 (SD 203.67), 4.00 × 102 PFU ml-1 (SD 115.47), 1.56 × 102 PFU ml-1 (SD 76.98) and 4.44 × 101 PFU ml-1 (SD 76.98) was recovered in exposures 2, 6, 18 and 54 s per side respectively. The germicidal device output and positioning was monitored and a minimum output of 5.43 mW cm-2 was maintained. Infectious SARS-CoV-2 was not detected by plaque assays (minimal level of detection is 67 PFU ml-1) on N95 respirator test coupons when irradiated for 120 s per side or longer suggesting 3.5 log reduction in 240 s of irradiation, 1.3 J cm-2. A scalable germicidal UVC device to deliver tailored UVC dose for rapid decontamination of SARS-CoV-2 was developed. UVC germicidal irradiation of N95 test coupons inoculated with SARS-CoV-2 for 120 s per side resulted in 3.5 log reduction of virus. These data support the reuse of N95 particle-filtrate apparatus upon irradiation with UVC and supports use of UVC-based decontamination of SARS-CoV-2 during the COVID-19 pandemic.

Scalable, effective, and rapid decontamination of SARS-CoV-2 contaminated N95 respirators using germicidal ultra-violet C (UVC) irradiation device.

IMPORTANCE: Particulate respirators such as N95 masks are an essential component of personal protective equipment (PPE) for front-line workers. This study describes a rapid and effective UVC irradiation system that would facilitate the safe re-use of N95 respirators and provides supporting information for deploying UVC for decontamination of SARS-CoV-2 during the COVID19 pandemic. OBJECTIVE: To assess the inactivation potential of the proposed UVC germicidal device as a function of time by using 3M 8211 - N95 particulate respirators inoculated with SARS-CoV-2. DESIGN: A germicidal UVC device to deliver tailored UVC dose was developed and snippets (2.5cm2) of the 3M-N95 respirator were inoculated with 106 plaque-forming units (PFU) of SARS-CoV-2 and were UV irradiated. Different exposure times were tested (0-164 seconds) by fixing the distance between the lamp (10 cm) and the mask while providing an exposure of at least 5.43 mWcm-2. SETTING: The current work is broadly applicable for healthcare-settings, particularly during a pandemic such as COVID-19. PARTICIPANTS: Not applicable. Main Outcome(s) and Measure(s): Primary measure of outcome was titration of infectious virus recovered from virus-inoculated respirator pieces after UVC exposure. Other measures included the method validation of the irradiation protocol, using lentiviruses (biosafety level-2 agent) and establishment of the germicidal UVC exposure protocol. RESULTS: An average of 4.38x103 PFUml-1(SD 772.68) was recovered from untreated masks while 4.44x102 PFUml-1(SD 203.67), 4.00x102 PFUml-1(SD 115.47), 1.56x102 PFUml-1(SD 76.98) and 4.44x101 PFUml-1(SD 76.98) was recovered in exposures 2s,6s,18s and 54 seconds per side respectively. The germicidal device output and positioning was monitored and a minimum output of 5.43 mWcm-2 was maintained. Infectious SARS-CoV-2 was not detected by plaque assays (minimal level of detection is 67 PFUml-1) on N95 respirator snippets when irradiated for 120s per side or longer suggesting 3.5 log reduction in 240 seconds of irradiation. CONCLUSIONS AND RELEVANCE: A scalable germicidal UVC device to deliver tailored UVC dose for rapid decontamination of SARS-CoV-2 was developed. UVC germicidal irradiation of N95 snippets inoculated with SARS-CoV-2 for 120s per side resulted in 100% (3.5 log in total) reduction of virus. These data support the reuse of N95 particle-filtrate apparatus upon irradiation with UVC and supports use of UVC-based decontamination of SARS-CoV-2 virus during the COVID19 pandemic.

Tuning Forkhead Box D3 overexpression to promote specific osteogenic differentiation of human embryonic stem cells while reducing pluripotency in a three-dimensional culture system.

Clinical use of human embryonic stem cells (hESCs) in bone regeneration applications requires that their osteogenic differentiation be highly controllable as well as time- and cost-effective. The main goals of the current work were thus (a) to assess whether overexpression of pluripotency regulator Forkhead Box D3 (FOXD3) can enhance the osteogenic commitment of hESCs seeded in three-dimensional (3D) scaffolds and (b) to evaluate if the degree of FOXD3 overexpression regulates the strength and specificity of hESC osteogenic commitment. In conducting these studies, an interpenetrating hydrogel network consisting of poly(ethylene glycol) diacrylate and collagen I was utilized as a 3D culture platform. Expression of osteogenic, chondrogenic, pluripotency, and germ layer markers by encapsulated hESCs was measured after 2 weeks of culture in osteogenic medium in the presence or absence doxycycline-induced FOXD3 transgene expression. Towards the first goal, FOXD3 overexpression initiated 24 hr prior to hESC encapsulation, relative to unstimulated controls, resulted in upregulation of osteogenic markers and enhanced calcium deposition, without promoting off-target effects. However, when initiation of FOXD3 overexpression was increased from 24 to 48 hr prior to encapsulation, hESC osteogenic commitment was not further enhanced and off-target effects were noted. Specifically, relative to 24-hr prestimulation, initiation of FOXD3 overexpression 48 hr prior to encapsulation yielded increased expression of pluripotency markers while reducing mesodermal but increasing endodermal germ layer marker expression. Combined, the current results indicate that the controlled overexpression of FOXD3 warrants further investigation as a mechanism to guide enhanced hESC osteogenic commitment.

Zinc inhibits Hedgehog autoprocessing: linking zinc deficiency with Hedgehog activation.

Zinc is an essential trace element with wide-ranging biological functions, whereas the Hedgehog (Hh) signaling pathway plays crucial roles in both development and disease. Here we show that there is a mechanistic link between zinc and Hh signaling. The upstream activator of Hh signaling, the Hh ligand, originates from Hh autoprocessing, which converts the Hh precursor protein to the Hh ligand. In an in vitro Hh autoprocessing assay we show that zinc inhibits Hh autoprocessing with a Ki of 2 µm. We then demonstrate that zinc inhibits Hh autoprocessing in a cellular environment with experiments in primary rat astrocyte culture. Solution NMR reveals that zinc binds the active site residues of the Hh autoprocessing domain to inhibit autoprocessing, and isothermal titration calorimetry provided the thermodynamics of the binding. In normal physiology, zinc likely acts as a negative regulator of Hh autoprocessing and inhibits the generation of Hh ligand and Hh signaling. In many diseases, zinc deficiency and elevated level of Hh ligand co-exist, including prostate cancer, lung cancer, ovarian cancer, and autism. Our data suggest a causal relationship between zinc deficiency and the overproduction of Hh ligand.

Modulation of FOXD3 activity in human embryonic stem cells directs pluripotency and paraxial mesoderm fates.

Transcription factor Foxd3 has been described in model systems as a key member of the pluripotency network in mice as well as being involved in the formation of many critical vertebrate cell types in vivo. Yet virtually nothing is known about roles of FOXD3 in human development and conflicting reports exist regarding its expression in human embryonic stem cells (hESCs). We find that FOXD3 is expressed at both the RNA and protein levels in undifferentiated hESCs and report a Foxd3 expression domain in paraxial mesoderm derivatives of wild-type mouse embryos. Furthermore, increasing FOXD3 activity in hESCs is sufficient for rapid and specific generation of mesenchymal cell types of the paraxial mesoderm, even under pluripotency maintenance conditions. Gene expression diagnostic of chondroblasts, skeletal myoblasts, osteoblasts, and adipoblast is observed within 48 hours of FOXD3 induction, as are morphological and genetic hallmarks of epithelial-to-mesenchymal transition. FOXD3-overexpressing cells can be maintained for several passages, while downregulation of the transgene leads to further differentiation. Loss-of-function also leads to differentiation, toward endoderm and mesoderm. Taken together, these data indicate that a balance of FOXD3 activity is required to maintain pluripotency.

The molecular circuitry underlying pluripotency in embryonic stem cells.

Cells in the pluripotent state have the ability to self-renew indefinitely and to differentiate to all the cells of the embryo. These cells provide an in vitro window into development, including human development, as well as holding extraordinary promise for cell-based therapies in regenerative medicine. The recent demonstration that somatic cells can be reprogrammed to the pluripotent state has raised the possibility of patient and disease-specific induced pluripotent cells. In this article, we review the molecular underpinning of pluripotency. We focus on the transcriptional and signaling networks that underlie the state of pluripotency and control differentiation. In general, the action of each of the molecular components and pathways is dose and context dependent highlighting the need for a systems approach to understanding pluripotency.

APOBEC2, a selective inhibitor of TGFß signaling, regulates left-right axis specification during early embryogenesis.

The specification of left-right asymmetry is an evolutionarily conserved developmental process in vertebrates. The interplay between two TGFß ligands, Derrière/GDF1 and Xnr1/Nodal, together with inhibitors such as Lefty and Coco/Cerl2, have been shown to provide the signals that lead to the establishment of laterality. However, molecular events leading to and following these signals remain mostly unknown. We find that APOBEC2, a member of the cytidine deaminase family of DNA/RNA editing enzymes, is induced by TGFß signaling, and that its activity is necessary to specify the left-right axis in Xenopus and zebrafish embryos. Surprisingly, we find that APOBEC2 selectively inhibits Derrière, but not Xnr1, signaling. The inhibitory effect is conserved, as APOBEC2 blocks TGFß signaling, and promotes muscle differentiation, in a mammalian myoblastic cell line. This demonstrates for the first time that a putative RNA/DNA editing enzyme regulates TGFß signaling and plays a major role in development.

Genetic ablation of neural crest cell diversification.

The neural crest generates multiple cell types during embryogenesis but the mechanisms regulating neural crest cell diversification are incompletely understood. Previous studies using mutant zebrafish indicated that foxd3 and tfap2a function early and differentially in the development of neural crest sublineages. Here, we show that the simultaneous loss of foxd3 and tfap2a function in zebrafish foxd3(zdf10);tfap2a(low) double mutant embryos globally prevents the specification of developmentally distinct neural crest sublineages. By contrast, neural crest induction occurs independently of foxd3 and tfap2a function. We show that the failure of neural crest cell diversification in double mutants is accompanied by the absence of neural crest sox10 and sox9a/b gene expression, and that forced expression of sox10 and sox9a/b differentially rescues neural crest sublineage specification and derivative differentiation. These results demonstrate the functional necessity for foxd3 and tfap2a for neural crest sublineage specification and that this requirement is mediated by the synergistic regulation of the expression of SoxE family genes. Our results identify a genetic regulatory pathway functionally discrete from the process of neural crest induction that is required for the initiation of neural crest cell diversification during embryonic development.

Zebrafish endzone regulates neural crest-derived chromatophore differentiation and morphology.

The development of neural crest-derived pigment cells has been studied extensively as a model for cellular differentiation, disease and environmental adaptation. Neural crest-derived chromatophores in the zebrafish (Danio rerio) consist of three types: melanophores, xanthophores and iridiphores. We have identified the zebrafish mutant endzone (enz), that was isolated in a screen for mutants with neural crest development phenotypes, based on an abnormal melanophore pattern. We have found that although wild-type numbers of chromatophore precursors are generated in the first day of development and migrate normally in enz mutants, the numbers of all three chromatophore cell types that ultimately develop are reduced. Further, differentiated melanophores and xanthophores subsequently lose dendricity, and iridiphores are reduced in size. We demonstrate that enz function is required cell autonomously by melanophores and that the enz locus is located on chromosome 7. In addition, zebrafish enz appears to selectively regulate chromatophore development within the neural crest lineage since all other major derivatives develop normally. Our results suggest that enz is required relatively late in the development of all three embryonic chromatophore types and is normally necessary for terminal differentiation and the maintenance of cell size and morphology. Thus, although developmental regulation of different chromatophore sublineages in zebrafish is in part genetically distinct, enz provides an example of a common regulator of neural crest-derived chromatophore differentiation and morphology.

Zebrafish foxd3 is selectively required for neural crest specification, migration and survival.

The vertebrate neural crest is a pluripotent cell population that generates a large variety of cell types, including peripheral neurons, cartilage and pigment cells. Mechanisms that control the patterning of the neural crest toward specific cell fates remain only partially understood. Zebrafish homozygous for the sympathetic mutation 1 (sym1) have defects in a subset of neural crest derivatives, such as peripheral neurons, glia and cartilage, but retain normal numbers of melanocytes. The sym1 mutation is a nucleotide deletion that disrupts the forkhead DNA-binding domain of the foxd3 gene, which encodes a conserved winged-helix transcription factor. We show that sym1 mutants have normal numbers of premigratory neural crest cells, but these cells express reduced levels of snai1b and sox10, implicating foxd3 as an essential regulator of these transcription factors in the premigratory neural crest. The onset of neural crest migration is also delayed in sym1 mutants, and there is a reduction in the number of migratory trunk neural crest cells, particularly along the medial migration pathway. TUNEL analysis revealed aberrant apoptosis localized to the hindbrain neural crest at the 15-somite stage, indicating a critical role for foxd3 in the survival of a subpopulation of neural crest cells. These results show that foxd3 selectively specifies premigratory neural crest cells for a neuronal, glial or cartilage fate, by inducing the expression of lineage-associated transcription factors in these cells and regulating their subsequent migration.

Defective skeletogenesis with kidney stone formation in dwarf zebrafish mutant for trpm7.

Development of the adult form requires coordinated growth and patterning of multiple traits in response to local gene activity as well as to global endocrine and physiological effectors. An excellent example of such coordination is the skeleton. Skeletal development depends on the differentiation and morphogenesis of multiple cell types to generate elements with distinct forms and functions throughout the body. We show that zebrafish touchtone/nutria mutants exhibit severe growth retardation and gross alterations in skeletal development in addition to embryonic melanophore and touch-response defects. These alterations include accelerated endochondral ossification but delayed intramembranous ossification, as well as skeletal deformities. We show that the touchtone/nutria phenotype results from mutations in trpm7, which encodes a transient receptor potential (TRP) family member that functions as both a cation channel and kinase. We find trpm7 expression in the mesonephric kidney and show that mutants develop kidney stones, indicating renal dysfunction. These results identify a requirement for trpm7 in growth and skeletogenesis and highlight the potential of forward genetic approaches to uncover physiological mechanisms contributing to the development of adult form.

Melanophore sublineage-specific requirement for zebrafish touchtone during neural crest development.

The specification, differentiation and maintenance of diverse cell types are of central importance to the development of multicellular organisms. The neural crest of vertebrate animals gives rise to many derivatives, including pigment cells, peripheral neurons, glia and elements of the craniofacial skeleton. The development of neural crest-derived pigment cells has been studied extensively to elucidate mechanisms involved in cell fate specification, differentiation, migration and survival. This analysis has been advanced considerably by the availability of large numbers of mouse and, more recently, zebrafish mutants with defects in pigment cell development. We have identified the zebrafish mutant touchtone (tct), which is characterized by the selective absence of most neural crest-derived melanophores. We find that although wild-type numbers of melanophore precursors are generated in the first day of development and migrate normally in tct mutants, most differentiated melanophores subsequently fail to appear. We demonstrate that the failure in melanophore differentiation in tct mutant embryos is due at least in part to the death of melanoblasts and that tct function is required cell autonomously by melanoblasts. The tct locus is located on chromosome 18 in a genomic region apparently devoid of genes known to be involved in melanophore development. Thus, zebrafish tct may represent a novel as well as selective regulator of melanoblast development within the neural crest lineage. Further, our results suggest that, like other neural crest-derived sublineages, melanogenic precursors constitute a heterogeneous population with respect to genetic requirements for development.