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The study presents the medical service activities in organizing the provision of troops (forces) with medical equipment during the operation to restore constitutional order in the Chechen Republic. To provide medical support to federal troops (forces), appropriate groupings of forces and medical services were created, functioning in three isolated areas. Thanks to the operational and professional work of military pharmaceutical specialists, a medical supply system was formed, involving the three main medical warehouses of the North Caucasus Military District. The paper also showed the work of medical supply units and institutions to provide federal troops (forces) with medical equipment. Issues of the functioning of the medical supply system, optimization of the composition of individual medical equipment of military personnel of the federal troops (forces), and peculiarities of storing medical equipment in the field were successfully resolved by involving the faculty of the Department of Military Medical Supply and Pharmacy of the Military Medical Academy of S.M. Kirov. Moreover, the paper presented the features of personnel provision and stages of medical evacuation with individual and group medical equipment, bags and sets of first aid, premedical, and medical kits. The system of supplying medical equipment to troops (forces) for special-purpose medical units operating in combat areas, evacuation centers, and military hospitals of the North Caucasus Military District was analyzed. Based on the medical support experience for the united grouping of troops (forces) during the operation to restore the constitutional order in the Chechen Republic, ways to improve the provision of complete equipment and technical medical service are proposed.

BACKGROUND Colon cancer cell lines are widely used for research and for the screening of drugs that specifically target the stem cell compartment of colon cancers. It was reported that colon cancer carcinoma specimens contain a subset of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-expressing stem cells, these so-called “tumour-initiating” cells, reminiscent in their properties of the normal intestinal stem cells (ISCs), may explain the apparent heterogeneity of colon cancer cell lines. Also, colon cancer is initiated by aberrant Wnt signaling in ISCs known to express high levels of LGR5. Furthermore, in vivo reports demonstrate the clonal expansion of intestinal adenomas from a single LGR5-expressing cell. AIM To investigate whether colon cancer cell lines contain cancer stem cells and to characterize these putative cancer stem cells. METHODS A portable fluorescent reporter construct based on a conserved fragment of the LGR5 promoter was used to isolate the cell compartments expressing different levels of LGR5 in two widely used colon cancer cell lines (Caco-2 and LoVo). These cells were then characterized according to their proliferation capacity, gene expression signatures of ISC markers, and their tumorigenic properties in vivo and in vitro. RESULTS The data revealed that the LGR5 reporter can be used to identify and isolate a classical intestinal crypt stem cell-like population from the Caco-2, but not from the LoVo, cell lines, in which the cancer stem cell population is more akin to B lymphoma Moloney murine leukemia virus insertion region 1 homolog (+4 crypt) stem cells. This sub-population within Caco-2 cells exhibits an intestinal cancer stem cell gene expression signature and can both self-renew and generate differentiated LGR5 negative progeny. Our data also show that cells expressing high levels of LGR5/enhanced yellow fluorescent protein (EYFP) from this cell line exhibit tumorigenic-like properties in vivo and in vitro. In contrast, cell compartments of LoVo that are expressing high levels of LGR5/EYFP did not show these stem cell-like properties. Thus, cells that exhibit high levels of LGR5/EYFP expression represent the cancer stem cell compartment of Caco-2 colon cancer cells, but not LoVo cells. CONCLUSION Our findings highlight the presence of a spectrum of different ISC-like compartments in different colon cancer cell lines. Their existence is an important consideration for their screening applications and should be taken into account when interpreting drug screening data. We have generated a portable LGR5-reporter that serves as a valuable tool for the identification and isolation of different colon cancer stem cell populations in colon cancer lines.

The market of smart products has been studied, the shortcomings of certain samples have been identified and ways of solving them with the help of microcontrollers have been put forward.

A device for automatic control and monitoring of heat under clothing based on DS18b20 sensors and an ESP32 microcontroller with the ability to control via a phone has been designed and created.

Virtual reality (VR) is widely used for medical purposes, particularly, for assessment and treatment of phobic anxiety disorders. The article provides a review and critical analysis of methods and tools for assessing phobic anxiety disorders via registration and digital processing of physiological signals, and adaptation of various VR scenes to the user's actual state. The article presents a technique for studying phobic anxiety disorders in VR environment that enables VR scenes to be adapted for a particular user, considering his individual features, and behavior parameters. Adaptation of VR scenes is carried out by selecting an optimal set of hidden signal patterns registered with electrocardiogram and electroencephalogram signals.

Patients with ataxia-telangiectasia (A-T) lack a functional ATM kinase protein and exhibit defective repair of DNA double-stranded breaks and response to oxidative stress. We show that CRISPR/Cas9-assisted gene correction combined with piggyBac (PB) transposon-mediated excision of the selection cassette enables seamless restoration of functional ATM alleles in induced pluripotent stem cells from an A-T patient carrying compound heterozygous exonic missense/frameshift mutations, and from a patient with a homozygous splicing acceptor mutation of an internal coding exon. We show that the correction of one allele restores expression of ~ 50% of full-length ATM protein and ameliorates DNA damage-induced activation (auto-phosphorylation) of ATM and phosphorylation of its downstream targets, KAP-1 and H2AX. Restoration of ATM function also normalizes radiosensitivity, mitochondrial ROS production and oxidative-stress-induced apoptosis levels in A-T iPSC lines, demonstrating that restoration of a single ATM allele is sufficient to rescue key ATM functions. Our data further show that despite the absence of a functional ATM kinase, homology-directed repair and seamless correction of a pathogenic ATM mutation is possible. The isogenic pairs of A-T and gene-corrected iPSCs described here constitute valuable tools for elucidating the role of ATM in ageing and A-T pathogenesis.

Marsupials have long attracted scientific interest because of their unique biological features and their position in mammalian evolution. Mesenchymal stem cells (MSCs) are of considerable research interest in translational medicine due to their immunomodulatory, anti-inflammatory, and regenerative properties. MSCs have been harvested from various tissues in numerous eutherian species; however, there are no descriptions of MSCs derived from a marsupial. In this study, we have generated Tasmanian devil (Sarcophilus harrisii) MSCs from devil induced pluripotent stem cells (iPSCs), thus providing an unlimited source of devil MSCs and circumventing the need to harvest tissues from live animals. Devil iPSCs were differentiated into MSCs (iMSCs) through both embryoid body formation assays (EB-iMSCs) and through inhibition of the transforming growth factor beta/activin signaling pathway (SB-iMSCs). Both EB-iMSCs and SB-iMSCs are highly proliferative and express the MSC-specific surface proteins CD73, CD90, and CD105, in addition to the pluripotency transcription factors OCT4/POU5F1, SOX2, and NANOG. Expression of the marsupial pluripotency factor POU5F3, a paralogue of OCT4/POU5F1, is significantly reduced in association with the transition from pluripotency to multipotency. Devil iMSCs readily differentiate along the adipogenic, osteogenic, and chondrogenic pathways in vitro, confirming their trilineage differentiation potential. Importantly, in vitro teratoma assays confirmed their multipotency, rather than pluripotency, since the iMSCs only formed derivatives of the mesodermal germ layer. Devil iMSCs show a tropism toward medium conditioned by devil facial tumor cells and express a range of immunomodulatory and anti-inflammatory factors. Therefore, devil iMSCs will be a valuable tool for further studies on marsupial biology and may facilitate the development of an MSC-based treatment strategy against Devil Facial Tumor Disease.

Altres ajuts: the E-Rare (ERA-Net for research programs on rare diseases) and EuroRETT (a European network on Rett syndrome, funded by the European Commission under its 6th Framework Program since 2006) When considering the clinical applications of autologous cell replacement therapy of human induced pluripotent stem cells (iPSC)-derived cells, there is a clear need to better understand what the immune response will be before we embark on extensive clinical trials to treat or model human disease. We performed a detailed assessment comparing human fibroblast cell lines (termed F1) reprogrammed into human iPSC and subsequently differentiated back to fibroblast cells (termed F2) or other human iPSC-derived cells including neural stem cells (NSC) made from either retroviral, episomal, or synthetic mRNA cell reprogramming methods. Global proteomic analysis reveals the main differences in signal transduction and immune cell protein expression between F1 and F2 cells, implicating wild type (WT) toll like receptor protein 3 (TLR3). Furthermore, global methylome analysis identified an isoform of the human TLR3 gene that is not epigenetically reset correctly upon differentiation to F2 cells resulting in a hypomethylated transcription start site in the TLR3 isoform promoter and overexpression in most human iPSC-derived cells not seen in normal human tissue. The human TLR3 isoform in human iPSC-NSC functions to suppress NF-KB p65 signaling pathway in response to virus (Poly IC), suggesting suppressed immunity of iPSC-derived cells to viral infection. The sustained WT TLR3 and TLR3 isoform overexpression is central to understanding the altered immunogenicity of human iPSC-derived cells calling for screening of human iPSC-derived cells for TLR3 expression levels before applications. Stem Cells 2019;37:476-488.

Background: With a view towards harnessing the therapeutic potential of canine mesenchymal stromal cells (cMSCs) as modulators of inflammation and the immune response, and to avoid the issues of the variable quality and quantity of harvested cMSCs, we examined the immunomodulatory properties of cMSCs derived from canine induced pluripotent stem cells (ciMSCs), and compared them to cMSCsharvested from adipose tissue (cAT-MSC) and bone marrow (cBM-MSC).Methods and results: Deep sequencing of the ciMSC transcriptome confirmed that ciMSCsexpress more genes in common with cBM-MSCsthan with the ciPSCs from which they were derived. Both ciMSCs and cBM-MSCsexpress a range of pluripotency factors in common withthe ciPSCsincluding NANOG, POU5F1 (OCT-4), SOX-2, KLF-4, LIN-28A, MYC, LIF, LIFR, and TERT. However, ESRRB and PRDM-14, both factors associated with naïve, rather than primed, pluripotency were expressed only in the ciPSCs. LOXL-2, which is involved in epithelial to mesenchymal transition (EMT), is also expressed in ciMSCs and cBM-MSCs but notciPSCs. ciMSCsconstitutively express the immunomodulatory factors iNOS, GAL-9, TGF-β1, PTGER-2αand VEGF, and the pro-inflammatory mediators COX-2,IL-1βand IL-8.When stimulated with the canine pro-inflammatory cytokines tumor necrosis factor-α (cTNF-α), interferon-γ (cIFN-γ), or a combination of both, ciMSCsupregulated their expression ofIDO,iNOS, GAL-9,HGF, TGF-β1, PTGER-2α, VEGF, COX-2, IL-1β andIL-8.When co-cultured with mitogen-stimulated lymphocytes, ciMSCsdownregulated their expression of iNOS, HGF, TGF-β1andPTGER-2α, while increasing their expression of COX-2, IDO and IL-1β. Conclusions: Taken together, these findings suggest that ciMSCs possess similar immunomodulatory capabilities as harvested cMSCs and support further investigation into the potential use ofciMSCsfor the management of canine immune-mediated and inflammatory disorders.

The platypus (Ornithorhynchus anatinus) is an egg-laying monotreme mammal whose ancestors diverged ∼166 million years ago from the evolutionary pathway that eventually gave rise to both marsupial and eutherian mammals. Consequently, its genome is an extraordinary amalgam of both ancestral reptilian and derived mammalian features. To gain insight into the evolution of mammalian pluripotency, we have generated induced pluripotent stem cells from the platypus (piPSCs). Deep sequencing of the piPSC transcriptome revealed that piPSCs robustly express the core eutherian pluripotency factors POU5F1/OCT4, SOX2, and NANOG. Given the more extensive role of SOX3 over SOX2 in avian pluripotency, our data indicate that between 315 and 166 million years ago, primitive mammals replaced the role of SOX3 in the vertebrate pluripotency network with SOX2. DAX1/NR0B1 is not expressed in piPSCs and an analysis of the platypus DAX1 promoter revealed the absence of a proximal SOX2-binding DNA motif known to be critical for DAX1 expression in eutherian pluripotent stem cells, suggesting that the acquisition of SOX2 responsiveness by DAX1 has facilitated its recruitment into the pluripotency network of eutherians. Using the RNAseq data, we were also able to demonstrate that in both fibroblasts and piPSCs, the expression ratio of X chromosomes to autosomes (X1-5 X1-5:AA) is approximately equal to 1, indicating that there is no upregulation of X-linked genes. Finally, the RNAseq data also allowed us to explore the process of X-linked gene inactivation in the platypus, where we determined that for any given gene, there is no preference for silencing of the maternal or paternal allele; that is, within a population of cells, the silencing of X-linked genes is not imprinted.

Midbrain dopamine (mDA) neurons can be replaced in patients with Parkinson's disease (PD) in order to provide long-term improvement in motor functions. The limited capacity for long-distance axonal growth in the adult brain means that cells are transplanted ectopically, into the striatal target. As a consequence, several mDA pathways are not re-instated, which may underlie the incomplete restoration of motor function in patients. Here, we show that viral delivery of GDNF to the striatum, in conjunction with homotopic transplantation of human pluripotent stem-cell-derived mDA neurons, recapitulates brain-wide mDA target innervation. The grafts provided re-instatement of striatal dopamine levels and correction of motor function and also connectivity with additional mDA target nuclei not well innervated by ectopic grafts. These results demonstrate the remarkable capacity for achieving functional and anatomically precise reconstruction of long-distance circuitry in the adult brain by matching appropriate growth-factor signaling to grafting of specific cell types.

While there is a body of experimental data concerning dimers formed by an aromatic molecule and its radical cation, information on the corresponding dimer radical anions (DRAs) is scarce. In this work, evidence for the formation of the DRAs of decafluorobiphenyl and 4-aminononafluorobiphenyl has been obtained by the optically detected electron paramagnetic resonance and the time-resolved magnetic field effect techniques. Theoretical investigation (DFT B3LYP-D3/6-31+G*) of these DRAs and the DRAs of octafluoronaphtalene and 1,2,4,5-tetrafluorobenzene previously detected by Werst has been undertaken to gain greater insight into the structure of the polyfluoroarene DRAs. Without substituents different from a fluorine atom, an extra electron is evenly delocalized over two fragments; the bonding interaction is π stacking. On the potential energy surfaces (PES), there are two minima of nearly equal energy corresponding to the structures of perfect and parallel displaced sandwiches. Such a PES structure is due to a conical intersection between two electronic states of different symmetry. The DRA of 4-aminononafluorobiphenyl is an ion-molecular associate stabilized by electrostatic interactions involving NH

Marsupials have long attracted scientific interest because of their unique biological features and their position in mammalian evolution. Mesenchymal stem cells (MSCs) are of considerable research interest in translational medicine due to their immunomodulatory, anti-inflammatory, and regenerative properties. MSCs have been harvested from various tissues in numerous eutherian species; however, there are no descriptions of MSCs derived from a marsupial. In this study, we have generated Tasmanian devil (

The indicators of growth and development, fattening and meat qualities of pigs of the breeding center “Lozovoe” CJSC “Plemzavod-Yubileyny” of the Tyumen region were evaluated. The indicators that characterize the growth and development of young animals were taken into account: live weight, age, average daily growth. When the live weight of 100 kg was reached, an ultrasound device was used to evaluate the thickness of bacon, the depth of muscles and the yield of lean meat, which are in the database of breeding records for 2011-2020. To characterize the meat qualities, 1144 Landrace piglets were slaughtered, 275 - large white breed, 129 - Pietren breed, 339 hybrids (LxKB), 159 hybrids (LxD), 460 hybrids obtained from boars of foreign selection. A comparative analysis of commercial hybrids of various variants of crossing pigs of domestic and foreign selection is carried out. Processing of the research results was carried out in the laboratory of Molecular diagnostics and Biotechnology of the Don State Agrarian University. The influence of the breed is established.

Precise and accurate gene correction is crucial for enabling iPSC-based therapies, and Cas9-Nickase based approaches are increasingly considered for in vivo correction of diseases such as beta-thalassemia. Here, we generate footprint-free induced pluripotent stem cells from a patient with a beta-thalassemia mutation (IVSII-1 G > A) and employ a double Cas9nickase-mediated correction strategy combined with a piggyBac transposon-modified donor vector for gene correction. Our approach further aimed to minimize the formation of adjacent single-strand breaks at the targeted allele through the destruction of the binding site for one guide and the use of a synonymous protospacer adjacent motif blocking mutation (canonical PAM sequence 5'-NGG-3' is changed to 5'-NCG-3', where N indicates any nucleobase) for the other guide. We show that this strategy indeed not only permits bi-allelic seamless repair of the beta-globin gene splice site mutation and negligible off-target mutagenesis or re-editing of the targeted allele but also results in unexpected on-target mutagenesis with some guide RNAs (gRNAs) in several targeted clones. This study thus not only validates a framework for seamless gene correction with enhanced specificity and accuracy but also highlights potential safety concerns associated with Cas9-nickase based gene correction.

In this article, the advantages of the unity of sensory and telecommunication approaches will be shown, using examples of monitoring systems for second-generation telecommunication passive optical networks (PON). In comparison with the TDM network architecture, its combination with the WDM architecture is much more efficient, both in terms of the number of channels per fiber, and in terms of the number of channels per laser. This architecture showed a high coverage of monitoring objects by sensors in optically efficient network organization with their small number. Theoretically, the number of sensors in the fiber optical sensor system (FOSS) can be expanded to 256 sensors using two fibers. Note that in the fiber optical telecommunication system (FOTS) the maintenance of 256 PON TDM-WDM terminals is a typical requirement. Thus, monitoring tasks for FOTS and FOSS PON are based on unified approaches. In general, when designing a FOTS, it is necessary to take into account the close relationship that exists between all the factors that determine the characteristics of the network elements, with the existing economic conditions, which will create an optimal network.

Horses are susceptible to a number of neurotropic viruses, including West Nile virus (WNV), which is a pathogen of global significance in both horses and humans. However, there are no in vitro models with which to study infectious neuropathic diseases in the horse. In an effort to redress this, we have generated neurons from equine induced pluripotent stem cells (equiPSCs) that express a range of cortical neuron-specific markers, in addition to the membrane-bound ligand ephrin B3, which plays an important role in axon guidance as well as functioning as the receptor through which henipaviruses, such as Hendra virus, enter mammalian neurons. EquiPSC-derived neurons spontaneously depolarize with waves of depolarization conducted unidirectionally to adjacent neurons. We sought to confirm that equiPSC-derived neurons are a possible in vitro model for viral neuropathic diseases in the horse by examining their susceptibility to infection with flaviviruses that are known to be neurotropic in horses, including WNV and Murray Valley encephalitis virus (MVEV), and to compare these to nonpathogenic flaviviruses such as Fitzroy River virus (FRV) and Bamaga virus (BgV). All three strains of WNV tested in this study grew to high titres in the equiPSC-derived neurons, inducing a strong cytopathic effect (cpe), as did MVEV. In contrast, FRV showed restricted replication, and no cpe, which is consistent with the observation that FRV infects, but does not cause disease, in horses. BgV, which is thought to infect only marsupials, did not replicate in the equiPSC-derived neurons. Hence, our equiPSC-derived neurons display virus-specific differences in terms of viral titre and cpe that are similar to observations made in vivo, thus supporting their use as an in vitro model for neurotropic viral infection in horses.

Uncaging promoter and enhancer dynamics In order to understand cellular differentiation, it is important to understand the timing of the regulation of gene expression. Arner et al. used cap analysis of gene expression (CAGE) to analyze gene enhancer and promoter activities in a number of human and mouse cell types. The RNA of enhancers was transcribed first, followed by that of transcription factors, and finally by genes that are not transcription factors. Science , this issue p. 1010

Scientific Data, 4, ISSN:2052-4463

The development of the technology of fiber-optic sensors is inextricably linked with the development of the technology of fiber-optic telecommunication systems (FOTS). The increased capabilities of telecommunication technologies allow the creation of spatially spaced and multidimensional sensor networks. On the other hand, the development of sensor technologies, in turn, allows the creation of highly efficient monitoring systems for telecommunications networks, in particular their selective elements, which include wave etalons, add/drop multiplexers, thin-film filters, arrayed waveguide gratings, widely used Fiber Bragg gratings (FBG), etc.

In this study we have generated canine mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, from canine induced pluripotent stem cells (ciPSCs) by small-molecule inhibition of the transforming growth factor beta (TGFβ)/activin signaling pathway. These ciPSC-derived MSCs (ciPSC-MSCs) express the MSC markers CD73, CD90, CD105, STRO1, cPDGFRβ and cKDR, in addition to the pluripotency factors OCT4, NANOG and REX1. ciPSC-MSCs lack immunostaining for H3K27me3, suggesting that they possess two active X chromosomes. ciPSC-MSCs are highly proliferative and undergo robust differentiation along the osteo-, chondro- and adipogenic pathways, but do not form teratoma-like tissues in vitro. Of further significance for the translational potential of ciPSC-MSCs, we show that these cells can be encapsulated and maintained within injectable hydrogel matrices that, when functionalized with bound pentosan polysulfate, dramatically enhance chondrogenesis and inhibit osteogenesis. The ability to efficiently derive large numbers of highly proliferative canine MSCs from ciPSCs that can be incorporated into injectable, functionalized hydrogels that enhance their differentiation along a desired lineage constitutes an important milestone towards developing an effective MSC-based therapy for osteoarthritis in dogs, but equally provides a model system for assessing the efficacy and safety of analogous approaches for treating human degenerative joint diseases.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) has opened the way for patient-specific disease modelling. Following their differentiation into neuronal cell types, iPSC have enabled the investigation of human neurodegenerative diseases, such as Alzheimer's disease (AD). While human iPSCs certainly provide great opportunities to repeatedly interrogate specific human brain cell types of individuals with familial and sporadic forms of the disease, the complex aetiology and timescale over which AD develops in humans poses particular challenges to iPSC-based AD models. Here, we discuss the current state-of-play in the context of these and other iPSC model-related challenges and elaborate on likely future developments in this field of research.

Expansion of pluripotent stem cells in defined media devoid of animal-derived feeder cells to generate multilayered three-dimensional (3D) bulk preparations or spheroids, rather than two-dimensional (2D) monolayers, is advantageous for many regenerative, biological or disease-modelling studies. Here we show that electrospun polymer matrices comprised of nanofibres that mimic the architecture of the natural fibrous extracellular matrix allow for feeder-free expansion of pluripotent human induced pluripotent stem cells (IPSCs) and human embryonic stem cells (HESCs) into multilayered 3D 'patty-like' spheroid structures in defined xeno-free culture medium. The observation that IPSCs and HESCs readily revert to 2D growth in the absence of the synthetic nanofibre membranes suggests that this 3D expansion behaviour is mediated by the physical microenvironment and artificial niche provided by the nanofibres only. Importantly, we could show that such 3D growth as patties maintained the pluripotency of cells as long as they were kept on nanofibres. The generation of complex multilayered 3D structures consisting of only pluripotent cells on biodegradable nanofibre matrices of the desired shape and size will enable both industrial-scale expansion and intricate organ–tissue engineering applications with human pluripotent stem cells, where simultaneous coupling of differentiation pathways of all germ layers from one stem cell source may be required for organ formation.

In this study we have reprogrammed dermal fibroblasts from an adult female horse into equine induced pluripotent stem cells (equiPSCs). These equiPSCs are dependent only on leukemia inhibitory factor (LIF), placing them in striking contrast to previously derived equiPSCs that have been shown to be co-dependent on both LIF and basic fibroblast growth factor (bFGF). These equiPSCs have a normal karyotype and have been maintained beyond 60 passages. They possess alkaline phosphatase activity and express eqNANOG, eqOCT4, and eqTERT mRNA. Immunocytochemistry confirmed that they produce NANOG, REX1, SSEA4, TRA1-60, and TRA1-81. While our equiPSCs are LIF dependent, bFGF co-stimulates their proliferation via the PI3K/AKT pathway. EquiPSCs lack expression of eqXIST and immunostaining for H3K27me3, suggesting that during reprogramming the inactive X chromosome has likely been reactivated to generate cells that have two active X chromosomes. EquiPSCs form embryoid bodies and in vitro teratomas that contain derivatives of all three germ layers. These LIF-dependent equiPSCs likely reflect a more naive state of pluripotency than equiPSCs that are co-dependent on both LIF and bFGF and so provide a novel resource for understanding pluripotency in the horse.

Use of human pluripotent stem cells (hPSCs) in regenerative medicine applications relies on control of cell fate decisions by exogenous factors. This control can be hindered by the use of undefined culture components, poorly understood autocrine/paracrine effects, spatiotemporal variations in microenvironmental composition inherent to static culture formats, and signal cross-talk between multiple factors. We recently described microbioreactor arrays that provide a full factorial spectrum of exogenous factors, and allow gradual accumulation of paracrine factors through serial culture chambers. We combined these with defined biochemical conditions, and in situ reporter gene- and immunofluorescence-based readouts to create an hPSC screening platform with enhanced data throughput and microenvironmental control. HES3-EOS-C(3+)-EiP reporter hESCs were screened against FGF-2, TGF-β1, and retinoic acid in a modified mTeSR-1 medium background. Differential pluripotency marker expression reflected mTeSR-1's maintenance capacity, and differentiation in response to removal of maintenance factors or addition of retinoic acid. Interestingly, pluripotency marker expression was downregulated progressively through serial chambers. Since downstream chambers are exposed to greater levels of paracrine factors under continuous flow, this effect is thought to result from secreted factors that negatively influence pluripotency. The microbioreactor array platform decodes factor interplay, and has a broad application in deciphering microenvironmental control of cell fate.

Down syndrome (DS) is a complex developmental disorder with diverse pathologies that affect multiple tissues and organ systems. Clear mechanistic description of how trisomy of chromosome 21 gives rise to most DS pathologies is currently lacking and is limited to a few examples of dosage-sensitive trisomic genes with large phenotypic effects. The recent advent of cellular reprogramming technology offers a promising way forward, by allowing derivation of patient-derived human cell types in vitro. We present general strategies that integrate genomics technologies and induced pluripotent stem cells to identify molecular networks driving different aspects of DS pathogenesis and describe experimental approaches to validate the causal requirement of candidate network defects for particular cellular phenotypes. This overall approach should be applicable to many poorly understood complex human genetic diseases, whose pathogenic mechanisms might involve the combined effects of many genes.

Genetic analysis has revealed that the dual specificity protein kinase DYRK1A has multiple roles in the development of the central nervous system. Increased DYRK1A gene dosage, such as occurs in Down syndrome, is known to affect neural progenitor cell differentiation, while haploinsufficiency of DYRK1A is associated with severe microcephaly. Using a set of known and newly synthesized DYRK1A inhibitors, along with CRISPR-mediated gene activation and shRNA knockdown of DYRK1A, we show here that chemical inhibition or genetic knockdown of DYRK1A interferes with neural specification of human pluripotent stem cells, a process equating to the earliest stage of human brain development. Specifically, DYRK1A inhibition insulates the self-renewing subpopulation of human pluripotent stem cells from powerful signals that drive neural induction. Our results suggest a novel mechanism for the disruptive effects of the absence or haploinsufficiency of DYRK1A on early mammalian development, and reveal a requirement for DYRK1A in the acquisition of competence for differentiation in human pluripotent stem cells.

We previously reported that synthetic vaccine particles (SVP) encapsulating antigens and TLR agonists resulted in augmentation of immune responses with minimal production of systemic inflammatory cytokines. Here we evaluated two different polymer formulations of SVP-encapsulated antigens and tested their ability to induce cytolytic T lymphocytes (CTL) in combination with SVP-encapsulated adjuvants. One formulation led to efficient antigen processing and cross-presentation, rapid and sustained CTL activity, and expansion of CD8+ T cell effector memory cells locally and centrally, which persisted for at least 1-2 years after a single immunization. SVP therapeutic dosing resulted in suppression of tumor growth and a substantial delay in mortality in several syngeneic mouse cancer models. Treatment with checkpoint inhibitors and/or cytotoxic drugs, while suboptimal on their own, showed considerable synergy with SVP immunization. SVP encapsulation of endosomal TLR agonists provided superior CTL induction, therapeutic benefit and/or improved safety profile compared to free adjuvants. SVP vaccines encapsulating mutated HPV-16 E7 and E6/E7 recombinant proteins led to induction of broad CTL activity and strong inhibition of TC-1 tumor growth, even when administered therapeutically 13-14 days after tumor inoculation in animals bearing palpable tumors. A pilot study in non-human primates showed that SVP-encapsulated E7/E6 adjuvanted with SVP-encapsulated poly(I:C) led to robust induction of antigen-specific T and B cell responses.

We generated double-transgenic mice carrying cointegrated tissue-specific Gal4 and Gal4 reporter transgenes to direct transgene overexpression in the mononuclear phagocyte system (MPS). A modified promoter of the Csf1r (c-fms) gene, containing a deletion of the trophoblast-specific promoter, was used to drive the expression of Gal4VP16 transcriptional activator specifically in macrophages. This module was cointegrated with a fluorescent reporter, enhanced cyan fluorescent protein (ECFP), driven by a Gal4-dependent promoter. ECFP fluorescence was first detected in forming blood islands of the yolk sac at 8 dpc, then in macrophages in the yolk sac and the embryo proper. In adult mice ECFP was detected primarily in monocytes, tissue macrophages, microglia, and dendritic cells, including Langerhans cells of the skin. Crossing of these mice to transgenics containing tagged protein under control of a Gal4-dependent promoter directed expression of that protein in mononuclear phagocytes of double-transgenic animals. The new mouse line provides a useful tool for overexpression of transgenes in cells of the myeloid lineage, while simultaneously labeling them by ECFP expression.

Human induced pluripotent stem cells (hiPSCs) have provided novel insights into the etiology of disease and are set to transform regenerative medicine and drug screening over the next decade. The generation of human iPSCs free of a genetic footprint of the reprogramming process is crucial for the realization of these potential uses. Here we describe in detail the generation of human iPSC from control and disease-carrying individuals' fibroblasts using episomal plasmids.

An analysis of gene expression variability can provide an insightful window into how regulatory control is distributed across the transcriptome. In a single cell analysis, the inter-cellular variability of gene expression measures the consistency of transcript copy numbers observed between cells in the same population. Application of these ideas to the study of early human embryonic development may reveal important insights into the transcriptional programs controlling this process, based on which components are most tightly regulated. Using a published single cell RNA-seq data set of human embryos collected at four-cell, eight-cell, morula and blastocyst stages, we identified genes with the most stable, invariant expression across all four developmental stages. Stably-expressed genes were found to be enriched for those sharing indispensable features, including essentiality, haploinsufficiency, and ubiquitous expression. The stable genes were less likely to be associated with loss-of-function variant genes or human recessive disease genes affected by a DNA copy number variant deletion, suggesting that stable genes have a functional impact on the regulation of some of the basic cellular processes. Genes with low expression variability at early stages of development are involved in regulation of DNA methylation, responses to hypoxia and telomerase activity, whereas by the blastocyst stage, low-variability genes are enriched for metabolic processes as well as telomerase signaling. Based on changes in expression variability, we identified a putative set of gene expression markers of morulae and blastocyst stages. Experimental validation of a blastocyst-expressed variability marker demonstrated that HDDC2 plays a role in the maintenance of pluripotency in human ES and iPS cells. Collectively our analyses identified new regulators involved in human embryonic development that would have otherwise been missed using methods that focus on assessment of the average expression levels; in doing so, we highlight the value of studying expression variability for single cell RNA-seq data., Author Summary In order to function properly, cells express specific sets of genes that are regulated via complex transcriptional programs. During early stages of development, when an embryo consists of only a handful of cells, it is vital that these cells work together so that the embryo can develop into a healthy baby. Single cell studies allow us to understand how each cell contributes to ensuring proper regulation of the embryo, as well as identify the critical genes whose expression is important for development. While we understand that regulation of a gene occurs through the timing of when it is expressed and also the quantity of its expression, more recently we have come to recognize that the variability of a gene’s expression across single cells may also contribute to the viability of the organism. In this study, we analyzed the gene expression variability of human embryos at different developmental stages. We discovered distinctive patterns of variability across cells in the embryo; some genes had extremely stable expression, and others were variable but with increased homogeneity in expression at a particular developmental stage. We validated one of these stage-specific markers and found that it played a role in the maintenance of pluripotency of human pluripotent stem cells. Overall, these results can help unlock additional clues into understanding how embryonic development is regulated in humans.