Immune-privileged tissues formed from immunologically cloaked mouse embryonic stem cells survive long term in allogeneic hosts.

The immunogenicity of transplanted allogeneic cells and tissues is a major hurdle to the advancement of cell therapies. Here we show that the overexpression of eight immunomodulatory transgenes (Pdl1, Cd200, Cd47, H2-M3, Fasl, Serpinb9, Ccl21 and Mfge8) in mouse embryonic stem cells (mESCs) is sufficient to immunologically 'cloak' the cells as well as tissues derived from them, allowing their survival for months in outbred and allogeneic inbred recipients. Overexpression of the human orthologues of these genes in human ESCs abolished the activation of allogeneic human peripheral blood mononuclear cells and their inflammatory responses. Moreover, by using the previously reported FailSafe transgene system, which transcriptionally links a gene essential for cell division with an inducible and cell-proliferation-dependent kill switch, we generated cloaked tissues from mESCs that served as immune-privileged subcutaneous sites that protected uncloaked allogeneic and xenogeneic cells from rejection in immune-competent hosts. The combination of cloaking and FailSafe technologies may allow for the generation of safe and allogeneically accepted cell lines and off-the-shelf cell products.

Sleep moderates symptom experience in combat veterans.

BACKGROUND: Though sleep disturbance has shown to negatively affect outcomes related to post-deployment conditions, it is unclear whether and how sleep disturbance affects mental health symptoms beyond these conditions. We evaluated the independent and moderating effects of sleep quality on posttraumatic stress disorder (PTSD), depressive, and neurobehavioral symptoms beyond mild traumatic brain injury (TBI) and PTSD diagnosis. METHODS: Participants were 274 US combat veterans who deployed after 9/11. All completed diagnostic TBI and PTSD interviews and self-report measures of sleep quality, as well as PTSD, depressive, and neurobehavioral symptoms. Only those who passed symptom validity were included in analyses. Hierarchical regression evaluated the contribution of sleep quality to outcomes beyond PTSD and mild TBI. Moderation analyses evaluated interactions between mild TBI, PTSD, and sleep quality on symptom outcomes. RESULTS: Mild TBI was only significantly associated with PTSD (p = .006) and neurobehavioral (p = .003) symptoms. PTSD diagnosis was associated with PTSD (p < .001), depressive (p < .001), and neurobehavioral symptoms (p < .001) beyond mild TBI. Sleep quality explained additional significant variance in all three outcome measures (p < .001), and also significantly moderated the effects of PTSD diagnosis on neurobehavioral symptoms (ΔR2 = .01, p = .023). LIMITATIONS: Sleep was evaluated subjectively and therefore must be interpreted in this context. CONCLUSIONS: These results provide support that sleep quality is an independent contributing factor to health outcomes in post-deployment veterans and should be considered in etiology of complaints.

Examining embedded validity indicators in Conners continuous performance test-3 (CPT-3).

OBJECTIVE: Prior research has identified a variety of embedded performance validity indicators on the Conners' Continuous Performance Test-II (CPT-II). The purpose of this study was to examine embedded validity indicators within the updated third edition of the Conners Continuous Performance Test (CPT-3). METHOD: This study used a retrospective chart review from an ADHD evaluation clinic at a Mid-Atlantic VA hospital. Participants were 197 military veterans who completed a clinical assessment for ADHD. All participants were consecutive referrals to the ADHD clinic who completed the CPT-3 and the Test of Memory Malingering, Trial 1 (TOMM1). RESULTS: Logistic regression analyses indicated that the following five variables were able to significantly predict validity status on the TOMM1: detectability (d'), omissions (OMI), commissions (COM), hit reaction time (HRT) standard deviation (SD), and HRT inter-stimulus interval (ISI) change. Among these measures, HRT SD and HRT ISI change were identified as the scores with the highest AUC values. Optimal cutoffs for all significant predictors were identified. A number of composite EVIs were created using various combinations of CPT-3 scores. All composite EVIs significantly differentiated between pass and fail status on the TOMM1. CONCLUSIONS: Several CPT-3 variables have clinical utility as embedded validity indicators; however, due to low sensitivity, they should not be used in isolation. These scores may be used as indicators of invalid performance but should not be used to rule out invalid performance. Identified CPT-3 scores may be useful as one component in a multivariate, multi-point continuous approach to performance validity sampling.

Observation of Fluorescent Proteins in Living Cells.

Frequently, there is a need for fluorescent protein detection in mouse cell cultures, including embryonic stem cells or their differentiated derivatives, primary and transformed cells. Here, cells expressing green fluorescent protein-labeled proteins are observed using fluorescent microscopy.

Observation of Fluorescent Proteins in Fixed Cells.

Fluorescent proteins (FPs) are popular reporters available for gene expression detection and determination of cellular identities in the mouse. This protocol can be used to detect green fluorescent protein spectral variants and proteins labeled with the fusion tag dsRed in fixed cells.

Preparation of Polymerase Chain Reaction Template DNA from Mouse Tail Tissue.

A simple cell or tissue lysate can provide a sufficient quality and amount of template DNA for polymerase chain reaction (PCR). In this protocol, a small piece from the tip of the tail is removed and processed using hot sodium hydroxide and Tris (HotSHOT).

Isolation of High-Molecular-Weight DNA from Mouse Tail Tips.

DNA samples are prepared from mouse tail tips by Proteinase K digestion and subsequently extracted. The resulting preparation is suitable for use in Southern blotting or polymerase chain reaction (PCR).

Isolation of High-Molecular-Weight DNA from Mouse Yolk Sacs and the Like.

Often, genotyping of mouse embryos is required, and a small part, such as the yolk sac, can be used for this purpose. Here, DNA samples are prepared from extra-embryonic tissues by digestion with Proteinase K and subsequent extraction. The yolk sac of mid-gestation or later-stage embryos provides a sufficient amount of DNA for Southern analysis. Small tissues of a few hundred cells are used for genotyping early postimplantation- and preimplantation-stage embryos by polymerase chain reaction (PCR).

In Vitro Screen to Identify Silent but Activatable (S/A) Integration Sites for a Tetracycline-Inducible Transgene in Mice.

To obtain ubiquitous or simply widespread transgene expression from a single stable integrant transgene is quite challenging because the random genomic integration sites of transgenes may create expression variation or frequent silencing. The tetracycline (Tet)-inducible system requires two reliable working transgenes, one for the tetracycline transactivators (tTA or rtTA) and one for the responder transgene driven by the tet-O promoter. Therefore, the challenge of getting this system working properly is a serious prospect. In this protocol, we describe how to identify a silent but highly activatable genomic site by taking advantage of transgenic lines reliably expressing the tetracycline transactivators from the Rosa-26 locus. These lines provide optimal Tet-inducible expression: There is minimal leakiness at the "off" state and a high level of induction in the presence of the inducer, doxycycline. The procedure requires (1) an embryonic stem (ES) cell line (germline competent) expressing rtTA from the Rosa-26 locus and (2) construction of a Tet-inducible transgene. The transgene contains the tet-O promoter followed by the gene of interest linked to a ßgeo gene (a fusion between lacZ and neo) through an internal ribosomal entry site (IRES) sequence, which allows the initiation of translation in a cap-independent manner.

Linking a cell-division gene and a suicide gene to define and improve cell therapy safety.

Human pluripotent cell lines hold enormous promise for the development of cell-based therapies. Safety, however, is a crucial prerequisite condition for clinical applications. Numerous groups have attempted to eliminate potentially harmful cells through the use of suicide genes1, but none has quantitatively defined the safety level of transplant therapies. Here, using genome-engineering strategies, we demonstrate the protection of a suicide system from inactivation in dividing cells. We created a transcriptional link between the suicide gene herpes simplex virus thymidine kinase (HSV-TK) and a cell-division gene (CDK1); this combination is designated the safe-cell system. Furthermore, we used a mathematical model to quantify the safety level of the cell therapy as a function of the number of cells that is needed for the therapy and the type of genome editing that is performed. Even with the highly conservative estimates described here, we anticipate that our solution will rapidly accelerate the entry of cell-based medicine into the clinic.

Subcutaneous Injection of Pluripotent Stem Cells in Mice.

This protocol describes the production of teratomas and teratocarcinomas used to determine the in vivo developmental potential of adult or embryonic tissues, including early-stage embryos, somites, and tail bud, as well as embryonic and other pluripotent stem cells. Resulting tumors are screened by histology and markers to assess differentiation of the tissues. Methods and sites may vary, depending on cell and tissue type. Subcutaneous injections are easy to perform and do not need surgical manipulations. Suspension of cells in Matrigel Basement Membrane (BD Biosciences) promotes their anatomical localization and is a commonly used, efficient, and reproducible approach.

Visualization of Fluorescent Protein Expression in Whole-Mount Postimplantation-Stage Mouse Embryos.

Fluorescent protein (FP)-expressing transgenic mice are powerful genetic resources for marking both live and fixed cells and tissues. Expression in live embryos requires only dissection and visualization using an appropriate microscope. Fixation can compromise FP activity. A simple tissue-clearing agent called Scale preserves FP activity while rendering the embryo or organ optically transparent.

Administration of Gonadotropins for Superovulation in Mice.

For experiments that require large numbers of preimplantation mouse embryos, such as microinjection of zygotes, gonadotropins are administered to females before mating to increase the number of oocytes that are ovulated (i.e., to induce superovulation). Pregnant mare serum gonadotropin (PMSG) is used to mimic the oocyte maturation effect of the endogenous follicle-stimulating hormone (FSH), and human chorionic gonadotropin (hCG) is used to mimic the ovulation induction effect of luteinizing hormone (LH).

Testing Serum Batches for Mouse Embryonic Stem Cell Culture.

The variability in embryonic stem (ES) cell culture is due primarily to serum. Serum is typically produced in large batches from many animals. However, samples may differ depending on the age and diet of the animals, the country of origin, and other factors creating lot-to-lot variations. Some vendors test FBS lots for compatibility with ES cell culture. Many laboratories prefer to test serum batches themselves to identify the lot giving optimal growth. In this protocol, small quantities of specific serum batches are obtained from different suppliers and tested for their ability to support ES cells in an undifferentiated state. A complete test includes the serum batches' influence on plating efficiency, cell morphology, toxicity, and, if possible, their ability to support generation of chimeras.

Preparation of DNA from Embryonic Stem Cells or Other Cultured Cells.

Characterization of embryonic stem (ES) cell-mediated genome alterations, including random insertional transgenesis, gene trapping, gene targeting, and site-specific recombinase-mediated changes, is performed mostly at the ES cell level, before the introduction of these alterations into a mouse. A detailed characterization requires a larger amount of DNA than is required for the initial detection of the candidates for the desired alteration. This protocol describes the preparation of DNA from a 10-cm tissue culture plate. The cells are trypsinized and lysed, and DNA is recovered from the lysate by organic extraction followed by ethanol precipitation.

Integrating piggyBac Transposon Transgenes into Mouse Fibroblasts by Electroporation.

Mouse embryonic fibroblasts can be reprogrammed to embryonic stem (ES) cell-like pluripotent stem cells by the forced expression of four transcription factors-OCT4, SOX2, KLF4, and c-MYC. The piggyBac transposon system has proven effective as a vehicle for the delivery of transgenes into fibroblasts and for successful reprogramming to induced pluripotent stem (iPS) cells. This protocol is designed for use with the Neon electroporation system. It can be adapted to other types of electroporation systems.

Integrating piggyBac Transposon Transgenes into Mouse Fibroblasts Using Chemical Methods.

Mouse embryonic fibroblasts can be reprogrammed to ES cell-like pluripotent stem cells by the forced expression of four transcription factors-OCT4, SOX2, KLF4, and c-MYC. The piggyBac transposon system has proven effective as a vehicle for the delivery of transgenes into fibroblasts and for successful reprogramming to induced pluripotent stem (iPS) cells. We found that FuGENE HD transfection reagent can be effective for mouse embryonic fibroblasts (MEFs) to generate induced pluripotent stem cells (iPSCs) with the piggyBac transposon transgenes. There are multitudes of cell transfection methods commercially available. Their efficiency, and thus their success, in inducing pluripotent cell types are cell-type-dependent.

Reprogramming Mouse Fibroblasts with piggyBac Transposons.

In 2006, Shinya Yamanaka and his student Kazutoshi Takahashi showed that the expression of only four specific genes is sufficient to reprogram fully differentiated somatic cells into pluripotent stem cells. These cells, termed induced pluripotent stem (iPS) cells, share many of their characteristics with embryonic stem (ES) cells. In this protocol, we describe one of the simplest ways of generating iPS cells from mouse fibroblasts. It combines an efficient transposon-mediated transfection and the tetracycline-inducible system to control the expression of the Yamanaka reprogramming factors.

Shipment of Live Preimplantation-Stage Mouse Embryos.

Sharing genetically modified mouse models is a very important part of collaboration between researchers. Shipping live animals around the world is inconvenient, expensive, and cumbersome because of the variety of international regulations and paperwork. The issue of health status differences between animal facilities is of great importance; traditionally, imported animals are quarantined to determine their health status and avoid the introduction of undesirable pathogens. The shipment of preimplantation-stage embryos for immediate transfer into pseudopregnant recipients upon arrival is a commonly used method for transportation. Time coordination on both sides is critical in this case, but the shipment can be done by any courier and the container does not need to be returned. This protocol has been used since the early 1990s to rederive dozens of mouse strains.