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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.

Within the vast panorama of multimodal systems for clinical diagnosis, PET/MRI has been receiving significant attention. In parallel, new technological solutions have been paving the way to simultaneous SPECT/MRI systems, whose development has been so far delayed due to the challenging compatibility between MRI, gamma-ray detectors, and collimators. This paper presents an silicon photomultiplier (SiPM)-based Anger camera designed for preclinical and clinical SPECT static inserts for standard MR scanners. The gamma-ray detector is based on a continuous CsI(Tl) scintillator readout by arrays of SiPMs and custom ASICs. The design has been adapted to fit with the limited space conditions (23 mm thickness), but also considering mutual compatibility issues. Despite these constraints and thanks to the adoption of an iterative statistical event estimation method simply requiring a flood image, the detector maintains state-of-the-art performance in terms of intrinsic spatial resolution (1.0 mm FWHM within the usable field of view) and an energy resolution below 14% FWHM at 140 keV. The experimental studies here performed show that the camera, designed to operate at 0 °C to reduce the dark current of SiPMs, can operate up to 10 °C without significant worsening of imaging performance.

A vast number of diseases could be treated with therapeutic cells derived from pluripotent stem cells (PSCs). However, cell products that come from non-autologous sources can be immune rejected by the recipient’s immune system. Here, we show that forced expression of eight immunomodulatory transgenes, includingCcl21, Pdl1, Fasl, Serpinb9, H2-M3, Cd47, Cd200, andMfge8, allows mouse embryonic stem cells (mESCs) and their derivatives to escape immune rejection in fully immunocompetent, allogeneic recipients. Despite no genetic alterations to major histocompatibility complex (MHC) genes, immune-modified C57BL/6 mESCs could generate long-term, allogeneic tissues in inbred FVB/N, C3H, and BALB/c, as well as outbred CD-1 recipients. Due to the tandem incorporation of our safe-cell suicide system, which allows tight and drug-inducible control over proliferationin vivo, these allotolerated cells can generate safe and dormant ectopic tissues in the host. We show that these ectopic tissues maintain high expression of all eight immunomodulatory transgenes and are immune-privileged sites that can host and protect unmodified mouse and human cells from rejection in allogeneic and xenogeneic settings, respectively. If translated to human clinical settings, we envision the development of a single pluripotent cell line that can be used to generate allo-tolerated, off-the-shelf cell products to serve all humankind, as well as immune-privileged ectopic tissues to host and immune-protect any kind of therapeutic cell product.

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).

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).

A preclinical insert for small animal simultaneous SPECT and MR imaging, in particular for imaging mouse brains, is presented. It consists of ten static magnetic resonance imaging (MRI)-compatible gamma cameras based on tiles of silicon photomultipliers readout by a multichannel ASIC and coupled to 5 cm $\boldsymbol \times $ 5 cm CsI(Tl) scintillators and to an MRI-compatible multipinhole collimator. Calibration and image reconstruction algorithm are illustrated. Mutual compatibility is demonstrated along with imaging performance that is comparable with other non-MR micro-SPECT systems: 0.9 mm tomographic spatial resolution across a transverse field of view of 15.6 mm, 12% energy resolution (at 140 keV), and 1105 cps/MBq sensitivity. Experimental results with phantoms (glass capillaries of 290 $ \mu \text{m}$ diameter and a mini Derenzo) are presented.

The first SiPM-based clinical SPECT ring for brain simultaneous imaging in standard MR scanners has been put into operation. Reliable operation of the instrument has been achieved by means of its optimization, such as the introduction of a dual-cooling system (liquid-gas) of the DAQ electronics. The static gantry is composed of 20 MRI-compatible γ-detection modules mounting 5 cm × 10 cm CsI(Tl) scintillators for a total of 1440 pixels (targeting a transaxial FoV of 20 cm). Here we present the results of a preliminary characterization showing 220 kcps max. count rate, 365cps/MBq sensitivity with the multislit-slat collimator, ~17% energy resolution and an average extrinsic spatial resolution of ~8 mm in the trans-axial plane.

The initial characterization of the first single-photon emission computed tomography (SPECT) insert for simultaneous human brain imaging inside magnetic resonance imaging (MRI) is presented. The instrument features a novel multimini-slit-slat collimator and a static ring of 20 detectors. The scanner architecture is modular and represents the scale-up of a preclinical version whose MRI-compatibility has been already successfully demonstrated. The detectors are based on CsI(Tl) crystals (50 mm $\times $ 100 mm $\times $ 8 mm) coupled to arrays of silicon photomultipliers and ASIC readout for a total of 1440 channels. The transaxial field of view (FoV) is 20 cm by 9 cm in the axial direction. The experimental results show a sensitivity of 362 cps/MBq, an energy resolution of ~17% (at 140 keV), and a tomographic extrinsic resolution in the transaxial plane of ~8 mm across the whole FoV.

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.

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.

The experimental characterization of a SiPM-based 10-module SPECT gamma imager suitable for insertion and operation inside unmodified MR scanners for simultaneous (and potentially multi-tracer) preclinical imaging is presented. The design challenges, here fulfilled, such as compactness while granting proper cooling, mutual compatibility and resilience to high (3-7 T) magnetic fields inside the bore, suitable energetic (better than 14% at 140 keV) and spatial resolution (better than 1 mm) are discussed. The carefully optimized detection module, featuring a $5\times5 cm^{2}$ CsI(Tl) scintillator coupled to arrays of SiPM conditioned by a 36-channel ASIC, and the digital daisy chain infrastructure are the basis for the realization of a larger (20-module ring) clinical insert. Moreover, planar image reconstruction based on advanced statistical approaches such as maximum likelihood and iteratively estimated light response function is reported.

The results of a systematic and quantitative experimental characterization of simultaneous operation of a preclinical SPECT insert inside an unmodified standard MR scanner (Philips 3T Achieva) are presented. The static gantry (20 cm diameter, 15 mm field of view) is composed of 10 MRI-compatible gamma cameras based on 8-mm thick CsI(Tl) scintillator (50 mm × 50mm) coupled with arrays of SiPMs. Mutual compatibility is achieved by proper design of the SPECT system: shielding, choice of materials, design of the analog boards. The measured energy resolution is 13% at 122 keV after correction of SiPM gain variability and local crystal luminosity. The intrinsic spatial resolution, after planar image statistical reconstruction by means of maximum likelihood, is 1mm, while the extrinsic resolution is below 0.9 mm across the entire transaxial FoV, measured with capillaries of 290 pm diameter. With the custom-designed 3 × 3 multi-pinhole collimator (whose minimal perturbation of the magnetic field can be reduced below 1ppm thanks to shimming), the measured sensitivity with 99mTc is 1100 cps/MBq. The MR image is not distorted by the operation of the SPECT, as well as SPECT performances are no degraded by MRI (T2W and gradient sequences). Simultaneous imaging of different phantoms (capillaries, Derenzo hot rods of 0.85-1.7 mm diameter) as well as a mouse brain are reported.

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

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.

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

A clinical SPECT insert for a commercial MRI scanner has been developed within the INSERT project, allowing simultaneous SPECT/MRI studies of the human brain. Here we present preliminary experimental results. The reconstructed resolution was 6-11 mm and the sensitivity 280-440 s-1/MBq. The INSERT is the first clinical SPECT prototype for simultaneous SPECT/MRI.

Embryonic stem (ES) cells can develop into many types of differentiated tissues if they are placed into a differentiating environment. This can occur in vivo when the ES cells are injected into or aggregated with an embryo, or in vitro if their culture conditions are modified to induce differentiation. There are an increasing number of differentiating culture conditions that can bias the differentiation of ES cells into desired cell types. Determining the mechanisms that control ES cell differentiation into therapeutically important cell types is a quickly growing area of research. Knowledge gained from these studies may eventually lead to the use of stem cells to repair specific damaged tissues. Many times ES cell differentiation proceeds through an intermediate stage called the embryoid body (EB). EBs are round structures composed of ES cells that have undergone some of the initial stages of differentiation. EBs can then be manipulated further to generate more specific cell types. This protocol describes a method to differentiate ES cells into EBs. It produces EBs of comparable size. This aspect is important because the differentiation processes taking place inside an EB are influenced by its size.

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.

Background & Aim Cell therapy-based regenerative medicine provides a powerful tool against diseases that have been intractable to treat with conventional medicine. Employing genome-editing strategies, we addressed the safety concerns associated with cell-based regenerative medicine through the development of the FailSafe (FS) Cell System. This proprietary technology inserts a suicide gene into a cell division essential locus, allowing selective elimination of proliferative cells through the administration of a pro-drug, whilst also protecting the suicide gene from inactivation. When incorporated into mouse embryonic stem cells (mESCs) and injected subcutaneously, the Fail-Safe system allows the proliferating component of a teratoma to be eliminated by brief treatment with the pro-drug for the suicide gene. The remaining non-proliferating component is induced into a stable and dormant ectopic tissue. To enable the development of economically feasible “off-the-shelf” cell products, we identified a set of eight immune-modulating genes involved in allograft tolerance and rejection. When expressed in FailSafe mESCs, the immune-cloaking system is sufficient to prevent rejection of ectopic tissues in immune-competent major histocompatibility complex-mismatched recipient mice. In the current study we sought to demonstrate the application of our technology for treatment of neurological diseases. Methods, Results & Conclusion By incorporating expression of enhanced firefly luciferase, we used bioluminescence imaging (BLI) to monitor the survival and proliferation of gene-edited mESCs after stereotaxic injection into the mouse brain. Small numbers (1 × 104) of mESCs injected into the lateral ventricles were detectable by BLI. Weekly monitoring of live animals revealed that the BL signal increased over time, indicative of the proliferation of injected cells. Histological examination of brain tissue confirmed that mESCs formed teratomas containing structures derived from the three primary germ layers. In mice that received gene-edited mESCs, teratoma development could be prevented by intraperitoneal administration of the pro-drug. Moreover, proliferating cells could be eliminated from developing teratomas, as shown by a decreased BL signal over time and Ki67 staining in tissue sections. Importantly, grafted cells that had undergone terminal differentiation remained intact. These technologies will therefore facilitate the advancement of cell-based regenerative therapies for neurological diseases.

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.

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).

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 βgeo 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.

We report on the results achieved with the first SPECT insert for general purpose MR scanners, developed within the INSERT project (INtegrated SPECT/MRI for Enhanced stratification of brain tumors in Radio-chemoTherapy). The present paper focuses on the implementation and performance assessment of a preclinical version of the SPECT. A clinical configuration is currently under development and will be optimized accordingly to the results from the preclinical system. The SPECT is composed by a full ring of gamma detection modules based on compact and MR compatible Silicon PhotoMultipliers (SiPMs). The intrinsic spatial resolution of the detection modules has been measured as 1.0 mm FWHM over 40 mm × 40 mm planar field of view (FOV), at 140 keV and at 4 °C. The energy resolution of the detector is 13.7 % for Tc-99m (140 keV, at 4 °C) and has been optimized to permit the simultaneous acquisition of multiple radiotracers. The main components, namely the detection modules, the collimator and custom RF coil, have been realized with a compact design to fit inside common MR bores. Moreover, mutual compatibility between SPECT system and MR has been studied and optimized for all the SPECT components. First tomographic images have been acquired from phantoms and in vivo animal models. The gamma detection modules have been tested in a 7 T MRI (MAGNETOM by Siemens) to measure induced disturbances on the electronic chain. The detection module negligibly affects MRI imaging. The outcomes of mutual compatibility tests provide enough confidence to proceed with the first synchronous SPECT/MR imaging experiments.

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).

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.

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.

Multipotent mesenchymal stromal cells (MSCs) are more and more frequently used to treat orthopedic injuries in horses. However, these cells are limited in their expandability and differentiation capacity. Recently, the first equine-induced pluripotent stem cell (iPSC) lines were reported by us [ 1 ]. In vitro differentiation of iPSCs into MSC-like cells is an attractive alternative to using MSCs derived from other sources, as a much larger quantity of patient-specific cells with broad differentiation potential could be generated. However, the differentiation capacity of iPSCs to MSCs and the potential for use in tissue engineering have yet to be explored. In this study, equine iPSCs were induced to differentiate into an MSC-like population. Upon induction, the iPSCs changed morphology toward spindle-shaped cells similar to MSCs. The ensuing iPSC-MSCs exhibited downregulation of pluripotency-associated genes and an upregulation of MSC-associated genes. In addition, the cells expressed the same surface markers as MSCs derived from equine umbilical cord blood. We then assessed the multilineage differentiation potential of iPSC-MSCs. Although chondrogenesis was not achieved after induction with transforming growth factor-beta 3 (TGFβ3) and/or bone morphogenic protein 4 (BMP-4) in 3D pellet culture, mineralization characteristic of osteogenesis and lipid droplet accumulation characteristic of adipogenesis were observed after chemical induction. We demonstrate a protocol for the derivation of MSC-like progenitor populations from equine iPS cells.

The discovery of induced pluripotent stem cells (iPSCs) has had a transforming effect on our understanding of biology and has brought an enormous promise to regenerative medicine. It has opened up a magnitude of unprecedented possibilities to study disease processes in vitro, model them in animal systems, and develop patient-specific cell-based regenerative therapies. iPSCs derived from other than the human species will be instrumental for bringing these prospects to fruition by providing preclinical models and novel treatments for veterinary medicine. In this chapter, we describe the derivation of iPSCs from equine embryonic fibroblasts using a non-viral method developed in our laboratory and originally applied to the murine and human systems (Woltjen et al., Nature 458:766-770, 2009). We will detail the procedures involved and discuss potential pitfalls as well as elaborate on possible variations and future improvements of this technique.

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.

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.

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.

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.

The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25-30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25-30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function.

The female mouse estrous cycle is divided into four phases: proestrus (development of ovarian follicles), estrus (ovulation), metestrus (formation of corpora lutea), and diestrus (beginning of follicle development for next ovulation and elimination of previous oocytes). The appearance of the epithelium of the external genitalia is used to identify the stage of the estrous cycle of a female mouse. This is usually easier to see in strains with either no or only light skin pigmentation. By examining the color, moistness, and degree of swelling of the vagina, females in estrus can readily be identified. To set up the matings, females are examined in the afternoon, and those in estrus are placed into the cages with males (one or two females in each cage with one male). Usually, 50% or more of the selected females will mate. The presence of a vaginal copulation plug next morning indicates that mating has occurred, but it does not mean that a pregnancy will result even if proven breeder fertile males were used. It is important to check vaginal plugs early in the morning because they fall out or are no longer detectable ~12 h after mating or sometimes earlier.

A major breakthrough in the past 5 years is the development of the ability to reprogram somatic cells to pluripotency. It has rejuvenated the field of stem cell research, providing regenerative medicine with new possibilities. In this paper, we discuss the progress made in the reprogramming field with focus on induction methodologies, the use of induced pluripotent stem cells (iPSCs) for drug discovery, and issues and precautions related to their use in regenerative medicine. Clinical Pharmacology & Therapeutics (2011) 89 5, 741–745. doi:10.1038/clpt.2011.37

The domesticated horse represents substantial value for the related sports and recreational fields, and holds enormous potential as a model for a range of medical conditions commonly found in humans. Most notable of these are injuries to muscles, tendons, ligaments and joints. Induced pluripotent stem (iPS) cells have sparked tremendous hopes for future regenerative therapies of conditions that today are not possible to cure. Equine iPS (EiPS) cells, in addition to bringing promises to the veterinary field, open up the opportunity to utilize horses for the validation of stem cell based therapies before moving into the human clinical setting. In this study, we report the generation of iPS cells from equine fibroblasts using a piggyBac (PB) transposon-based method to deliver transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, expressed in a temporally regulated fashion. The established iPS cell lines express hallmark pluripotency markers, display a stable karyotype even during long-term culture, and readily form complex teratomas containing all three embryonic germ layer derived tissues upon in vivo grafting into immunocompromised mice. Our EiPS cell lines hold the promise to enable the development of a whole new range of stem cell-based regenerative therapies in veterinary medicine, as well as aid the development of preclinical models for human applications. EiPS cell could also potentially be used to revive recently extinct or currently threatened equine species.

Experimental mouse chimeras have served as immensely important research tools for studying many aspects of mammalian development ever since they first were produced over 50 years ago. Chimera studies have served as crucial assays in the era of modern mouse genetics that was triggered by the advent of mouse embryonic stem cells. Lately, chimeras are also used as proof of pluripotency and normality of induced pluripotent stem cells. With this long history in mind, it may seem surprising that chimeras now have an ever-increasing role to play. The high-throughput mouse gene targeting projects are in the process of producing ES cell lines with a mutation in each of the close to 20,000 known protein coding genes. These will all be waiting for germline transmission through chimeras. Such a large-scale approach calls for simplified methods for generating germline transmitting chimeras. In this chapter, we will describe the currently most cost efficient and simple method; the aggregation of pluripotent stem cells with diploid or tetraploid mouse embryos. Since most of the large knockout projects are using the C57BL/6 background, we will pay special attention to cell lines derived from this inbred strain.

The discovery that it is possible to render somatic cells pluripotent by the exogenous expression of a set of transcription factors provides an experimental model for studying the molecular nature of cellular identity.

Erratum to: Stem Cell Rev and Rep DOI 10.1007/s12015-011-9239-5 Please note the correction to the above mentioned article: In the Materials and Methods section, the concentration of MEK inhibitor StemGent #PD0325901 was published as 0.5M. The correct concentration should be 0.5 M. We apologize for any confusion caused by this error.

Summary Vascular endothelial growth factor A (VEGF) is highly expressed in adipose tissue. Its role, however, has not been fully elucidated. Here, we reveal the metabolic role of adipose-VEGF by studying mice with deletion (VEGF AdΔ ) or doxycycline-inducible overexpression of a VEGF transgene (VEGF AdTg ) in the adipose tissue. VEGF AdΔ mice have reduced adipose vascular density and show adipose hypoxia, apoptosis, inflammation, and metabolic defects on a high-fat diet. In contrast, induction of VEGF expression in VEGF AdTg mice leads to increased adipose vasculature and reduced hypoxia. The latter changes are sufficient to counteract an established compromising effect of high-fat diet on the metabolism, indicating that metabolic misbalance is reversible by adipose vessel density increase. Our data clearly show the essential role of VEGF signaling for adequate adipose function. Besides revealing insights into the molecular mechanisms of obesity-related metabolic diseases, this study points to the therapeutic potential of increased adipose angiogenesis.