Wendy Y. Zhang

The Role of SIRT6 Protein in Aging and Reprogramming of Human Induced Pluripotent Stem Cells

Background: Human dermal fibroblasts (HDFs) from older subjects are known to be more resistant to reprogramming. Results: Inclusion of SIRT6 can significantly improve the reprogramming efficiency. Conclusion: Changes in SIRT6 expression and its posttranscriptional regulation may be relevant in aging. Significance: MiR-766-mediated posttranscriptional regulation of SIRT6 has implications in human aging. Aging is known to be the single most important risk factor for multiple diseases. Sirtuin 6, or SIRT6, has recently been identified as a critical regulator of transcription, genome stability, telomere integrity, DNA repair, and metabolic homeostasis. A knockout mouse model of SIRT6 has displayed dramatic phenotypes of accelerated aging. In keeping with its role in aging, we demonstrated that human dermal fibroblasts (HDFs) from older human subjects were more resistant to reprogramming by classic Yamanaka factors than those from younger human subjects, but the addition of SIRT6 during reprogramming improved such efficiency in older HDFs substantially. Despite the importance of SIRT6, little is known about the molecular mechanism of its regulation. We show, for the first, time posttranscriptional regulation of SIRT6 by miR-766 and inverse correlation in the expression of this microRNA in HDFs from different age groups. Our results suggest that SIRT6 regulates miR-766 transcription via a feedback regulatory loop, which has implications for the modulation of SIRT6 expression in reprogramming of aging cells.

Genome Editing of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells With Zinc Finger Nucleases for Cellular Imaging

Rationale: Molecular imaging has proven to be a vital tool in the characterization of stem cell behavior in vivo. However, the integration of reporter genes has typically relied on random integration, a method that is associated with unwanted insertional mutagenesis and positional effects on transgene expression. Objective: To address this barrier, we used genome editing with zinc finger nuclease (ZFN) technology to integrate reporter genes into a safe harbor gene locus (PPP1R12C, also known as AAVS1) in the genome of human embryonic stem cells and human induced pluripotent stem cells for molecular imaging. Methods and Results: We used ZFN technology to integrate a construct containing monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase reporter genes driven by a constitutive ubiquitin promoter into a safe harbor locus for fluorescence imaging, bioluminescence imaging, and positron emission tomography imaging, respectively. High efficiency of ZFN-mediated targeted integration was achieved in both human embryonic stem cells and induced pluripotent stem cells. ZFN-edited cells maintained both pluripotency and long-term reporter gene expression. Functionally, we successfully tracked the survival of ZFN-edited human embryonic stem cells and their differentiated cardiomyocytes and endothelial cells in murine models, demonstrating the use of ZFN-edited cells for preclinical studies in regenerative medicine. Conclusion: Our study demonstrates a novel application of ZFN technology to the targeted genetic engineering of human pluripotent stem cells and their progeny for molecular imaging in vitro and in vivo.

Effects of Ionizing Radiation on Self Renewal and Pluripotency of Human Embryonic Stem Cells

Human embryonic stem cells (hESC) present a novel platform for in vitro investigation of the early embryonic cellular response to ionizing radiation. Thus far, no study has analyzed the genome-wide transcriptional response to ionizing radiation in hESCs, nor has any study assessed their ability to form teratomas, the definitive test of pluripotency. In this study, we use microarrays to analyze the global gene expression changes in hESCs after low-dose (0.4 Gy), medium-dose (2 Gy), and high-dose (4 Gy) irradiation. We identify genes and pathways at each radiation dose that are involved in cell death, p53 signaling, cell cycling, cancer, embryonic and organ development, and others. Using Gene Set Enrichment Analysis, we also show that the expression of a comprehensive set of core embryonic transcription factors is not altered by radiation at any dose. Transplantation of irradiated hESCs to immune-deficient mice results in teratoma formation from hESCs irradiated at all doses, definitive proof of pluripotency. Further, using a bioluminescence imaging technique, we have found that irradiation causes hESCs to initially die after transplantation, but the surviving cells quickly recover by 2 weeks to levels similar to control. To conclude, we show that similar to somatic cells, irradiated hESCs suffer significant death and apoptosis after irradiation. However, they continue to remain pluripotent and are able to form all three embryonic germ layers. Studies such as this will help define the limits for radiation exposure for pregnant women and also radiotracer reporter probes for tracking cellular regenerative therapies.

Mechanisms of Ligand Transfer by the Hepatic Tocopherol Transfer Protein

α-Tocopherol is a member of the vitamin E family that functions as the principal fat-soluble antioxidant in vertebrates. Body-wide distribution of tocopherol is regulated by the hepatic α-tocopherol transfer protein (αTTP), which stimulates secretion of the vitamin from hepatocytes to circulating lipoproteins. This biological activity of αTTP is thought to stem from its ability to facilitate the transfer of vitamin E between membranes, but the mechanism by which the protein exerts this activity remains poorly understood. Using a fluorescence energy transfer methodology, we found that the rate of tocopherol transfer from lipid vesicles to αTTP increases with increasing αTTP concentration. This concentration dependence indicates that ligand transfer by αTTP involves direct protein-membrane interaction. In support of this notion, equilibrium analyses employing filtration, dual polarization interferometry, and tryptophan fluorescence demonstrated the presence of a stable αTTP-bilayer complex. The physical association of αTTP with membranes is markedly sensitive to the presence of vitamin E in the bilayer. Some naturally occurring mutations in αTTP that cause the hereditary disorder ataxia with vitamin E deficiency diminish the effect of tocopherol on the protein-membrane association, suggesting a possible mechanism for the accompanying pathology.

Costimulation-adhesion blockade is superior to cyclosporine A and prednisone immunosuppressive therapy for preventing rejection of differentiated human embryonic stem cells following transplantation.

Rationale: Human embryonic stem cell (hESC) derivatives are attractive candidates for therapeutic use. The engraftment and survival of hESC derivatives as xenografts or allografts require effective immunosuppression to prevent immune cell infiltration and graft destruction. Objective: To test the hypothesis that a short‐course, dual‐agent regimen of two costimulation‐adhesion blockade agents can induce better engraftment of hESC derivatives compared to current immunosuppressive agents. Methods and Results: We transduced hESCs with a double fusion reporter gene construct expressing firefly luciferase (Fluc) and enhanced green fluorescent protein, and differentiated these cells to endothelial cells (hESC‐ECs). Reporter gene expression enabled longitudinal assessment of cell engraftment by bioluminescence imaging. Costimulation‐adhesion therapy resulted in superior hESC‐EC and mouse EC engraftment compared to cyclosporine therapy in a hind limb model. Costimulation‐adhesion therapy also promoted robust hESC‐EC and hESC‐derived cardiomyocyte survival in an ischemic myocardial injury model. Improved hESC‐EC engraftment had a cardioprotective effect after myocardial injury, as assessed by magnetic resonance imaging. Mechanistically, costimulation‐adhesion therapy is associated with systemic and intragraft upregulation of T‐cell immunoglobulin and mucin domain 3 (TIM3) and a reduced proinflammatory cytokine profile. Conclusions: Costimulation‐adhesion therapy is a superior alternative to current clinical immunosuppressive strategies for preventing the post‐transplant rejection of hESC derivatives. By extending the window for cellular engraftment, costimulation‐adhesion therapy enhances functional preservation following ischemic injury. This regimen may function through a TIM3‐dependent mechanism. Stem Cells 2013;31:2354–2363

Imaging Cardiac Stem Cell Therapy: Translations to Human Clinical Studies

Stem cell therapy promises to open exciting new options in the treatment of cardiovascular diseases. Although feasible and clinically safe, the in vivo behavior and integration of stem cell transplants still remain largely unknown. Thus, the development of innovative non-invasive imaging techniques capable of effectively tracking such therapy in vivo is vital for a more in-depth investigation into future clinical applications. Such imaging modalities will not only generate further insight into the mechanisms behind stem cell-based therapy, but also address some major concerns associated with translational cardiovascular stem cell therapy. In the present review, we summarize the principles underlying three major stem cell tracking methods: (1) radioactive labeling for positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging, (2) iron particle labeling for magnetic resonance imaging (MRI), and (3) reporter gene labeling for bioluminescence, fluorescence, MRI, SPECT, and PET imaging. We then discuss recent clinical studies that have utilized these modalities to gain biological insights into stem cell fate.

Prolonged survival of transplanted stem cells after ischaemic injury via the slow release of pro-survival peptides from a collagen matrix

Stem-cell-based therapies hold considerable promise for regenerative medicine. However, acute donor-cell death within several weeks after cell delivery remains a critical hurdle for clinical translation. Co-transplantation of stem cells with pro-survival factors can improve cell engraftment, but this strategy has been hampered by the typically short half-lives of the factors and by the use of Matrigel and other scaffolds that are not chemically defined. Here, we report a collagen–dendrimer biomaterial crosslinked with pro-survival peptide analogues that adheres to the extracellular matrix and slowly releases the peptides, significantly prolonging stem cell survival in mouse models of ischaemic injury. The biomaterial can serve as a generic delivery system to improve functional outcomes in cell-replacement therapy.The slow release of pro-survival peptide analogues crosslinked to an injectable collagen–dendrimer biomaterial significantly prolongs the engraftment and survival of transplanted stem cells in mouse models of ischaemic injury.

Brief Report: External Beam Radiation Therapy for the Treatment of Human Pluripotent Stem Cell-Derived Teratomas

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs), have great potential as an unlimited donor source for cell‐based therapeutics. The risk of teratoma formation from residual undifferentiated cells, however, remains a critical barrier to the clinical application of these cells. Herein, we describe external beam radiation therapy (EBRT) as an attractive option for the treatment of this iatrogenic growth. We present evidence that EBRT is effective in arresting growth of hESC‐derived teratomas in vivo at day 28 post‐implantation by using a microCT irradiator capable of targeted treatment in small animals. Within several days of irradiation, teratomas derived from injection of undifferentiated hESCs and hiPSCs demonstrated complete growth arrest lasting several months. In addition, EBRT reduced reseeding potential of teratoma cells during serial transplantation experiments, requiring irradiated teratomas to be seeded at 1 × 103 higher doses to form new teratomas. We demonstrate that irradiation induces teratoma cell apoptosis, senescence, and growth arrest, similar to established radiobiology mechanisms. Taken together, these results provide proof of concept for the use of EBRT in the treatment of existing teratomas and highlight a strategy to increase the safety of stem cell‐based therapies. Stem Cells 2017;35:1994–2000

External Beam Radiation Therapy for the Treatment of Human Pluripotent Stem Cell-Derived Teratomas

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs), have great potential as an unlimited donor source for cell‐based therapeutics. The risk of teratoma formation from residual undifferentiated cells, however, remains a critical barrier to the clinical application of these cells. Herein, we describe external beam radiation therapy (EBRT) as an attractive option for the treatment of this iatrogenic growth. We present evidence that EBRT is effective in arresting growth of hESC‐derived teratomas in vivo at day 28 post‐implantation by using a microCT irradiator capable of targeted treatment in small animals. Within several days of irradiation, teratomas derived from injection of undifferentiated hESCs and hiPSCs demonstrated complete growth arrest lasting several months. In addition, EBRT reduced reseeding potential of teratoma cells during serial transplantation experiments, requiring irradiated teratomas to be seeded at 1 × 103 higher doses to form new teratomas. We demonstrate that irradiation induces teratoma cell apoptosis, senescence, and growth arrest, similar to established radiobiology mechanisms. Taken together, these results provide proof of concept for the use of EBRT in the treatment of existing teratomas and highlight a strategy to increase the safety of stem cell‐based therapies. Stem Cells 2017;35:1994–2000

Abstract 4332: Diagnostic applications of fatty acid synthase monoclonal antibodies

Fatty acid synthase (FASN), is an enzyme capable of de novo fatty acid synthesis and highly expressed and activated in most human carcinomas. Fatty acid synthase is a multi-enzyme protein that catalyzes fatty acid synthesis and is not a single enzyme but a whole enzymatic system composed of two identical 272 KDa multifunctional polypeptides. Fatty-acid synthesis is now associated with clinically aggressive tumor behavior, tumor-cell growth and is associated with poor prognosis in prostate and breast cancer. Its inhibition is selectively cytotoxic to human cancer cells. Thus, FASN and fatty acid metabolism have become an important focus for the diagnostic and treatment of cancer. We have developed a panel of anti-human FASN Mab for application to several diagnostic platforms. Recombinant full length intact FASN protein (rFASN) was used to immunized C57BL/6 mice, sera were collected from pre- and post-immunized mice and tested by ELISA and Western blot on rFASN protein or rFASN immobilized on Western blot. Spleens from mice with the highest anti-FASN antibody titer was fused with a mouse myeloma cell line Sp2/0-Ag14 for hybridoma production. Among the panel of positive anti-FASN clones selected, two clones with isotype of IgG1kappa have shown strong reactivity to the rFASN and native FASN protein on Western analysis. Upon further Western analyses with cell lysates, we have demonstrated specific detection of FASN protein in human embryonic kidney cells (HEK), HEK cells overexpressing the rFASN protein, HOP-62 cells which express low levels of FASN and MALME cells, which express high levels of FASN. Additional biochemical characterization of these antibodies is ongoing, and includes epitope mapping and reactivity to various FASN protein fragments. These anti-FASN antibodies will be tested by IHC, ELISA to compare tissues expression levels of FAS by IHC or circulating levels of the FASN in both normal and cancer patients. With the increasing development of FAS inhibitors these antibodies will be tested as Companion Diagnostics and tested to see if the rise and fall of the FASN circulating levels correspond with cancer progression or therapy response. Citation Format: Walter P. Carney, Wendy Zhang, David Jarosz, Patrick Muraca, Sunny TAM. Diagnostic applications of fatty acid synthase monoclonal antibodies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4332. doi:10.1158/1538-7445.AM2014-4332