Estudio de los mecanismos de inducción de pluripotencialidad usando un modelo experimental de células germinales primordiales de ratón

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 20 de Febrero de 2013

Primordial Germ Cell Reprogramming

Primordial germ cells (PGCs) are the embryonic precursors of the gametes. Thus, they are unipotent cells. However, PGCs share some common features with pluripotent stem cells. Among them, PGCs show alkaline phosphatase activity and express stage-specific embryonic antigens and pluripotency factors Lin28, Oct4, Sox2, and Nanog. Under specific conditions, they undergo spontaneous reprogramming in vivo. Moreover, they can be easily reprogrammed in vitro into pluripotent embryonic germ cells (EGCs) by culturing them in the presence of basic fibroblast growth factor or the epigenetic modulator trichostatin A. Previous work in our laboratory has also proven that hypoxia alone can reprogram PGCs into hypoxia-induced embryonic germ-like cells, which have a pluripotent phenotype but which do not show self-renewal capacity. Therefore, PGCs are an interesting model to further comprehensively understand the process of cell reprogramming. This chapter reviews various methods to achieve PGC reprogramming, as well as the molecular pathways involved. We focus on soluble factors and genetic strategies to obtain pluripotent cells from PGCs. Special emphasis will be given to factors implied in energetic metabolism, epigenetics, and cell signaling transduction, both in vitro and in vivo

Iodotyrosines are biomarkers for preclinical stages of iodine-deficient hypothyroidism in Dehal1 knockout mice

BACKGROUND: Iodine is required for the synthesis of thyroid hormone (TH), but its natural availability is limited. Dehalogenase1 (Dehal1) recycles iodine from mono- and di-iodotyrosines (MIT, DIT) to sustain TH synthesis when iodine supplies are scarce, but its role in the dynamics of storage and conservation of iodine is unknown. METHODS: Dehal1 knockout mice (Dehal1KO) were generated by gene-trapping and the timing of expression and distribution was investigated by X-Gal staining and immunofluorescence using recombinant Dehal1-Betagalactosidase protein produced in fetuses and adult mice. Adult Dehal1KO and wild-type (Wt) animals were fed normal and iodine-deficient diets for 1 month and plasma, urine, and tissues were isolated for analyses. TH status was monitored including T4, T3, MIT, DIT, and urinary iodine concentration using a novel LC-MS-MS method and the Sandell-Kolthoff (S-K) technique throughout the experimental period. RESULTS: Dehal1 is highly expressed in the thyroid, is also present in kidneys, liver, and, unexpectedly, the choroid plexus. In vivo transcription of Dehal1 was induced by iodine deficiency only in the thyroid tissue. Under normal iodine intake, Dehal1KO mice were euthyroid but they showed negative iodine balance due to a continuous loss of iodotyrosines in the urine. Counter-intuitively, the urinary iodine concentration of Dehal1KO mice is two-fold higher than in Wt mice, indicating S-K measures both inorganic and organic iodine. Under iodine restriction, Dehal1KO mice rapidly develop profound hypothyroidism while Wt mice remain euthyroid, suggesting reduced retention of iodine in Dehal1KO mice. Urinary and plasma iodotyrosines were continually elevated throughout their life cycle, including the neonatal period, when pups are still euthyroid. CONCLUSIONS: Plasma and urine iodotyrosine elevation occurs in Dehal1-deficient mice throughout life. Therefore, measurement of iodotyrosines predicts an eventual iodine shortage and development of hypothyroidism in the pre-clinical phase. The prompt establishment of hypothyroidism upon the start of iodine restriction suggests that Dehal1KO mice have low iodine reserves in their thyroid glands, pointing to defective capacity for iodine storage

SSTR2 as an anatomical imaging marker and a safety switch to monitor and manage CAR T cell toxicity

Abstract The ability to image adoptively transferred T cells in the body and to eliminate them to avoid toxicity will be vital for chimeric antigen receptor (CAR) T cell therapy, particularly against solid tumors with higher risk of off-tumor toxicity. Previously, we have demonstrated the utility of somatostatin receptor 2 (SSTR2) for CAR T cell imaging, illustrating the expansion and contraction of CAR T cells in tumor as well as off-tumor expansion. Using intercellular adhesion molecule 1 (ICAM-1)-specific CAR T cells that secrete interleukin (IL)-12 as a model, herein we examined the potential of SSTR2 as a safety switch when combined with the SSTR2-specific maytansine-octreotate conjugate PEN-221. Constitutive secretion of IL-12 led to continuous expansion of CAR T cells after rapid elimination of tumors, causing systemic toxicity in mice with intact MHC expression. Treatment with PEN-221 rapidly reduced the abundance of CAR T cells, decreasing the severity of xenogeneic graft-versus-host disease (GvHD), and prolonged survival. Our study supports the development of SSTR2 as a single genetic marker for CAR T cells that is readily applicable to humans both for anatomical detection of T cell distribution and an image-guided safety switch for rapid elimination of CAR T cells

Chimeric Antigen Receptor T Cell Therapy Targeting Epithelial Cell Adhesion Molecule in Gastric Cancer: Mechanisms of Tumor Resistance

Epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen that is frequently overexpressed in various carcinomas. We have developed chimeric antigen receptor (CAR) T cells specifically targeting EpCAM for the treatment of gastric cancer. This study sought to unravel the precise mechanisms by which tumors evade immune surveillance and develop resistance to CAR T cell therapy. Through a combination of whole-body CAR T cell imaging and single-cell multiomic analyses, we uncovered intricate interactions between tumors and tumor-infiltrating lymphocytes (TILs). In a gastric cancer model, tumor-infiltrating CD8 T cells exhibited both cytotoxic and exhausted phenotypes, while CD4 T cells were mainly regulatory T cells. A T cell receptor (TCR) clonal analysis provided evidence of CAR T cell proliferation and clonal expansion within resistant tumors, which was substantiated by whole-body CAR T cell imaging. Furthermore, single-cell transcriptomics showed that tumor cells in mice with refractory or relapsing outcomes were enriched for genes involved in major histocompatibility complex (MHC) and antigen presentation pathways, interferon-γ and interferon-α responses, mitochondrial activities, and a set of genes (e.g., CD74, IDO1, IFI27) linked to tumor progression and unfavorable disease prognoses. This research highlights an approach that combines imaging and multiomic methodologies to concurrently characterize the evolution of tumors and the differentiation of CAR T cells