Hypoxia Increases the Efficiencies of Cellular Reprogramming and Oncogenic Transformation in Human Blood Cell Subpopulations In Vitro and In Vivo.

Patients with chronic hypoxia show a higher tumor incidence; however, no primary common cause has been recognized. Given the similarities between cellular reprogramming and oncogenic transformation, we directly compared these processes in human cells subjected to hypoxia. Mouse embryonic fibroblasts were employed as controls to compare transfection and reprogramming efficiency; human adipose-derived mesenchymal stem cells were employed as controls in human cells. Easily obtainable human peripheral blood mononuclear cells (PBMCs) were chosen to establish a standard protocol to compare cell reprogramming (into induced pluripotent stem cells (iPSCs)) and oncogenic focus formation efficiency. Cell reprogramming was achieved for all three cell types, generating actual pluripotent cells capable for differentiating into the three germ layers. The efficiencies of the cell reprogramming and oncogenic transformation were similar. Hypoxia slightly increased the reprogramming efficiency in all the cell types but with no statistical significance for PBMCs. Various PBMC types can respond to hypoxia differently; lymphocytes and monocytes were, therefore, reprogrammed separately, finding a significant difference between normoxia and hypoxia in monocytes in vitro. These differences were then searched for in vivo. The iPSCs and oncogenic foci were generated from healthy volunteers and patients with chronic obstructive pulmonary disease (COPD). Although higher iPSC generation efficiency in the patients with COPD was found for lymphocytes, this increase was not statistically significant for oncogenic foci. Remarkably, a higher statistically significant efficiency in COPD monocytes was demonstrated for both processes, suggesting that physiological hypoxia exerts an effect on cell reprogramming and oncogenic transformation in vivo in at least some cell types.

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 diiodotyrosines (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 (Dehal1KO) mice were generated by gene trapping. The timing of expression and distribution was investigated by X-Gal staining and immunofluorescence using recombinant Dehal1-beta-galactosidase 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 thyroxine, triiodothyronine, MIT, DIT, and urinary iodine concentration (UIC) using a novel liquid chromatography with tandem mass spectrometry method and the Sandell-Kolthoff (S-K) technique throughout the experimental period. Results: Dehal1 is highly expressed in the thyroid and is also present in the 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. Counterintuitively, the UIC of Dehal1KO mice is twofold higher than that of Wt mice, indicating that 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 the thyroids of Dehal1KO mice. Urinary and plasma iodotyrosines were continually elevated throughout the life cycles of Dehal1KO mice, including the neonatal period, when pups were 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 preclinical 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.

Inhibition of PKCε induces primordial germ cell reprogramming into pluripotency by HIF1&2 upregulation and histone acetylation.

Historically, primordial germ cells (PGCs) have been a good model to study pluripotency. Despite their low numbers and limited accessibility in the mouse embryo, they can be easily and rapidly reprogrammed at high efficiency with external physicochemical factors and do not require transcription factor transfection. Employing this model to deepen our understanding of cell reprogramming, we specifically aimed to determine the relevance of Ca2+ signal transduction pathway components in the reprogramming process. Our results showed that PGC reprogramming requires a normal extracellular [Ca2+] range, in contrast to neoplastic or transformed cells, which can continue to proliferate in Ca2+-deficient media, differentiating normal reprogramming from neoplastic transformation. Our results also showed that a spike in extracellular [Ca2+] of 1-3 mM can directly reprogram PGC. Intracellular manipulation of Ca2+ signal transduction pathway components revealed that inhibition of classical Ca2+ and diacylglycerol (DAG)-dependent PKCs, or intriguingly, of only the novel DAG-dependent PKC, PKCε, were able to induce reprogramming. PKCε inhibition changed the metabolism of PGCs toward glycolysis, increasing the proportion of inactive mitochondria. This metabolic switch from oxidative phosphorylation to glycolysis is mediated by hypoxia-inducible factors (HIFs), given we found upregulation of both HIF1α and HIF2α in the first 48 hours of culturing. PKCε inhibition did not change the classical pluripotency gene expression of PGCs, Oct4, or Nanog. PKCε inhibition changed the histone acetylation of PGCs, with histones H2B, H3, and H4 becoming acetylated in PKCε-inhibited cultures (markers were H2BacK20, H3acK9, and H4acK5K8, K12, K16), suggesting that reprogramming by PKCε inhibition is mediated by histone acetylation.

Treatment of corneal endothelial damage in a rabbit model with a bioengineered graft using human decellularized corneal lamina and cultured human corneal endothelium.

OBJECTIVE: We aimed to investigate the functionality of human decellularized stromal laminas seeded with cultured human corneal endothelial cells as a tissue engineered endothelial graft (TEEK) construct to perform endothelial keratoplasty in an animal model of corneal endothelial damage. METHODS: Engineered corneal endothelial grafts were constructed by seeding cultured human corneal endothelial cell (hCEC) suspensions onto decellularized human corneal stromal laminas with various coatings. The functionality and survival of these grafts with cultured hCECs was examined in a rabbit model of corneal endothelial damage after central descemetorhexis. Rabbits received laminas with and without hCECs (TEEK and control group, respectively). RESULTS: hCEC seeding over fibronectin-coated laminas provided an optimal and consistent endothelial cell count density and polygonal shape on the decellularized laminas, showing active pump fuction. Surgery was performed uneventfully as standard Descemet stripping automated endothelial keratoplasty (DSAEK). Corneal transparency gradually recovered in the TEEK group, whereas haze and edema persisted for up to 4 weeks in the controls. Histologic examination showed endothelial cells of human origin covering the posterior surface of the graft in the TEEK group. CONCLUSIONS: Grafting of decellularized stroma carriers re-surfaced with human corneal endothelial cells ex vivo can be a readily translatable method to improve visual quality in corneal endothelial diseases.