Potential Role of Induced Pluripotent Stem Cells (IPSCs) for Cell-Based Therapy of the Ocular Surface

The integrity and normal function of the corneal epithelium are crucial for maintaining the cornea's transparency and vision. The existence of a cell population with progenitor characteristics in the limbus maintains a dynamic of constant epithelial repair and renewal. Currently, cell-based therapies for bio replacement cultured limbal epithelial transplantation (CLET) and cultured oral mucosal epithelial transplantation (COMET) present very encouraging clinical results for treating limbal stem cell deficiency (LSCD) and restoring vision. Another emerging therapeutic approach consists of obtaining and implementing human progenitor cells of different origins in association with tissue engineering methods. The development of cell-based therapies using stem cells, such as human adult mesenchymal or induced pluripotent stem cells (IPSCs), represent a significant breakthrough in the treatment of certain eye diseases, offering a more rational, less invasive, and better physiological treatment option in regenerative medicine for the ocular surface. This review will focus on the main concepts of cell-based therapies for the ocular surface and the future use of IPSCs to treat LSCD

Xenofree generation of limbal stem cells for ocular surface advanced cell therapy.

Background: Limbal stem cells (LSC) sustain the corneal integrity and homeostasis. LSC deficiency (LSCD) leads to loss of corneal transparency and blindness. A clinical approach to treat unilateral LSCD comprises autologous cultured limbal epithelial stem cell transplantation (CLET). CLET uses xenobiotic culture systems with potential zoonotic transmission risks, and regulatory guidelines make necessary to find xenofree alternatives. Methods: We compared two xenofree clinical grade media and two feeder layers. We used CnT07, a defined commercial medium for keratinocytes, and a modified xenofree supplemented hormonal epithelial medium with human serum (XSHEM). Optimal formulation was used to compare two feeder layers: the gold standard 3T3 murine fibroblasts and human processed lipoaspirate cells (PLA). We tested the expressions of ΔNp63α and cytokeratin 3 and 12 by qPCR and immunofluorescence. Morphology, viability, clonogenicity, proliferation, and cell growth assays were carried out. We also evaluated interleukin 6 (IL-6) and stromal-derived factor 1 (SDF-1) by qPCR and ELISA. Results: XSHEM maintained better LSC culture viability and morphology than CnT07. Irradiated PLA feeder cells improved the undifferentiated state of LSC and enhanced their growth and clonogenicity stimulating IL-6 secretion and SDF-1 expression, as well as increased proliferation and cell growth when compared with irradiated 3T3 feeder cells. Conclusions: The combination of XSHEM and PLA feeder cells efficiently sustained LSC xenofree cultures for clinical application. Moreover, PLA feeder layers were able to improve the LSC potential characteristics. Our results would have direct clinical application in CLET for advanced therapy

Priming human adipose-derived mesenchymal stem cells for corneal surface regeneration.

Limbal stem cells (LSC) maintain the transparency of the corneal epithelium. Chemical burns lead the loss of LSC inducing an up-regulation of pro-inflammatory and pro-angiogenic factors, triggering corneal neovascularization and blindness. Adipose tissue-derived mesenchymal stem cells (AT-MSC) have shown promise in animal models to treat LSC deficiency (LSCD), but there are not studies showing their efficacy when primed with different media before transplantation. We cultured AT-MSC with standard medium and media used to culture LSC for clinical application. We demonstrated that different media changed the AT-MSC paracrine secretion showing different paracrine effector functions in an in vivo model of chemical burn and in response to a novel in vitro model of corneal inflammation by alkali induction. Treatment of LSCD with AT-MSC changed the angiogenic and inflammatory cytokine profile of mice corneas. AT-MSC cultured with the medium that improved their cytokine secretion, enhanced the anti-angiogenic and anti-inflammatory profile of the treated corneas. Those corneas also presented better outcome in terms of corneal transparency, neovascularization and histologic reconstruction. Priming human AT-MSC with LSC specific medium can potentiate their ability to improve corneal wound healing, decrease neovascularization and inflammation modulating paracrine effector functions in an in vivo optimized rat model of LSCD

Electron energy-loss spectroscopic tomography of FexCo(3-x)O4 impregnated Co3O4 mesoporous particles: unraveling the chemical information in three dimensions

Electron energy-loss spectroscopy-spectrum image (EELS-SI) tomography is a powerful tool to investigate the three dimensional chemical configuration in nanostructures. Here, we demonstrate, for the first time, the possibility to characterize the spatial distribution of Fe and Co cations in a complex FexCo(3-x)O4/Co3O4 ordered mesoporous system. This hybrid material is relevant because of the ferrimagnetic/antiferromagnetic coupling and high surface area. We unambiguously prove that the EELS-SI tomography shows a sufficiently high resolution to simultaneously unravel the pore structure and the chemical signal

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Characterization of Ocular Surface Epithelial and Progenitor Cell Markers in Human Adipose Stromal Cells Derived from Lipoaspirates

The expression of putative stem cell markers and cytokeratins supports the hypothesis that mesenchymal stem cells derived from adult human adipose tissue (ADS cells) have a self-renewal capacity and an intrinsic plasticity that enable them to acquire some epithelial-like characteristics

Xenofree generation of limbal stem cells for ocular surface advanced cell therapy

Limbal stem cells (LSC) sustain the corneal integrity and homeostasis. LSC deficiency (LSCD) leads to loss of corneal transparency and blindness. A clinical approach to treat unilateral LSCD comprises autologous cultured limbal epithelial stem cell transplantation (CLET). CLET uses xenobiotic culture systems with potential zoonotic transmission risks, and regulatory guidelines make necessary to find xenofree alternatives. We compared two xenofree clinical grade media and two feeder layers. We used CnT07, a defined commercial medium for keratinocytes, and a modified xenofree supplemented hormonal epithelial medium with human serum (XSHEM). Optimal formulation was used to compare two feeder layers: the gold standard 3T3 murine fibroblasts and human processed lipoaspirate cells (PLA). We tested the expressions of ΔNp63α and cytokeratin 3 and 12 by qPCR and immunofluorescence. Morphology, viability, clonogenicity, proliferation, and cell growth assays were carried out. We also evaluated interleukin 6 (IL-6) and stromal-derived factor 1 (SDF-1) by qPCR and ELISA. XSHEM maintained better LSC culture viability and morphology than CnT07. Irradiated PLA feeder cells improved the undifferentiated state of LSC and enhanced their growth and clonogenicity stimulating IL-6 secretion and SDF-1 expression, as well as increased proliferation and cell growth when compared with irradiated 3T3 feeder cells. The combination of XSHEM and PLA feeder cells efficiently sustained LSC xenofree cultures for clinical application. Moreover, PLA feeder layers were able to improve the LSC potential characteristics. Our results would have direct clinical application in CLET for advanced therapy

Polymorphisms at phase I-metabolizing enzyme and hormone receptor loci influence the response to anti-TNF therapy in rheumatoid arthritis patients

© Springer Nature Limited 2018The aim of this case-control study was to evaluate whether 47 single-nucleotide polymorphisms (SNPs) in steroid hormone-related genes are associated with the risk of RA and anti-TNF drug response. We conducted a case-control study in 3 European populations including 2936 RA patients and 2197 healthy controls. Of those, a total of 1985 RA patients were treated with anti-TNF blockers. The association of potentially interesting markers in the discovery population was validated through meta-analysis with data from DREAM and DANBIO registries. Although none of the selected variants had a relevant role in modulating RA risk, the meta-analysis of the linear regression data with those from the DREAM and DANBIO registries showed a significant correlation of the CYP3A4rs11773597 and CYP2C9rs1799853 variants with changes in DAS28 after the administration of anti-TNF drugs (P = 0.00074 and P = 0.006, respectively). An overall haplotype analysis also showed that the ESR2GGG haplotype significantly associated with a reduced chance of having poor response to anti-TNF drugs (P = 0.0009). Finally, a ROC curve analysis confirmed that a model built with eight steroid hormone-related variants significantly improved the ability to predict drug response compared with the reference model including demographic and clinical variables (AUC = 0.633 vs. AUC = 0.556; PLR_test = 1.52 × 10-6). These data together with those reporting that the CYP3A4 and ESR2 SNPs correlate with the expression of TRIM4 and ESR2 mRNAs in PBMCs (ranging from P = 1.98 × 10-6 to P = 2.0 × 10-35), and that the CYP2C9rs1799853 SNP modulates the efficiency of multiple drugs, suggest that steroid hormone-related genes may have a role in determining the response to anti-TNF drugs.KEY POINTS• Polymorphisms within the CYP3A4 and CYP2C9 loci correlate with changes in DAS28 after treatment with anti-TNF drugs.• A haplotype including eQTL SNPs within the ESR2 gene associates with better response to anti-TNF drugs.• A genetic model built with eight steroid hormone-related variants significantly improved the ability to predict drug response.This work was supported by grants from FIBAO foundation (Granada, Spain), Novo Nordisk Fonden (NNF15OC0016932), Knud og Edith Eriksens Mindefond and Gigtforeningen (A2037, A3570).info:eu-repo/semantics/publishedVersio