11 research outputs found
Optimization of human limbal epithelial stem cell expansion under chemically defined culture conditions
Limbal epithelial stem cells (LESC’s) are responsible for corneal epithelium regeneration due to corneal damage, or LESC deficiency. One of the major challenges of LESC’s culture is their optimal derivation and expansion in a chemically defined culture media; with the purpose to generate enough amounts of cells with the correct genotype and phenotype. Chemically defined conditions would avoid cells that could compromise the security and efficiency of cell therapy. The need for alternative options to cadaveric corneas will continue to grow in importance as a result of increasing incidence of keratoplasties, hence if we investigate the use of different proteins as extracellular matrix (Laminin, Fibronectin, Collagen type I and Collagen Type IV) during the expansion of LESC’s, we could optimize a “selective expansion of LESC’s”. LESC’s isolation was carried out from human sclera-corneal rims remaining after penetrating keratoplasty procedure. After disinfection, rims were treated by enzymatic digestion followed by a mechanical separation. We investigated different proteins (Laminin, Fibrin, Collagen type I and Collagen type IV) as extracellular matrix in order to culture the cells obtained from enzymatic digestion. Culture plates were pre-treated with 50μg•mL
-1 of each protein for 24h prior to use. The absence of extracellular matrix was included as control. Cell culture and expansion was performed at standard culture conditions (5% CO
2 , 37º C) using chemically defined culture media Epilife
10 (Invitrogen). The presence of Keratin 3 and P63-α proteins, were used to identify epithelial cells and limbal stem cells (respectively) by Immunofluorescence analyses; and mRNA detection by PCR. Cell expansion was assessed by cell count, viability and flow cytometry identifying limbal stem cells (P63-α+) and epithelial cells (CK3+). Once we identified the protein that allows an optimal expansion of LESC, we compared two different culture media (chemical defined Epilife
10 medium and medium SHEM). The use of collagen Type I as extracellular matrix, allowed the “selective expansion” of LESC, obtaining 90% of cells expressing p63-α+ (p=0.0023). Meanwhile Epithelial cells (CK3+) did not show a selective expansion with the use of any protein as extracellular matrix. Previous reports suggest the use of collagen Type I as an “adhesion gradient” by culturing cells only for 24 h post enzymatic digestions of sclera-corneal rims. However we found that culturing cells for the whole period (up to 15 days) of expansion under chemically defined conditions, allows the expansion exclusively of LESC. Growth kinetics showed that the exponential phase of LESC expansion is between 216h to 260h (9 – 12 days). When comparing two different culture media, the non-defined medium SHEM results in expansion of LESC and fibroblast cells; meanwhile the use of chemically defined Epilife
10 (Invitrogen) mediaavoids the presence of fibroblast cell phenotype. Our results suggest that Collagen Type I can be used as extracellular matrix for the selective expansion of limbal epithelial stem cells, reaching the maximum cell number between 216 h and 260 h of culture. Using this strategy, it is possible to obtain up to 1,760,000 cells per rim after 576 hours of culture. This expansion procedure, delivers enough amount of LESC in order to obtain at least 3 corneal substitutes (through 3D culture strategy) from sclera-corneal rims derived and cultured under chemically defined conditions. References
Thoft R., Friend J. (1983). Invest Ophthalmol Vis Sci. 24 (10): 1442- 1443.
Schlötzer U., Kruse F. (2005). Experimental Eye Research. 81 (3): 247- 264.
Daniels J., Harris A., Mason C. (2006). Stem Cell Reviews. 6 (3): 247- 254.
Shortt A., Secker G., Rajan M., Meligonis G., Dart J., Tuft S., Daniels J. (2008). Ophtalmology. 115 (11): 1899-1997.
Ghezzi E., Rnjak-Kovacina J., Kaplan D. (2015) Tissue Eng Part B Rev. 21 (3): 278-8
Directed differentiation of inner ear hair cells from mouse embryonic stem cells (E14Tg2a)
The hair cell plays an essential role in the transmission of the acoustic waves from the air to the auditory neurons in the brain. These cells are found inside the cochlea, a bone spirally duct fully of perilymph, a liquid with high concentrations of K+. It has been reported around 25,000 hairs cells in the human ear. Deafness is a condition quite common; about 90% is due to neurosensorial condition and involves the loss of hair cells and their associated neurons (Rivolta, 2013; Chen et al., 2009). Age, genetic abnormalities and environmental factors (for example, noise and ototoxic drugs such aminoglycosides) are most common causes of deafness. Due to the lack of endogenous regeneration, and to the limitations of available therapies; the potential to develop a system based on the introduction of exogenous specialized cells offers new alternatives as deafness treatment. Embryonic stem cells are excellent candidates for biological implantation, as they have the potential to proliferate and differentiate (Rivolta, 2013). Protocols currently reported to differentiate hair cells from embryonic stem cells use “embryonic –bodies” and co-culture techniques or viral transfection. The use of these techniques results in spontaneous differentiation and low control over early differentiation. This work aims to establish a model of directed differentiation through monolayer culture, using chemically defined media, feeder-cell free, avoiding the use of embryoid bodies and the use of fetal bovine serum in order to obtain higher control over the whole differentiation process. Mouse Embryonic Stem Cells (E14Tg2a) were cultured in GMEM added with Leukemia Inhibitory Factor (LIF). Passage of mES was performed during the exponential phase. To start the differentiation process, 1 x 104 cells·cm-2 were inoculated and cultured for 16 hours in GMEM free of fetal bovine serum and LIF, in order to promote the adhesion. The proposed differentiation method was modified from Li et al. 2003 and involves three stages (all in chemically defined medium) as monolayer culture: 1) Generation of Otic Plate Precursors Cells, culture in GMEM added whit IGF-1 (50 ng·mL-1), EGF (20 ng·mL-1) and N2 supplement, renewing the media every 48 hours. The cells were maintained under these conditions for 240 hours; 2) Expansion of Otic Plate Precursors Cells, the media was replaced by GMEM added with IGF-1 (50 ng·mL-1), EGF (20 ng·mL-1), bFGF (10 ng·mL-1) and N2 supplement, renewing the media every 48 hours during 192 hours. 3) Specialization of Otic Plate Precursors Cells into Hair Cells, all growth factors were removed from the media to promote the specialization, cultured in GMEM added only with N2 supplement during 240 hours. The evaluation of the pluripotent state and cell differentiation was assessed by flow cytometry and inmunocytochemistry. The expression of pluripotency markers for OCT3/4 was 96%, whereas for Nanog was 95% for mES maintenance. The expression of Pax2, Myosin VIIa and Math1 is essential for the development and maturation of hair cells, these markers were detected during the differentiation of embryonic stem cells as follows: In the generation of otic progenitors (168 hours) we observed Pax2 (64%), Myosin VIIa (86%) and Math1 (58%). Meanwhile the expression of these proteins during the specialization of hair cells (at 672 hours of culture) was Pax2 (46%), Myosin VIIa (53%), Math1 (38%). According to the proposed protocol, it is feasible to generate inner ear - hair cells through monolayer culture, feeder-cell free and using chemically defined medium. The expression in an early stage of Pax2 indicated the generation of Otic Precursors Cells, whereas coexpression of Myosin VIIa and Math1 it allowed us to determinate the presence of hair cells.
References
- Chen W., Jhonson S., Marcotti W., Andrews P., Moore H., Rivolta M. Human Fetal Auditory Stem Cells Can Be Expanded In Vitro and Differentiate Into Functional Auditory Neurons and Hair Cell-Like Cells. Stem Cells 2009; 27: 1196 – 1204.
- Li H., Roblin G., Liu H., Heller S. Generation of hair cells by stepwise differentiation of embryonic stem cells.P NatlAcadSci USA 2003; 100(3): 13495-13500.
- Rivolta M. N. New strategies for the restoration of hearing loss: challenges and opportunities. Brit Med Bull 2013; 105: 69 – 8
Mecanismos de señalización intracelular en cáncer de tiroides
ResumenAntecedentesEl cáncer de tiroides es el tumor maligno más frecuente del sistema endocrino; la variante papilar representa entre el 80 y el 90% de todos los casos diagnosticados. En el desarrollo del cáncer papilar de tiroides están afectados principalmente 2genes llamados BRAF y RAS, que alteran el sistema de señalización intracelular de las proteínas conocidas como «cinasas de proteínas activadas por mitógenos» (MAPK, por sus siglas en inglés para mitogen-activated protein kinase) y que se componen de «módulos» de proteínas de señalización interna (Receptor/Ras/Raf/MEK/ERK), que van de la membrana celular al núcleo y que en el cáncer de tiroides regulan diversos procesos celulares, como la diferenciación, crecimiento, desarrollo y apoptosis. Tienen un papel importante en la patogénesis del cáncer de tiroides, debido a que son usados como biomarcadores moleculares, como elementos de diagnóstico, pronóstico y como posibles blancos terapéuticos moleculares.ObjetivoRevisar los mecanismos moleculares que intervienen en las vías de señalización, en las que están involucradas las proteínas de los genes BRAF y RAS, en el cáncer de tiroides.ConclusionesLas mutaciones en el gen BRAF han sido correlacionadas con una pobre respuesta al tratamiento con quimioterapia tradicional, además de ser un índice de mal pronóstico. La terapia molecular es de gran interés en este tipo de cáncer, ya que se han desarrollado medicamentos que actúan inhibiendo alguno de los componentes de la vía de señalización (RET/PTC)/Ras/Raf/MEK/ERK, con especial énfasis en la sección (RET/PTC)/Ras/Raf, que constituye un efector principal de la vía ERK.AbstractBackgroundThyroid cancer is the most common malignancy of the endocrine system, the papillary variant accounts for 80-90% of all diagnosed cases. In the development of papillary thyroid cancer, BRAF and RAS genes are mainly affected, resulting in a modification of the system of intracellular signaling proteins known as «protein kinase mitogen-activated» (MAPK) which consist of «modules» of internal signaling proteins (Receptor/Ras/Raf/MEK/ERK) from the cell membrane to the nucleus. In thyroid cancer, these signanling proteins regulate diverse cellular processes such as differentiation, growth, development and apoptosis. MAPK play an important role in the pathogenesis of thyroid cancer as they are used as molecular biomarkers for diagnostic, prognostic and as possible therapeutic molecular targets. Mutations in BRAF gene have been correlated with poor response to treatment with traditional chemotherapy and as an indicator of poor prognosis.ObjectiveTo review the molecular mechanisms involved in intracellular signaling of BRAF and RAS genes in thyroid cancer.ConclusionsMolecular therapy research is in progress for this type of cancer as new molecules have been developed in order to inhibit any of the components of the signaling pathway (RET/PTC)/Ras/Raf/MEK/ERK; with special emphasis on the (RET/PTC)/Ras/Raf section, which is a major effector of ERK pathway
Spontaneous Bacterial Peritonitis: Physiopathological Mechanism and Clinical Manifestations
Changes in intestinal permeability have been determined to influence secondary inflammatory reactions and clinical manifestations such as spontaneous bacterial peritonitis (SBP) secondary to cirrhosis. As of yet, no in-depth exploration of the changes in the microbiota and how this influences cirrhosis to differ from clinically more severe cases than others has not begun. However, at the level of pathophysiological mechanism, it must be taken into account that due to the abuse of substances such as alcohol and chronic fatty liver disease, changes in the bacterial composition and intestinal permeability are induced. This set of changes in the bacterial composition (microbiome) and modification of the intestinal permeability could be related to the presence of ascites and spontaneous peritonitis secondary to cirrhosis, being of relevance the knowledge of the mechanisms underlying this phenomenon, as well as clinical manifestation. Prophylaxis and antibiotic treatment of SBP requires clinical knowledge for the treatment decisions based mainly on the presence of ascitic fluid, accompanied of risk factors, laboratory indexes such as PMN count and culture results, in order to determine the kind of molecule that will help to the SBP recovery or to amelioration symptoms, always taking care of not exceed the antibiotic consumption and restoring the microbiome imbalance
Effect of Norelgestromin and Ethinylestradiol in Transdermal Patches on the Clinical Outcomes and Biochemical Parameters of COVID-19 Patients: A Clinical Trial Pilot Study
The disease caused by SARS-CoV-2 is still considered a global pandemic. Transdermal patches (TP) with immunoregulators such as estrogen and progesterone compounds could be a feasible option to treat COVID-19 because of their accessibility and relative safety. The objective of the current study was to evaluate the additional treatment with norelgestromin and ethinylestradiol in TP on the clinical and biochemical evolution of COVID-19 patients. The present is a clinical-trial pilot study that included subjects diagnosed with COVID-19, randomized into two groups; the experimental Evra® TP (norelgestromin 6 mg and ethinylestradiol 0.60 mg) was administered such that it was applied on arrival and replaced at day 8 and day 15. The control continued with the conventional COVID-19 treatment protocol. A blood sample was taken each week in order to evaluate relevant biochemical parameters, clinical signs, and evolution. In total, 44 subjects participated in this study, 30 in the experimental group and 14 in the control group. Both groups were homogeneous in terms of age and comorbidities. The experimental group had a significantly lower hospital stay (p = 0.01), high flow supplemental oxygen (p = 0.001), mechanical ventilation (p = 0.003), and intubation (p = 0.01), and the oxygen saturation significantly increased (p = 0.01) in comparison with control group when patients were exposed to room air. A decrease in ferritin (p < 0.05) was observed, with no significant increase in ESR (p > 0.05), D dimer (p > 0.05) and platelets (p > 0.05) in an auto-controlled analysis in the experimental group. Norelgestromin and ethinylestradiol TP could be a safe and effective treatment for moderate and severe COVID-19 patients
Analysis of Impulse Control Disorders (ICDs) and Factors Associated with Their Development in a Parkinson’s Disease Population
Parkinson’s Disease (PD) is a neurodegenerative disease in which non-motor symptoms may appear before motor phenomena, which include Impulse Control Disorders (ICDs). The objective of this study is to identify factors associated with the development of ICDs in PD. An analytical, cross-sectional study was conducted using clinical records from patients diagnosed with PD, both genders, from 40 to 80 years old. Clinical and demographic data were collected: 181 patients were recruited; 80 of them showed PD and ICDs, and they constituted the study group, whereas 101 patients with PD without ICDs constituted the control reference group. The duration of PD was longer in the group with ICDs (p < 0.008), and all patients showed at least one ICD: binge eating (61.29%), compulsive shopping (48.75%), hypersexuality (23.75%), gambling behavior (8.75%), and punding (3.75%). After logistic regression analysis, only the use of dopamine agonists remained associated with ICDs (p < 0.001), and the tremorgenic form was suggested to be a protective factor (p < 0.001). Positive associations were observed between the rigid-akinetic form and compulsive shopping (p < 0.007), between male and hypersexuality (p < 0.018), and between dopamine agonists and compulsive shopping (p < 0.004), and negative associations were observed between motor fluctuations and compulsive shopping (p < 0.031), between Deep Brain Stimulation and binge eating (p < 0.046), and between levodopa consumption and binge eating (p < 0.045). Binge eating, compulsive shopping, and hypersexuality were the most frequent ICDs. Complex forms and motor complications of PD were associated with the development of ICDs
Non-Invasive Biomarkers for Duchenne Muscular Dystrophy and Carrier Detection
Non-invasive biological indicators of the absence/presence or progress of the disease that could be used to support diagnosis and to evaluate the effectiveness of treatment are of utmost importance in Duchenne Muscular Dystrophy (DMD). This neuromuscular disorder affects male children, causing weakness and disability, whereas female relatives are at risk of being carriers of the disease. A biomarker with both high sensitivity and specificity for accurate prediction is preferred. Until now creatine kinase (CK) levels have been used for DMD diagnosis but these fail to assess disease progression. Herein we examined the potential applicability of serum levels of matrix metalloproteinase 9 (MMP-9) and matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinases 1 (TIMP-1), myostatin (GDF-8) and follistatin (FSTN) as non-invasive biomarkers to distinguish between DMD steroid naïve patients and healthy controls of similar age and also for carrier detection. Our data suggest that serum levels of MMP-9, GDF-8 and FSTN are useful to discriminate DMD from controls (p < 0.05), to correlate with some neuromuscular assessments for DMD, and also to differentiate between Becker muscular dystrophy (BMD) and Limb-girdle muscular dystrophy (LGMD) patients. In DMD individuals under steroid treatment, GDF-8 levels increased as FSTN levels decreased, resembling the proportions of these proteins in healthy controls and also the baseline ratio of patients without steroids. GDF-8 and FSTN serum levels were also useful for carrier detection (p < 0.05). Longitudinal studies with larger cohorts are necessary to confirm that these molecules correlate with disease progression. The biomarkers presented herein could potentially outperform CK levels for carrier detection and also harbor potential for monitoring disease progression
Epidemiological Profile and Social Welfare Index as Factors Associated with COVID-19 Hospitalization and Severity in Mexico City: A Retrospective Analysis
Epidemiological data indicate that Mexico holds the 19th place in cumulative cases (5506.53 per 100,000 inhabitants) of COVID-19 and the 5th place in cumulative deaths (256.14 per 100,000 inhabitants) globally and holds the 4th and 3rd place in cumulative cases and deaths in the Americas region, respectively, with Mexico City being the most affected area. Several modifiable and non-modifiable risk factors have been linked to a poor clinical outcome in COVID-19 infection; however, whether socioeconomic and welfare factors are associated with clinical outcome has been scanty addressed. This study tried to investigate the association of Social Welfare Index (SWI) with hospitalization and severity due to COVID-19. A retrospective analysis was conducted at the Centro Médico Nacional “20 de Noviembre”—ISSSTE, based in Mexico City, Mexico. A total of 3963 patients with confirmed or suspected COVID-19, registered from March to July 2020, were included, retrieved information from the Virology Analysis and Reference Unit Database. Demographic, symptoms and clinical data were analyzed, as well as the SWI, a multidimensional parameter based on living and household conditions. An adjusted binary logistic regression model was performed in order to compare the outcomes of hospitalization, mechanical ventilation requirement (MVR) and mortality between SWI categories: Very high (VHi), high (Hi), medium (M) and low (L). The main findings show that lower SWI were independently associated with higher probability for hospital entry: VHi vs. Hi vs. M vs. L-SWI (0 vs. +0.24 [OR = 1.24, CI95% 1.01–1.53] vs. +0.90 [OR = 1.90, CI95% 1.56–2.32] vs. 0.73 [OR = 1.73, CI95% 1.36–2.19], respectively); Mechanical Ventilation Requirement: VHi vs. M vs. L-SWI (0 vs. +0.45 [OR = 1.45, CI95% 1.11–1.87] vs. +0.35 [OR = 1.35, CI95% 1.00–1.82]) and mortality: VHi vs. Hi vs. M (0 vs. +0.54 [OR = 1.54, CI95% 1.22–1.94] vs. +0.41 [OR = 1.41, CI95% 1.13–1.76]). We concluded that SWI was independently associated with the poor clinical outcomes in COVID-19, beyond demographic, epidemiological and clinical characteristics
Endothelial Progenitor Cells May Be Related to Major Amputation after Angioplasty in Patients with Critical Limb Ischemia
Background: Critical limb ischemia represents an advanced stage of peripheral arterial disease. Angioplasty improves blood flow to the limb; however, some patients progress irreversibly to lower limb amputation. Few studies have explored the predictive potential of biomarkers during postangioplasty outcomes. Aim: To evaluate the behavior of endothelial progenitor cells in patients with critical limb ischemia, in relation to their postangioplasty outcome. Methods: Twenty patients with critical limb ischemia, candidates for angioplasty, were enrolled. Flow-mediated dilation, as well as endothelial progenitor cells (subpopulations CD45+/CD34+/CD133+/CD184+ and CD45+/CD/34+/KDR[VEGFR-2]+ estimated by flow cytometry) from blood flow close to vascular damage, were evaluated before and after angioplasty. Association with lower limb amputation during a 30-day follow-up was analyzed. Results: Endothelial progenitor cells were related with flow-mediated dilation. A higher number of baseline EPCs CD45+CD34+KDR+, as well as an impaired reactivity of endothelial progenitor cells CD45+CD34+CD133+CD184+ after angioplasty, were observed in cases further undergoing major limb amputation, with a significant discrimination ability and risk (0.75, specificity 0.83 and RR 4.5 p +CD34+KDR+, as well as an impaired reactivity of subpopulation CD45+CD34+CD133+CD184+ after angioplasty, showed a predictive ability for major limb amputation in patients with critical limb ischemia