Restored glyoxylate metabolism after AGXT gene correction and direct reprogramming of primary hyperoxaluria type 1 fibroblasts.

Primary hyperoxaluria type 1 (PH1) is a rare inherited metabolic disorder characterized by oxalate overproduction in the liver, resulting in renal damage. It is caused by mutations in the AGXT gene. Combined liver and kidney transplantation is currently the only permanent curative treatment. We combined locus-specific gene correction and hepatic direct cell reprogramming to generate autologous healthy induced hepatocytes (iHeps) from PH1 patient-derived fibroblasts. First, site-specific AGXT corrected cells were obtained by homology directed repair (HDR) assisted by CRISPR-Cas9, following two different strategies: accurate point mutation (c.731T>C) correction or knockin of an enhanced version of AGXT cDNA. Then, iHeps were generated, by overexpression of hepatic transcription factors. Generated AGXT-corrected iHeps showed hepatic gene expression profile and exhibited in vitro reversion of oxalate accumulation compared to non-edited PH1-derived iHeps. This strategy set up a potential alternative cellular source for liver cell replacement therapy and a personalized PH1 in vitro disease model.

Soluble mutant huntingtin drives early human pathogenesis in Huntington's disease.

Huntington's disease (HD) is an incurable inherited brain disorder characterised by massive degeneration of striatal neurons, which correlates with abnormal accumulation of misfolded mutant huntingtin (mHTT) protein. Research on HD has been hampered by the inability to study early dysfunction and progressive degeneration of human striatal neurons in vivo. To investigate human pathogenesis in a physiologically relevant context, we transplanted human pluripotent stem cell-derived neural progenitor cells (hNPCs) from control and HD patients into the striatum of new-born mice. Most hNPCs differentiated into striatal neurons that projected to their target areas and established synaptic connexions within the host basal ganglia circuitry. Remarkably, HD human striatal neurons first developed soluble forms of mHTT, which primarily targeted endoplasmic reticulum, mitochondria and nuclear membrane to cause structural alterations. Furthermore, HD human cells secreted extracellular vesicles containing mHTT monomers and oligomers, which were internalised by non-mutated mouse striatal neurons triggering cell death. We conclude that interaction of mHTT soluble forms with key cellular organelles initially drives disease progression in HD patients and their transmission through exosomes contributes to spread the disease in a non-cell autonomous manner.

Lack of EGFR catalytic activity in hepatocytes improves liver regeneration following DDC-induced cholestatic injury by promoting a pro-restorative inflammatory response.

Despite the well-known hepatoprotective role of the epidermal growth factor receptor (EGFR) pathway upon acute damage, its specific actions during chronic liver disease, particularly cholestatic injury, remain ambiguous and unresolved. Here, we analyzed the consequences of inactivating EGFR signaling in the liver on the regenerative response following cholestatic injury. For that, transgenic mice overexpressing a dominant negative mutant human EGFR lacking tyrosine kinase activity (ΔEGFR) in albumin-positive cells were submitted to liver damage induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), an experimental model resembling human primary sclerosing cholangitis. Our results show an early activation of EGFR after 1-2 days of a DDC-supplemented diet, followed by a signaling switch-off. Furthermore, ΔEGFR mice showed less liver damage and a more efficient regeneration following DDC injury. Analysis of the mechanisms driving this effect revealed an enhanced activation of mitogenic/survival signals, AKT and ERK1/2-MAPKs, and changes in cell turnover consistent with a quicker resolution of damage in response to DDC. These changes were concomitant with profound differences in the profile of intrahepatic immune cells, consisting of a shift in the M1/M2 balance towards M2 polarity, and the Cd4/Cd8 ratio in favor of Cd4 lymphocytes, overall supporting an immune cell switch into a pro-restorative phenotype. Interestingly, ΔEGFR livers also displayed an amplified ductular reaction, with increased expression of EPCAM and an increased number of CK19-positive ductular structures in portal areas, demonstrating an overexpansion of ductular progenitor cells. In summary, our work supports the notion that hepatocyte-specific EGFR activity acts as a key player in the crosstalk between parenchymal and non-parenchymal hepatic cells, promoting the pro-inflammatory response activated during cholestatic injury and therefore contributing to the pathogenesis of cholestatic liver disease. © 2022 The Pathological Society of Great Britain and Ireland.

Efficient and Fast Generation of Relevant Disease Mouse Models by In Vitro and In Vivo Gene Editing of Zygotes.

Knockout mice for human disease-causing genes provide valuable models in which new therapeutic approaches can be tested. Electroporation of genome editing tools into zygotes, in vitro or within oviducts, allows for the generation of targeted mutations in a shorter time. We have generated mouse models deficient in genes involved in metabolic rare diseases (Primary Hyperoxaluria Type 1 Pyruvate Kinase Deficiency) or in a tumor suppressor gene (Rasa1). Pairs of guide RNAs were designed to generate controlled deletions that led to the absence of protein. In vitro or in vivo ribonucleoprotein (RNP) electroporation rendered more than 90% and 30% edited newborn animals, respectively. Mice lines with edited alleles were established and disease hallmarks have been verified in the three models that showed a high consistency of results and validating RNP electroporation into zygotes as an efficient technique for disease modeling without the need to outsource to external facilities.

Diabetes mellitus y su influencia en la sexualidad de pacientes varones / Diabetes mellitus and its influence on the sexuality of male patients

RESUMEN Objetivo Estudiar cómo la diabetes mellitus tipo 2 ha influenciado la sexualidad de 54 varones mellitus que acudieron a consulta a dos hospitales de Taxco, Guerrero. Materiales y Métodos Se aplicó una encuesta a un total de 54 hombres que acudieron a consulta a dos hospitales de Taxco, Guerrero, México. Se consideró a hombres con diabetes mellitus tipo 2, con vida sexual activa, con más de 5 años de evolución de la enfermedad, y que acudieron a consulta periódica. Los casos se eligieron tomando en cuenta los planteamientos sobre muestras por conveniencia. El trabajo fue cuantitativo no probabilístico. El cuestionario de encuesta incluyó 15 variables. Resultados Entre los hallazgos alentadores y positivos se destaca que el 76% de los casos manifestó que nunca ha tenido una disminución en el desempeño sexual, y el 80% de los casos expresó nunca haber presentado dolor o molestia después del coito. Sobre los porcentajes adversos y negativos, el 33% manifestó haber tenido siempre un cambió (de manera nociva o dañina) en el interés ante la sexualidad. Conclusiones Diversos autores destacan cómo la diabetes mellitus es una enfermedad epidémica en México, y que provoca incapacidad, mortalidad prematura, y afecta de manera grave la sexualidad de pacientes. En el presente estudio las respuestas indican, tanto hallazgos positivos, como también, un impacto adverso y negativo en la sexualidad de algunos varones encuestados.

ABSTRACT Objective To study and analyze how type 2 diabetes mellitus has influenced the sexuality of male patients. Material and Methods A survey was applied to a total of 54 men who attended in two hospitals in Taxco, Guerrero, México. Men with type 2 diabetes mellitus, with an active sexual life, with more than 5 years of evolution of the disease, and who attended periodic consultations were considered. The cases were chosen taking into account the approaches on convenience samples. The work was quantitative, not probabilistic. The survey questionnaire included 15 variables. Results Among the findings encouraging and positive, it is highlighted that 76% of the cases reported never having had a decrease in sexual performance. Likewise, and 80% of the cases expressed never having presented pain or discomfort after inter-course. Regarding the adverse and negative percentages, the 33% stated that they had always had a change (in a harmful way) in their interest in sexuality. Conclusions Several authors highlight how diabetes mellitus is an epidemic disease in Mexico, which can cause disability and premature mortality, and seriously affect the sexuality of patients. In the present study the responses indicate both positive findings, as well as an adverse and negative impact on the sexuality of some male respondents.

Preclinical studies of efficacy thresholds and tolerability of a clinically ready lentiviral vector for pyruvate kinase deficiency treatment.

Pyruvate kinase deficiency (PKD) is a rare autosomal recessive disorder caused by mutations in the PKLR gene. PKD is characterized by non-spherocytic hemolytic anemia of variable severity and may be fatal in some cases during early childhood. Although not considered the standard of care, allogeneic stem cell transplantation has been shown as a potentially curative treatment, limited by donor availability, toxicity, and incomplete engraftment. Preclinical studies were conducted to define conditions to enable consistent therapeutic reversal, which were based on our previous data on lentiviral gene therapy for PKD. Improvement of erythroid parameters was identified by the presence of 20%-30% healthy donor cells. A minimum vector copy number (VCN) of 0.2-0.3 was required to correct PKD when corrected cells were transplanted in a mouse model for PKD. Biodistribution and pharmacokinetics studies, with the aim of conducting a global gene therapy clinical trial for PKD patients (RP-L301-0119), demonstrated that genetically corrected cells do not confer additional side effects. Moreover, a clinically compatible transduction protocol with mobilized peripheral blood CD34+ cells was optimized, thus facilitating the efficient transduction on human cells capable of repopulating the hematopoiesis of immunodeficient mice. We established conditions for a curative lentiviral vector gene therapy protocol for PKD.

Impacto de la diabetes en la sexualidad de pacientes mujeres / Impact of diabetes on the sexuality of female patients

RESUMEN Objetivo Estudiar el impacto de la diabetes mellitus tipo 2 en la sexualidad de pacientes mujeres. Se analiza el efecto que ha tenido la diabetes mellitus tipo 2 en la sexualidad de 93 mujeres que recibieron consulta en dos hospitales de la comunidad de Taxco, Guerrero. Metodología Se aplicó una encuesta de 15 variables a un total de 93 pacientes mujeres que acudieron a consulta a dos hospitales de Gobierno en la ciudad de Taxco, Guerrero, México. Se contempló a mujeres con diabetes mellitus tipo 2, con vida sexual activa, con más de cinco años de evolución de la enfermedad. Los casos se eligieron tomando en cuenta el concepto de muestras por conveniencia. El diseño fue cuantitativo, no probabilístico. Resultados La diabetes tuvo un impacto desfavorable en la sexualidad de una proporción importante de las mujeres consideradas. Respecto a los porcentajes negativos, se documentó cómo el 32% de las mujeres nunca tuvieron deseo de tener relaciones sexuales. El 25% del universo manifestó que siempre hubo un cambio de interés por las relaciones después el diagnóstico (de forma perjudicial y adversa). El 17% confirmó siempre presentar irritación vaginal. Y el 42% de las pacientes manifestaron nunca experimentar orgasmos. Finalmente, para el 23% de las mujeres nunca fue satisfactoria la frecuencia de dichas relaciones. Conclusiones Se destacan hallazgos nocivos en algunas de las variables que ratifican cómo la diabetes ha tenido un impacto dañino en la sexualidad de un porcentaje importante de las mujeres encuestadas.

ABSTRACT Objective To study the impact that type 2 diabetes mellitus has had on the sexuality of 93 women. This work analyzes the effect that type 2 diabetes mellitus has had on the sexuality of 93 female patients who received consultations in two hospitals in the city of Taxco, Guerrero. Methodology A survey of 15 variables was applied to a total of 93 female patients who attended in two Hospitals in the city of Taxco, Guerrero, Mexico. Women with type 2 diabetes mellitus, with an active sexual life, with more than 5 years of evolution of the disease, and who attended periodic consultations were considered. The cases were chosen keeping the concept of convenience samples in mind. The design was quantitative, not probabilistic. Results The diabetes had an unfavorable impact on the sexuality of a significant pro-portion of the women considered. Regarding the negative percentages, it was documented how 32% of the women never had a desire to have sexual intercourse. The 25% of the universe stated that there was always a change in interest in relationships after the diagnosis (in adverse way). The 17% always confirmed having vaginal irritation or dryness. And 42% patients reported never experiencing orgasms. Finally, for 23% of the women the frequency of such relationships was never satisfactory. Conclusions They highlighted harmful findings in some of the variables, which confirm how diabetes has had a harmful impact on the sexuality of a significant percentage of the women surveyed.

Human Pluripotent Stem Cell-Derived Neurons Are Functionally Mature In Vitro and Integrate into the Mouse Striatum Following Transplantation.

Human pluripotent stem cells (hPSCs) are a powerful tool for modelling human development. In recent years, hPSCs have become central in cell-based therapies for neurodegenerative diseases given their potential to replace affected neurons. However, directing hPSCs into specific neuronal types is complex and requires an accurate protocol that mimics endogenous neuronal development. Here we describe step-by-step a fast feeder-free neuronal differentiation protocol to direct hPSCs to mature forebrain neurons in 37 days in vitro (DIV). The protocol is based upon a combination of specific morphogens, trophic and growth factors, ions, neurotransmitters and extracellular matrix elements. A human-induced PSC line (Ctr-Q33) and a human embryonic stem cell line (GEN-Q18) were used to reinforce the potential of the protocol. Neuronal activity was analysed by single-cell calcium imaging. At 8 DIV, we obtained a homogeneous population of hPSC-derived neuroectodermal progenitors which self-arranged in bi-dimensional neural tube-like structures. At 16 DIV, we generated hPSC-derived neural progenitor cells (NPCs) with mostly a subpallial identity along with a subpopulation of pallial NPCs. Terminal in vitro neuronal differentiation was confirmed by the expression of microtubule associated protein 2b (Map 2b) by almost 100% of hPSC-derived neurons and the expression of specific-striatal neuronal markers including GABA, CTIP2 and DARPP-32. HPSC-derived neurons showed mature and functional phenotypes as they expressed synaptic markers, voltage-gated ion channels and neurotransmitter receptors. Neurons displayed diverse spontaneous activity patterns that were classified into three major groups, namely "high", "intermediate" and "low" firing neurons. Finally, transplantation experiments showed that the NPCs survived and differentiated within mouse striatum for at least 3 months. NPCs integrated host environmental cues and differentiated into striatal medium-sized spiny neurons (MSNs), which successfully integrated into the endogenous circuitry without teratoma formation. Altogether, these findings demonstrate the potential of this robust human neuronal differentiation protocol, which will bring new opportunities for the study of human neurodevelopment and neurodegeneration, and will open new avenues in cell-based therapies, pharmacological studies and alternative in vitro toxicology.

c-Met Signaling Is Essential for Mouse Adult Liver Progenitor Cells Expansion After Transforming Growth Factor-ß-Induced Epithelial-Mesenchymal Transition and Regulates Cell Phenotypic Switch.

Adult hepatic progenitor cells (HPCs)/oval cells are bipotential progenitors that participate in liver repair responses upon chronic injury. Recent findings highlight HPCs plasticity and importance of the HPCs niche signals to determine their fate during the regenerative process, favoring either fibrogenesis or damage resolution. Transforming growth factor-ß (TGF-ß) and hepatocyte growth factor (HGF) are among the key signals involved in liver regeneration and as component of HPCs niche regulates HPCs biology. Here, we characterize the TGF-ß-triggered epithelial-mesenchymal transition (EMT) response in oval cells, its effects on cell fate in vivo, and the regulatory effect of the HGF/c-Met signaling. Our data show that chronic treatment with TGF-ß triggers a partial EMT in oval cells based on coexpression of epithelial and mesenchymal markers. The phenotypic and functional profiling indicates that TGF-ß-induced EMT is not associated with stemness but rather represents a step forward along hepatic lineage. This phenotypic transition confers advantageous traits to HPCs including survival, migratory/invasive and metabolic benefit, overall enhancing the regenerative potential of oval cells upon transplantation into a carbon tetrachloride-damaged liver. We further uncover a key contribution of the HGF/c-Met pathway to modulate the TGF-ß-mediated EMT response. It allows oval cells expansion after EMT by controlling oxidative stress and apoptosis, likely via Twist regulation, and it counterbalances EMT by maintaining epithelial properties. Our work provides evidence that a coordinated and balanced action of TGF-ß and HGF are critical for achievement of the optimal regenerative potential of HPCs, opening new therapeutic perspectives. Stem Cells 2019;37:1108-1118.

Medición de parámetros asociados al síndrome metabólico en alumnos de enfermería en Taxco, México / Measurement of parameters associated with metabolic syndrome in nursing students from Taxco, México / Medição de parâmetros associados à síndrome metabólica em estudantes de enfermagem em Taxco, México

RESUMEN Objetivo Medir los parámetros relacionados con el síndrome metabólico (historias clínicas, medidas antropométricas y muestras bioquímicas) de 50 estudiantes de enfermería. Material y Métodos En esta obra participaron bajo su consentimiento informado, 50 alumnos del primer año de la Licenciatura en Enfermería en la Unidad Académica No. 4 de la Universidad Autónoma de Guerrero, México. Se elaboraron historias clínicas, se registraron variables antropométricas (peso, índice de masa corporal, y medición de cintura), se tomaron muestras bioquímicas (colesterol HDL, triglicéridos y glucosa), y se anotó la tensión arterial del grupo en cuestión. Finalmente, se utilizó estadística descriptiva para explicar los resultados. Resultados Se encontraron tres parámetros que sobrepasaron los límites estándares normales establecidos; mismos que se encuentran asociados al síndrome metabólico. Éstos parámetros fueron el índice de masa corporal con sobrepeso y obesidad en el 62% del universo (31 casos), las medidas de la cintura altos en el 52% del total (26 estudiantes), y el colesterol HDL bajo en el 72% de los casos (36 alumnos). Conclusiones Finalmente, los resultados analizados confirman cómo el 26% (13 estudiantes) presentaron el síndrome metabólico, ya que registraron tres o más parámetros de forma conjunta fuera del rango permitido o normal. Estos alumnos pueden encontrarse en riesgo de contraer, a mediano y largo plazo, afecciones crónicas graves relacionadas con dicho síndrome, tales como las enfermedades cardiovasculares, y/o diabetes.(AU)

ABSTRACT Objective To measure the parameters and results related to metabolic syndrome (clinical records, anthropometric measurements and biochemical samples) in the group of students in question. Material and Methods Fifty first-year students of the Bachelor of Nursing at the Academic Unit No. 4 of the Universidad Autónoma de Guerrero, México, participated in this study and provided their informed consent. Medical records were prepared, while anthropometric variables (weight, body mass index and waist measurement) were recorded. Biochemical samples (HDL cholesterol, triglycerides and glucose) and blood pressure were taken. Finally, descriptive statistics were used to explain the results. Results Regarding the findings, three parameters exceeded the normal standards limits. These parameters were associated with metabolic syndrome and included overweight and obesity in 62% of the sample (n=31), high waist measurements in 52% (n=26), and low HDL cholesterol in 72% (n=36). Conclusions The analyzed results confirm that 26% (13 students) of the sample presented with metabolic syndrome, as they had three or more parameters above the normal range. These students may be at medium- and long-term risk for serious chronic conditions related to the metabolic syndrome, such as cardiovascular diseases and/or diabetes.(AU)

RESUMO Objetivo Medir os parâmetros relacionados à síndrome metabólica (prontuários, medidas antropométricas e amostras bioquímicas) de 50 estudantes de enfermagem. Material e métodos Neste trabalho, com seu consentimento informado, participaram 50 estudantes do primeiro ano do Curso de Enfermagem da Unidade Acadêmica nº 4 da Universidade Autônoma de Guerrero, México. Foram elaborados históricos médicos, anotadas as variáveis ​​antropométricas (peso, índice de massa corporal e medida da cintura), colhidas amostras bioquímicas (colesterol HDL, triglicerídeos e glicose) e anotada a pressão arterial do grupo em questão. Por fim, a estatística descritiva foi utilizada para explicar os resultados. Resultados Foram encontrados três parâmetros que ultrapassaram os limites do padrão normal estabelecido; os mesmos que estão associados à síndrome metabólica. Esses parâmetros foram índice de massa corporal sobrepeso e obesidade em 62% do universo (31 casos), medidas de cintura alta em 52% do total (26 alunos) e baixo colesterol HDL em 72% dos os casos (36 alunos). Conclusões Por fim, os resultados analisados ​​confirmam como 26% (13 alunos) apresentaram a síndrome metabólica, pois registraram três ou mais parâmetros juntos fora da faixa permitida ou normal. Esses alunos podem estar sob risco de contrair, a médio e longo prazo, condições crônicas graves relacionadas a essa síndrome, como doenças cardiovasculares e / ou diabetes.(AU)

[Measurement of parameters associated with metabolic syndrome in nursing students from Taxco, México]. / Medición de parámetros asociados al síndrome metabólico en alumnos de enfermería en Taxco, México.

OBJECTIVE: To measure the parameters and results related to metabolic syndrome (clinical records, anthropometric measurements and biochemical samples) in the group of students in question. MATERIAL AND METHODS: Fifty first-year students of the Bachelor of Nursing at the Academic Unit No. 4 of the Universidad Autónoma de Guerrero, México, participated in this study and provided their informed consent. Medical records were prepared, while anthropometric variables (weight, body mass index and waist measurement) were recorded. Biochemical samples (HDL cholesterol, triglycerides and glucose) and blood pressure were taken. Finally, descriptive statistics were used to explain the results. RESULTS: Regarding the findings, three parameters exceeded the normal standards limits. These parameters were associated with metabolic syndrome and included overweight and obesity in 62% of the sample (n=31), high waist measurements in 52% (n=26), and low HDL cholesterol in 72% (n=36). CONCLUSIONS: The analyzed results confirm that 26% (13 students) of the sample presented with metabolic syndrome, as they had three or more parameters above the normal range. These students may be at medium- and long-term risk for serious chronic conditions related to the metabolic syndrome, such as cardiovascular diseases and/or diabetes.

OBJETIVO: Medir los parámetros relacionados con el síndrome metabólico (historias clínicas, medidas antropométricas y muestras bioquímicas) de 50 estudiantes de enfermería. MATERIAL Y MÉTODOS: En esta obra participaron bajo su consentimiento informado, 50 alumnos del primer año de la Licenciatura en Enfermería en la Unidad Académica No. 4 de la Universidad Autónoma de Guerrero, México. Se elaboraron historias clínicas, se registraron variables antropométricas (peso, índice de masa corporal, y medición de cintura), se tomaron muestras bioquímicas (colesterol HDL, triglicéridos y glucosa), y se anotó la tensión arterial del grupo en cuestión. Finalmente, se utilizó estadística descriptiva para explicar los resultados. RESULTADOS: Se encontraron tres parámetros que sobrepasaron los límites estándares normales establecidos; mismos que se encuentran asociados al síndrome metabólico. Éstos parámetros fueron el índice de masa corporal con sobrepeso y obesidad en el 62% del universo (31 casos), las medidas de la cintura altos en el 52% del total (26 estudiantes), y el colesterol HDL bajo en el 72% de los casos (36 alumnos). CONCLUSIONES: Finalmente, los resultados analizados confirman cómo el 26% (13 estudiantes) presentaron el síndrome metabólico, ya que registraron tres o más parámetros de forma conjunta fuera del rango permitido o normal. Estos alumnos pueden encontrarse en riesgo de contraer, a mediano y largo plazo, afecciones crónicas graves relacionadas con dicho síndrome, tales como las enfermedades cardiovasculares, y/o diabetes.

Insulin receptor isoform A ameliorates long-term glucose intolerance in diabetic mice.

Type 2 diabetes mellitus is a complex metabolic disease and its pathogenesis involves abnormalities in both peripheral insulin action and insulin secretion. Previous in vitro data showed that insulin receptor isoform A, but not B, favours basal glucose uptake through its specific association with endogenous GLUT1/2 in murine hepatocytes and beta cells. With this background, we hypothesized that hepatic expression of insulin receptor isoform A in a mouse model of type 2 diabetes could potentially increase the glucose uptake of these cells, decreasing the hyperglycaemia and therefore ameliorating the diabetic phenotype. To assure this hypothesis, we have developed recombinant adeno-associated viral vectors expressing insulin receptor isoform A (IRA) or isoform B (IRB) under the control of a hepatocyte--specific promoter. Our results demonstrate that in the long term, hepatic expression of IRA in diabetic mice is more efficient than IRB in ameliorating glucose intolerance. Consequently, it impairs the induction of compensatory mechanisms through beta cell hyperplasia and/or hypertrophy that finally lead to beta cell failure, reverting the diabetic phenotype in about 8 weeks. Our data suggest that long-term hepatic expression of IRA could be a promising therapeutic approach for the treatment of type 2 diabetes mellitus.

Silencing of hepatic fate-conversion factors induce tumorigenesis in reprogrammed hepatic progenitor-like cells.

BACKGROUND: Several studies have reported the direct conversion of mouse fibroblasts to hepatocyte-like cells with different degrees of maturation by expression of hepatic fate-conversion factors. METHODS: We have used a combination of lentiviral vectors expressing hepatic fate-conversion factors with Oct4, Sox2, Klf4, and Myc to convert mouse embryonic fibroblasts into hepatic cells. RESULTS: We have generated hepatic cells with progenitor-like features (iHepL cells). iHepL cells displayed basic hepatocyte functions but failed to perform functions characteristic of mature hepatocytes such as significant Cyp450 or urea cycle activities. iHepL cells expressed multiple hepatic-specific transcription factors and functional genes characteristic of immature hepatocytes and cholangiocytes, as well as high levels of Foxl1, Cd24a, and Lgr5, specific markers of hepatic progenitor cells. When transplanted into partial hepatectomized and hepatic irradiated mice, they differentiated into hepatocytes and cholangiocytes. However, iHepL cells formed malignant non-teratoma cell aggregations in one out of five engrafted livers and five out of five xenografts assays. All the cells in these tumors had silenced key hepatic fate-conversion factors, and lost hepatic features. CONCLUSIONS: This study highlights the dangers of using pluripotency factors in reprogramming strategies when fate-conversion factors are silenced in vivo, and urges us to perform extensive tumorigenic tests in reprogrammed cells.

Safe and Efficient Gene Therapy for Pyruvate Kinase Deficiency.

Pyruvate kinase deficiency (PKD) is a monogenic metabolic disease caused by mutations in the PKLR gene that leads to hemolytic anemia of variable symptomatology and that can be fatal during the neonatal period. PKD recessive inheritance trait and its curative treatment by allogeneic bone marrow transplantation provide an ideal scenario for developing gene therapy approaches. Here, we provide a preclinical gene therapy for PKD based on a lentiviral vector harboring the hPGK eukaryotic promoter that drives the expression of the PKLR cDNA. This therapeutic vector was used to transduce mouse PKD hematopoietic stem cells (HSCs) that were subsequently transplanted into myeloablated PKD mice. Ectopic RPK expression normalized the erythroid compartment correcting the hematological phenotype and reverting organ pathology. Metabolomic studies demonstrated functional correction of the glycolytic pathway in RBCs derived from genetically corrected PKD HSCs, with no metabolic disturbances in leukocytes. The analysis of the lentiviral insertion sites in the genome of transplanted hematopoietic cells demonstrated no evidence of genotoxicity in any of the transplanted animals. Overall, our results underscore the therapeutic potential of the hPGK-coRPK lentiviral vector and provide high expectations toward the gene therapy of PKD and other erythroid metabolic genetic disorders.

Dissecting the role of epidermal growth factor receptor catalytic activity during liver regeneration and hepatocarcinogenesis.

UNLABELLED: Different data support a role for the epidermal growth factor receptor (EGFR) pathway during liver regeneration and hepatocarcinogenesis. However, important issues, such as the precise mechanisms mediating its actions and the unique versus redundant functions, have not been fully defined. Here, we present a novel transgenic mouse model expressing a hepatocyte-specific truncated form of human EGFR, which acts as negative dominant mutant (ΔEGFR) and allows definition of its tyrosine kinase-dependent functions. Results indicate a critical role for EGFR catalytic activity during the early stages of liver regeneration. Thus, after two-thirds partial hepatectomy, ΔEGFR livers displayed lower and delayed proliferation and lower activation of proliferative signals, which correlated with overactivation of the transforming growth factor-ß pathway. Altered regenerative response was associated with amplification of cytostatic effects of transforming growth factor-ß through induction of cell cycle negative regulators. Interestingly, lipid synthesis was severely inhibited in ΔEGFR livers after partial hepatectomy, revealing a new function for EGFR kinase activity as a lipid metabolism regulator in regenerating hepatocytes. In spite of these profound alterations, ΔEGFR livers were able to recover liver mass by overactivating compensatory signals, such as c-Met. Our results also indicate that EGFR catalytic activity is critical in the early preneoplastic stages of the liver because ΔEGFR mice showed a delay in the appearance of diethyl-nitrosamine-induced tumors, which correlated with decreased proliferation and delay in the diethyl-nitrosamine-induced inflammatory process. CONCLUSION: These studies demonstrate that EGFR catalytic activity is critical during the initial phases of both liver regeneration and carcinogenesis and provide key mechanistic insights into how this kinase acts to regulate liver pathophysiology. (Hepatology 2016;63:604-619).

Cell fusion reprogramming leads to a specific hepatic expression pattern during mouse bone marrow derived hepatocyte formation in vivo.

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-ß1 (TGF-ß(1)) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation.

Smoking but not homozygosity for CYP1A2 g-163A allelic variant leads to earlier disease onset in patients with sporadic porphyria cutanea tarda.

Porphyria cutanea tarda (PCT) results from decreased activity of hepatic uroporphyrinogen decarboxylase (UROD). Both sporadic and familial forms are characterised by typical cutaneous lesions triggered by genetic/environmental factors. Studies in rodents showed that cytochrome P4501A2 (CYP1A2) plays a central role in the synthesis of a competitive inhibitor of hepatic UROD, but there is little evidence in humans. The impact of smoking and CYP1A2 g-163C > A allelic variant upon first appearance of clinical signs was investigated in 102 patients (80 sporadic-PCT) and 150 healthy donors from Spain. We found an increase in the frequency of CYP1A2 g-163A allele in patients with PCT when compared with controls, although the more inducible A/A genotype had no effect on the onset age. In sporadic-PCT, smoking leads to earlier onset of clinically overt disease in moderate-to-heavy smokers (>or=10 cigarettes/day). In conclusion, this study provides evidence that smoking hastens the onset of cutaneous symptoms in sporadic-PCT patients.

Bone marrow-derived cells promote liver regeneration in mice with erythropoietic protoporphyria.

BACKGROUND: Bone marrow transplantation can reverse hepatic protoporphyrin accumulation and prevent the hepatobiliary complications characteristic of erythropoietic protoporphyria. The aim of this study was to assess the recruitment capacity of bone marrow cells in the damaged liver and their possible contribution to the improved or recovered hepatic function in a murine model of erythropoietic protoporphyria (EPP). METHODS: Lethally irradiated female EPP mice were transplanted with bone marrow cells from healthy male mice and were monitored during 12 or 36 weeks. Two groups of animals killed 12 weeks after transplant were also treated with granulocyte colony-stimulating factor. RESULTS: Cell transplantation decreased porphyrin contents in erythrocytes and liver. Improved hepatic structure and function and reduced hepatic fibrosis were observed, especially 36 weeks after transplant. Bone marrow-derived cells (22%-35%) were identified in the liver of recipient mice by means of fluorescence in situ hybridization (chrY-FISH) or green fluorescent protein staining and were characterized by immunofluorescence staining. The livers of recipients contained 20% to 30% myofibroblasts (alpha-smooth muscle actin-positive cells), 40% CK19-positive cells, and 10% to 28% hepatocytes (albumin-positive cells) derived from the donor bone marrow. CONCLUSIONS: Bone marrow-derived cells play a significant role in restoring and regenerating hepatic tissue in EPP mice. Hepatic repair was associated with fibrogenesis, enhanced by granulocyte colony-stimulating factor treatment, and almost normal liver structure and function was observed in the long term (36 weeks posttransplant).

Bone marrow transplantation into hemochromatotic mice decreases hepatic and duodenal iron overload.

Human hereditary hemochromatosis is a disorder of iron homeostasis characterized by increased absorption of iron and its deposition in parenchymal organs. The maintenance of iron homeostasis is regulated by molecules involved in the absorption, transport, storage and redox of iron. The potential of hematopoietic stem cell therapy for liver diseases has been studied in some experimental animal models. Our objective was to evaluate the effect of bone marrow transplantation from wild type mice on the status of iron overload in Hfe knockout hemochromatotic mice (Hfe(-/-)). The transplanted cells were detected in the liver (11% of the total cells) and characterized as hepatocytes and myofibroblasts. They were also detected in the duodenum and characterized as myofibroblasts. The iron content in the Hfe(-/-) mice descended 2.9-fold in the liver and 2.4-fold in the duodenum 6 months after transplantation. Non-significant changes of relative mRNA abundance of genes of iron metabolism were observed in the liver and duodenum of Hfe(-/-) transplanted mice. At 6 months after transplantation the proportion of Hfe mRNA in Hfe(-/-) mice reached 3.8% of the levels in wild type mice in the liver and 1.6% in the duodenum. In conclusion, adult stem cells from bone marrow transplant were able to differentiate into hepatocytes and myofibroblasts in hemochromatotic mice. Bone marrow transplantation assisted in reducing the iron overload in this murine model of hemochromatosis. These findings might contribute to the knowledge of pathways involved in the regulatory system of iron homeostasis.

Effects of repeated administration with CP-55,940, a cannabinoid CB1 receptor agonist on the metabolism of the hepatic heme.

Drugs metabolised by cytochrome P450 (CYP) such as analgesics may induce acute attacks in patients with hepatic porphyrias. In recent years, preclinical and clinical studies have suggested that cannabinoid pharmaceutical preparations may be potentially useful in the treatment of pain. The purpose of the study was to examine the effects of CP-55,940, a cannabinoid CB1 receptor agonist, on the hepatic heme metabolism in mice. To this end, hepatic activities of aminolevulinic acid synthase (ALAS), heme oxygenase (HO) and CYP levels were determined in mice treated with CP-55,940 (0.5 mg/kg/day; i.p.; 5 or 24 days). Results showed that treatment with CP-55,940 decreased CYP concentrations by 80% and increased HO activity by 158%. However, ALAS activity also decreased by 37%, suggesting that regulatory free heme pool was not modified. Our findings indicate that CP-55,940 and its metabolites do not behave as porphyrinogenic drugs and may potentially be safe for treating pain in patients with acute porphyrias.