Overcoming the Variability of iPSCs in the Manufacturing of Cell-Based Therapies.

Various factors are known to contribute to the diversity of human induced pluripotent stem cells (hiPSCs). Among these are the donor's genetic background and family history, the somatic cell source, the iPSC reprogramming method, and the culture system of choice. Moreover, variability is seen even in iPSC clones, generated in a single reprogramming event, where the donor, somatic cell type, and reprogramming platform are the same. The diversity seen in iPSC lines often translates to epigenetic differences, as well as to differences in the expansion rate, iPSC line culture robustness, and their ability to differentiate into specific cell types. As such, the diversity of iPSCs presents a hurdle to standardizing iPSC-based cell therapy manufacturing. In this review, we will expand on the various factors that impact iPSC diversity and the strategies and tools that could be taken by the industry to overcome the differences amongst various iPSC lines, therefore enabling robust and reproducible iPSC-based cell therapy manufacturing processes.

Long-Term Stability and Differentiation Potential of Cryopreserved cGMP-Compliant Human Induced Pluripotent Stem Cells.

The clinical effectiveness of human induced pluripotent stem cells (iPSCs) is highly dependent on a few key quality characteristics including the generation of high quality cell bank, long-term genomic stability, post-thaw viability, plating efficiency, retention of pluripotency, directed differentiation, purity, potency, and sterility. We have already reported the establishment of iPSC master cell banks (MCBs) and working cell banks (WCBs) under current good manufacturing procedure (cGMP)-compliant conditions. In this study, we assessed the cellular and genomic stability of the iPSC lines generated and cryopreserved five years ago under cGMP-compliant conditions. iPSC lines were thawed, characterized, and directly differentiated into cells from three germ layers including cardiomyocytes (CMs), neural stem cells (NSCs), and definitive endoderm (DE). The cells were also expanded in 2D and 3D spinner flasks to evaluate their long-term expansion potential in matrix-dependent and feeder-free culture environment. All three lines successfully thawed and attached to the L7TM matrix, and formed typical iPSC colonies that expressed pluripotency markers over 15 passages. iPSCs maintained their differentiation potential as demonstrated with spontaneous and directed differentiation to the three germ layers and corresponding expression of specific markers, respectfully. Furthermore, post-thaw cells showed normal karyotype, negative mycoplasma, and sterility testing. These cells maintained both their 2D and 3D proliferation potential after five years of cryopreservation without acquiring karyotype abnormality, loss of pluripotency, and telomerase activity. These results illustrate the long-term stability of cGMP iPSC lines, which is an important step in establishing a reliable, long-term source of starting materials for clinical and commercial manufacturing of iPSC-derived cell therapy products.

A rare case of Kearns-Sayre syndrome in a 17-year-old Venezuelan male with bilateral ptosis as the initial presentation.

Kearns-Sayre syndrome (KSS) was first described in 1958 as 'a rare neuromuscular disorder defined by a characteristic triad of progressive external ophthalmoplegia, pigmentary retinopathy, atrioventricular block and cerebellar ataxia'. The prevalence rate of KSS is ∼1-3 per 100 000 individuals. Here, we report a rare case of a 17-year-old Venezuelan male with KSS.

Estrés en padres de recién nacidos prematuros de muy bajo peso hospitalizados en unidades de cuidados intensivos neonatales. Estudio multicéntrico / Stress in parents of very low birth weight preterm infants hospitalized in neonatal intensive care units. A multicenter study

Introducción. El nacimiento de un hijo prematuro es un evento estresante para sus padres. El objetivo de este estudio fue determinar el estrés inicial de padres de recién nacidos de muy bajo peso de nacimiento (RNMBPN) hospitalizados en 12 unidades de cuidados intensivos neonatales en una red neonatal sudamericana, identificar los factores asociados y comparar el nivel de estrés parental en centros públicos vs. privados. Población y métodos. Estudio transversal en madres/padres de RNMBPN (de 500 a 1500 g). El estrés parental inicial se midió utilizando la Escala de Estrés Parental en una escala de 1 (bajo estrés) a 5 (alto estrés). Las características sociodemográficas de las madres/padres y de los neonatos fueron recolectadas y asociadas a los niveles de estrés parental. Resultados. Participaron del estudio 273 padres / madres de un total de 218 RNMBPN. La encuesta fue aplicada en el 5,9 ± 2,0 días de vida del recién nacido. El estrés parental total promedio fue de 3,1 ± 0,8, y la subescala rol parental fue aquella que puntuó más alto (3,6). Tener un menor nivel educacional, estar desempleado, no haber tomado al recién nacido en brazos y el requerimiento de apoyo ventilatorio se asociaron a mayor estrés parental. El estrés fue mayor en madres que en padres y en centros públicos que en privados. Conclusiones. En padres de RNMBPN, se encontró un estrés inicial moderado. El factor más relevante fue la alteración en su rol parental. El estrés parental fue mayor en las madres y en los centros públicos. Se requiere una mayor sensibilización, investigación e intervención en esta área.

Introduction. The birth of a premature baby is a stressful event for parents. The objective of this study was to determine early stress in parents of very low birth weight infants (VLBWIs) hospitalized in 12 neonatal intensive care units from a South American Neonatal Network, to identify associated factors, and to compare the level of parental stress in public versus private healthcare facilities. Population and Methods. Cross-sectional study in mothers/fathers of VLBWIs (500 to 1500 g). Early parental stress was measured using the Parental Stressor Scale, with a score from 1 (low stress) to 5 (high stress). The sociodemographic characteristics of parents and newborn infants were collected and associated with levels of parental stress. Results. The study included273 fathers/mothers of a total of218 VLBW preterm infants. The survey was administered at 5.9 ± 2.0 days of life. The average total parental stress was 3.1 ± 0.8, and the highest score was obtained for the parental role subscale (3.6). A lower education level, unemployment, not having held the newborn infant, and respiratory support requirement were associated with higher parental stress levels. Stress was higher among mothers than fathers, and at public facilities versus private ones. Conclusions. Among parents of VLBWIs, a moderate early parental stress was observed. Parental role alteration was the most relevant factor. Parental stress was higher among mothers and at public healthcare facilities. A greater sensitization, further research and interventions in this area are required.

Estrés en padres de recién nacidos prematuros de muy bajo peso hospitalizados en unidades de cuidados intensivos neonatales. Estudio multicéntrico / Stress in parents of very low birth weight preterm infants hospitalized in neonatal intensive care units. A multicenter study

Introducción. El nacimiento de un hijo prematuro es un evento estresante para sus padres. El objetivo de este estudio fue determinar el estrés inicial de padres de recién nacidos de muy bajo peso de nacimiento (RNMBPN) hospitalizados en 12 unidades de cuidados intensivos neonatales en una red neonatal sudamericana, identificar los factores asociados y comparar el nivel de estrés parental en centros públicos vs. privados. Población y métodos. Estudio transversal en madres/padres de RNMBPN (de 500 a 1500 g). El estrés parental inicial se midió utilizando la Escala de Estrés Parental en una escala de 1 (bajo estrés) a 5 (alto estrés). Las características sociodemográficas de las madres/padres y de los neonatos fueron recolectadas y asociadas a los niveles de estrés parental. Resultados. Participaron del estudio 273 padres / madres de un total de 218 RNMBPN. La encuesta fue aplicada en el 5,9 ± 2,0 días de vida del recién nacido. El estrés parental total promedio fue de 3,1 ± 0,8, y la subescala rol parental fue aquella que puntuó más alto (3,6). Tener un menor nivel educacional, estar desempleado, no haber tomado al recién nacido en brazos y el requerimiento de apoyo ventilatorio se asociaron a mayor estrés parental. El estrés fue mayor en madres que en padres y en centros públicos que en privados. Conclusiones. En padres de RNMBPN, se encontró un estrés inicial moderado. El factor más relevante fue la alteración en su rol parental. El estrés parental fue mayor en las madres y en los centros públicos. Se requiere una mayor sensibilización, investigación e intervención en esta área.(AU)

Introduction. The birth of a premature baby is a stressful event for parents. The objective of this study was to determine early stress in parents of very low birth weight infants (VLBWIs) hospitalized in 12 neonatal intensive care units from a South American Neonatal Network, to identify associated factors, and to compare the level of parental stress in public versus private healthcare facilities. Population and Methods. Cross-sectional study in mothers/fathers of VLBWIs (500 to 1500 g). Early parental stress was measured using the Parental Stressor Scale, with a score from 1 (low stress) to 5 (high stress). The sociodemographic characteristics of parents and newborn infants were collected and associated with levels of parental stress. Results. The study included273 fathers/mothers of a total of218 VLBW preterm infants. The survey was administered at 5.9 ± 2.0 days of life. The average total parental stress was 3.1 ± 0.8, and the highest score was obtained for the parental role subscale (3.6). A lower education level, unemployment, not having held the newborn infant, and respiratory support requirement were associated with higher parental stress levels. Stress was higher among mothers than fathers, and at public facilities versus private ones. Conclusions. Among parents of VLBWIs, a moderate early parental stress was observed. Parental role alteration was the most relevant factor. Parental stress was higher among mothers and at public healthcare facilities. A greater sensitization, further research and interventions in this area are required.(AU)

Stress in parents of very low birth weight preterm infants hospitalized in neonatal intensive care units. A multicenter study.

INTRODUCTION: The birth of a premature baby is a stressful event for parents. The objective of this study was to determine early stress in parents of very low birth weight infants (VLBWIs) hospitalized in 12 neonatal intensive care units from a South American Neonatal Network, to identify associated factors, and to compare the level of parental stress in public versus private healthcare facilities. POPULATION AND METHODS: Cross-sectional study in mothers/fathers of VLBWIs (500 to 1500 g). Early parental stress was measured using the Parental Stressor Scale, with a score from 1 (low stress) to 5 (high stress). The sociodemographic characteristics of parents and newborn infants were collected and associated with levels of parental stress. RESULTS: The study included 273 fathers/mothers of a total of 218 VLBW preterm infants. The survey was administered at 5.9 ± 2.0 days of life. The average total parental stress was 3.1 ± 0.8, and the highest score was obtained for the parental role subscale (3.6). A lower education level, unemployment, not having held the newborn infant, and respiratory support requirement were associated with higher parental stress levels. Stress was higher among mothers than fathers, and at public facilities versus private ones. CONCLUSIONS: Among parents of VLBWIs, a moderate early parental stress was observed. Parental role alteration was the most relevant factor. Parental stress was higher among mothers and at public healthcare facilities. A greater sensitization, further research and interventions in this area are required.

Dynamic changes in replication timing and gene expression during lineage specification of human pluripotent stem cells.

Duplication of the genome in mammalian cells occurs in a defined temporal order referred to as its replication-timing (RT) program. RT changes dynamically during development, regulated in units of 400-800 kb referred to as replication domains (RDs). Changes in RT are generally coordinated with transcriptional competence and changes in subnuclear position. We generated genome-wide RT profiles for 26 distinct human cell types, including embryonic stem cell (hESC)-derived, primary cells and established cell lines representing intermediate stages of endoderm, mesoderm, ectoderm, and neural crest (NC) development. We identified clusters of RDs that replicate at unique times in each stage (RT signatures) and confirmed global consolidation of the genome into larger synchronously replicating segments during differentiation. Surprisingly, transcriptome data revealed that the well-accepted correlation between early replication and transcriptional activity was restricted to RT-constitutive genes, whereas two-thirds of the genes that switched RT during differentiation were strongly expressed when late replicating in one or more cell types. Closer inspection revealed that transcription of this class of genes was frequently restricted to the lineage in which the RT switch occurred, but was induced prior to a late-to-early RT switch and/or down-regulated after an early-to-late RT switch. Analysis of transcriptional regulatory networks showed that this class of genes contains strong regulators of genes that were only expressed when early replicating. These results provide intriguing new insight into the complex relationship between transcription and RT regulation during human development.

Directed differentiation of human pluripotent cells to neural crest stem cells.

Multipotent neural crest stem cells (NCSCs) have the potential to generate a wide range of cell types including melanocytes; peripheral neurons; and smooth muscle, bone, cartilage and fat cells. This protocol describes in detail how to perform a highly efficient, lineage-specific differentiation of human pluripotent cells to a NCSC fate. The approach uses chemically defined media under feeder-free conditions, and it uses two small-molecule compounds to achieve efficient conversion of human pluripotent cells to NCSCs in ~15 d. After completion of this protocol, NCSCs can be used for numerous applications, including the generation of sufficient cell numbers to perform drug screens, for the development of cell therapeutics on an industrial scale and to provide a robust model for human disease. This protocol can be also be applied to patient-derived induced pluripotent stem cells and thus used to further the knowledge of human disease associated with neural crest development, for example, Treacher-Collins Syndrome.

Signaling network crosstalk in human pluripotent cells: a Smad2/3-regulated switch that controls the balance between self-renewal and differentiation.

A general mechanism for how intracellular signaling pathways in human pluripotent cells are coordinated and how they maintain self-renewal remain to be elucidated. In this report, we describe a signaling mechanism where PI3K/Akt activity maintains self-renewal by restraining prodifferentiation signaling through suppression of the Raf/Mek/Erk and canonical Wnt signaling pathways. When active, PI3K/Akt establishes conditions where Activin A/Smad2,3 performs a pro-self-renewal function by activating target genes, including Nanog. When PI3K/Akt signaling is low, Wnt effectors are activated and function in conjunction with Smad2,3 to promote differentiation. The switch in Smad2,3 activity after inactivation of PI3K/Akt requires the activation of canonical Wnt signaling by Erk, which targets Gsk3ß. In sum, we define a signaling framework that converges on Smad2,3 and determines its ability to regulate the balance between alternative cell states. This signaling paradigm has far-reaching implications for cell fate decisions during early embryonic development.

Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells.

Neural crest stem cells can be isolated from differentiated cultures of human pluripotent stem cells, but the process is inefficient and requires cell sorting to obtain a highly enriched population. No specific method for directed differentiation of human pluripotent cells toward neural crest stem cells has yet been reported. This severely restricts the utility of these cells as a model for disease and development and for more applied purposes such as cell therapy and tissue engineering. In this report, we use small-molecule compounds in a single-step method for the efficient generation of self-renewing neural crest-like stem cells in chemically defined media. This approach is accomplished directly from human pluripotent cells without the need for coculture on feeder layers or cell sorting to obtain a highly enriched population. Critical to this approach is the activation of canonical Wnt signaling and concurrent suppression of the Activin A/Nodal pathway. Over 12-14 d, pluripotent cells are efficiently specified along the neuroectoderm lineage toward p75(+) Hnk1(+) Ap2(+) neural crest-like cells with little or no contamination by Pax6(+) neural progenitors. This cell population can be clonally amplified and maintained for >25 passages (>100 d) while retaining the capacity to differentiate into peripheral neurons, smooth muscle cells, and mesenchymal precursor cells. Neural crest-like stem cell-derived mesenchymal precursors have the capacity for differentiation into osteocytes, chondrocytes, and adipocytes. In sum, we have developed methods for the efficient generation of self-renewing neural crest stem cells that greatly enhance their potential utility in disease modeling and regenerative medicine.

Artritis idiopática juvenil: caso clínico y revisión bibliográfica / Juvenile idiopathic arthritis: clinical case and bibliographic revision

La artritis reumatoidea juvenil (AIJ)es una inflamación crónica de la sinovial, de etiología desconocida, que comienza antes de los 16 años de edad, es más frecuente en niñas; la principal lesión se produce en los cartílagos, mismos que se destruyen progresivamente; los anticuerpos antinucleares son positivos especialmente cuando el evento cursa con iridociclitis. La AIJ se clasifica en 5 tipos básicos en concordancia con el número de articulaciones afectadas durante los 6 primeros meses de la enfermedad y que son: la oligoarticular, poliarticular, sistémica, la relacionada con entesitis y la artritis psoriásica. El diagnóstico es más clínico, con buen pronóstico si es diagnosticada a tiempo pues así se evitarán las deformaciones a nivel de las articulaciones; las radiografías son de ayuda para valorar articulaciones afectadas; y, un tratamiento oportuno permitirá evitar las complicaciones. Se presenta paciente de 4 años de edad con cuadro clínico de aproximadamente 4 semanas de evolución, caracterizado por fiebre vespertina, escalofríos, se le administró antitérmicos y antibióticos; además presentó artralgias de tobillos y rodillas, con dificultad para la marcha y la consecuente limitación funcional. Al examen físico paciente activo; piel, lesiones hipercrómicas en el dorso de las manos; normocéfalo; orofaringe, amígdalas normales; cardiorrespiratorio, normal; abdomen sin megalias. Extremidades: artrosis en muñecas, tobillos; engrosamiento de dorso de manos y pies, limitación funcional para la marcha.

Juvenile Rheumatoid arthritis is a chronic inflammation of the synovia, of unknown etiology, which begins before the age of 16, and is more recurrent in girls. The main injury takes place in the cartilages, which get damaged progressively. The antinuclear antibodies are positive especially when the event is accompanied with iridocyclitis. There are 3 types of JIA that are: oligoarticular, polyarticular and systemic. The diagnosis is more clinical, with good prognosis if it is diagnosed in time because the deformations in the joints will be avoided. X-rays help to evaluate affected joints; and, an opportune treatment will allow the prevention of complications. A 4-year-old patient is admitted. He is diagnosed to have approximately 4 weeks of evolution. He has fever in the mornings, shivers, which do not improve after the administration of anti-fever medication and antibiotics. In addition, arthralgia in ankles and knees, which make it difficult to move and consequently, there is functional limitation. According to the physical examination the patient is active. He has hyperchromic injuries on the back of his hands; normocephalous; oro-pharynx, normal tonsils; cardiorespiratory, normal; abdomen without megalies. Extremities: arthrosis in wrists, ankles; thickening of back of hands and feet, functional limitation for movement.

Epigenetic changes within the promoter region of the HLA-G gene in ovarian tumors.

BACKGROUND: Previous findings have suggested that epigenetic-mediated HLA-G expression in tumor cells may be associated with resistance to host immunosurveillance. To explore the potential role of DNA methylation on HLA-G expression in ovarian cancer, we correlated differences in HLA-G expression with methylation changes within the HLA-G regulatory region in an ovarian cancer cell line treated with 5-aza-deoxycytidine (5-aza-dC) and in malignant and benign ovarian tumor samples and ovarian surface epithelial cells (OSE) isolated from patients with normal ovaries. RESULTS: A region containing an intact hypoxia response element (HRE) remained completely methylated in the cell line after treatment with 5-aza-dC and was completely methylated in all of the ovarian tumor (malignant and benign) samples examined, but only variably methylated in normal OSE samples. HLA-G expression was significantly increased in the 5-aza-dC treated cell line but no significant difference was detected between the tumor and OSE samples examined. CONCLUSION: Since HRE is the binding site of a known repressor of HLA-G expression (HIF-1), we hypothesize that methylation of the region surrounding the HRE may help maintain the potential for expression of HLA-G in ovarian tumors. The fact that no correlation exists between methylation and HLA-G gene expression between ovarian tumor samples and OSE, suggests that changes in methylation may be necessary but not sufficient for HLA-G expression in ovarian cancer.

Identification of candidate methylation-responsive genes in ovarian cancer.

BACKGROUND: Aberrant methylation of gene promoter regions has been linked to changes in gene expression in cancer development and progression. Genes associated with CpG islands (CGIs) are especially prone to methylation, but not all CGI-associated genes display changes in methylation patterns in cancers. RESULTS: In order to identify genes subject to regulation by methylation, we conducted gene expression profile analyses of an ovarian cancer cell line (OVCAR-3) before and after treatment with the demethylating agent 5-aza-deoxycytidine (5-aza-dC). An overlapping subset of these genes was found to display significant differences in gene expression between normal ovarian surface epithelial cells and malignant cells isolated from ovarian carcinomas. While 40% of all human genes are associated with CGIs, > 94% of the overlapping subset of genes is associated with CGIs. The predicted change in methylation status of genes randomly selected from the overlapping subset was experimentally verified. CONCLUSION: We conclude that correlating genes that are upregulated in response to 5-aza-dC treatment of cancer cell lines with genes that are down-regulated in cancer cells may be a useful method to identify genes experiencing epigenetic-mediated changes in expression over cancer development.

The immunohistochemical localization of neuronal nitric oxide synthase in the basal forebrain of the dog.

The present work describes for the first time the anatomical distribution of neuronal nitric oxide synthase (nNOS) immunoreactivity and NADPH-d activity in the basal forebrain of the dog. As in other species, small, intensely nNOS-immunoreactive cells were seen within the olfactory tubercle, caudate nucleus, putamen, nucleus accumbens and amygdala. In addition, a population of mixed large and small nNOS positive cells was found in the medial septum, diagonal band and nucleus basalis overlapping the distribution of the magnocellular cholinergic system of the basal forebrain. Our results show that the distribution of NOS containing neurons in these nuclei in the dog is more extensive and uniform than that reported in rodents and primates. When double labeling of nNOS and NADPH-d was performed in the same tissue section most neurons were double labeled. However, a considerable number of large perikarya in the diagonal band and nucleus basalis appeared to be single labeled for nNOS. Thought a certain degree of interference between the two procedures could not be completely excluded, these findings suggest that NADPH-d histochemistry, which is frequently used to show the presence of NOS, underestimates the potential of basal forebrains neurons to produce nitric oxide. In addition, a few neurons mainly localized among the fibers of the internal capsule, appeared to be labeled only for NADPH-d. These neurons could be expressing a different isoform of NOS, not recognized by our anti-nNOS antibody, as has been reported in healthy humans and AD patients.

L1 and HERV-W retrotransposons are hypomethylated in human ovarian carcinomas.

Wide-spread hypomethylation of CpG dinucleotides is characteristic of many cancers. Retrotransposons have been identified as potential targets of hypomethylation during cellular transformation. We report the results of an preliminary examination of the methylation status of CpG dinucleotides associated with the L1 and HERV-W retrotransposons in benign and malignant human ovarian tumors. We find a reduction in the methylation of CpG dinucleotides within the promoter regions of these retroelements in malignant relative to non-malignant ovarian tissues. Consistent with these results, we find that relative L1 and HERV-W expression levels are elevated in representative samples of malignant vs. non-malignant ovarian tissues.

Perfil epidemiológico social del personal de enfermería: determinaciones en su salud mental / Profile epidemiologist social the personal of nursing: determinations in his health mental

La prevención y la asistencia en materia de salud laboral debe considerar la integridad física, psíquicas y social de los trabajadores. La lectura del presente trabajo cuyo objetivo principal es: determinar las influencias que el medio ambiente y las condiciones de trabajo tiene sobre la salud mental del personal de enfermería en los servicios hospitalarios, puede contribuir a través de un proceso de toma de conciencia a prevenir y cuidar la salud de un sector de trabajadores en permanente situación de riesgo: el personal de enfermería.

Perfil epidemiológico social del personal de enfermería: determinaciones en su salud mental / Profile epidemiologist social the personal of nursing: determinations in his health mental

La prevención y la asistencia en materia de salud laboral debe considerar la integridad física, psíquicas y social de los trabajadores. La lectura del presente trabajo cuyo objetivo principal es: determinar las influencias que el medio ambiente y las condiciones de trabajo tiene sobre la salud mental del personal de enfermería en los servicios hospitalarios, puede contribuir a través de un proceso de toma de conciencia a prevenir y cuidar la salud de un sector de trabajadores en permanente situación de riesgo: el personal de enfermería. (AU)