Pluripotent stem cells: induction and self-renewal.

Pluripotent stem cells (PSCs) lie at the heart of modern regenerative medicine due to their properties of unlimited self-renewal in vitro and their ability to differentiate into cell types representative of the three embryonic germ layers-mesoderm, ectoderm and endoderm. The derivation of induced PSCs bypasses ethical concerns associated with the use of human embryonic stem cells and also enables personalized cell-based therapies. To exploit their regenerative potential, it is essential to have a firm understanding of the molecular processes associated with their induction from somatic cells. This understanding serves two purposes: first, to enable efficient, reliable and cost-effective production of excellent quality induced PSCs and, second, to enable the derivation of safe, good manufacturing practice-grade transplantable donor cells. Here, we review the reprogramming process of somatic cells into induced PSCs and associated mechanisms with emphasis on self-renewal, epigenetic control, mitochondrial bioenergetics, sub-states of pluripotency, naive ground state, naive and primed. A meta-analysis identified genes expressed exclusively in the inner cell mass and in the naive but not in the primed pluripotent state. We propose these as additional biomarkers defining naive PSCs.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

Innovative encapsulation platform based on pancreatic extracellular matrix achieve substantial insulin delivery.

Cell-based therapies for the treatment of diabetes, generally aim to provide long-term glucose regulated-insulin delivery using insulin producing cells. The delivery platform is crucial for the therapeutic outcome as well as for immunoisolation of the entrapped cells. We have developed a novel artificial pancreas encapsulation platform for the treatment of diabetes that is based on solubilized whole porcine pancreatic extracellular matrix (ECM). These unique capsules were used to entrap human liver cells and mesenchymal stem cells that were induced to differentiate into glucose-regulated insulin-producing cells. We demonstrate that the ECM-microcapsule platform provides a natural fibrous 3D niche, supporting cell viability and differentiation, while significantly improving insulin delivery. In vivo, ECM-encapsulated cells were shown to be non-immunogenic, and most importantly, to significantly improve the glycemic control in diabetic mouse preclinical model, thus establishing a proof-of-concept for this new cell-based insulin delivery platform.

Final results from 18 years of the International Lamotrigine Pregnancy Registry.

OBJECTIVE: To monitor for a signal for major teratogenicity following in utero lamotrigine exposure. METHODS: Health care providers reported lamotrigine exposure during pregnancy, and subsequent outcomes, on a voluntary basis. Prospective reporting early in pregnancy was encouraged. Major congenital malformations (MCMs) were classified according to the Centers for Disease Control and Prevention (CDC) criteria and were reviewed by a pediatrician on the Registry's Scientific Advisory Committee. The proportion of infants with MCMs was calculated by trimester and therapy type and descriptively compared to population-based reference estimates. RESULTS: Over an 18-year period, 35 infants with MCMs were observed among 1,558 first-trimester monotherapy exposures: 2.2%(95% confidence interval [CI] 1.6%-3.1%). This was similar to estimates from general population-based cohorts. The observed proportion of infants with MCMs among 150 lamotrigine/valproate polytherapy exposures was 10.7% (95% CI 6.4%-17.0%) and was 2.8% (95% CI 1.5%-5.0%) among 430 infants exposed to lamotrigine polytherapy without valproate. No consistent pattern of malformation type, or malformation frequency by dose, was observed. DISCUSSION: The Registry did not detect an appreciable increase in MCM frequency following first-trimester lamotrigine monotherapy exposure. With over 1,500 first-trimester monotherapy exposures, the Registry was powered to detect major teratogenicity. The proportion of infants with MCMs following lamotrigine/valproate polytherapy exposure was high, but similar to that previously reported with valproate monotherapy. The Registry failed to observe an increased MCM frequency with increasing lamotrigine dose. Monitoring of specific malformations among lamotrigine-exposed pregnancies will continue through case-control surveillance in the European Congenital Anomalies and Twins Registers network.

Use of humour in primary care: different perceptions among patients and physicians.

PURPOSE: (1) To explore the frequency with which humorous behaviour and statements occur in family medicine practice in Israel, and (2) to quantitatively assess the correlation between the subjective perceptions of humour in medical encounters between patients and physicians. METHOD: In a cross sectional study, two populations (doctors and patients) were surveyed with paired structured questionnaires completed immediately after primary care practice visits. Two hundred and fifty consecutive encounters from 15 practices were sampled. The physician questionnaire was self administered, and patient questionnaire was administered by a trained research assistant. RESULTS: A mean of 16.7 questionnaires was completed per physician (range 6-20). The physicians reported having used some humour in only 95 encounters (38%), whereas almost 60% of patients agreed with the statement, "The doctor used some humour during the visit". At the same time, for specific encounters, the agreement between patients' perception and physicians' perceptions on the use of humour, although not completely by chance (p = 0.04), is low (kappa = 0.115). Patient characteristics (age, education, gender, family status, mother tongue, self perceived heath status, stress, mood, and expectations) were not related to the degree of agreement between the patients' and physicians' perceptions. CONCLUSION: Humour was used in a large proportion of encounters, independently of patient characteristics. Patients seem to be more sensitised to humour than physicians, probably because of their high stress level during medical encounters. Cultural differences may also play a part. Physicians should be made aware of this magnifying effect, and the issue should be discussed in medical schools.

Eye movements tell only half the story.

The dramatic improvements of neglect symptoms after prism adaptation (PA) have been interpreted as evidence that PA reorganizes higher levels of spatial representation. Here the authors demonstrate that while the exploratory eye movements of a patient with neglect were clearly shifted toward the left after PA, he still showed no awareness for the left side of the stimuli he was now actively exploring. PA modulates functions of the parietal lobe, such as eye movement control, but fails to influence the underlying mechanisms of neglect.

Prognosis of contraversive pushing.

Stroke patients with 'pusher syndrome' actively push away from the non-hemiparetic side leading to a loss of postural balance and falling towards the paralysed side. The behaviour is due to an altered perception of the body's orientation in relation to gravity. Here, we studied the prognosis of the disorder. Twelve pusher patients first investigated immediately after the stroke were re-examined 6 months later. Pusher symptoms had nearly completely recovered. The aim for physiotherapy of patients with contraversive pushing thus is to shorten the period of necessary treatment and enable earlier discharge from residential care.

IPF1/PDX1 deficiency and beta-cell dysfunction in Psammomys obesus, an animal With type 2 diabetes.

The homeodomain transcription factor IPF1/PDX1 is required in beta-cells for efficient expression of insulin, glucose transporter 2, and prohormone convertases 1/3 and 2. Psammomys obesus, a model of diet-responsive type 2 diabetes, shows markedly depleted insulin stores when given a high-energy (HE) diet. Despite hyperglycemia, insulin mRNA levels initially remained unchanged and then decreased gradually to 15% of the basal level by 3 weeks. Moreover, insulin gene expression was not increased when isolated P. obesus islets were exposed to elevated glucose concentrations. Consistent with these observations, no functional Ipf1/Pdx1 gene product was detected in islets of newborn or adult P. obesus using immunostaining, Western blot, DNA binding, and reverse transcriptase-polymerase chain reaction analyses. Other beta-cell transcription factors (e.g., ISL-1, Nkx2.2, and Nkx6.1) were expressed in P. obesus islets, and the DNA binding activity of the insulin transcription factors RIPE3b1-Act and IEF1 was intact. Ipf1/Pdx1 gene transfer to isolated P. obesus islets normalized the defect in glucose-stimulated insulin gene expression and prevented the rapid depletion of insulin content after exposure to high glucose. Taken together, these results suggest that the inability of P. obesus islets to adapt to dietary overload, with depletion of insulin content as a consequence, results from IPF1/PDX1 deficiency. However, because not all animals become hyperglycemic on HE diet, additional factors may be important for the development of diabetes in this animal model.

Tissue-specific gene therapy directed to tumor angiogenesis.

Gene therapy directed specifically to the vascular wall, particularly to angiogenic endothelial cells is a prerequisite in vascular disease treatment. Angiogenesis is a major feature in many pathological conditions including wound healing, solid tumors, developing metastases, ischemic heart diseases and diabetic retinopathy. In the present study we developed a tissue-specific gene therapy to the angiogenic blood vessels of tumor metastasis using an adeno-based vector containing the murine preproendothelin-1 (PPE-1) promoter. Genes activated by the PPE-1 promoter were highly expressed in bovine aortic endothelial cells in vitro. Systemic injection of the adenoviral vectors AdPPE-1-luciferase and AdCMV-luciferase to normal C57BL/6 mice, resulted in higher activity of PPE-1 promoter compared with CMV promoter in the aorta and vascularized tissues such as heart, kidney, lung and pancreas. Systemic administration of the adenoviral vector, in mice bearing Lewis lung carcinoma, resulted in high and specific activity of PPE-1 in the new vasculature of primary tumors and lung metastasis. Cellular distribution of the delivered gene revealed highest expression of GFP in angiogenic endothelial cells of the metastasis. We expect that this approach of 'vascular-directed' gene therapy will be applicable to both vascular diseases and cancer.

Spatial awareness is a function of the temporal not the posterior parietal lobe.

Our current understanding of spatial behaviour and parietal lobe function is largely based on the belief that spatial neglect in humans (a lack of awareness of space on the side of the body contralateral to a brain injury) is typically associated with lesions of the posterior parietal lobe. However, in monkeys, this disorder is observed after lesions of the superior temporal cortex, a puzzling discrepancy between the species. Here we show that, contrary to the widely accepted view, the superior temporal cortex is the neural substrate of spatial neglect in humans, as it is in monkeys. Unlike the monkey brain, spatial awareness in humans is a function largely confined to the right superior temporal cortex, a location topographically reminiscent of that for language on the left. Hence, the decisive phylogenetic transition from monkey to human brain seems to be a restriction of a formerly bilateral function to the right side, rather than a shift from the temporal to the parietal lobe. One may speculate that this lateralization of spatial awareness parallels the emergence of an elaborate representation for language on the left side.

Sporadic cerebellar ataxia associated with gluten sensitivity.

A total of 104 patients with sporadic cerebellar ataxia were tested for antigliadin and antiendomysium antibodies. Twelve individuals (11.5%) with gluten sensitivity underwent duodenal biopsy and extensive clinical, electrophysiological, neuropsychological, radiological and laboratory investigations including human leucocyte antigen (HLA) typing. Two patients showed typical changes of gluten-sensitive enteropathy with crypt hyperplasia and mucosal flattening. In five patients, the intraepithelial lymphocyte count was elevated. Sporadic ataxia with gluten sensitivity was found to be tightly linked to the HLA DQB1*0201 haplotype (70%). Neurological symptoms were not related to hypovitaminosis or inflammatory CSF changes. The clinical syndrome was dominated by progressive cerebellar ataxia with ataxia of stance and gait (100%), dysarthria (100%) and limb ataxia (97%). Oculomotor abnormalities were gaze-evoked nystagmus (66.7%), spontaneous nystagmus (33.3%), saccade slowing (25%) and upward gaze palsy (16.7%). Extracerebellar features also included deep sensory loss (58.3%), bladder dysfunction (33.3%) and reduced ankle reflexes (33.3%). In accordance with clinical findings, electrophysiological investigations revealed prominent axonal neuropathy with reduced amplitudes (50%) and abnormal evoked potentials (58.3%). On neuropsychological testing, patients presented with moderate verbal memory and executive dysfunction. All patients had evidence of cerebellar atrophy on MRI. We conclude that sporadic ataxia may be associated with positive antibodies against gliadin. Nevertheless, mucosal pathology does not represent an obligatory condition of ataxia with gluten sensitivity. The fact that the disease is strongly associated with the same HLA haplotypes found in coeliac disease not only demonstrates coeliac disease and ataxia with gluten sensitivity to be part of the same disease entity but supports the hypothesis of an immunological pathogenesis of cerebellar degeneration.

Size perception in hemianopia and neglect.

Some debate remains as to whether underestimation of the horizontal size of objects in the left part of visual space is a general disturbance in spatial neglect. The issue is unclear because size perception may be influenced by factors other than neglect, e.g. visual field defects such as hemianopia. To disentangle these effects, we compared the performance of patients with pure neglect, pure hemianopia or both on the same size-comparison test. Whereas pure neglect was accompanied by misperception of horizontal object size, patients with pure hemianopia showed an even greater impairment of size perception. Accordingly, the area of maximum lesion overlap of these patients with impaired size perception was not centred around the parietotemporal cortex, which is typically associated with spatial neglect, but rather was found in the occipital lobe (Brodmann areas 17 and 18). The results suggest that spatial neglect is not an inevitable consequence of distorted size perception. The perceptual distortion of objects in the leftward parts of visual space is not sufficient to account for the occurrence of visuospatial neglect.

How to assess spatial neglect--line bisection or cancellation tasks?

Spatial neglect is usually assessed using cancellation tests or line bisection. A recent comparison of these tests has revealed a double dissociation, in which one neglect patient was impaired in line bisection but not in star cancellation whereas another showed the reverse deficit. This dissociation has prompted the question whether 'neglect' is still a meaningful theoretical entity. We compared line bisection and cancellation tasks regarding their accuracy in detecting spatial neglect. We tested 35 patients with well-defined spatial neglect using a line bisection task and four different cancellation tasks. The line bisection test missed 40% of our neglect patients. Far superior were the letter cancellation and bells tests, each of which missed only 6% of the cases. A deviation in line bisection is not fundamentally related to spatial neglect, but may also arise from other causes (e.g., hemianopia, or which hand is used), and therefore, should be treated with caution in clinical diagnosis. Cancellation tests, such as the bells test and letter cancellation, are more helpful tools to detect spatial neglect.

The origin of contraversive pushing: evidence for a second graviceptive system in humans.

BACKGROUND: Stroke patients may exhibit the peculiar behavior of actively pushing away from the nonhemiparetic side, leading to lateral postural imbalance and a tendency to fall toward the paralyzed side. This phenomenon has been called the "pusher syndrome." OBJECTIVE: The current study analyzes the mechanism leading to contraversive pushing. METHODS: The subjective postural vertical (SPV) and subjective visual vertical (SVV) were determined in five consecutively admitted patients with severe contraversive pushing and in controls. Whereas adjustment of the SPV reflects the perceived upright orientation of the body, the SVV provides a sensitive and direction-specific measurement of peripheral and central vestibular dysfunction. RESULTS: The deficit leading to contraversive pushing is an altered perception of the body's orientation in relation to gravity. Pusher patients experience their body as oriented "upright" when it is tilted 18 degrees to the nonhemiparetic, ipsilesional side. In contrast, perception of the SVV was undisturbed. CONCLUSIONS: A separate pathway seems to be present in humans for sensing the orientation of gravity apart from the one for orientation perception of the visual world. This second graviceptive system decisively contributes to humans' control of upright body posture. Contraversive pushing occurring after stroke lesions may represent the behavioral correlate of a disturbed neural representation of this system.

The neural representation of postural control in humans.

Lesion of the "vestibular cortex" in the human posterior insula leads to a tilted perception of visual vertical but not to tilted body posture and loss of lateral balance. However, some stroke patients show the reverse pattern. Although their processing of visual and vestibular inputs for orientation perception of the visual world is undisturbed, they push away actively from the ipsilesional side (the side of lesion location), leading to a contraversive tilt of the body (tilt toward the side opposite to the lesion) and falling to that side. Recently, the origin of contraversive pushing was identified as an altered perception of the body's orientation in relation to gravity. These patients experience their body as oriented "upright" when actually tilted enormously to the ipsilesional side (18 degrees on average). The findings argued for a separate pathway in humans for sensing body orientation in relation to gravity apart from the one projecting to the vestibular cortex. The present study aimed at identifying this brain area. The infarcted brain regions of 23 consecutively admitted patients with severe contraversive pushing were projected onto a template MRI scan, which had been normalized to Talairach space. The overlapping area of these infarctions centered on the posterolateral thalamus. Our finding necessitates reinterpretation of this area as being only a "relay structure" of the vestibular pathway on its way from the brainstem to the vestibular cortex. The ventral posterior and lateral posterior nuclei of the posterolateral thalamus (and probably its cortical projections) rather seem to be fundamentally involved in the neural representation of a second graviceptive system in humans decisive for our control of upright body posture.

Can we create new organs from our own tissues?

The many new technologies of the past few years have set the stage for novel human therapeutic methods. Identification of pluripotent stem cells as being capable of generating various cell types in the body, together with advanced genetic and cell engineering techniques, may enable the design of custom tissues and organs and thus solve the problem of donor organ scarcity and the need for immune compatibility and immunosuppression to avoid graft rejection. One of the most prevalent metabolic disorders that will benefit from such technologies is insulin-dependent diabetes mellitus. The purpose of our study is to review potential future methods of curing metabolic disorders such as diabetes, and analyze the capacity to genetically manipulate the developmental fate of a tissue in vivo using "master regulator" genes. We systemically delivered the homeobox gene Pancreatic and Duodenal Homeobox gene-1 to liver of mice, by recombinant adenovirus technology, and analyzed whether it induces a developmental shift toward a beta cell phenotype. We demonstrated that PDX-1 is sufficient to activate the endogenous, otherwise silent, mouse insulin 1 and 2 and pro-insulin convertase gene expression in liver. PDX-1 expression in liver resulted in a 25-fold increase in hepatic immunoreactive insulin content and a threefold increase in plasma immunoreactive insulin levels, as compared to control adenovirus-treated mice. Hepatic immunoreactive insulin, induced by PDX-1, was processed to mature mI-1 and mI-2 and was biologically active; it ameliorated hyperglycemia in streptozotocin-treated diabetic mice. PDX-1 has the capacity to reprogram extra-pancreatic tissue toward a beta cell phenotype. The data provide a valuable approach to generate "self" surrogate beta cells that are suitable for replacing impaired islet cell function in diabetics.

Neuronal representation of object orientation.

The dissociation between object identity and object orientation observed in six patients with brain damage, has been taken as evidence for a view-invariant model of object recognition. However, there was also some indication that these patients were not generally agnosic for object orientation but were able to gain access to at least some information about objects' canonical upright. We studied a new case (KB) with spared knowledge of object identity and impaired perception of object orientation using a forced choice paradigm to contrast directly the patient's ability to perceive objects' canonical upright vs non-upright orientations. We presented 2D-pictures of objects with unambiguous canonical upright orientations in four different orientations (0 degrees, -90 degrees, +90 degrees, 180 degrees ). KB showed no impairment in identifying letters, objects, animals, or faces irrespective of their given orientation. Also, her knowledge of upright orientation of stimuli was perfectly preserved. In sharp contrast, KB was not able to judge the orientation when the stimuli were presented in a non-upright orientation. The findings give further support for a distributed view-based representation of objects in which neurons become tuned to the features present in certain views of an object. Since we see more upright than inverted animals and familiar objects, the statistics of these images leads to a larger number of neurons tuned for objects in an upright orientation. We suppose that probably for this reason KB's knowledge of upright orientation was found to be more robust against neuronal damage than knowledge of other orientations.

Implantation of rat insulinoma cell line into cyclosporine treated rats. Effect of the in vivo environment on beta-cell specific gene expression.

BACKGROUND: Transplantation of engineered beta cell-derived lines is a promising modality for cell-based therapy of diabetes mellitus. The in vivo environment and antirejection and other medications may have significant effects on the differentiation and proliferation of the transplanted beta cells, thus affecting their function. The effect of the in vivo environment on expression of genes encoding proteins involved in insulin production, secretion, and glucose sensing were analyzed in the RIN 104638 cell line. METHODS: RIN 104638 cells, were used for s.c. implantation in cyclosporine treated rats and for parallel in vitro culture. The differential expression of the insulin, PDX-1, GLUT-2, and glucokinase genes were assessed by quantitative reverse transcription polymerase chain reaction. RESULTS: The in vivo environment of cyclosporine-treated rats, preserved most of the differentiated characteristics of the implanted cells. Insulin and glucokinase gene expression were maintained at high levels, although GLUT-2 expression decreased. This was in contrast to the substantial decrease of all the three genes expression when cultured in vitro. Cyclosporine treatment reduced insulin and GLUT-2 gene expression in in vitro culture. CONCLUSIONS: Beta cell implantation in cyclosporine-treated rats induces alteration in expression of genes pivotal to insulin production and secretion and the glucose sensing abilities. The normal in vivo environment improves the implanted b cell function by increasing the insulin gene expression and content. Furthermore, it reverses some of the dedifferentiating changes caused by the in vitro culture. This may have a positive effect on the therapeutic efficiency of this cell line.

Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia.

Insulin gene expression is restricted to islet beta cells of the mammalian pancreas through specific control mechanisms mediated in part by specific transcription factors. The protein encoded by the pancreatic and duodenal homeobox gene 1 (PDX-1) is central in regulating pancreatic development and islet cell function. PDX-1 regulates insulin gene expression and is involved in islet cell-specific expression of various genes. Involvement of PDX-1 in islet-cell differentiation and function has been demonstrated mainly by 'loss-of-function' studies. We used a 'gain-of-function' approach to test whether PDX-1 could endow a non-islet tissue with pancreatic beta-cell characteristics in vivo. Recombinant-adenovirus-mediated gene transfer of PDX-1 to the livers of BALB/C and C57BL/6 mice activated expression of the endogenous, otherwise silent, genes for mouse insulin 1 and 2 and prohormone convertase 1/3 (PC 1/3). Expression of PDX-1 resulted in a substantial increase in hepatic immunoreactive insulin content and an increase of 300% in plasma immunoreactive insulin levels, compared with that in mice treated with control adenovirus. Hepatic immunoreactive insulin induced by PDX-1 was processed to mature mouse insulin 1 and 2 and was biologically active; it ameliorated hyperglycemia in diabetic mice treated with streptozotocin. These data indicate the capacity of PDX-1 to reprogram extrapancreatic tissue towards a beta-cell phenotype, may provide a valuable approach for generating 'self' surrogate beta cells, suitable for replacing impaired islet-cell function in diabetics.

Parietal and occipital lobe contributions to perception of straight ahead orientation.

OBJECTIVES: Several studies have investigated how peripheral stimulation affects the perception of body orientation in healthy subjects. The studies showed that opposing stimulation of two different input modalities can cancel out, leaving perception of body orientation unchanged. It was ascertained whether a comparable phenomenon could be found in brain damaged patients with two distinct disorders which individually lead to opposing shifts of the perceived midline. METHODS: The visual subjective straight ahead was measured in patients with pure neglect, pure hemianopia, or a combination of neglect and hemianopia. RESULTS: As in previous studies, patients with pure neglect displayed an ipsilesional displacement of the perceived straight ahead. Patients with pure hemianopia showed a contralesional shift. By contrast, no significant midline shift occurred in the patients with both neglect and hemianopia. CONCLUSIONS: Neglect and hemianopia interact so that opposing biases in the perception of body orientation neutralise each other. Both parietal and occipital areas seem to contribute to the perception of straight ahead body orientation and seem to have counteracting effects when lesioned in the same hemisphere.