Quantitative changes in gene transcription during induction of differentiation in porcine neural progenitor cells

PurposeDifferentiation of neural stem/progenitor cells involves changes in the gene expression of these cells. Less clear is the extent to which incremental changes occur and the time course of such changes, particularly in non-rodents.MethodsUsing porcine genome microarrays, we analyzed changes in the expression of 23,256 genes in porcine neural progenitor cells (pNPCs) subject to two established differentiation protocols. In addition, we performed sequential quantitative assessment of a defined transcription profile consisting of 15 progenitor- and lineage-associated genes following exposure to the same treatment protocols, to examine the temporal dynamics of phenotypic changes following induction of differentiation. Immunocytochemistry was also used to examine the expression of seven of these phenotypically important genes at the protein level. Initial primary isolates were passaged four times in proliferation medium containing 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF) before differentiation was induced. Differentiation was induced by medium without EGF or bFGF and containing either 10 ng/ml ciliary neurotrophic factor or 10% fetal bovine serum (FBS). Cultures were fed every two days and harvested on days 0, 1, 3, and 5 for quantitative real-time PCR.ResultsThe microarray results illustrated and contrasted the global shifts in the porcine transcriptome associated with both treatment conditions. PCR confirmed dramatic upregulation of transcripts for myelin basic protein (up to 88 fold), claudin 11 (up to 32 fold), glial fibrillary acidic protein (GFAP; up to 26 fold), together with notable (>twofold) increases in message for microtubule associated protein 2 (MAP2) and C-X-C chemokine receptor type 4 (CXCR4), Janus kinase 1 (Jak1), signal transducer and activator of transcription 1 (STAT1), and signal transducer and activator of transcription 3 (STAT3). Transcripts for nestin and Krüppel-like factor 4 (KLF4) decreased sharply (>twofold). The specific dynamics of expression changes varied according to the transcript and treatment condition over the five days examined following induction. The magnitude of neuronal marker induction was greater for the ciliary neurotrophic factor condition while glial fibrillary acidic protein induction was greater for the FBS condition.ConclusionsThe transient dynamic of CXCR4 expression during induction of differentiation, as well as the upregulation of several major histocompatibility complex (MHC) transcripts, has implications in terms of graft integration and tolerance, respectively. These data confirm and extend in the pig the findings previously reported with murine retinal progenitors and support the use of this large animal model for translational development of regenerative approaches to neurologic diseases

The tail-elicited tail withdrawal reflex of Aplysia is mediated centrally at tail sensory-motor synapses and exhibits sensitization across multiple temporal domains

The defensive withdrawal reflexes of Aplysia californica have provided powerful behavioral systems for studying the cellular and molecular basis of memory formation. Among these reflexes the tail-elicited tail withdrawal reflex (T-TWR) has been especially useful. In vitro studies examining the monosynaptic circuit for the T-TWR, the tail sensory-motor (SN-MN) synapses, have identified the induction requirements and molecular basis of different temporal phases of synaptic facilitation that underlie sensitization in this system. They have also permitted more recent studies elucidating the role of synaptic and nuclear signaling during synaptic facilitation. Here we report the development of a novel, compartmentalized semi-intact T-TWR preparation that allows examination of the unique contributions of processing in the SN somatic compartment (the pleural ganglion) and the SN-MN synaptic compartment (the pedal ganglion) during the induction of sensitization. Using this preparation we find that the T-TWR is mediated entirely by central connections in the synaptic compartment. Moreover, the reflex is stably expressed for at least 24 h, and can be modified by tail shocks that induce sensitization across multiple temporal domains, as well as direct application of the modulatory neurotransmitter serotonin. This preparation now provides an experimentally powerful system in which to directly examine the unique and combined roles of synaptic and nuclear signaling in different temporal domains of memory formation

Porcine Neural Progenitor Cells Derived from Tissue at Different Gestational Ages Can Be Distinguished by Global Transcriptome.

The impact of gestational age on mammalian neural progenitor cells is potentially important for both an understanding of neural development and the selection of donor cells for novel cell-based treatment strategies. In terms of the latter, it can be problematic to rely entirely on rodent models in which the gestational period is significantly shorter and the brain much smaller than is the case in humans. Here, we analyzed pig brain progenitor cells (pBPCs) harvested at 2 different gestational ages (E45 and E60) using gene expression profiles, obtained by microarray analysis and quantitative polymerase chain reaction (qPCR), across time in culture. Comparison of the global transcriptome of pBPCs from age-matched transgenic green flourescent protein (GFP)-expressing fetuses versus non- GFP-expressing fetuses did not reveal significant differences between the 2 cell types, whereas comparison between E45 and E60 pBPCs did show separation between the data sets by principle component analysis. Further examination by qPCR showed evidence of relative downregulation of proliferation markers and upregulation of glial markers in the gestationally older (E60) cells. Additional comparisons were made. This study provides evidence of age-related changes in the gene expression of cultured fetal porcine neural progenitors that are potentially relevant to the role of these cells during development and as donor cells for transplantation studies

Porcine Neural Progenitor Cells Derived from Tissue at Different Gestational Ages Can Be Distinguished by Global Transcriptome.

The impact of gestational age on mammalian neural progenitor cells is potentially important for both an understanding of neural development and the selection of donor cells for novel cell-based treatment strategies. In terms of the latter, it can be problematic to rely entirely on rodent models in which the gestational period is significantly shorter and the brain much smaller than is the case in humans. Here, we analyzed pig brain progenitor cells (pBPCs) harvested at 2 different gestational ages (E45 and E60) using gene expression profiles, obtained by microarray analysis and quantitative polymerase chain reaction (qPCR), across time in culture. Comparison of the global transcriptome of pBPCs from age-matched transgenic green flourescent protein (GFP)-expressing fetuses versus non- GFP-expressing fetuses did not reveal significant differences between the 2 cell types, whereas comparison between E45 and E60 pBPCs did show separation between the data sets by principle component analysis. Further examination by qPCR showed evidence of relative downregulation of proliferation markers and upregulation of glial markers in the gestationally older (E60) cells. Additional comparisons were made. This study provides evidence of age-related changes in the gene expression of cultured fetal porcine neural progenitors that are potentially relevant to the role of these cells during development and as donor cells for transplantation studies

Immunosuppressive Treatment Can Alter Visual Performance in the Royal College of Surgeons Rat.

PurposeImmunosuppression is frequently employed to enhance survival of xenografted human cells as part of translational proof-of-concept studies. However, the potential effects of this treatment are easily overlooked.MethodsAs part of baseline testing in the dark-eyed variant of the dystrophic Royal College of Surgeons (RCS) rat, we documented the time course of retinal degenerative changes versus Long Evans controls using bright field retinal imaging, fluorescein angiography, and histology and examined the impact of immunosuppression on visual function. Rats received either no treatment or systemic immunosuppression with oral cyclosporine A and injectable dexamethasone and subsequently underwent functional evaluation by optomotor response testing and electroretinography (ERG) at multiple intervals from P45 to P180.ResultsImmunosuppressed RCS animals demonstrated poorer performance on functional tests than age-matched untreated rats during the earlier stages of degeneration, including significantly lower spatial acuities on optomotor threshold testing and significantly lower b-wave amplitudes on scotopic and photopic ERGs. Retinal imaging documented the progression of degenerative changes in the RCS fundus and histologic evaluation of the RCS retina confirmed progressive thinning of the outer nuclear layer.ConclusionsA standard regimen of cyclosporine A plus dexamethasone, administered to RCS rats, results in demonstrable systemic side effects and depressed scores on behavioral and electrophysiological testing at time points before P90. The source of the functional impairment was not identified. This finding has implications for the interpretation of data generated using this commonly used translational model

Impact of warfarin persistence on health-care utilization and costs among patients with atrial fibrillation managed in anticoagulation clinics in the United States

Warfarin is a recommended therapy to reduce the risk of stroke in patients with nonvalvular atrial fibrillation (NVAF). The objectives of this study were to identify potential factors associated with warfarin persistence and evaluate the impact of warfarin persistence on health-care resource utilization and costs among patients with NVAF in the United States. Patients (18 years) with 1 inpatient or 2 outpatient diagnoses of AF without valvular disease were identified from an electronic medical record database (January 1, 2004, to January 31, 2015). The patients with NVAF were grouped into 2 cohortspersistent with warfarin therapy and not persistent (warfarin discontinuation i