Overexpression of hTERT increases stem-like properties and decreases spontaneous differentiation in human mesenchymal stem cell lines

To overcome loss of stem-like properties and spontaneous differentiation those hinder the expansion and application of human mesenchymal stem cells (hMSCs), we have clonally isolated permanent and stable human MSC lines by ectopic overexpression of primary cell cultures of hMSCs with HPV 16 E6E7 and human telomerase reverse transcriptase (hTERT) genes. These cells were found to have a differentiation potential far beyond the ordinary hMSCs. They expressed trophoectoderm and germline specific markers upon differentiation with BMP4 and retinoic acid, respectively. Furthermore, they displayed higher osteogenic and neural differentiation efficiency than primary hMSCs or hMSCs expressed HPV16 E6E7 alone with a decrease in methylation level as proven by a global CpG island methylation profile analysis. Notably, the demethylated CpG islands were highly associated with development and differentiation associated genes. Principal component analysis further pointed out the expression profile of the cells converged toward embryonic stem cells. These data demonstrate these cells not only are a useful tool for the studies of cell differentiation both for the mesenchymal and neurogenic lineages, but also provide a valuable source of cells for cell therapy studies in animal models of skeletal and neurological disorders

Efficacy of first-line doxorubicin and ifosfamide in myxoid liposarcoma

<p>Abstract</p> <p>Background</p> <p>Myxoid liposarcoma (MLS) is a soft tissue sarcoma with adipocytic differentiation characterized by a unique chromosome rearrangement, t(12;16)(q13;p11). The exact efficacy of chemotherapy in MLS has not been clearly established.</p> <p>Patients and methods</p> <p>We retrospectively analyzed the records of 37 histologically confirmed MLS patients who were treated at the University of Texas MD Anderson Cancer Center from January 2000 to December 2009 with doxorubicin 75-90 mg/m²over 72 hours combined with ifosfamide 10 gm/m²in the first-line setting. Response was assessed using RECIST and Choi criteria. The Kaplan-Meier method and log-rank test was used to estimate clinical outcomes.</p> <p>Results</p> <p>The median follow-up period was 50.1 months. The overall response rates were 43.2% using RECIST and 86.5% using the Choi criteria. The 5-year disease-free survival rate was 90% for patients with resectable tumors. Median time to progression and overall survival time for the advanced-disease group were 23 and 31.1 months, respectively.</p> <p>Conclusion</p> <p>Our study demonstrates that doxorubicin-ifosfamide combination therapy has a role in the treatment of MLS. The Choi criteria may be more sensitive in evaluating response to chemotherapy in MLS.</p

Transcriptional Responses of Cultured Rat Sympathetic Neurons during BMP-7-Induced Dendritic Growth

Dendrites are the primary site of synapse formation in the vertebrate nervous system; however, relatively little is known about the molecular mechanisms that regulate the initial formation of primary dendrites. Embryonic rat sympathetic neurons cultured under defined conditions extend a single functional axon, but fail to form dendrites. Addition of bone morphogenetic proteins (BMPs) triggers these neurons to extend multiple dendrites without altering axonal growth or cell survival. We used this culture system to examine differential gene expression patterns in naïve vs. BMP-treated sympathetic neurons in order to identify candidate genes involved in regulation of primary dendritogenesis.To determine the critical transcriptional window during BMP-induced dendritic growth, morphometric analysis of microtubule-associated protein (MAP-2)-immunopositive processes was used to quantify dendritic growth in cultures exposed to the transcription inhibitor actinomycin-D added at varying times after addition of BMP-7. BMP-7-induced dendritic growth was blocked when transcription was inhibited within the first 24 hr after adding exogenous BMP-7. Thus, total RNA was isolated from sympathetic neurons exposed to three different experimental conditions: (1) no BMP-7 treatment; (2) treatment with BMP-7 for 6 hr; and (3) treatment with BMP-7 for 24 hr. Affymetrix oligonucleotide microarrays were used to identify differential gene expression under these three culture conditions. BMP-7 significantly regulated 56 unique genes at 6 hr and 185 unique genes at 24 hr. Bioinformatic analyses implicate both established and novel genes and signaling pathways in primary dendritogenesis.This study provides a unique dataset that will be useful in generating testable hypotheses regarding transcriptional control of the initial stages of dendritic growth. Since BMPs selectively promote dendritic growth in central neurons as well, these findings may be generally applicable to dendritic growth in other neuronal cell types

Getting Down to Specifics: Profiling Gene Expression and Protein-DNA Interactions in a Cell Type-Specific Manner.

The majority of multicellular organisms are comprised of an extraordinary range of cell types, with different properties and gene expression profiles. Understanding what makes each cell type unique, and how their individual characteristics are attributed, are key questions for both developmental and neurobiologists alike. The brain is an excellent example of the cellular diversity expressed in the majority of eukaryotes. The mouse brain comprises of approximately 75 million neurons varying in morphology, electrophysiology, and preferences for synaptic partners. A powerful process in beginning to pick apart the mechanisms that specify individual characteristics of the cell, as well as their fate, is to profile gene expression patterns, chromatin states, and transcriptional networks in a cell type-specific manner, i.e. only profiling the cells of interest in a particular tissue. Depending on the organism, the questions being investigated, and the material available, certain cell type-specific profiling methods are more suitable than others. This chapter reviews the approaches presently available for selecting and isolating specific cell types and evaluates their key features

A Guide to the Brain Initiative Cell Census Network Data Ecosystem

Characterizing cellular diversity at different levels of biological organization and across data modalities is a prerequisite to understanding the function of cell types in the brain. Classification of neurons is also essential to manipulate cell types in controlled ways and to understand their variation and vulnerability in brain disorders. The BRAIN Initiative Cell Census Network (BICCN) is an integrated network of data-generating centers, data archives, and data standards developers, with the goal of systematic multimodal brain cell type profiling and characterization. Emphasis of the BICCN is on the whole mouse brain with demonstration of prototype feasibility for human and nonhuman primate (NHP) brains. Here, we provide a guide to the cellular and spatial approaches employed by the BICCN, and to accessing and using these data and extensive resources, including the BRAIN Cell Data Center (BCDC), which serves to manage and integrate data across the ecosystem. We illustrate the power of the BICCN data ecosystem through vignettes highlighting several BICCN analysis and visualization tools. Finally, we present emerging standards that have been developed or adopted toward Findable, Accessible, Interoperable, and Reusable (FAIR) neuroscience. The combined BICCN ecosystem provides a comprehensive resource for the exploration and analysis of cell types in the brain

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Control of mitochondrial physiology and cell death by the Bcl-2 family proteins Bax and Bok

Neuronal cell death is often triggered by events that involve intracellular increases in Ca(2+). Under resting conditions, the intracellular Ca(2+) concentration is tightly controlled by a number of extrusion and sequestering mechanisms involving the plasma membrane, mitochondria, and ER. These mechanisms act to prevent a disruption of neuronal ion homeostasis. As these processes require ATP, excessive Ca(2+) overloading may cause energy depletion, mitochondrial dysfunction, and may eventually lead to Ca(2+)-dependent cell death. Excessive Ca(2+) entry though glutamate receptors (excitotoxicity) has been implicated in several neurologic and chronic neurodegenerative diseases, including ischemic stroke, epilepsy, and Alzheimer\u27s disease. Recent evidence has revealed that excitotoxic cell death is regulated by the B-cell lymphoma-2 (Bcl-2) family of proteins. Bcl-2 proteins, comprising of both pro-apoptotic and anti-apoptotic members, have been shown to not only mediate the intrinsic apoptosis pathway by controlling mitochondrial outer membrane (MOM) integrity, but to also control neuronal Ca(2+) homeostasis and energetics. In this review, the role of Bcl-2 family proteins in the regulation of apoptosis, their expression in the central nervous system and how they control Ca(2+)-dependent neuronal injury are summarized. We review the current knowledge on Bcl-2 family proteins in the regulation of mitochondrial function and bioenergetics, including the fusion and fission machinery, and their role in Ca(2+) homeostasis regulation at the mitochondria and ER. Specifically, we discuss how the \u27pro-apoptotic\u27 Bcl-2 family proteins, Bax and Bok, physiologically expressed in the nervous system, regulate such \u27non-apoptotic/daytime\u27 functions

Comparative cellular analysis of motor cortex in human, marmoset and mouse

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties