Sequence-Defined Cationic Lipo-Oligomers Containing Unsaturated Fatty Acids for Transfection.
Abstract Sequence-defined cationic lipo-oligomers containing unsaturated fatty acids are potent nucleic acid carriers that are produced by solid-phase supported synthesis. However, the trifluoroacetic acid (TFA)-mediated removal of acid-labile protecting groups and cleavage from the resin can be accompanied by side products caused by an addition of TFA to the double bonds of unsaturated fatty acids. These TFA adducts are converted into hydroxylated derivatives under aqueous conditions. Here we describe an optimized cleavage protocol (precooling cleavage solution to 4 °C, 20 min cleavage at 22 &d...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Reinhard S, Wagner E Tags: Methods Mol Biol Source Type: research

Near-Infrared Optical Imaging of Nucleic Acid Nanocarriers In Vivo.
Abstract Noninvasive, real-time optical imaging methods are well suited to follow the in vivo distribution of nucleic acid nanocarriers, their dissociation and the resulting gene expression or inhibition. Indeed, most small animal imaging devices are performing bioluminescence and fluorescence measurements without moving the animal, allowing a simple, rapid, and cost-effective method of investigation of several parameters at a time, in longitudinal experiments that can last for days or weeks.Here we help the reader in choosing adapted near-infrared (NIR) fluorophores or pairs of fluorophores for FRET assays, imagi...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Rome C, Gravier J, Morille M, Divita G, Bolcato-Bellemin AL, Josserand V, Coll JL Tags: Methods Mol Biol Source Type: research

Microbubbles for Nucleic Acid Delivery in Liver Using Mild Sonoporation.
Abstract Ultrasound-mediated gene delivery is an interesting approach, which could help in increasing gene transfer in deep tissues. Moreover, it allows for performing experiments guided by the image to determine which elements are required. Microbubbles complexed with a eukaryotic expression cassette are excellent agents as they are responsive to ultrasounds and, upon oscillation, can destabilize membranes to enhance gene transfer. Here, we describe the preparation of positively charged microbubbles, plasmid free of antibiotic resistance marker, their combination and the conditions of ultrasound-mediated liver tr...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Mignet N, Marie C, Delalande A, Manta S, Bureau MF, Renault G, Scherman D, Pichon C Tags: Methods Mol Biol Source Type: research

The effect of AP-2 δ on transcription of the Prestin gene in HEI-OC1 cells upon oxidative stress.
Conclusions: In conclusion, oxidative stress inhibits the expression of Prestin protein, and the transcription mechanism is triggered to compensate for the loss of Prestin protein. AP-2δ is one of the important TFs that suppresses transcription of the Prestin gene, and AP-2δ suppression further boosted Prestin mRNA activation under oxidative stress. PMID: 31297132 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Luo X, Xia Y, Li XD, Wang JY Tags: Cell Mol Biol Lett Source Type: research

MiR-216a-5p targets TCTN1 to inhibit cell proliferation and induce apoptosis in esophageal squamous cell carcinoma.
Conclusions: Our results demonstrate that miR-216a-5p might serve as a tumor suppressor in ESCC cells through negatively regulating TCTN1 expression, indicating the possibility that miR-216a-5p and TCTN1 might be attractive targets for ESCC therapeutic intervention. PMID: 31297133 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Chai L, Yang G Tags: Cell Mol Biol Lett Source Type: research

Correction to: Manipulation of Developmental Function in Turtles with Notes on Alligators.
Abstract The author added a sentence to this chapter. The text has been added to the chapter opening page. PMID: 31290130 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 12, 2019 Category: Molecular Biology Authors: Moustakas-Verho JE, McLennan R, Spengler J, Kulesa PM, Cebra-Thomas JA Tags: Methods Mol Biol Source Type: research

Correction to: Molecular Interaction Networks to Select Factors for Cell Conversion.
Abstract This chapter was published without including the "Conflict of Interest" section given by the author along with the corrected proof. PMID: 31290135 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 12, 2019 Category: Molecular Biology Authors: Ouyang JF, Kamaraj US, Polo JM, Gough J, Rackham OJL Tags: Methods Mol Biol Source Type: research

The Astrocyte-Neuron Interface: An Overview on Molecular and Cellular Dynamics Controlling Formation and Maintenance of the Tripartite Synapse.
Abstract Astrocytes are known to provide trophic support to neurons and were originally thought to be passive space-filling cells in the brain. However, recent advances in astrocyte development and functions have highlighted their active roles in controlling brain functions by modulating synaptic transmission. A bidirectional cross talk between astrocytic processes and neuronal synapses define the concept of tripartite synapse. Any change in astrocytic structure/function influences neuronal activity which could lead to neurodevelopmental and neurodegenerative disorders. In this chapter, we briefly overview the met...
Source: Mol Biol Cell - July 10, 2019 Category: Molecular Biology Authors: Hasan U, Singh SK Tags: Methods Mol Biol Source Type: research

Role of Astrocytes in the Neurogenic Niches.
Abstract In the mammalian brain, highly specialized astrocytes serve as neural stem cells (NSCs) that divide and give rise to new neurons, in a process called neurogenesis. During embryonic development NSCs generate almost all neurons of the brain. Soon after birth the neurogenic potential of NSCs is highly reduced, and neurogenesis occurs only in two specialized brain regions called the neurogenic niches. Niche cells are essential to stem cells as they provide structural and nutritional support, and control fundamental stem cell decisions. Astrocytes, major components of the adult neurogenic niches, are evolving ...
Source: Mol Biol Cell - July 10, 2019 Category: Molecular Biology Authors: Schneider J, Karpf J, Beckervordersandforth R Tags: Methods Mol Biol Source Type: research

Isolation and Culture of Astrocytes from Postnatal and Adult Mouse Brains.
Abstract Astrocytes play fundamental roles in development and plasticity of the central nervous system. Dysfunction of astrocytes contributes to most of neurological diseases ranging from neurodegenerative diseases to psychological disorders. To better understand the involvement of astrocytes in both physiological and pathological conditions, and further elucidate their underlying mechanisms, it is critical to establish a reliable isolation and culture method for the cells. In this chapter, we describe the isolation and culture protocols of astrocytes from postnatal and adult mouse brains. PMID: 30617971 [Pub...
Source: Mol Biol Cell - July 10, 2019 Category: Molecular Biology Authors: He Y, Taylor N, Bhattacharya A Tags: Methods Mol Biol Source Type: research

Fluorescence-Activated Cell Sorting-Based Isolation and Characterization of Neural Stem Cells from the Adult Zebrafish Telencephalon.
Abstract Adult mammalian brain, including humans, has rather limited addition of new neurons and poor regenerative capacity. In contrast, neural stem cells (NSC) with glial identity and neurogenesis are highly abundant throughout the adult zebrafish brain. Importantly, the activation of NSC and production of new neurons in response to injuries lead to the brain regeneration in zebrafish brain. Therefore, understanding of the molecular pathways regulating NSC behavior in response to injury is crucial in order to set the basis for experimental modification of these pathways in glial cells after injury in the mammali...
Source: Mol Biol Cell - July 10, 2019 Category: Molecular Biology Authors: Di Giaimo R, Aschenbroich S, Ninkovic J Tags: Methods Mol Biol Source Type: research

Monitoring Interneuron-Astrocyte Signaling and Its Consequences on Synaptic Transmission.
Abstract Whole-cell patch clamp allows the characterization of synaptic transmission in neurons. It is possible to manipulate astrocytic activity and record how these glial cells affect neuronal networks. Here we describe the methodology to monitor the endogenously activation of astrocytes by inhibitory synaptic activity. Afterward, such glial activation will let us study the consequences of interneuron-astrocyte signaling on excitatory neurotransmission at hippocampal synapses. PMID: 30617977 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 10, 2019 Category: Molecular Biology Authors: Mederos S, Perea G Tags: Methods Mol Biol Source Type: research

Astrocytes and Circadian Rhythms: An Emerging Astrocyte-Neuron Synergy in the Timekeeping System.
Abstract Animals have an internal timekeeping system to anticipate daily changes associated with the transition of day to night, which is deeply involved in the regulation and maintenance of behavioral and physiological processes. Prevailing knowledge associated the control of circadian clocks to a network of neurons in the central pacemaker, the suprachiasmatic nucleus (SCN), but astrocytes are rapidly emerging as key cellular contributors to the timekeeping system. However, how these glial cells impact the neuronal clock to modulate rhythmic neurobehavioral outputs just begin to be investigated. Astrocyte-neuron...
Source: Mol Biol Cell - July 10, 2019 Category: Molecular Biology Authors: Barca Mayo O, Berdondini L, De Pietri Tonelli D Tags: Methods Mol Biol Source Type: research

In Vitro Engulfment Assay to Measure Phagocytic Activity of Astrocytes Using Synaptosomes.
Abstract Astrocytes eliminate unnecessary synapses, neural debris, and pathogenic proteins such as amyloid β plaque. Although the emerging evidences suggest that the phagocytic roles of astrocytes are critical in maintaining brain homeostasis during development as well as pathogenic conditions, the efficient assay for measuring phagocytic capacity and kinetics of astrocytes has been lacking. Here we present in vitro engulfment assay using purified astrocytes and synaptosomes. Based on imaging methods, either fluorescent or pH indicator-conjugated synaptosomes can be used in this assay. PMID: 30617979 [Pu...
Source: Mol Biol Cell - July 10, 2019 Category: Molecular Biology Authors: Byun YG, Chung WS Tags: Methods Mol Biol Source Type: research

Quantifying Compartment-Specific Protein Translocation in Astrocytes by Object-Oriented Image Analysis: Mitochondrial Translocation of PKC δ.
Quantifying Compartment-Specific Protein Translocation in Astrocytes by Object-Oriented Image Analysis: Mitochondrial Translocation of PKCδ. Methods Mol Biol. 2019;1938:169-186 Authors: Farid KMN, Derouiche A Abstract Assessing the amount and subcellular distribution of protein expression is a key component in modern cell biological and medical research. We studied protein kinase Cδ (PKCδ) as a potential regulator of mitochondrial metabolism in astrocytes, and sought to evaluate mitochondrial translocation of PKCδ since this is an important determinant of its function. Apart fr...
Source: Mol Biol Cell - July 10, 2019 Category: Molecular Biology Authors: Farid KMN, Derouiche A Tags: Methods Mol Biol Source Type: research

Detection of Protein Uptake in In Vitro Cultured Astrocytes Exemplified by the Uptake of the Serine Protease, Tissue Plasminogen Activator.
Abstract Astrocytes are heterogeneous cells of the central nervous system whose uptake of neurotransmitters and neuromodulators can influence synaptic signaling. Any malfunction in this process can lead to serious defects in synaptic transmission found in, for example, neurodegenerative diseases like Alzheimer's or epilepsy.Here we describe how to visualize the uptake of an extracellularly located protein by in vitro cultured astrocytes on the example of tissue plasminogen activator, a serine protease tightly involved in long-term potentiation and seizure generation. PMID: 30617982 [PubMed - indexed for MEDLI...
Source: Mol Biol Cell - July 10, 2019 Category: Molecular Biology Authors: Bres EE, Faissner A Tags: Methods Mol Biol Source Type: research

Native Chromatin Immunoprecipitation (N-ChIP) in Primary Cortical Rat Astrocytes.
Abstract Chromatin immunoprecipitation (ChIP) in conjunction with qPCR or next generation sequencing (ChIP-seq) is used to detect protein-DNA interaction. Typically, DNA bound to a protein of interest is captured with an antibody against this protein, and DNA is then purified from DNA-protein complexes. Here, we describe a native Chromatin immunoprecipitation (N-ChIP) approach which is an efficient ChIP method with high resolution for histone modifications and a number of transcription factors. This protocol has been tailored for cultured primary rat astrocytes, and we included the preparation of astrocytic cell c...
Source: Mol Biol Cell - July 10, 2019 Category: Molecular Biology Authors: Malik VA, Di Benedetto B, Jakovcevski M Tags: Methods Mol Biol Source Type: research

Imaging and Manipulating Astrocyte Function In Vivo in the Context of CNS Injury.
Abstract Astrocytes are glial cells carrying out complex homeostatic functions in the healthy and diseased central nervous system (CNS). It has so far been impossible to reliably culture adult astrocytes and the results of studies on astrocytes outside of their normal environment are challenging to interpret. Consequently, most culture studies use astrocytes isolated from postnatal rodents. Yet cultured astrocytes do not display their complex three-dimensional in vivo morphology, and transcriptomes of cultured astrocytes vary significantly from those of acutely isolated astrocytes (Cahoy et al., J Neurosci 28:264-...
Source: Mol Biol Cell - July 10, 2019 Category: Molecular Biology Authors: Shandra O, Robel S Tags: Methods Mol Biol Source Type: research

Investigating Age-Related Changes in Proliferation and the Cell Division Repertoire of Parenchymal Reactive Astrocytes.
Abstract Reactive gliosis is a complicated process involving all types of glial cells and is the therapeutic target of efforts to treat several types of neuropathologies. Parenchymal astrocytes continuously survey their microenvironment to identify even tiny abnormalities in the central nervous system (CNS) homeostasis and react rapidly to brain damage, such as following ischemia, trauma, or neurodegenerative diseases, to prevent propagation of tissue damage. Aging can play causal roles in certain astroglial dysfunctions, however, still little is known to what extent the heterogeneous reaction of astrocytes at the...
Source: Mol Biol Cell - July 10, 2019 Category: Molecular Biology Authors: Heimann G, Sirko S Tags: Methods Mol Biol Source Type: research

High-Throughput Screening for Drug Combinations.
Abstract The identification of drug combinations as alternatives to single-agent therapeutics has traditionally been a slow, largely manual process. In the last 10 years, high-throughput screening platforms have been developed that enable routine screening of thousands of drug pairs in an in vitro setting. In this chapter, we describe the workflow involved in screening a single agent versus a library of mechanistically annotated, investigation, and approved drugs using a full dose-response matrix scheme using viability as the readout. We provide details of the automation required to run the screen and the inf...
Source: Mol Biol Cell - July 10, 2019 Category: Molecular Biology Authors: Shinn P, Chen L, Ferrer M, Itkin Z, Klumpp-Thomas C, McKnight C, Michael S, Mierzwa T, Thomas C, Wilson K, Guha R Tags: Methods Mol Biol Source Type: research

Drug Effect Prediction by Integrating L1000 Genomic and Proteomic Big Data.
Abstract The library of integrated Network-Based Cellular Signatures (LINCS) project aims to create a network-based understanding of biology by cataloging changes in gene expression and signal transduction. Gene expression and proteomic data in LINCS L1000 are cataloged for human cancer cells treated with compounds and genetic reagents. For understanding the related cell pathways and facilitating drug discovery, we developed binary linear programming (BLP) to infer cell-specific pathways and identify compounds' effects using L1000 gene expression and phosphoproteomics data. A generic pathway map for the MCF7 breas...
Source: Mol Biol Cell - July 10, 2019 Category: Molecular Biology Authors: Chen W, Zhou X Tags: Methods Mol Biol Source Type: research

The effects of CRISPR-Cas9 knockout of the TGF- β1 gene on antler cartilage cells in vitro.
Conclusions: TGF-β1 is a newly identified regulatory factor of rapid growth in sika deer antler. PMID: 31285745 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 10, 2019 Category: Molecular Biology Authors: Liu M, Han X, Liu H, Chen D, Li Y, Hu W Tags: Cell Mol Biol Lett Source Type: research

Methods for Analysis of Keratinocyte Migration.
Abstract Reepithelialization of wounds involves several keratinocyte functions: proliferation, migration, and differentiation. The key step is keratinocyte migration, an essential aspect for understanding chronic non-healing wounds. Scratch wounding assay and cell motility assay are used to evaluate keratinocyte migration in vitro. The ability of keratinocyte migration is measured by the dynamic change of the wound closure area over time under a living-cell workstation. NIH ImageJ software is used to quantify the motility ability of single cell. The following protocols offer a general guideline for keratinocyte mi...
Source: Mol Biol Cell - July 9, 2019 Category: Molecular Biology Authors: Liu J, Zhang J Tags: Methods Mol Biol Source Type: research

A novel individualized drug repositioning approach for predicting personalized candidate drugs for type 1 diabetes mellitus.
This study conducts drug discovery via detecting the functional connections among disease and drug action, on a personalized or customized basis. Our framework provides a rational way for systematic personalized drug discovery of complex diseases and contributes to the future application of custom therapeutic decisions. PMID: 31287801 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 9, 2019 Category: Molecular Biology Authors: Zheng H Tags: Stat Appl Genet Mol Biol Source Type: research

Introduction to Genome Biology and Diversity.
Abstract Organisms display astonishing levels of cell and molecular diversity, including genome size, shape, and architecture. In this chapter, we review how the genome can be viewed as both a structural and an informational unit of biological diversity and explicitly define our intended meaning of genetic information. A brief overview of the characteristic features of bacterial, archaeal, and eukaryotic cell types and viruses sets the stage for a review of the differences in organization, size, and packaging strategies of their genomes. We include a detailed review of genetic elements found outside the primary ch...
Source: Mol Biol Cell - July 8, 2019 Category: Molecular Biology Authors: Youssef N, Budd A, Bielawski JP Tags: Methods Mol Biol Source Type: research

Overview on Emotional Behavioral Testing in Rodent Models of Pediatric Epilepsy.
Abstract Psychiatric and cognitive disturbances are the most common comorbidities of epileptic disorders in children. The successful treatment of these comorbidities faces many challenges including their etiologically heterogonous nature. Translational neurobehavioral research in age-tailored and clinically relevant rodent seizure models offers a controlled setting to investigate emotional and cognitive behavioral disturbances, their causative factors, and potentially novel treatment interventions. In this review, we propose a conceptual framework that provides a nonsubjective approach to rodent emotional behavior...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Medlej Y, Salah H, Wadi L, Saad S, Asdikian R, Karnib N, Ghazal D, Bashir B, Allam J, Obeid M Tags: Methods Mol Biol Source Type: research

Y-Shaped Maze to Test Spontaneous Object Recognition and Temporal Order Memory After Traumatic Brain Injury.
Abstract Traumatic brain injury (TBI) is one of the most frequent causes of brain damage. Cognitive deficits have been reported in the literature after mild-to-severe TBI affecting memory, language, executive functions, attention, and information processing speed. In this chapter, we describe a method to characterize cognitive impairment in rats following TBI of various intensities. The focus will be on spontaneous object recognition and temporal order memory in rats. These tests are performed in a Y-shaped maze. We have previously identified using this method persistent spontaneous object recognition and temporal...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Darwish H, Hasan H Tags: Methods Mol Biol Source Type: research

Methods in Emotional Behavioral Testing in Immature Epilepsy Rodent Models.
Abstract Pediatric epilepsy is associated with prominent comorbid psychiatric and cognitive disturbances. Neurobehavioral testing is employed to characterize the cognitive and emotional behavioral derangements that accompany seizures in age-tailored and clinically relevant immature rodent seizure models. In addition to dissecting the causes of the etiologically multifaceted psychiatric and cognitive comorbidities of the epilepsies, neurobehavioral panels are essential in investigating potential neuroprotective strategies, especially during neurodevelopment. Here we describe a battery of behavioral testing panels t...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Salah H, Medlej Y, Karnib N, Darwish N, Asdikian R, Wehbe S, Makki G, Obeid M Tags: Methods Mol Biol Source Type: research

Methods in Electrode Implantation and Wiring for Long-Term Continuous EEG Monitoring in Rodent Models of Epilepsy and Behavioral Disturbances.
Abstract Rodent seizure models that pathologically and behaviorally recapitulate age-tailored epileptic disorders are used by us and others to advance our understanding of the chronobiology and mechanisms of epileptic seizure emergence and their comorbidities and to investigate potential novel treatment strategies. Obtaining prolonged continuous electroencephalogram (EEG) tracings over months is essential in this line of translational research, particularly to assess the relation between electrographic changes and the development of seizures and their various psychiatric and cognitive comorbidities in models where...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Medlej Y, Salah H, Wadi L, Atoui Z, Fadlallah Y, Asdikian R, Khalil RB, Hashash R, Obeid M Tags: Methods Mol Biol Source Type: research

The Contribution of Inflammation to Autism Spectrum Disorders: Recent Clinical Evidence.
Abstract Autism comprises a complex and heterogeneous spectrum of neurodevelopmental disorders, usually termed autism spectrum disorders (ASD). It is more prevalent in males than females, and genetic and environmental factors are believed to account in similar percentages to the development of ASD. In recent years, the contribution of inflammation and inflammatory mediators to disease aetiology and perpetuation has been the object of intense research. In this chapter, inflammatory aspects that contribute to ASD are discussed, including abnormal microglia activation and polarization phenotypes, increased systemic l...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Prata J, Machado AS, von Doellinger O, Almeida MI, Barbosa MA, Coelho R, Santos SG Tags: Methods Mol Biol Source Type: research

MeCP2 Dysfunction in Rett Syndrome and Neuropsychiatric Disorders.
Abstract Elucidating the functions of a particular gene is paramount to the understanding of how its dysfunction contributes to disease. This is especially important when the gene is implicated in multiple different disorders. One such gene is methyl-CpG-binding protein 2 (MECP2), which has been most prominently associated with the neurodevelopmental disorder Rett syndrome, as well as major neuropsychiatric disorders such as autism and schizophrenia. Being initially identified as a transcriptional regulator that modulates gene expression and subsequently also shown to be involved in other molecular events, dysfunc...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Chin EWM, Goh ELK Tags: Methods Mol Biol Source Type: research

The Role of Epigenetics in Addiction: Clinical Overview and Recent Updates.
Abstract Addiction is an international public health problem. It is a polygenic disorder best understood by accounting for the interplay between genetic and environmental factors. A recent way of perceiving this interaction is through epigenetics, which help grasp the neurobiological changes that occur in addiction and explain its relapsing-remitting nature. It is now known that every cell has a different way of expressing its phenotype, despite a universal DNA sequence. This is particularly true in the central nervous system where environmental factors influence this expression. Three major epigenetic processes h...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Beayno A, El Hayek S, Noufi P, Tarabay Y, Shamseddeen W Tags: Methods Mol Biol Source Type: research

Chromatin Immunoprecipitation Assay for Analyzing Transcription Factor Activity at the Level of Peripheral Myelin Gene Promoters.
Abstract Disruption of epigenetic regulators of transcription is a central mechanism of oncogenesis. Differential gene expression is facilitated by transcriptional regulatory mechanisms and chromatin modifications through DNA-protein interactions. One of the widely used assays to study this is chromatin immunoprecipitation (ChIP) assay, which enables the analysis of association between regulatory molecules, specific promoters, and histone modifications within the context of the cell. This is of immense value as ChIP assays can provide a glimpse of the regulatory mechanisms involved in gene expression in vivo. It i...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Makoukji J Tags: Methods Mol Biol Source Type: research

Isolation and Molecular Characterization of Progenitor Cells from Human Umbilical Cord.
Abstract Mesenchymal stem cells (MSCs) are multipotent precursor cells which have been isolated from different vascularized tissue sources. Due to their paracrine function of secreting trophic and immunomodulatory molecules, MSCs are successfully used in cell-based transplantations and provide an alternative medical paradigm for treating a variety of devastating disorders. Umbilical cord is a medical waste with a large, readily available donor pool. Since umbilical cord is a fetal tissue, MSCs derived from it are considered more primitive with proliferative and differentiation advantages over adult MSCs. We define...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Goyal U, Sen A, Ta M Tags: Methods Mol Biol Source Type: research

Assessment of β-Cell Replication in Isolated Rat Islets of Langerhans.
Assessment of β-Cell Replication in Isolated Rat Islets of Langerhans. Methods Mol Biol. 2019;2029:25-35 Authors: Dalgaard LT Abstract Pancreatic β-cells in the islets of Langerhans secrete insulin in response to the rise in glucose levels following food intake. The hypoglycemic action of insulin applies a strong evolutionary brake on β-cell division. However, under some conditions β-cells can be stimulated to enter cell cycle progression and divide, for example following exposure to increased glucose levels or during pregnancy. Here, a protocol is described for the isolation of ra...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Dalgaard LT Tags: Methods Mol Biol Source Type: research

Droplet Digital PCR for Measuring Absolute Copies of Gene Transcripts in Human Islet-Derived Progenitor Cells.
Abstract Transcript analysis is a routinely used method to assess the expression profile of progenitor cells at different stages starting from their isolation to differentiation into specific lineages. It is a powerful way to understand similarities and differences between different cell types as well to estimate successful differentiation process. Transcript measurement is most commonly done using polymerase chain reaction (PCR) but other methods such as in situ hybridization, RNA sequencing are available. The quantitative PCR using TaqMan chemistry is a highly sensitive and reproducible method that measures gene...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Maynard CL, Wong WKM, Hardikar AA, Joglekar MV Tags: Methods Mol Biol Source Type: research

Isolation and In Vitro Culture of Human Gut Progenitor Cells.
Abstract The gastrointestinal epithelium is a highly regenerative organ, where each cell is replaced in a cycle of 4-6 days, depending on the mammalian species. This highly proliferative state is driven by gastrointestinal stem and progenitor cells, located at the base of crypts. These cells give rise to at least six types of differentiated epithelial cells, each with a distinct function in maintaining homeostasis between the intestinal interface and the luminal environment. The isolation and culture of these cells from mammalian gastrointestinal tissue is a novel technique, which allows for the generation an...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Bruce J, Kaiko GE, Keely S Tags: Methods Mol Biol Source Type: research

Isolation and Characterization of Colony-Forming Progenitor Cells from Adult Pancreas.
Abstract Obtaining, growing, and analysis of pancreatic progenitor cells. Adult stem and progenitor cells have been successfully used for cell-based therapies such as transplantation of hematopoietic stem cells for various diseases. Whether stem and progenitor cells in the adult pancreas can be identified and used for replacement therapy has been a highly controversial topic. To address this controversy, our laboratory has developed in vitro colony assays to detect and characterize individual pancreatic stem and progenitor-like cells. We found that a subpopulation of ductal cells in the adult murine pancreas has t...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Quijano JC, Tremblay JR, Rawson J, Ku HT Tags: Methods Mol Biol Source Type: research

A Novel Gene Delivery Approach Using Metal Organic Frameworks in Human Islet-Derived Progenitor Cells.
Abstract The ability to regenerate insulin-producing β cells is the ultimate goal for treatment of type 1 diabetes. Several sources of stem cells have been investigated by studying their differential potential to form insulin-producing β cells that can be used for replacement therapy. Progenitor cells derived from human islets that are lineage committed have been shown to be better alternatives with regard to their differentiation capabilities for the generation of insulin-producing β-like cells. Controlling the differentiation of progenitor cells is a vital approach in exploiting cellular expansion...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Poddar A, Joglekar MV, Hardikar AA, Shukla R Tags: Methods Mol Biol Source Type: research

Encapsulation and Transplantation of Pancreatic Progenitor Cells.
Abstract Type 1 diabetes, characterized by autoimmune destruction of pancreatic beta cells, affects 41 million people worldwide. Beta cell replacement therapies have immense potential as a treatment option because pancreatic progenitors derived from human pluripotent stem cells can provide a near limitless supply of transplantable tissue. The key limitation of this approach is the need for lifelong use of immunosuppressive drugs that have undesirable side effects. Microencapsulation is an option for providing protection for transplanted cells from mechanical stress and immune attack. Traditionally, pluripotent cel...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Carroll L, Mridha AR, Tuch BE Tags: Methods Mol Biol Source Type: research

Differentiation of Urothelium from Mouse Embryonic Stem Cells in Chemically Defined Conditions.
Abstract The urothelium of the bladder and urethra are derived from the definitive endoderm during development. Cellular signaling molecules important to the developmental specification of the urothelium are also implicated in the dysregulation of the tissue repair mechanism characteristic of bladder disease. Hence, a complete understanding of the regulation of urothelium development is central to understanding the processes of bladder disease, and in development of simple chemically defined methods for use in regenerative medicine. Key to this is a suitable in vitro model that readily allows for the prosecution o...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Boumelhem BB, Fraser ST, Assinder SJ Tags: Methods Mol Biol Source Type: research

Identification and Analysis of Mouse Erythroid Progenitor Cells.
Abstract The most common cell type in the human body, the red blood cell or erythrocyte, has a life span of approximately 3 months. To compensate for this massive cellular requirement and short life span, the major blood producing tissues contain vast numbers of erythroid progenitor cells. Erythroid progenitors differentiate progressively from hematopoietic stem cells to committed erythroid progenitors to reticulocytes lacking a nucleus and finally to functionally mature erythrocytes in the circulation. Different erythroid progenitor activity, representative of distinct stages of erythropoiesis, can be observ...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Colonne CK, Yeo JH, McKenzie CV, Fraser ST Tags: Methods Mol Biol Source Type: research

Single-Cell Assays Using Hematopoietic Stem and Progenitor Cells.
Abstract Hematopoietic stem cells (HSCs) undergo division, making two daughter cells with unique fate decision choices, that is, whether to self-renew to maintain stemness or differentiate to committed progenitors. Since HSCs are heterogeneous in nature understanding this phenomenon at the single cell level is important. In vitro single-cell assays like the paired-daughter cell and myeloid multilineage differentiation are useful to understand this unique stem cell process. Both assays are performed using cytokine combination which allows four-lineage myeloid differentiation-neutrophil, erythroid, macrophage/monocy...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Hinge AS, Filippi MD Tags: Methods Mol Biol Source Type: research

Mitochondrial Assays Using Cardiac Stem Cells.
Abstract Ischemic heart disease is the leading cause of death worldwide. Stem cell therapy to repair and regenerate the infarcted myocardium is a promising approach to address this unmet medical need. However, the poor survival of transplanted cells in the hostile ischemic myocardium has been a major hurdle in achieving an effective cell therapy against myocardial infarction. As such, novel strategies to promote the survival of transplanted cells are highly sought after. Mitochondria are intimately involved in cell survival and have been the main organelles being targeted for cytoprotection. Mitochondrial morpholo...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Rosdah AA, Delbridge LMD, Lim SY Tags: Methods Mol Biol Source Type: research

High-Efficiency Lentiviral Gene Modification of Primary Murine Bone-Marrow Mesenchymal Stem Cells.
Abstract Lentiviral vectors are the method of choice for stable gene modification of a variety of cell types. However, the efficiency with which they transduce target cells varies significantly, in particular their typically poor capacity to transduce primary stem cells. Here we describe the isolation and enrichment of murine bone-marrow mesenchymal stem cells (MSCs) via fluorescence-activated cell sorting (FACS); the cloning, production, and concentration of high-titer second generation lentiviral vectors via combined tangential flow filtration (TFF) and ultracentrifugation; and the subsequent high-efficiency gen...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Gerace D, Ren B, Martiniello-Wilks R, Simpson AM Tags: Methods Mol Biol Source Type: research

Bone Marrow-Derived Progenitor Cells Mediate Immune Cell Regulation.
Abstract Human bone marrow (BM) derived mesenchymal stem cells (MSC) have high capacity to propagate ex vivo with superior reparative, immunosuppressive, and anti-inflammatory properties. Here we describe standardized protocols and culture conditions that enable the isolation, expansion and maintenance of a highly purified and homogenous population of human MSC. These third party-derived off-the-shelf MSC from healthy human bone marrow donors can potently inhibit mitogenically or allogeneically activated human T cells in proliferation assays. The standard operating procedures described in this chapter can be appli...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Sivanathan KN, Coates PT Tags: Methods Mol Biol Source Type: research

Flow Cytometry and Cell Sorting Using Hematopoietic Progenitor Cells.
Abstract Flow cytometry is a widely used laser-based technology for rapid analysis of the expression of cell surface antigens and intracellular molecules in various cell types including hematopoietic stem/progenitor cells (HSPCs). Multiparametric analysis of individual cells within a short time frame makes this tool attractive and indispensable in the field of stem cell research. This is accomplished by harnessing the specific light scattering ability of the cell type, which determines its size and internal complexity. In addition, use of fluorescently conjugated antibodies allows the detection of a specific surfa...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Vadakke-Madathil S, Limaye LS, Kale VP, Chaudhry HW Tags: Methods Mol Biol Source Type: research

Isolation of Epidermal Progenitor Cells from Rat Tympanic Membrane.
Abstract The eardrum is an important structural component for hearing, but it is delicate and subject to traumatic injury and disease. Healing mechanisms are activated after injury but sometimes healing fails and chronic perforations develop, requiring surgical intervention. To model the wound healing responses we established a simple method for isolating keratinocytes and progenitors from individual eardrums. The central region of the eardrum contains epidermal proliferative centers that produce keratinocytes which migrate to cover the eardrum surface. We dissected out the central region and explanted it to the p...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Liew LJ, Wang AY, Dilley RJ Tags: Methods Mol Biol Source Type: research

Measurement of Store-Operated Calcium Entry in Human Neural Cells: From Precursors to Differentiated Neurons.
Abstract Calcium imaging in an ex-vivo setup is used to understand the calcium status of isolated cells or tissue. In this chapter we explain the use of the ratiometric chemical indicator Fura-2 which can be loaded into isolated cells in the form of lipophilic acetomethyl (AM) esters. Fura-2 is a combination of calcium chelator and fluorophore, and can be used with dual wavelength excitation (340/380 nm) for quantitative calcium concentrations. The cells can then be viewed using a fluorescence microscope and captured by a CCD camera. We specifically discuss the technique involved in understanding the end...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Gopurappilly R, Deb BK, Chakraborty P, Hasan G Tags: Methods Mol Biol Source Type: research

Embryoid Body Differentiation of Mouse Embryonic Stem Cells into Neurectoderm and Neural Progenitors.
Abstract Mouse embryonic stem cells (mESCs) are pluripotent cells capable of differentiating in vitro to form the ~200 types of cells of the developing embryo and adult, including cells of the nervous system. This makes mESCs a useful tool for studying the molecular mechanisms of mammalian embryonic development. Many protocols involving the use of growth factors and small molecules to differentiate mESCs into neural progenitors and neurons currently exist. However, there is a paucity of protocols available that recapitulate the developmental process. Our laboratory has developed a protocol to recapitulate mammalia...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Shparberg RA, Glover HJ, Morris MB Tags: Methods Mol Biol Source Type: research