Glial Differentiation Of Human Umbilical Stem Cells In 2d And 3d Environments

During differentiation stem cells are exposed to a range of microenvironmental chemical and physical cues. In this study, human multipotent progenitor cells (hMLPCs) were differentiated from umbilical cord into oligodendrocytes and astrocytes. Chemical cues were represented by a novel defined differentiation medium containing the neurotransmitter norepinephrine (NE). In traditional 2 dimensional (2D) conditions, the hMLPCs differentiated into oligodendrocyte precursors, but did not progress further. However, in a constructed 3 dimensional (3D) environment, the hMLPCs differentiated into committed oligodendrocytes that expressed MBP. When co-cultured with rat embryonic hippocampal neurons (EHNs), hMLPCs developed in astrocytes or oligodendrocytes, based on presence of growth factors in the differentiation medium. In co-culture, physical cues provided by axons were essential for complete differentiation of both astrocytes and oligodendrocytes. This study presents a novel method of obtaining glia from human MLPCs that could eliminate many of the difficulties associated with their differentiation from embryonic stem cells. In addition, it reveals the complex interplay between physical cues and biomolecules on stem cell differentiation

Characterization of a novel and specific inhibitor for the pro-apoptotic protease Omi/HtrA2

Omi/HtrA2 is a mammalian serine protease with high homology to bacterial HtrA chaperones. Omi/HtrA2 is localized in mitochondria and is released to the cytoplasm in response to apoptotic stimuli. Omi/HtrA2 induces cell death in a caspase-dependent manner by interacting with the inhibitor of apoptosis protein as well as in a caspase-independent manner that relies on its protease activity. We describe the identification and characterization of a novel compound as a specific inhibitor of the proteolytic activity of Omi/HtrA2. This compound (ucf-101) was isolated in a high throughput screening of a combinatorial library using bacterially made Omi-(134-458) protease and fluorescein-casein as a generic substrate. ucf-101 showed specific activity against Omi/HtrA2 and very little activity against various other serine proteases. This compound has a natural fluorescence that was used to monitor its ability to enter mammalian cells. ucf-101, when tested in caspase-9 (-/-) null fibroblasts, was found to inhibit Omi/HtrA2-induced cell death

Characterization Of A Human Fetal Spinal Cord Stem Cell Line, Nsi-566Rsc, And Its Induction To Functional Motoneurons

Specific neuronal subtypes, especially motoneurons (MNs), derived from human stem cells provide a significant therapeutic potential for spinal cord diseases, such as amyotrophic lateral sclerosis (ALS) and spinal cord injury. So far, in vitro, MNs have only been successfully induced from embryonic stem cells (hESC) and human fetal cortical progenitors. Although neural progenitors from spinal cord would be a likely source for generating MNs, there has been no study reporting successful in vitro differentiation of MNs from spinal cord progenitors. This study first characterized a polyclonal spinal cord stem cell line isolated from an 8 week-old fetus. Then a paradigm was introduced to successfully induce MNs from this cell line, which was demonstrated by immunostaining using the MN markers HB9, Islet1 and choline acetyl transferase (ChAT). The combination of HB9 and ChAT immunostainings indicated that ∼20% of the cells were MNs after this induction protocol. The presence of other cell types in the differentiated culture was also analysed. Finally, the electrophysiological properties of these differentiated MNs were characterized to confirm their functional integrity. The majority of these MNs fired repetitive action potentials (APs), which is an indicator of functional maturation. The recordings of spontaneous excitatory postsynaptic currents (EPSCs) confirmed the formation of synapses onto these MNs. This study reports the first successful differentiation of MNs from human spinal cord stem cells in vitro, providing a novel approach for obtaining functional MNs when designing the therapeutic strategy for spinal cord diseases or injuries. Copyright © 2009 John Wiley & Sons, Ltd

Small Molecule Induction Of Human Umbilical Stem Cells Into Myelin Basic Protein Positive Oligodendrocytes In A Defined Three-Dimensional Environment

The molecular level damage to DNA is important due to DNAs susceptibility to free radical attacks and crucial roles in maintaining cell functions. Although a panel of techniques can be used to detect DNA damages, most of them are limited due to low sensitivity, low throughput, incompatibility for automated data analysis, and labor-intensive operations. We have developed a cell array based DNA damage assay in which mammalian cells are attached on an array of microfabricated patterns through electrostatic interactions. After trapping patterned cells inside gels, damaged DNA fragment can diffuse out of the nucleus and form a halo around each cell inside gels. The halo array can be observed fluorescently after labeling DNA with ethidium bromide. DNA damages can be determined sensitively at the single cell level, accurately due to the symmetric shape of the halo, and automatically due to the spatial registry of each cell and the nonoverlapping halos surrounding cells. The HaloChip can be used to detect DNA damages caused by chemicals and ultraviolet and X-ray irradiations with high efficiency. A major advantage of HaloChip is the ability to increase throughout by spatially encoding multiple dosing conditions on the same chip. Most importantly, the method can be used to measure variations in response to DNA damaging agents within the same cell population. Compared with halo assay or comet assay alone, this method allows automated analysis of a million cells without an overlapping issue. Compared with the microwell array based comet assay, this method can selectively capture and analyze cells, and the results can be easily analyzed to provide precise information on DNA damage. This method can be used in a broad range of clinical, epidemiological, and experimental settings. © 2011 American Chemical Society

Rat Cortical Oligodendrocyte-Embryonic Motoneuron Co-Culture: An In Vitro Axon-Oligodendrocyte Interaction Model

Mechanisms that control the differentiation and function of oligodendrocytes in the central nervous system are complex and involve multiple inputs from the surrounding environment, including localized concentrations of growth factors and the extracellular matrix. Dissection and analysis of these inputs are key to understanding the pathology of central nervous system demyelinating diseases such as multiple sclerosis, where the differentiation of myelinating oligodendrocytes from their precursors underlies the remission phase of the disease. In vitro co-culture models provide a mechanism for the study of factors that regulate differentiation of oligodendrocyte precursors but have been difficult to develop due to the complex nature of central nervous system myelination. This study describes development of an in vitro model that merges a defined medium with a chemically modified substrate to study aspects of myelination in the central nervous system. We demonstrate that oligodendrocyte precursors co-cultured with rat embryonic motoneurons on non-biological substrate (diethylenetriamine trimethoxy-silylpropyldiethylenetriamine), can be induced to differentiate into mature oligodendrocytes that express myelin basic protein, using a serum-free medium. This defined and reproducible model of in vitro myelination could be a valuable tool for the development of treatments for demyelinating diseases such as multiple sclerosis. © 2012 American Scientific Publishers. All rights reserved

A Phenotypic Culture System For The Molecular Analysis Of Cns Myelination In The Spinal Cord

Studies of central nervous system myelination lack defined in vitro models which would effectively dissect molecular mechanisms of myelination that contain cells of the correct phenotype. Here we describe a co-culture of purified motoneurons and oligodendrocyte progenitor cells, isolated from rat embryonic spinal cord using a combination of immunopanning techniques. This model illustrates differentiation of oligodendrocyte progenitors into fully functional mature oligodendrocytes that myelinate axons. It also illustrates a contribution of axons to the rate of oligodendrocyte maturation and myelin gene expression. The defined conditions used allow molecular analysis of distinct stages of myelination and precise manipulation of inductive cues affecting axonal-oligodendrocyte interactions. This phenotypic in vitro myelination model can provide valuable insight into our understanding of demyelinating disorders, such as multiple sclerosis and traumatic diseases such as spinal cord injury where demyelination represents a contributing factor to the pathology of the disorder. © 2014 Elsevier Ltd

A phenotypic culture system for the molecular analysis of CNS myelination in the spinal cord

Studies of central nervous system myelination lack defined in vitro models which would effectively dissect molecular mechanisms of myelination that contain cells of the correct phenotype. Here we describe a co-culture of purified motoneurons and oligodendrocyte progenitor cells, isolated from rat embryonic spinal cord using a combination of immunopanning techniques. This model illustrates differentiation of oligodendrocyte progenitors into fully functional mature oligodendrocytes that myelinate axons. It also illustrates a contribution of axons to the rate of oligodendrocyte maturation and myelin gene expression. The defined conditions used allow molecular analysis of distinct stages of myelination and precise manipulation of inductive cues affecting axonal–oligodendrocyte interactions. This phenotypic in vitro myelination model can provide valuable insight into our understanding of demyelinating disorders, such as multiple sclerosis and traumatic diseases such as spinal cord injury where demyelination represents a contributing factor to the pathology of the disorder