This work was supported by the Australian National Health and Medical Research Council (NHMRC fellowship #454330 to JX, project grant #628761 to SM and APP1058647 to JX), Multiple Sclerosis Research Australia (MSRA #12070 to JX), the University of Melbourne Research Grant Support Scheme and Melbourne Research CI Fellowship to JX as well as Australia Postgraduate Scholarships to HP and AF.&#160;We would like to acknowledge the Operational Infrastructure Scheme of the Department of Innovation, Industry and Regional Development, Victoria Australia.

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	<li>Baumann, N., Pham-Dinh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+oligodendrocyte+and+myelin+in+the+mammalian+central+nervous+system.">Biology of oligodendrocyte and myelin in the mammalian central nervous system.</a> Physiol Rev. 81 (2), 871-927 (2001).</li><li>Watkins, T. A., Emery, B., Mulinyawe, S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+stages+of+myelination+regulated+by+gamma-secretase+and+astrocytes+in+a+rapidly+myelinating+CNS+coculture+system.">Distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating CNS coculture system.</a> Neuron. 60 (4), 555-569 (2008).</li><li>Lee, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDGAs+interact+selectively+with+neuroligin-2+but+not+other+neuroligins+to+regulate+inhibitory+synapse+development.">MDGAs interact selectively with neuroligin-2 but not other neuroligins to regulate inhibitory synapse development.</a> Proc Natl Acad Sci U S A. 110 (1), 336-341 (2013).</li><li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDNF+exerts+contrasting+effects+on+peripheral+myelination+of+NGF-dependent+and+BDNF-dependent+DRG+neurons.">BDNF exerts contrasting effects on peripheral myelination of NGF-dependent and BDNF-dependent DRG neurons.</a> J Neurosci. 29 (13), 4016-4022 (2009).</li><li>Chan, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGF+controls+axonal+receptivity+to+myelination+by+Schwann+cells+or+oligodendrocytes.">NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes.</a> Neuron. 43 (2), 183-191 (2004).</li><li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-Derived+Neurotrophic+Factor+Promotes+Central+Nervous+System+Myelination+via+a+Direct+Effect+upon+Oligodendrocytes.">Brain-Derived Neurotrophic Factor Promotes Central Nervous System Myelination via a Direct Effect upon Oligodendrocytes.</a> Neurosignals. 18 (3), 186-202 (2010).</li><li>Lundgaard, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuregulin+and+BDNF+induce+a+switch+to+NMDA+receptor-dependent+myelination+by+oligodendrocytes.">Neuregulin and BDNF induce a switch to NMDA receptor-dependent myelination by oligodendrocytes.</a> PLoS Biology. 11 (12), e1001743 (2013).</li><li>Kleitman, N., W, P. M., Bunge, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tissue culture methodes for the study of myelination. , (1991).</li><li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+signal-regulated+kinase+1/2+signaling+promotes+oligodendrocyte+myelination+in+vitro.">Extracellular signal-regulated kinase 1/2 signaling promotes oligodendrocyte myelination in vitro.</a> J Neurochem. 122 (6), 1167-1180 (2012).</li><li>Wong, A. W., Xiao, J., Kemper, D., Kilpatrick, T. J., Murray, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendroglial+expression+of+TrkB+independently+regulates+myelination+and+progenitor+cell+proliferation.">Oligodendroglial expression of TrkB independently regulates myelination and progenitor cell proliferation.</a> The Journal of Neuroscience. 33 (11), 4947-4957 (2013).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning,+Characterization+and+Expression+of+miR-15a-3p+and+miR-15b-3p+in+Dairy+Cattle.">Molecular cloning, Characterization and Expression of miR-15a-3p and miR-15b-3p in Dairy Cattle.</a> Molecular and Cellular Probes. , (2014).</li><li>Emery, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+gene+regulatory+factor+is+a+critical+transcriptional+regulator+required+for+CNS+myelination.">Myelin gene regulatory factor is a critical transcriptional regulator required for CNS myelination.</a> Cell. 138 (1), 172-185 (2009).</li><li>Murai, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+receptor+TLX+stimulates+hippocampal+neurogenesis+and+enhances+learning+and+memory+in+a+transgenic+mouse+model.">Nuclear receptor TLX stimulates hippocampal neurogenesis and enhances learning and memory in a transgenic mouse model.</a> Proc Natl Acad Sci U S A. 111 (25), 9115-9120 (2014).</li></ol>

The authors declare that there is no conflict of interest regarding this research.

Myelination of axons is a crucial process for the optimal function of both the central and peripheral nervous systems of vertebrates. The generation and maintenance of myelinated axons is a complex and coordinated process involving molecular interactions between neuronal, glial (from Schwann cells or oligodendrocytes) and extra cellular matrix proteins. The significance and applicability of this protocol is that it allows manipulation of proteins in one specific cell type within the mixed co-culture settings. As multiple...

Myelination of axons is crucial for the fast and efficient transmission of action potentials in both the central and peripheral nervous systems. Specialized cells, Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system, wrap around and ensheathe axons in myelin, effectively insulating the nerve and facilitating saltatory conduction1. The process of myelination can be studied in vitro using retinal ganglion neurons2, engineered nanofibers3, or dorsal root ganglion neurons co-cultured with either Schwann cells4 or oligodendrocytes5-7. The in vitro myelination assay is an established model for studying nervous system myelination and it replicates many of the fundamental processes that occur during myelination in vivo5-8. The assay involves the coculture of purified populations of Dorsal Root Ganglion (DRG) neurons, with OPCs (for CNS myelination) or Schwann cells (for PNS myelination). Under specific conditions these myelinating cells ensheathe DRG axons in the ordered, ultra structurally verified, multi-lamellar sheet of insulating plasma membrane that express the same complement of myelin specific proteins present in vivo.
The most commonly used cell model of studying CNS myelination in vitro is the co-cultures of DRG neurons and OPCs, which have been successfully used to study the effect that exogenous factors such as the neurotrophins exert on CNS myelination in vitro5,6. Exogenous factors such as growth factors or small molecule pharmacological inhibitors have been widely used to study the role of signaling pathways in myelination using the DRG-OPC coculture model7,9. However, in the mixed co-culture settings that contain both the neurons and oligodendrocytes, it remained formally possible that either the growth factors or the pharmacological inhibitors could have exerted effects upon both the DRG neurons and oligodendrocytes (OL). This does offer the ability to specifically dissect the roles that the proteins expressed only by DRGs or oligodendroglia exerts upon myelination using this dual cell system. To unequivocally confirm that the signaling pathway in oligodendroglial directly regulates myelination, lentiviral transduction of OPCs, prior to seeding onto DRG neurons for the in vitro myelination assay, has proven to be an elegant way to overexpress both wild-type and mutant proteins, as well as knockdown expression of constitutively expressed proteins by oligodendrocytes. Thus this approach offers an avenue to specifically interrogate and manipulate signaling pathways within oligodendrocytes for studying myelination9,10.
In this paper, we report methods that we have developed to overexpress a protein of interest selectively in oligodendrocytes via a lentiviral approach for studying myelination in vitro. The technique begins with the generation of expression vectors containing the gene of interest, be it in a wild type, constitutively active or dominant negative form which are then subsequently cloned into the pENTR vector (pENTR L1-L2 pENTR4IRES2GFP). This vector (containing the gene of interest), the CMV promotor donor (pENTR L4-R1 pENTR-pDNOR-CMV) and the 2K7 lentivector are combined in an enzyme reaction to produce a 2K7 vector containing CMV promoter, the gene of interest, an internal ribosomal entry site and GFP (Figure 1). This Gateway cloned 2K7 construct combined with the PMD2.G virus envelope and the pBR8.91 virus package can be co-transfected into HEK293T cells to generate lentivirus that can subsequently be used to transduce OPCs. Once infected with the lentivirus the OPCs express a high level of the protein of interest. These OPCs can then be seeded onto DRG neuron cultures and the effect that expression of high levels of the desired protein exerts on myelination can be interrogated. The co-cultures are assessed for myelin protein expression by western blot analysis and visualized for the formation of myelinated axonal segments by immunocytochemistry.

<table><tbody><tr><td>2K7 lentivector&amp;nbsp;</td><td></td><td></td><td>Kind gift from Dr Suter&lt;sup&gt;9&lt;/sup&gt;</td></tr><tr><td>5-Fluoro-2&amp;prime;-deoxyuridine</td><td>Sigma-Aldrich</td><td>F0503-100mg</td><td></td></tr><tr><td>Alexa Fluor 488 Goat anti-mouse IgG</td><td>Jackson Immunoresearch</td><td>115545205</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-rabbit IgG (H+L)</td><td>Life Technologies</td><td>A11008</td><td></td></tr><tr><td>Alexa Fluor 594 goat anti-mouse IgG (H+L)</td><td>Life Technologies</td><td>A11005</td><td></td></tr><tr><td>Alexa Fluor 594 goat anti-rabbit IgG (H+L)</td><td>Life Technologies</td><td>A11012</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A9518-5G</td><td></td></tr><tr><td>B27 - NeuroCul SM1 Neuronal Supplement</td><td>Stem Cell Technologies&amp;nbsp;</td><td>5711</td><td></td></tr><tr><td>BDNF (Human)</td><td>Peprotech</td><td>PT450021000</td><td></td></tr><tr><td>Biotin (d-Biotin)</td><td>Sigma Aldrich</td><td>B4639</td><td></td></tr><tr><td>Bradford Reagent</td><td>Sigma Aldrich</td><td>B6916-500ML&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A4161</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma-Aldrich</td><td>C0378-100G</td><td></td></tr><tr><td>CNTF</td><td>Peprotech</td><td>450-13020</td><td></td></tr><tr><td>DAKO fluoresence mounting media</td><td>DAKO</td><td>S302380-2</td><td></td></tr><tr><td>DMEM, high glucose, pyruvate, no glutamine</td><td>Life Technologies</td><td>10313039</td><td></td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025-375KU</td><td></td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190250</td><td></td></tr><tr><td>DPBS, calcium, magnesium</td><td>Life Technologies</td><td>14040182</td><td></td></tr><tr><td>EBSS</td><td>Life Technologies</td><td>14155063</td><td></td></tr><tr><td>EcoRI-HF</td><td>NEB</td><td>R3101</td><td></td></tr><tr><td>Entry vectors for promoter and gene of interest</td><td></td><td></td><td>Generate as per protocols 1-2</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>12003C</td><td></td></tr><tr><td>Forskolin&amp;nbsp;</td><td>Sigma Aldrich</td><td>F6886-50MG</td><td></td></tr><tr><td>Glucose (D-glucose)</td><td>Sigma-Aldrich</td><td>G7528</td><td></td></tr><tr><td>Glycerol</td><td>Chem Supply</td><td>GL010-500M</td><td>See stock solutions</td></tr><tr><td>Goat Anti-Mouse IgG</td><td>Jackson ImmunoResearch</td><td>115005003</td><td></td></tr><tr><td>Goat Anti-Mouse IgM&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>115005020</td><td></td></tr><tr><td>Goat Anti-Rat IgG</td><td>Jackson ImmunoResearch</td><td>112005167</td><td></td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H3570</td><td></td></tr><tr><td>Igepal</td><td>Sigma Aldrich</td><td>I3021-100ML&amp;nbsp;</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma Aldrich</td><td>I6634&amp;nbsp;</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma-Aldrich</td><td>60615</td><td></td></tr><tr><td>Laminin&amp;nbsp;</td><td>Life Technologies</td><td>23017015</td><td></td></tr><tr><td>LB Medium</td><td></td><td></td><td>See stock solutions</td></tr><tr><td>LB-Agar</td><td></td><td></td><td>See stock solutions</td></tr><tr><td>L-Cysteine</td><td>Sigma-Aldrich</td><td>C-7477</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium</td><td>Life Technologies</td><td>11415064</td><td></td></tr><tr><td>L-Glutamate</td><td>Sigma-Aldrich</td><td>G1626</td><td></td></tr><tr><td>L-Glutamine- 200 mM (100x) liquid</td><td>Life Technologies</td><td>25030081</td><td></td></tr><tr><td>LR Clonase II Plus enzyme</td><td>Life Technologies</td><td>12538-120</td><td></td></tr><tr><td>MEM, NEAA, no Glutamine</td><td>Life Technologies</td><td>10370088</td><td></td></tr><tr><td>Mouse &amp;alpha; &amp;beta;III Tubulin&amp;nbsp;</td><td>Promega</td><td>G7121</td><td></td></tr><tr><td>Mouse &amp;alpha;MBP (monoclonal)</td><td>Millipore</td><td>MAB381</td><td></td></tr><tr><td>Na pyruvate&amp;nbsp;</td><td>Life Technologies</td><td>11360-070</td><td></td></tr><tr><td>NAC</td><td>Sigma Aldrich</td><td>A8199</td><td></td></tr><tr><td>NcoI-HF</td><td>NEB</td><td>R3193S</td><td></td></tr><tr><td>NEBuffer 4</td><td>NEB</td><td>B7004S</td><td></td></tr><tr><td>Neurobasal medium</td><td>Life Technologies</td><td>21103049</td><td></td></tr><tr><td>NGF (mouse)</td><td>Alomone Labs</td><td>N-100</td><td></td></tr><tr><td>NT-3</td><td>Peprotech</td><td>&amp;nbsp;450-03</td><td></td></tr><tr><td>O1 antibody - Mouse anti-O1</td><td>Millipore</td><td>MAB344</td><td>Alternative if O1 hybridoma cells are unavailable</td></tr><tr><td>O1 hybridoma cells</td><td></td><td></td><td>Conditioned medium containing anti-O1 antibody to be used for immunopanning</td></tr><tr><td>O4 antibody - Mouse anti-O4</td><td>Millipore</td><td>MAB345</td><td>Alternative if O4 hybridoma cells are unavailable</td></tr><tr><td>O4 hybridoma cells</td><td></td><td></td><td>Conditioned medium containing anti-O4 antibody to be used for immunopanning</td></tr><tr><td>Competent cells</td><td>Life Technologies</td><td>A10460</td><td></td></tr><tr><td>One Shot Stbl competent cells</td><td>Life Technologies</td><td>C7373-03</td><td></td></tr><tr><td>Papain Suspension</td><td>Worthington/Cooper</td><td>LS003126</td><td></td></tr><tr><td>pBR8.91</td><td></td><td></td><td>Kind gift from Dr Denham&lt;sup&gt;10&lt;/sup&gt;</td></tr><tr><td>PDGF-AA (Human)</td><td>Peprotech</td><td>PT10013A500&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Penicillin-streptomycin 100x solution</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>pENTRY4IRES2GFP</td><td>Invitrogen</td><td>11818-010&amp;nbsp;</td><td></td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>12259</td><td></td></tr><tr><td>Poly-D-lysine</td><td>Sigma</td><td>P6407-5MG</td><td></td></tr><tr><td>Polyethylenimine (PEI)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>408727-100ML</td><td></td></tr><tr><td>Poly-L-ornithine&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>P3655</td><td></td></tr><tr><td>Progesterone&amp;nbsp;</td><td>Sigma Aldrich</td><td>P8783</td><td></td></tr><tr><td>Protease inhibitor tablet (Complete mini)</td><td>Roche</td><td>11836153001</td><td></td></tr><tr><td>Proteinase K</td><td></td><td></td><td>Supplied with Clonase enzyme</td></tr><tr><td>Putrescine</td><td>Sigma Aldrich</td><td>P-5780</td><td></td></tr><tr><td>Rabbit &amp;alpha; neurofilament</td><td>Millipore</td><td>AB1987&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Rabbit &amp;alpha;MBP (polyclonal)</td><td>Millipore</td><td>AB980</td><td></td></tr><tr><td>Ran2 hybridoma cells</td><td>ATCC</td><td>TIB-119</td><td>Conditioned medium containing anti-Ran2 antibody to be used for immunopanning</td></tr><tr><td>Rat anti CD140A/PDGFRa antibody</td><td>BD Pharmingen</td><td>558774</td><td></td></tr><tr><td>SacII</td><td>NEB</td><td>R0157</td><td></td></tr><tr><td>SOC medium</td><td></td><td></td><td>Supplied with competent bacteria</td></tr><tr><td>Sodium selenite&amp;nbsp;</td><td>Sigma Aldrich</td><td>S5261</td><td></td></tr><tr><td>Spe I</td><td>NEB</td><td>R0133S</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>NEB</td><td>M0202S</td><td></td></tr><tr><td>T4 DNA Ligase Buffer</td><td>NEB</td><td>B0202S</td><td></td></tr><tr><td>TE buffer pH8</td><td></td><td></td><td>See stock solutions</td></tr><tr><td>TNE lysis buffer</td><td></td><td></td><td></td></tr><tr><td>Trace Elements B</td><td>Cellgro&amp;nbsp;</td><td>99-175-CI</td><td></td></tr><tr><td>Transferrin (apo-Transferrin human)</td><td>Sigma-Aldrich</td><td>T1147</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284&amp;nbsp;</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T9201-1G</td><td></td></tr><tr><td>Trypsin Inhibitor From Chicken Egg White</td><td>Roche</td><td>10109878001</td><td></td></tr><tr><td>Trypsin-EDTA (1x), phenol red (0.05%)</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Unconjugated Griffonia Simplicifolia Lectin BSL-1</td><td>Vector laboratories&amp;nbsp;</td><td>L-1100</td><td></td></tr><tr><td>Uridine</td><td>Sigma-Aldrich</td><td>U3003-5G</td><td></td></tr></tbody></table>

production and use of lentivirus to selectively transduce primary oligodendrocyte precursor cells for in vitro myelination assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

The flag-tagged extracellular signal-related kinase 1(Flag-Erk1) construct used for lentivirus production is verified by restriction enzyme digest of the constructs used, including both the 2K7 constructs and the packaging and accessory constructs required for virus production (Figure 3).
<img alt="Figure 3" src="/files/ftp_upload/52179/52179fig3highres.jpg" /> 
Figure 3: DNA construct verification. All DNA constructs...

Watch this Scientific Journal Video about Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays at JoVE.com

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

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Developmental Biology

14.4K Views.  The University of Melbourne. Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

Video: Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Genetics

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

Immunology and Infection

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Neuroscience

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Biology

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

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