Name: isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay
Uploaded: 2011-10-30T14:00:00
Duration: 9 min 24 s
Description: Watch this Scientific Journal Video about Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay at JoVE.com

This work was supported by Grants from the Florida Centre for Brain Tumor Research; Preston A. Wells Jr. Center for Brain Tumor Therapy.

1. Collection of Primary GBM Tissue
<ol>
	<li>A glioblastoma mutliforme (GBM) tumor is obtained from a patient diagnosed with the cancer and undergoing surgery. </li>
	<li>Arrangements must be made with neurosurgery team. The neurosurgeon will place the resected GBM tumor in an appropriate size tube containing neural stem cell (NSC) basal medium supplemented with 10-15% antibiotics (Peniciline/Streptomycine). Cold medium must be provided to operating room by lab. Alternatively, cold HEPES-buffered minimum essential medium (HEM) or phosphate buffered saline (PBS) with high concentration of antibiotics can also be used for this purpose.</li>
	<li>The resected tumor tissue is delivered to the lab on ice and placed under the hood.</li>
</ol>
2. Dissociation of Primary Tumor into Single-Cell Suspension
<ol>
	<li>Excess of the original medium/PBS is removed from the falcon tube and the sample is washed 2-3 times with 5-10ml of PBS/NSC basal medium to remove blood and debris. The PBS/Medium is removed and the GBM tumor tissue is placed in a Petri dish. </li>
	<li>The tissue is cut into small pieces and minced with a No. 10 scalpel blade into tiny pieces to increase the surface area for trypsinization process. Mincing can take 1-3 minutes depending on the size of the tumor. </li>
	<li>The minced tissue is trypsinized in 3-5ml of pre-warmed %0.05 trypsin-EDTA for 10-15 minutes at a 37&deg;C water bath. An electronic pipette is used to transfer the tiny tumor pieces and trypsin into a 15ml Falcon tube. </li>
	<li>An equal volume of soybean trypsin inhibitor is added to stop the enzymatic trypsin reaction after the incubation period.</li>
	<li>Trypsin inactivation is ensured by pipetting the suspension up and down several times. Then, the suspension is pelleted down by centrifuging at 800rpm (110g) for 5min.</li>
	<li>The supernatant is discarded and the tissue pieces are resuspended in 1ml of sterile NSC basal medium. The clumps are dissociated by gently pipetting up and down (3-7 times) until a smooth milky single cell suspension is achieved. The number of pipetting steps directly depends on the size of particles in the minced tissue. Lengthy and vigorous mechanical dissociation should be avoided as it might result in cell death and a reduction in sphere formation.</li>
	<li>To remove un-dissociated pieces and debris, 10-15 ml of basal medium is added to the tube and the cell suspension is filtered through a 40 micron cell strainer into a 50ml tube.</li>
	<li>The filtered suspension is centrifuged at 800rpm (110g) for 5min. The supernatant is discarded afterwards.</li>
	<li>Pelletted cells are then resuspended in 1-2ml of complete NSC medium for cell counting. </li>
</ol>
3. Cell Count and Plating
<ol>
	<li>10 &mu;L of the cell suspension is added to 90 &mu;L of 0.04% Trypan blue in a 1ml eppendorf tube. 
	Note: other appropriate cell dilutions may also be used.</li>
	<li>Pipette up and down to mix the suspension. 10 &mu;L of the cells/trypan blue mixture is transferred to hemocytometer in order to count cell density.</li>
	<li>Cells are plated in complete NSC medium (a mixture of NSC basal medium and NSC proliferation supplement at a 9:1 ratio) supplemented with 20ng/ml EGF, 10ng/ml bFGF and 1&mu;l/ml of 0.2% heparin (2&mu;g/ml) in appropriate tissue culture vessels. 5, 20 and 40 ml of medium is used for T25, T80 and T175 flasks, respectively. Antibiotics may be added to the medium at a concentration of 1:100 to decrease chance of contamination.</li>
	<li>The flask is placed in an incubator set at 37&deg;C and 5% CO2.</li>
</ol>
4. Passaging and expansion of GBM derived spheres:
<ol>
	<li>When the neurospheres reached an average size of 150-200 &mu;m in diameter, the culture is ready for subculture. The content of each flask is removed and placed in an appropriate size sterile tissue culture tube, and centrifuged at 800 rpm (110 g) for 5 min at room temperature.</li>
	<li>The supernatant is removed and the pellet is resuspended in 1 ml of %0.05 trypsin-EDTA.</li>
	<li>To achieve an optimal trypsinization, the cell suspension is incubated at 37&deg;C in a water bath for 2-3 min. To stop the trypsin activity an equal volume of soybean trypsin inhibitor is added to the cell suspension and the cell suspension is gently pipetted up and down.</li>
	<li>The cell suspension is centrifuged at 800 rpm (110g) for 5 min. Then, the supernatant is removed and the cells are resuspended in 1 ml of NSC medium. 	</li>
	<li>A cell count is performed as described earlier.</li>
	<li>The cells are plated at a concentration of 5x104 cells/ml in complete NSC medium supplemented with growth factors and plated in appropriate size tissue culture vessels as described in previous part.</li>
	<li>Secondary neurospheres are formed in 7-10 days when incubated at 37&deg; C in a humidified incubator with 5% CO2.</li>
</ol>
5. Representative Results:
After plating the single cells harvested from GBM tumor tissue, tumor stem-like cells proliferate and generate small clusters of cells composed of few cells in 3-4 days (see the video and Figure 1). As these clusters grow, they acquire a more spherical shape so that by 7-8 days, proper phase bright spheres with an average diameter of 150-200 microns form (See the video and Figure 2). At higher magnification, healthy spheres usually demonstrate microspikes at their periphery. Having large sphere like clusters in 1-2 days after culture initiation is due to existence of non-dissociated clumps at the binging of the culture and should not be mistaken as true spheres. The amount of debris in primary tumor sphere culture varies depending on the initial source of tissue and whether or not it includes any surrounding brain tissue. Proper tissue preparation techniques including enzymatic and mechanical dissociation, and subsequent filtration of the sample with sufficient amount of medium can result in less debris in culture.
<img src="/files/ftp_upload/3633/3633fig1.jpg" alt="Figure 1" /> 
Figure 1. Primary GBM sphere culture 4 days after plating. Tumor stem-like cells proliferate and generate small clusters of cells in 3-4 days. Original Magnification; 20x.
<img src="/files/ftp_upload/3633/3633fig2.jpg" alt="Figure 2" /> 
Figure 2. Passage one GBM sphere culture 8 days after plating. Original Magnification; 20x.

<ol>
	<li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Siebzehnrubl, F. A., Vedam-Mai, V., Azari, H., Reynolds, B. A., Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Adult+Neural+Stem+Cells.">Isolation and Characterization of Adult Neural Stem Cells.</a> Methods Mol Biol. 750, 61-77 (2011).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, 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=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J. Vis. Exp. (45), e2393-e2393 (2010).</li><li>Azari, H., Sharififar, S., Rahman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J. Vis. Exp. (47), e2457-e2457 (2011).</li><li>Eramo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+expansion+of+the+tumorigenic+lung+cancer+stem+cell+population.">Identification and expansion of the tumorigenic lung cancer stem cell population.</a> Cell Death Differ. 15, 504-514 (2008).</li><li>Patrawala, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+purified+CD44++prostate+cancer+cells+from+xenograft+human+tumors+are+enriched+in+tumorigenic+and+metastatic+progenitor+cells.">Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells.</a> Oncogene. 25, 1696-1708 (2006).</li><li>Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+resistance+of+tumorigenic+breast+cancer+cells+to+chemotherapy.">Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy.</a> J Natl. Cancer. Inst. 100, 672-679 (2008).</li><li>Galli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+tumorigenic,+stem-like+neural+precursors+from+human+glioblastoma.">Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.</a> Cancer Res. 64, 7011-7021 (2004).</li><li>Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+label-retaining+tumour-initiating+cells+in+human+glioblastoma.">Evidence for label-retaining tumour-initiating cells in human glioblastoma.</a> Brain. 134, 1331-1343 (2011).</li></ol>

No conflicts of interest declared.

To isolate neural stem and progenitor cells from normal adult and fetal brains1, 2, 3, 4 and also tumor stem-like cells from cancer tissues such as lung5, prostate6, breast7 and brain8, 9 the neurosphere assay has been frequently used as the method of choice. Using this simple and reproducible assay, one can generate an indefinite number of cells from resected tumor tissue that show similar characteristics as somatic stem cells; ex vivo multipotency, the ability to create new tumors upon implantation, and self-renewal. These cells could be used to study the basic cancer cell biology including cell-to-cell interactions, and differentiation, migration, invasion and cell death. In addition, isolated tumor stem-like cells provide an invaluable tool to study how tumors form, progress, and relapse and also to unravel the underlying deriving cellular mechanisms that eventually could provide insights to therapeutic options.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Type</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NeuroCult NSC Basal Medium (Human)</td>
			<td>Medium</td>
			<td>Stem Cell Technologies</td>
			<td>05750</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NeuroCult NSC Proliferation Supplements (Human)</td>
			<td>Medium supplement</td>
			<td>Stem Cell Technologies</td>
			<td>05753</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>%0.05 	trypsin-EDTA</td>
			<td>Reagent</td>
			<td>Gibco</td>
			<td>25300-062</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>*MEM</td>
			<td>Reagent</td>
			<td>Gibco</td>
			<td>41500-018</td>
			<td>HEM component</td>
		</tr>
		<tr>
			<td>*HEPES</td>
			<td>Reagent</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td>HEM component</td>
		</tr>
		<tr>
			<td>*Distilled water</td>
			<td>Reagent </td>
			<td>Gibco</td>
			<td>15230-147</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>**DNase I</td>
			<td>Reagent</td>
			<td>Roche</td>
			<td>104159</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>**Soybean trypsin inhibitor </td>
			<td>Reagent</td>
			<td>Sigma</td>
			<td>T6522</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen/Strep </td>
			<td>Reagent</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>No. 10 scalpel blade</td>
			<td>Surgical tool</td>
			<td>BD</td>
			<td>371610</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Culture ware</td>
			<td>BD Falcon</td>
			<td>353003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Small forceps</td>
			<td>Surgical tools</td>
			<td>Fine Science Tools</td>
			<td>11050-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell strainer </td>
			<td>Sieve</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T25 flask</td>
			<td>Culture ware</td>
			<td>Nalge Nunc international</td>
			<td>136196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T80 flask</td>
			<td>Culture ware</td>
			<td>Nalge Nunc international</td>
			<td>178905</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>Culture ware</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Culture ware</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Growth factor</td>
			<td>R&amp;D</td>
			<td>2028-EG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>b-FGF</td>
			<td>Growth factor</td>
			<td>R&amp;D</td>
			<td>3139-FB</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Growth factor</td>
			<td>Sigma</td>
			<td>H4784</td>
			<td>Reconstituted in PBS</td>
		</tr>
	</tbody>
</table>
* To make HEM, mix 1x10L packet of MEM and160ml of 1M HEPES and bring the volume to 8.75 L using distilled water. Set the final PH to 7.4 and store it at 4&deg;C.
** To prepare trypsin inhibitor solution, first make 10 ml of DNase I solution (100 mg DNase dissolved in 100 ml of HEM) and then add 0.14 g of trypsin inhibitor to DNase solution and finally make the volume up to 1 Liter using HEM. Keep aliquots of the final products in -20&deg;C freezer.

isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

Watch this Scientific Journal Video about Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay at JoVE.com

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

isolation-expansion-human-glioblastoma-multiforme-tumor-cells-using

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Neuroscience

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...