We thank the UW-Madison flow cytometry core (P30 CA014520 and 1S10RR025483-01), and members of the Moore lab and UW-Madison community for their input. We thank our funding sources: NIH T32 T32GM008688 (to C.S.M.), Diana Jacobs Kalman Fellowship from AFAR (to C.S.M.), Wisconsin Graduate Fellowship (to C.S.M.), DP2 NIH New Innovator Award (1DP2OD025783, to D.L.M.), Vallee Scholar Award (to D.L.M.), NIH 1R56NS130450 (to D.L.M and M.C.S.), R01 CA185747 (to M.C.S.), R01 CA205101 (to M.C.S.), R01 CA211082 (to M.C.S.), and the National Science Foundation Grant No. CBET-1642287 (to M.C.S.).

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R., Mizrak, D., Codega, P., Doetsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+niche+signals+from+the+choroid+plexus+regulate+adult+neural+stem+cells.">Age-dependent niche signals from the choroid plexus regulate adult neural stem cells.</a> Cell Stem Cell. 19 (5), 643-652 (2016).</li><li>Urban, N., Blomfield, I. M., Guillemot, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quiescence+of+adult+mammalian+neural+stem+cells:+A+highly+regulated+rest.">Quiescence of adult mammalian neural stem cells: A highly regulated rest.</a> Neuron. 104 (5), 834-848 (2019).</li><li>Urban, N., 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=Return+to+quiescence+of+mouse+neural+stem+cells+by+degradation+of+a+proactivation+protein.">Return to quiescence of mouse neural stem cells by degradation of a proactivation protein.</a> Science. 353 (6296), 292-295 (2016).</li><li>Kalamakis, G., 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=Quiescence+modulates+stem+cell+maintenance+and+regenerative+capacity+in+the+aging+brain.">Quiescence modulates stem cell maintenance and regenerative capacity in the aging brain.</a> Cell. 176 (6), 1407-1419 (2019).</li><li>Walsh, A. 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=Classification+of+T-cell+activation+via+autofluorescence+lifetime+imaging.">Classification of T-cell activation via autofluorescence lifetime imaging.</a> Nat Biomed Eng. 5 (1), 77-88 (2021).</li><li>Sagar, M. A. 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=Microglia+activation+visualization+via+fluorescence+lifetime+imaging+microscopy+of+intrinsically+fluorescent+metabolic+cofactors.">Microglia activation visualization via fluorescence lifetime imaging microscopy of intrinsically fluorescent metabolic cofactors.</a> Neurophotonics. 7 (3), 035003 (2020).</li><li>Knobloch, M., 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=Metabolic+control+of+adult+neural+stem+cell+activity+by+Fasn-dependent+lipogenesis.">Metabolic control of adult neural stem cell activity by Fasn-dependent lipogenesis.</a> Nature. 493 (7431), 226-230 (2013).</li><li>Knobloch, M., 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=A+fatty+acid+oxidation-dependent+metabolic+shift+regulates+adult+neural+stem+cell+activity.">A fatty acid oxidation-dependent metabolic shift regulates adult neural stem cell activity.</a> Cell Rep. 20 (9), 2144-2155 (2017).</li><li>Knobloch, M., 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=SPOT14-positive+neural+stem/progenitor+cells+in+the+hippocampus+respond+dynamically+to+neurogenic+regulators.">SPOT14-positive neural stem/progenitor cells in the hippocampus respond dynamically to neurogenic regulators.</a> Stem Cell Reports. 3 (5), 735-742 (2014).</li><li>Shin, 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=Single-cell+RNA-Seq+with+waterfall+reveals+molecular+cascades+underlying+adult+neurogenesis.">Single-cell RNA-Seq with waterfall reveals molecular cascades underlying adult neurogenesis.</a> Cell Stem Cell. 17 (3), 360-372 (2015).</li><li>Leeman, D. S., 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=Lysosome+activation+clears+aggregates+and+enhances+quiescent+neural+stem+cell+activation+during+aging.">Lysosome activation clears aggregates and enhances quiescent neural stem cell activation during aging.</a> Science. 359 (6381), 1277-1283 (2018).</li><li>Beckervordersandforth, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+metabolism-mediated+regulation+of+adult+neurogenesis.">Mitochondrial metabolism-mediated regulation of adult neurogenesis.</a> Brain Plast. 3 (1), 73-87 (2017).</li><li>Morrow, C. S., 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=Autofluorescence+is+a+biomarker+of+neural+stem+cell+activation+state.">Autofluorescence is a biomarker of neural stem cell activation state.</a> Cell Stem Cell. , (2024).</li><li>Kolenc, O. I., Quinn, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+cell+metabolism+through+autofluorescence+imaging+of+NAD(P)H+and+FAD.">Evaluating cell metabolism through autofluorescence imaging of NAD(P)H and FAD.</a> Antioxid Redox Signal. 30 (6), 875-889 (2019).</li><li>Mira, H., 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=Signaling+through+BMPR-IA+regulates+quiescence+and+long-term+activity+of+neural+stem+cells+in+the+adult+hippocampus.">Signaling through BMPR-IA regulates quiescence and long-term activity of neural stem cells in the adult hippocampus.</a> Cell Stem Cell. 7 (1), 78-89 (2010).</li><li>Morrow, C. S., 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=Vimentin+coordinates+protein+turnover+at+the+aggresome+during+neural+stem+cell+quiescence+exit.">Vimentin coordinates protein turnover at the aggresome during neural stem cell quiescence exit.</a> Cell Stem Cell. 26 (4), 558-568 (2020).</li><li>Martynoga, 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=Epigenomic+enhancer+annotation+reveals+a+key+role+for+NFIX+in+neural+stem+cell+quiescence.">Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescence.</a> Genes Dev. 27 (16), 1769-1786 (2013).</li><li>Bin Imtiaz, M. K., Jessberger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+adult+mouse+hippocampal+neural+stem+cells+for+fluorescence+loss+in+photobleaching+assays.">Isolation of adult mouse hippocampal neural stem cells for fluorescence loss in photobleaching assays.</a> STAR Protoc. 2 (3), 100695 (2021).</li><li>Guo, W., Patzlaff, N. E., Jobe, E. M., Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+multipotent+neural+stem+or+progenitor+cells+from+both+the+dentate+gyrus+and+subventricular+zone+of+a+single+adult+mouse.">Isolation of multipotent neural stem or progenitor cells from both the dentate gyrus and subventricular zone of a single adult mouse.</a> Nat Protoc. 7 (11), 2005-2012 (2012).</li><li>Lakowicz, J. R., Szmacinski, H., Nowaczyk, K., Johnson, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+lifetime+imaging+of+free+and+protein-bound+NADH.">Fluorescence lifetime imaging of free and protein-bound NADH.</a> Proc Natl Acad Sci U S A. 89 (4), 1271-1275 (1992).</li><li>Blacker, T. S., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+mitochondrial+redox+state+using+NADH+and+NADPH+autofluorescence.">Investigating mitochondrial redox state using NADH and NADPH autofluorescence.</a> Free Radic Biol Med. 100, 53-65 (2016).</li><li>Datta, R., Heaster, T. M., Sharick, J. T., Gillette, A. A., Skala, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+lifetime+imaging+microscopy:+fundamentals+and+advances+in+instrumentation,+analysis,+and+applications.">Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications.</a> J Biomed Opt. 25 (7), 1-43 (2020).</li><li>. ImageJ Available from: <a target="_blank" href="https://imagej.nih.gov/ij/">https://imagej.nih.gov/ij/</a> (2021)</li><li>. Becker &amp; Hichl Handbooks Available from: <a target="_blank" href="https://imagej.nih.gov/ij/">https://imagej.nih.gov/ij/</a> (2024)</li><li>. GitHub Materials Available from: <a target="_blank" href="https://github.com/chrismorrow5/Neural-Stem-Cell-Autofluorescence-Analysis/">https://github.com/chrismorrow5/Neural-Stem-Cell-Autofluorescence-Analysis/</a> (2024)</li><li>Codega, P., 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=Prospective+identification+and+purification+of+quiescent+adult+neural+stem+cells+from+their+in+vivo+niche.">Prospective identification and purification of quiescent adult neural stem cells from their in vivo niche.</a> Neuron. 82 (3), 545-559 (2014).</li><li>Llorens-Bobadilla, E., 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=Single-cell+transcriptomics+reveals+a+population+of+dormant+neural+stem+cells+that+become+activated+upon+brain+injury.">Single-cell transcriptomics reveals a population of dormant neural stem cells that become activated upon brain injury.</a> Cell Stem Cell. 17 (3), 329-340 (2015).</li></ol>

The authors declare no competing interests.

This protocol describes a live-cell, label-free, non-destructive, single-cell resolution technique that allows for the classification of NSC cell-state in vitro through imaging of autofluorescent signals in NSCs. This approach detects metabolic shifts that occur during NSC quiescence exit, which influence the optical properties of metabolic cofactors, such as NAD(P)H, and offers many advantages over existing technologies to study NSC quiescence. For example, many conventional techniques for studying qNSCs and aN...

NSCs create newborn neurons throughout life in many organisms in a process referred to as adult neurogenesis1,2. To produce newborn neurons, a qNSC first must activate, entering the cell cycle to&#160;expand the population&#160;and produce neural progenitors3,4,5,6. Although there is much known about NSC quiescence, our ability to fully identify the drivers and regulators of NSC quiescence is constrained by technical limitations that exist to isolate and identify qNSCs and their transition to activation. Autofluorescence imaging has previously been successful in studying changes in cell state in many different cell types, such as microglia and T-cells, by resolving metabolic remodeling, which influences the optical properties of autofluorescent metabolic cofactors such as nicotinamide adenine dinucleotide phosphate (NAD(P)H) and flavin adenine dinucleotide (FAD)7,8. NSCs substantially remodel their metabolic networks as they undergo quiescence exit9,10,11,12,13,14. Thus, to take advantage of these differences, NSC autofluorescence was recently used to identify and enrich the NSC activation state by detecting shifts in autofluorescence attributed to the metabolic remodeling that occurs as NSCs exit quiescence15. Imaging autofluorescence provides several technical advantages: i) it does not require the addition of exogenous labels, which can impact cell behavior; ii) it can provide high-resolution single-cell data on the NSC activation state; and iii) it does not require the destruction of the cell7,16. This protocol outlines three strategies for harnessing NSC autofluorescence to study NSC quiescent and activated cell states15.
Recently, NSCs isolated from 6-week-old male mice from the subgranular zone of the hippocampus, cultured and reversibly put into quiescence in vitro10,13,17,18,19,20,21, were found to exhibit increased levels of punctate autofluorescence (PAF) that excite between 400-600 nm and emit between 500-700 nm. This signal was specific to qNSCs compared to activated, cycling NSCs15. The ability to visually separate these two populations without the use of additional antibody markers or reporters is useful for many experimental questions on the nature of qNSCs and quiescence exits. Thus, first, this protocol describes strategies to image the PAF in qNSCs using a confocal microscope, which can be used to identify&#160;NSC activation state. Second, this protocol describes strategies to detect the PAF using fluorescence-activated cell sorting (FACS) and further describes how to sort based on this signal to enrich qNSCs or aNSCs. These strategies provide one measure that can be used to cluster and separate NSCs based on cell state.
To develop a higher resolution method of separating NSCs not only in distinct states but also as they transition through quiescence exit towards full activation, fluorescence lifetime imaging (FLIM) was performed using a multiphoton microscope to image NAD(P)H (termed Channel 1) autofluorescence and green autofluorescence (termed Channel 2; which detects both FAD autofluorescence and PAF in qNSCs) lifetimes together with their intensity. This approach capitalizes on the fact that the optical properties of molecules in the cell are dependent on their physical properties16,22. For example, NAD(P) (NAD and NADP are optically indistinguishable, and thus NAD(P) is used to refer to both species) is not autofluorescent in the oxidized state but is autofluorescent in its reduced state (NAD(P)H)23. Further, additional physical properties of autofluorescent molecules, such as their binding status to enzymes, can be extrapolated by performing fluorescence lifetime imaging7,22,24. For example, NAD(P)H has a shorter fluorescence lifetime when not bound to an enzyme22. As autofluorescent molecules such as NAD(P)H, which is involved in hundreds of metabolic reactions, are used differently by cells progressing through different states or cell behaviors, these shifts can be detected and quantified using a multiphoton microscope detecting autofluorescence lifetime23. Together with the abundance, or intensity, of the autofluorescence, these measures provide multi-dimensional information to separate NSCs into one cell state or the other and through the dynamic transitions between states. Third, this protocol describes performing, analyzing, and interpreting FLIM and intensity measures of Channel 1 (NAD(P)H) and Channel 2 (PAF) signals using a multiphoton microscope. In summary, this protocol describes a live-cell, label-free toolkit for studying NSC quiescence that provides high-resolution single-cell data on&#160;NSC state.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>40x Water objective lens</td>
			<td>Nikon</td>
			<td>MRD77410</td>
			<td>Objective lens used in multiphoton microscope in Part 3</td>
		</tr>
		<tr>
			<td>8 well cuvette</td>
			<td>Ibidi</td>
			<td>80826-90</td>
			<td>For imaging aNSCs/qNSCs</td>
		</tr>
		<tr>
			<td>Analog power meter</td>
			<td>Thorlabs</td>
			<td>PM100A</td>
			<td>Used in multiphoton microscope in Part 3</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X) (PSF)</td>
			<td>Thermo Fisher</td>
			<td>15240062</td>
			<td>Antibiotic for NSC media</td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td>Nutrient supplement for NSC media</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>Fisher Scientific</td>
			<td>5020BP010</td>
			<td>Factor for inducing quiescence</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A4919-25G</td>
			<td>For making BMP4</td>
		</tr>
		<tr>
			<td>Chameleon ultrafast laser</td>
			<td>Coherent</td>
			<td>N/A</td>
			<td>Laser used in multiphoton microscope in Part 3</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>C2</td>
			<td>Microscope used for Part 1</td>
		</tr>
		<tr>
			<td>DMEM/F-12 (without GlutaMAX)</td>
			<td>Invitrogen</td>
			<td>11320033</td>
			<td>Base media for NSCs</td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma</td>
			<td>D5025-15KU</td>
			<td>Added to trypsin inhibitor</td>
		</tr>
		<tr>
			<td>EdU assay kit</td>
			<td>Invitrogen</td>
			<td>C10337</td>
			<td>Proliferation assay for cell culture</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>PeproTech</td>
			<td>AF-100-15-500UG</td>
			<td>Growth factor for NSC media</td>
		</tr>
		<tr>
			<td>FGF</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td>Growth factor for NSC media</td>
		</tr>
		<tr>
			<td>Fluorescent activated cell sorter</td>
			<td>BD</td>
			<td>FACSAria</td>
			<td>Fluorescent Activated Cell Sorter used for Part 2</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149-50KU</td>
			<td>Additive for NSC media</td>
		</tr>
		<tr>
			<td>L-15</td>
			<td>Invitrogen</td>
			<td>21083027</td>
			<td>For preparing trypsin inhibitor solution</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020-1MG</td>
			<td>For coating glassware</td>
		</tr>
		<tr>
			<td>Nikon TiE inverted microscope</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td>Microscope frame Used for Part 3</td>
		</tr>
		<tr>
			<td>PLO</td>
			<td>Sigma</td>
			<td>P3655-100MG</td>
			<td>For coating glassware</td>
		</tr>
		<tr>
			<td>SPC-150 Single photon counting electronics</td>
			<td>Becker and Hickl</td>
			<td>N/A</td>
			<td>Used in multiphoton microscope in Part 3</td>
		</tr>
		<tr>
			<td>Trypsin (for trypsinizing pellets of aNSCs that were growing as spheres or monolayers)</td>
			<td>Gibco</td>
			<td>15090046</td>
			<td>For trypsinizing neurospheres or adherent aNSCs</td>
		</tr>
		<tr>
			<td>Trypsin (for trypsinizing qNSCs)</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td>For trypsinizing adherent qNSCs</td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T6522-100MG</td>
			<td>For inhibiting trypsinization of aNSCs</td>
		</tr>
		<tr>
			<td>Urea crystals</td>
			<td>Sigma</td>
			<td>U5128-5G</td>
			<td>Used to collect an IRF</td>
		</tr>
		<tr>
			<td>Versene</td>
			<td>Thermo Fisher</td>
			<td>15040066</td>
			<td>For preparing trypsin</td>
		</tr>
	</tbody>
</table>

classification of neural stem cell activation state in vitro using autofluorescence

Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily quiescent, a reversible cell state where they have exited the cell cycle (G0) yet remain responsive to the environment. In the first step of adult neurogenesis, quiescent NSCs (qNSCs) receive a signal and activate, exiting quiescence and re-entering the cell cycle. Thus, understanding the regulators of NSC quiescence and quiescence exit is critical for future strategies targeting adult neurogenesis. However, our understanding of NSC quiescence is limited by technical constraints in identifying quiescent NSCs (qNSCs) and activated NSCs (aNSCs). This protocol describes a new approach to identify and enrich qNSCs and aNSCs generated in in vitro cultures by imaging NSC autofluorescence. First, this protocol describes how to use a confocal microscope to identify autofluorescent markers of qNSCs and aNSCs to classify NSC activation state using autofluorescence intensity. Second, this protocol describes how to use a fluorescent activated cell sorter (FACS) to classify NSC activation state and enrich samples for qNSCs or aNSCs using autofluorescence intensity. Third, this protocol describes how to use a multiphoton microscope to perform fluorescence lifetime imaging (FLIM) at single-cell resolution, classify NSC activation state, and track the dynamics of quiescent exit using both autofluorescence intensities&#160;and fluorescence lifetimes. Thus, this protocol provides a live-cell, label-free, single-cell resolution toolkit for studying NSC quiescence and quiescence exit.

Confocal autofluorescence imaging to separate NSC cell state (Figure 1) 
To use confocal microscopy to resolve the NSC activation state, qNSCs, and aNSCs were generated in vitro using either an activation medium or quiescence medium, as described previously10,13,17,18. To detect PAF in NSCs, live qNSCs and aNSCs were imaged using the ...

Read this Scientific Article Protocol about Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence at JoVE.com

Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence

This protocol describes strategies to identify and enrich for cell-state in primary adult mouse neural stem cell cultures by autofluorescence imaging using i) a confocal microscope, ii) a fluorescent activated cell sorter to perform intensity imaging, or iii) a multiphoton microscope to perform fluorescence lifetime imaging.

Classification of Neural Stem Cell Activation State In Vitro Using Autofluorescence

Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily ...

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506 Views.  University of Wisconsin-Madison. This protocol describes strategies to identify and enrich for cell-state in primary adult mouse neural stem cell cultures by autofluorescence imaging using i) a confocal microscope, ii) a fluorescent activated cell sorter to perform intensity imaging, or iii) a multiphoton microscope to perform fluorescence lifetime imaging.

Video: Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence

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 ...

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...