Name: Classification of Neural Stem Cell Activation State In Vitro Using Autofluorescence (Video) | JoVE Journal
Uploaded: 2024-04-12T12:30:00.000+00:00
Duration: 6 min 56 s
Description: Learn to classify neural stem cell activation states in vitro using autofluorescence imaging and FACS. This label-free method enables live cell analysis of quiescent and activated NSCs in adult mouse cultures.

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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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<li>Morrow, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autofluorescence+is+a+biomarker+of+neural+stem+cell+activation+state%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">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/pubmed?term=((Evaluating+cell+metabolism+through+autofluorescence+imaging+of+NAD%28P%29H+and+FAD%5BTitle%5D)+AND+%22Antioxid+Redox+Signal%22%5BJournal%5D)">Evaluating cell metabolism through autofluorescence imaging of NAD(P)H and FAD.</a> Antioxid Redox Signal. 30 (6), 875-889 (2019).</li>
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<li>Morrow, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vimentin+coordinates+protein+turnover+at+the+aggresome+during+neural+stem+cell+quiescence+exit%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Vimentin coordinates protein turnover at the aggresome during neural stem cell quiescence exit.</a> Cell Stem Cell. 26 (4), 558-568 (2020).</li>
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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 aNSCs, such as labeling with cell cycle markers like EdU, require the fixation of samples. Methods that currently exist that are capable of studying live NSCs are further limited by requiring the introduction of exogenous labels, typically by generating fluorescent protein-encoding transgenes. These tools are limited both in the resources required to generate them and many technical caveats. For example, the intermediate filament proteins nestin and Glial Fibrillary Acidic Protein (GFAP) are commonly used as markers of qNSCs and aNSCs12,13,18,28,29. However, nestin and GFAP are known to be differentially expressed between qNSCs and aNSCs12,13,18,28,29. Further, autofluorescence imaging provides high-resolution single-cell data, which can unravel single-cell heterogeneity that is lost or complicated to interpret in experiments that culminate in a bulk analysis of a population of cells.
However, autofluorescence imaging also has several limitations. Many of the autofluorescent signals become lost upon cell fixation. Thus, autofluorescence imaging is largely limited to the study of living cells. Autofluorescence imaging also relies upon a lack of exogenously introduced fluorophores, which can bleed into autofluorescent channels. Although many fluorophores can be compatible with autofluorescence imaging in specific situations, pairing autofluorescence imaging with many existing tools is not always possible. Live-cell imaging can also induce phototoxicity. One iteration of imaging using this protocol does not induce sufficient phototoxicity to reduce NSC viability. However, repeated imaging of cells in a time course at more frequent intervals many times per day may generate significant phototoxicity and, thus, is not a viable strategy for studying NSC quiescence. Lastly, although autofluorescence imaging can be used to study NSCs from 6-week-old male mice from either the hippocampus or the lateral ventricles, it is unclear how broadly this technique may be used to study NSCs from other sources, ages, or other stem cell types.
The protocol described here also assumes accurate production of qNSCs and aNSCs in vitro using previously established protocols10,13,17,18,19. If reproducing the results of this protocol is challenging, ensure that qNSCs and aNSCs are being properly generated by probing for the presence or absence of various markers of qNSCs and aNSCs, as previously described10,13,17,18,19. Taken together, autofluorescence imaging provides a novel technical approach to identifying qNSCs and aNSCs and studying NSC quiescence exits.

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><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

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 same exposure on a confocal microscope (Ex: 405 nm, Em: 580-620 nm). qNSCs exhibited a higher number of PAF compared to aNSCs (Figure 1A,B). This finding illustrates how autofluorescence properties can be used as markers to identify the cell state of qNSCs and aNSCs.
FACS enrichment of NSC cell state using autofluorescence (Figure 2) 
To enrich for cell cycle state using FACS, qNSCs and aNSCs were generated in vitro10,13,17,18,19 as described in this protocol and pre-labeled with EdU for 1 h prior to trypsinization and analysis in the flow cytometer to label cells progressing through S-phase. qNSCs and aNSCs were then analyzed by flow cytometry either separately or mixed at a ratio of 1 qNSC:1 aNSC (Figure 2). Gates were drawn to enrich for aNSCs or qNSCs from the mixed population, and cells were then sorted based on these gates. After FACS, cells in each sample were plated onto PLO-, laminin-coated glassware and allowed to adhere to the dish for 3 h before being fixed, stained, and analyzed for %EdU+ cells. Expectedly, cells sorted from the high autofluorescence gate were less EdU+ than the Mix sample, and samples sorted through the low autofluorescence gate were more proliferative than the Mix sample (Figure 2C). This finding confirms the capacity to enrich for NSC activation state from a heterogeneous mixture of qNSCs and aNSCs using FACS.
Multiphoton fluorescence lifetime imaging to classify NSC cell state (Figure 3) 
qNSCs and aNSCs were generated in vitro and then imaged using a multiphoton microscope to perform FLIM on Channel 1 autofluorescence (Ex: 750 nm (2P), Em: 360-520 nm) and Channel 2 (Ex: 890 nm (2P), Em: 450-650 nm) (Figure 3A, Table 2). qNSCs and aNSCs exhibited autofluorescent profiles that were largely significantly different. For example, qNSCs had a higher Channel 1 fluorescence mean lifetime τm, but a lower α1 compared with aNSCs. To evaluate the capacity of NSC FLIM autofluorescence data to predict NSC activation state, a logistic regression model was generated with Channel 1 intensity, α1, τ1, τ2 and Channel 2 intensity, α1, τ1, τ2. A receiver operator curve illustrates that these data are sufficient to create a near-perfect model (Area under the curve = 0.963), accurately predicting the NSC activation state. Together, these data illustrate the capacity of FLIM and autofluorescence to be used to classify the NSC cell state.
<img alt="Neural stem cell analysis; microscopy, excitation, emission data; PAF visualization, graph results." class="xfigimg" src="/files/ftp_upload/63110/63110fig01.jpg" data-altupdatedat="1788282455000" data-alt="Figure 1"> 
Figure 1: Confocal imaging resolves autofluorescent biomarkers of NSC activation state. (A-B) qNSCs (purple) and aNSCs (black) were imaged using the same exposure on a confocal microscope (red; Ex. 405 nm, Em 580-620 nm) and analyzed for the number of PAF (N = 3, Mann-Whitney test, mean ± SD). White dashed lines denote the edge of the cell, and blue dashed lines denote nuclei. (C) Autofluorescence in the same qNSC was imaged using various excitation and emission conditions, as indicated in the figure, with identical laser power and gain. Scale bars: 10 µm. **** p &lt; 0.0001. <a href="https://www.jove.com/files/ftp_upload/63110/63110fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Autofluorescence histograms for neural stem cells; analysis of proliferative states; three-part graph." class="xfigimg" src="/files/ftp_upload/63110/63110fig02.jpg" data-altupdatedat="1788282455000" data-alt="Figure 2"> 
Figure 2: FACS can enrich for NSC activation state. (A-C) qNSCs, aNSCs or a qNSCs: aNSCs mix (1:1) were treated with EdU for 1 h, trypsinized, and then analyzed by flow cytometry (Ex: 405 nm, Em: 580-620 nm). Mix cells (1:1) were then sorted by FACS for cells that had either low or high autofluorescence, plated, and analyzed for proliferation by measuring the percentage of cells that were EdU+. Arbitrary units is abbreviated as "A.U." (N = 4, two-way ANOVA with post hoc Tukey's test, mean ± SD). **** p &lt; 0.0001. <a href="https://www.jove.com/files/ftp_upload/63110/63110fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence lifetime imaging graph; Violin plots; ROC curve; analyze aNSC and qNSC data." class="xfigimg" src="/files/ftp_upload/63110/63110fig03.jpg" data-altupdatedat="1788282455000" data-alt="Figure 3"> 
Figure 3: Multiphoton fluorescence lifetime imaging reveals biomarkers of NSC activation state. (A) Schematic depicting curve fitting analysis of acquired FLIM data. (B) Channel 1 and Channel 2 FLIM measurements, including the intensity, mean lifetime (Tm), and fractional contribution (α1) values for qNSC (purple) and aNSC (black) data (n = 501 cells, two-sided logistic regression, generalized linear model). (C) Receiver operator curve demonstrating a logistic regression model generated using Channel 1 intensity, α1, τ1, τ2, and Channel 2 intensity, α1, τ1, τ2 to classify NSCs as aNSCs or qNSCs. *** p &lt; 0.001. <a href="https://www.jove.com/files/ftp_upload/63110/63110fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Media and solutions. Recipes for all solutions used in this protocol. <a href="https://www.jove.com/files/ftp_upload/63110/Table%201%20-%20Solution%20Recipes.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Example data. Representative qNSC and aNSC FLIM data for Channel 1 and Channel 2 autofluorescence. <a href="https://www.jove.com/files/ftp_upload/63110/Table%202%20-%20Example%20Data.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1: manual_segmentation.cpproj <a href="https://www.jove.com/files/ftp_upload/63110/Supplementary%20File%201.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 2: R_ASCtoTIFF.rmd <a href="https://www.jove.com/files/ftp_upload/63110/Supplementary%20File%202.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 3: test_key <a href="https://www.jove.com/files/ftp_upload/63110/Supplementary%20File%203.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 4: Integrate decay matrices and cytoplasmic masks.rmd <a href="https://www.jove.com/files/ftp_upload/63110/Supplementary%20File%204.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 5: autofluorescence FLIM data.rmd <a href="https://www.jove.com/files/ftp_upload/63110/Supplementary%20File%205.zip" target="_blank">Please click here to download this File.</a>

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.

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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Research

JoVE Journal

Neuroscience

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

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We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

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Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

Learn to classify neural stem cell activation states in vitro using autofluorescence imaging and FACS. This label-free method enables live cell analysis of quiescent and activated NSCs in adult mouse cultures.

Classification of Neural Stem Cell Activation State In Vitro Using Autofluorescence

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