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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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. 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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

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&#160;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&#160;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&#160;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 &#964;m, but a lower &#945;1 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, &#945;1, &#964;1, &#964;2 and Channel 2 intensity, &#945;1, &#964;1, &#964;2. 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="Figure 1" class="xfigimg" src="/files/ftp_upload/63110/63110fig01.jpg" /> 
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 &#177; 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 &#181;m. **** p &#60; 0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63110/63110fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63110/63110fig02.jpg" /> 
Figure 2: FACS can enrich for&#160;NSC activation state. (A-C) qNSCs, aNSCs or a qNSCs: aNSCs mix (1:1) were treated with EdU for 1 h, trypsinized,&#160;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,&#160;and analyzed for proliferation by measuring the percentage of cells that were EdU+. Arbitrary units is abbreviated as &#34;A.U.&#34; (N = 4, two-way ANOVA with post hoc Tukey&#39;s test, mean &#177; SD). **** p &#60; 0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63110/63110fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63110/63110fig03.jpg" /> 
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 (&#945;1) 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, &#945;1, &#964;1, &#964;2, and Channel 2 intensity, &#945;1, &#964;1, &#964;2 to classify NSCs as aNSCs or qNSCs. *** p &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/files/ftp_upload/63110/Table 1 - Solution Recipes.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.remotexs.ntu.edu.sg/files/ftp_upload/63110/Table 2 - Example Data.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1: manual_segmentation.cpproj <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63110/Supplementary File 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 2: R_ASCtoTIFF.rmd <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63110/Supplementary File 2.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 3: test_key <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63110/Supplementary File 3.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.remotexs.ntu.edu.sg/files/ftp_upload/63110/Supplementary File 4.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 5: autofluorescence FLIM data.rmd <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63110/Supplementary File 5.zip" target="_blank">Please click here to download this File.</a>

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 ...

classification-neural-stem-cell-activation-state-vitro-using

Nanyang Technological University

Research

JoVE Journal

Neuroscience

540 Views.  University of Wisconsin-Madison. Dit protocol biedt een nieuwe benadering om de activeringstoestand van neurale stamcellen te classificeren met een cohort van technische voordelen. Met behulp van dit protocol kunnen we een analyse van de resolutie van neurale stamcellen zonder live cellabel van neurale stamcellen bereiken. Dit protocol kan worden gebruikt om licht te werpen op mechanismen die neurogenese bij volwassenen reguleren.Autofluorescentie is veel zwakker dan veel fluoroforen ...

Video: Classificatie van neurale stamcelactiveringstoestand in vitro met behulp van autofluorescentie

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. 
The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. 
The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

Cancer Research

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

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.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Biology

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 ...

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS). 
The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine1 and TH is also present in a subset of adult pelvic ganglia neurons2-4 . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc1. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP. 
To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine2-6. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (&gt;70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.

An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

An optimized procedure to purify neural crest-derived neuronal progenitors from fetal mouse tissues is described. This method takes advantage of expression from fluorescent reporter alleles to isolate discrete populations by fluorescence-activated cell sorting (FACS). The technique can be applied to isolate neuronal subpopulations throughout development or from adult tissues.

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the ...

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

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.

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

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. ...

Chemogenetic Regulation in Reprogrammed Stem Cell-derived Precursor Cells in Treating Neurodegenerative Diseases

The integration of designer receptors exclusively activated by designer drugs (DREADD) with stem cell-based therapies presents an advanced strategy for precise neuronal modulation. Here, we utilized CRISPR-engineered human reprogrammed stem cells expressing excitatory (hM3Dq) or inhibitory (hM4Di) DREADD receptors to evaluate the functional integration and modulation of transplanted dopaminergic precursors in a murine model of Parkinson&#39;s disease (PD). Key steps included generating non-fusion DREADD-expressing stem cell lines, differentiating them into midbrain dopaminergic precursors, and transplanting these cells into the striatum of 6-hydroxydopamine (6-OHDA)-lesioned mice. We conducted behavioral assessments and electrophysiological recordings to analyze the effects of the transplanted cells. Behavioral tests, such as the cylinder test, demonstrated significant modulation of motor function following clozapine-N-oxide (CNO) administration. Specifically, activation of hM4Di reduced contralateral forelimb movement, whereas activation of hM3Dq was associated with enhanced motor behavior. Electrophysiological recordings revealed distinct synaptic responses. hM4Di activation increased interevent intervals and decreased peak amplitudes of spontaneous excitatory postsynaptic currents (sEPSCs), whereas hM3Dq activation decreased interevent intervals and increased peak amplitudes, reflecting enhanced excitatory signaling. In summary, the integration of behavioral and electrophysiological assessments validates the precise functional incorporation of engineered chemically reprogrammed stem cells into host neural circuits.

Here, we describe a protocol for engineering chemically reprogrammed stem cells to achieve precise neuronal modulation by differentiating these cells into dopaminergic precursor cells, transplanting them into mouse models of Parkinson's disease, and evaluating behavioral and electrophysiological outcomes to confirm the successful integration and functional effectiveness of the transplanted cells.

The integration of designer receptors exclusively activated by designer drugs (DREADD) with stem cell-based therapies presents an advanced strategy for ...

Immunofluorescence Microscopy of Neural Cells Differentiated from Peripheral Blood-Derived Cells 

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/61634/gm-free-generation-of-blood-derived-neuronal-cells'>Source: Becker-Koji&#263;, Z. A., et. al., GM-Free Generation of Blood-Derived Neuronal Cells. J. Vis. Exp. (2021)</a>The video demonstrates a protocol for preparing neural cells derived from reprogrammed peripheral blood cells for fluorescence microscopy, showcasing distinct cell types and developmental stages through immunofluorescence imaging.

Source: Becker-Koji&#263;, Z. A., et. al., GM-Free Generation of Blood-Derived Neuronal Cells. J. Vis. Exp. (2021)The video demonstrates a protocol for ...

Classification of Neural Stem Cell Activation State In Vitro Using Autofluorescence

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