Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence

Summary

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.

Abstract

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 and fluorescence lifetimes. Thus, this protocol provides a live-cell, label-free, single-cell resolution toolkit for studying NSC quiescence and quiescence exit.

Introduction

NSCs create newborn neurons throughout life in many organisms in a process referred to as adult neurogenesis^1,2. To produce newborn neurons, a qNSC first must activate, entering the cell cycle to expand the population and produce neural progenitors^3,4,5,6. 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 exit^{9,10,11,12,13,14}. 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 cell^7,16. 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 vitro^{10,13,17,18,19,20,21}, 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 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 properties^16,22. 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 imaging^7,22,24. 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 NSC state.

Protocol

All procedures in this protocol are approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Wisconsin-Madison. 1. Using a confocal microscope to image PAF in qNSCs and aNSCs to identify NSC cell-state Generate qNSCs and aNSCs in vitro from primary NSC cultures. Culture NSCs purified from adult mouse brains in a proliferative medium for expansion (Table 1)18,<su…

Representative Results

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 …

Discussion

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…

Materials

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