The authors wish to thank Dr. Diana Carlone (Boston Children&#39;s Hospital, Harvard Medical School) and Dr. Matthew Lynes (Joslin Diabetes Center; Maine Medical Center Research Institute) for guidance utilizing mTert-rtTA animals.

The authors have nothing to disclose.

A method for the creation, utilization, and analysis of a triple transgenic lineage tracing mouse line marking stem cells within the adult mouse brain in vivo is described. When combined with immunostaining or brain clearing, the identification and characterization of traced fluorescent cells across the brain can be accomplished. This technique offers the ability to understand the plastic/regenerative/remodeling potential of labeled stem cells as they activate, migrate, differentiate, or proliferate. While previ...

Value of a lineage tracing mouse line 
Analysis of stem cells in vivo can be difficult since many assays that examine such cells only focus on characterizing these cells at the time of death of the animal, which represents a terminal snapshot in time. To better understand the processes of proliferation, differentiation, and migration of progenitor, intermediate/transition cell types, and mature cells over time, a longitudinal analysis approach is required. This can be achieved with lineage tracing studies whereby stem/progenitor cells are marked indelibly and can be followed for any length of time afterwards1.
In the adult mammalian brain, the process of neurogenesis by which adult-born neurons are created from stem and progenitor cells was first analyzed via label retention with thymidine-H3&#160;2,3,4 or 5-bromo-2&#39;-deoxyuridine (BrdU)5,6,7,8. In these studies, proliferative cells were marked with the thymine analog, which was incorporated into the cells&#39; DNA during replication and cell division/proliferation. The marked cell, as well as their progeny, therefore contained this analog, which were then identified post-mortem. However, while thymidine-H3 and BrdU allowed for the marking of proliferative cells and their progeny in brain regions where stem cells were poorly understood, these studies were hindered by the downsides of these tools. Thymidine-H3 induces cell-cycle arrest, apoptosis, and dose-dependent DNA synthesis inhibition9, while BrdU marks cells at varying levels depending on the route of administration10 and is taken up by cells during repair or apoptosis, as well as during cell division11. More efficient labeling methods have been utilized recently, such as labeling with 5-ethynyl-2&#39;-deoxyuridine (EdU), but many of the same issues as BrdU still remain12. These approaches are also limiting in that they mark any proliferative cell, not just stem cells, and thus interpretation of results can be confounded. In the neurogenic lineage, all stem and progenitor cells retain mitotic capacity, and only terminal/mature neurons are not proliferative. For astrocytes and microglia, but not oligodendrocytes, proliferation is maintained indefinitely13,14,15.
Transgenic mice used to lineage trace only cells that express a protein of interest, such as one confirmed to identify adult stem cells as we use here, have therefore become more common when investigating stem cells and their progeny. While difficult to generate through mouse transgenic approaches, lineage-tracing mouse lines allow for the tracing of specifically marked cells in the brain, and do not rely on proliferation alone. In the Tet-On transgenic mouse system, administration of tetracycline or doxycycline (a tetracycline derivative) induces Cre-recombinase expression in cells that have been engineered with a reverse tetracycline transactivator (rtTA), which is transcribed by a promoter of interest. The Tet-inducible Cre-driven recombination will then activate the expression of an indelible fluorescent or luminescent protein in the cells of interest, depending on the Rosa-reporter mouse employed. These indelibly marked cells continue to express this reporter after division, differentiation, or migration, allowing the tracking of these cells and their progeny over time or after different interventions16. Advantages of transgenic lineage-tracing approaches include: 1) specificity of tracing to a particular cell lineage or progenitor/stem cell marked by the rtTA, 2) indelible expression of the fluorescent or luminescent protein despite cell turnover or differentiation, 3) low toxicity, 4) conditional activation during any point in the animal&#39;s life cycle, and 5) ease of use with common assays, including immunostaining/immunofluorescence16.
Other methods of temporally inducible reporter tools in mice include the use of Cre-ERT2 mice, which can be paired with ROSA-GFP, ROSA-mTmG, or other fluorescent reporter transgenes. In these animals, a cell-specific regulatory element, such as a promoter or enhancer region of interest, drives the production of Cre recombinase, which can only be activated via the administration of tamoxifen. While Cre-ERT2 mouse lines allow for the induction of Cre in specific cell lines, there is a wealth of knowledge detailing the effects of tamoxifen on adult neurogenesis17,18. Additionally, there exists many ROSA-driven fluorescent reporter genes that can be utilized instead of GFP or mTmG, including YFP or CFP, which would allow for alternate fluorescent labeling in other fluorescent wavelengths. These fluorescent reporter genes can be used with Tet-On or Cre-ERT2 systems.
Telomerase reverse transcriptase (TERT) is the rate-limiting component of the holoenzyme telomerase, which works to extend telomeres after they are shortened during cell division19,20. TERT has been identified as a marker of adult tissue stem cells in the intestine21,22, bone marrow21, liver23, adipose24, endometrium25,26, and bone1. Whether TERT expression in these adult stem cells is solely for telomerase-extending activity or to perform non-canonical TERT roles27 is still unknown. TERT-expressing quiescent adult stem cells (qASCs) have been identified and traced throughout the body with transgenic lineage tracing mice to study the multipotency and self-renewal ability of these stem cells, as well as their potential to activate, proliferate, differentiate, and migrate1,22,23,28. The creation of the Tert-rtTA transgene has been performed previously1. In this paper, we will describe the use of a lineage tracing TERT mouse line to study TERT + qASCs which we have identified as a novel ASC population in the adult mouse brain.
Generation of a transgenic lineage tracing mouse line 
To generate a lineage-tracing mouse line using the Tet-On system, three transgenes must be combined within a single animal through mouse mating. The first is a rtTA, expressed under the control of the promoter of the gene of interest (ours is TERT-rtTA). In a cell that expresses this gene of interest, the rtTA will therefore be expressed. The second is an oTet-Cre gene, which contains a tetracycline response element (TRE) that will allow for the transcription of Cre recombinase in the presence of both a rtTA fusion transcript and tetracycline or doxycycline. Tetracycline or doxycycline can be administered to an animal via drinking water or chow29. Finally, there must be a gene that will be activated by Cre recombinase cleavage. In this manuscript, the labeling gene we will describe is the Rosa26-mTmG gene, which until Cre recombination will ubiquitously transcribe mTomato (membrane red fluorescence in all cells). However, if a cell expresses the rtTA transcript and contains tetracycline or doxycycline, Cre recombination of a set of lox P sites between the mTomato and mGFP sites will change the R26 site and cause the cell to produce membrane EGFP (mGFP, or membrane green fluorescence) instead of membrane tomato. The indelible nature of this mGFP signal will allow for in vivo labeling and tracing of cells as they proliferate, migrate, and differentiate. Following the trace, cells can be analyzed for expression of GFP via immunofluorescence in standard cryosections or thicker optically cleared brains.
In this paper, we will describe the use of the specific mouse line, mTert-rtTA::oTet-Cre::Rosa-mTmG. To create this triple transgenic mouse line, we first mated oTet-Cre animals (The Jackson Lab strain #006234) to Rosa-mTmG animals (The Jackson Lab strain #007676) to create oTet-Cre::Rosa-mTmG double transgenic mice. These animals were genotyped with primers and PCR templates indicated in Supplementary Tables 1 and 2. mTert-rtTA mice (created by David Breault1) were mated to oTet-Cre::Rosa-mTmG animals to create mTert-rtTA::oTet-Cre::Rosa-mTmG mice (Figure 1A)1,30. The mechanism of action of the mTert-rtTA::oTet-Cre::Rosa-mTmG mouse line is illustrated in Figure 1B.
Doxycycline induction and pulse-chase design 
When planning experiments, it is important to consider several factors that will affect the outcome of lineage tracing experiments, including the age of the animal at doxycycline induction, the length of doxycycline administration (the &#34;pulse&#34; period), the length of time after doxycycline removal before tissue collection (the &#34;chase&#34; period), and the timing of any interventions during these processes. These study design considerations are essential for understanding the labeled cells and their progeny at the conclusion of the experiment. The first step in the process is to identify the age of interest of the animal and the length of the doxycycline administration. Longer pulse periods will allow for the potential for more cells to express the rtTA-linked promoter to be expressed and more cells of interest to be indelibly marked. A cell type that exhibits transient expression of the gene of interest may require a longer pulse period than cells that continuously express the gene of interest. However, if the goal of the study is to understand short-term effects of the cell of interest or an acute intervention, the pulse period cannot be too long, as once a cell is marked, it will be traced through any number of changes during the remainder of the pulse period. In some of our studies, a 2 day pulse with no chase period was used to more closely mimic a direct-reporter for TERT. This is because the minimum length of time for recombination of the Rosa-mTmG gene is 2 days31.
The next essential period in a pulse-chase experiment is the length between removal of doxycycline and perfusion of the animal, also known as the &#39;chase&#39;. The half-life of doxycycline in mice is approximately 170 minutes regardless of administration route, allowing for the conclusion that doxycycline induction is likely no longer occurring several hours after removal32. However, it is important to note that doxycycline metabolism is slower in aged mice, which may lead to longer effective &#39;pulse&#39; periods than young animals33.
During the chase period, labeled cells will continue to be labeled, even after proliferation, differentiation, or migration. The progeny of these cells will also be labeled. The goal of the study will shape the length of the pulse-chase paradigm. If the goal of the study is to understand the regeneration of a tissue over a long period of time with the cells of interest, a long chase period may be required. Finally, the timing of any interventions or treatments must be decided. Administration during the pulse period will affect the cells expressing the gene of interest as well as any progeny created during this time, whereas administration during the chase period may affect mostly progeny of the cells of interest, although this will depend on how quickly the labeled cells divide or differentiate. Examples of pulse-chase experimental designs we have utilized are outlined in Figure 2A.
It is also important to be aware of the possibility of background GFP signal due to leaky expression. Leaky expression occurs as a result of inherent binding potential between rtTA and the Otet sequences in the absence of doxycycline, and residual activity of the Otet transgene in the absence of rtTA (reviewed in34). Leaky expression represents one weakness of the Tet systems, and although the leak is often an acceptable downside to the system, situations where the leaky expression can lead to toxicity and mortality, such as with diptheria toxin (DTA) in Otet-DTA animals can limit the use of these systems35.
Brain processing, sectioning, and immunofluorescence of thin sections for microscopy 
To analyze the brains of mice after a lineage tracing experiment, mice must first be perfused via transcardial perfusion to remove blood and CSF that can lead to high autofluorescence in the brain. There are two commonly used fixatives that the animal may be perfused with: Histochoice Tissue Fixative, a glyoxal fixative (GF), or 4% paraformaldehyde (PFA). Glyoxal fixatives allow for a relatively gentle fixation process with less cross-linking than PFA. This reduces tissue stiffness, reduces the need for antigen retrieval, and allows for less concentrated antibody solutions during immunostaining. However, if they contain methanol this may serve to increase autofluorescence36. On the other hand, PFA will often allow for a more robust fixation, and while antigen retrieval will be required during immunostaining, there are antibodies that will only work with PFA-fixed tissues, and perfusion with 4% PFA is required for the optical clearing technique described later.
Following perfusion is a post-fixation step, in which brains are immersed in the same type of fixative utilized during the perfusion overnight. This allows for any brain regions that may not have been fully fixed during the perfusion to continue to fix. Next, brains will be incubated in sucrose in phosphate buffered saline (PBS) to remove as much water as possible before the freezing step to prevent ice formation within the tissue (&#34;cryo-preservation&#34;). Brains may then be cut into any number of sagittal, coronal, or transverse tissue sections for processing. For both precision and reproducibility, calibrated brain blocks need to be used in a way that ensures consistent bregma cuts between animals. The most important factor that can affect how brains are sectioned is the brain region(s) of interest. For example, while a sagittal cut may allow for more brain regions to be analyzed in one tissue section, this cut will not allow analysis of neuro/gliogenic regions of the ventricular-subventricular zone (V-SVZ) as the lateral and medial sides of the lateral ventricle will be impossible to discern in that dimension and are better observed in the coronal plane. This can be an important distinction to make in adult neurogenesis studies, as the lateral side of the lateral ventricle contains the neurogenic V-SVZ, while the medial wall of the lateral ventricle is a more gliogenic niche37.
After tissues have been divided into coronal, sagittal, or transverse sections, they will be frozen into blocks using Optimal Cooling Temperature (OCT) embedding solution. Here, brains are frozen within this embedding material, with the use of a mixture of dry ice and ethanol. If thin section analysis is required, blocks will be sliced on a cryostat, and depending on the downstream analysis required can be adhered to positively charged glass slides as thin sections (&#60;20 &#181;m). Glass slides that are not charged can also be used, but the use of uncharged slides may result in tissues becoming unstuck from the slides38. If medium-thickness free-floating tissue sections (&#62;20 &#181;m) are required, tissues will be collected as free-floating sections. If thick sections (0.5-4 mm) are required, slicing non-frozen brain sections with a vibratome is required. It is important to note the storage differences between sections on slides, which require freezing at -20 to -80 &#176;C and free-floating sections, which will be stored at 4 &#176;C.
Brain clearing and whole mount immunofluorescent confocal microscopy 
If a broader, yet less detailed analysis of the lineage-traced cells in the mouse brain is desired, a comprehensive analysis of thicker segments of brain tissue is possible with iDISCO brain clearing39 followed by immunostaining and tiled z-stack confocal microscopy or light-sheet microscopy. Here, large sections of mouse brain will be optically cleared (a process of delipidation39), which better allows for fluorescent signal imaging across a 1 mm tissue section. The downsides of this process are high autofluorescence and a diminished ability to obtain high resolution images of cell morphology and detailed co-staining analysis due to the increased working distances required when compared to more traditional methods (thin cryosections or free-floating sections). For this reason, we recommend brain clearing to analyze the large-scale lineage tracing results throughout multiple brain areas, instead of attempting to characterize or analyze individual cells.

<table><tbody><tr><td>Antibody diluent</td><td>Agilent</td><td>S080983-2</td><td></td></tr><tr><td>Antigen Retrieval Solution</td><td>Agilent</td><td>S2367</td><td></td></tr><tr><td>anti-GFP antibody</td><td>Invitrogen</td><td>A2311</td><td>Use at 1:500-1:000</td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A18-4</td><td></td></tr><tr><td>B6.Cg-Tg(tet0-cre)1Jaw/J</td><td>The Jackson Laboratory</td><td>JAX:006234</td><td></td></tr><tr><td>B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J</td><td>The Jackson Laboratory</td><td>JAX:007676</td><td></td></tr><tr><td>Blocking Solution (IHC)</td><td>Millipore Sigma</td><td>20773</td><td></td></tr><tr><td>Blunt needle</td><td>BSTEAN</td><td>X0012SYHIV</td><td></td></tr><tr><td>Coronal brain block</td><td>Braintree</td><td>BS-2000C</td><td></td></tr><tr><td>Cover slip</td><td>Corning</td><td>2850-22</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM1900</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D564</td><td></td></tr><tr><td>Dibenzyl ether</td><td>Millipore Sigma</td><td>33630</td><td></td></tr><tr><td>Dichloromethane</td><td>Sigma-Aldrich</td><td>270997</td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Donkey Serum</td><td>Sigma-Aldrich</td><td>D9663</td><td></td></tr><tr><td>Doxycycline Hyclate</td><td>Sigma-Aldrich</td><td>D9891</td><td></td></tr><tr><td>Glycine</td><td>Bio-Rad</td><td>161-0718</td><td></td></tr><tr><td>Heparin Sodium Salt from Porcine Mucosa</td><td>Sigma-Aldrich</td><td>H3393</td><td></td></tr><tr><td>Histochoice Molecular Biology Tissue Fixative</td><td>Amresco</td><td>H120</td><td>This product has since been discontinued, but can be replaced with Glyo-Fixx (Thermo Scientific, 6764265)</td></tr><tr><td>Hydrogen Peroxide Solution</td><td>Sigma-Aldrich</td><td>516813</td><td></td></tr><tr><td>IHC Select TBS Rinse Buffer</td><td>Millipore Sigma</td><td>20845</td><td></td></tr><tr><td>Ketamine</td><td>Westward</td><td>0143-95095-10</td><td>This product requires a DEA license for storage and use.</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A452-4</td><td></td></tr><tr><td>Microscope Slides</td><td>Fisherbrand</td><td>12-550-15</td><td></td></tr><tr><td>Milipore Block</td><td>Millipore</td><td>20773</td><td></td></tr><tr><td>Mounting medium</td><td>Millipore Sigma</td><td>5013</td><td></td></tr><tr><td>Optimal Cutting Temperature Solution</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>PBS Solution (10X)</td><td>Teknova</td><td>P0496</td><td></td></tr><tr><td>Sagittal brain block</td><td>Braintree</td><td>RBM-2000S</td><td></td></tr><tr><td>Saline</td><td>Braun</td><td>S8004-5264</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S7903</td><td></td></tr><tr><td>Sudan (Typogen) Black</td><td>Millipore Sigma</td><td>199664</td><td></td></tr><tr><td>Tetrahydrofuran</td><td>Sigma-Aldrich</td><td>186562</td><td></td></tr><tr><td>Tissue cartridge</td><td>Simport</td><td>M512</td><td></td></tr><tr><td>Triton X-100</td><td>Bio-Rad</td><td>1610407</td><td></td></tr><tr><td>Tween-20</td><td>Millipore Sigma</td><td>655205</td><td></td></tr><tr><td>Xylazine</td><td>AnaSed</td><td>sc-362950Rx</td><td></td></tr></tbody></table>

lineage tracing of inducible fluorescently-labeled stem cells in the adult mouse brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

While the fluorescent signal resulting from a Tet-On system with a fluorescent reporter will vary depending on the promoter of interest and fluorophore(s) used, we will describe how findings were analyzed with mTert-rtTA::oTet-Cre::Rosa-mTmG animals. Analysis of adult brains after a pulse-chase resulted in membrane GFP-expressing cells throughout various anatomical niches. The difference in fluorescent intensity between various cell types must be noted. One variable regarding fluorescent intensity is the size of the cell...

Watch this Scientific Journal Video about Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain at JoVE.com

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

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

lineage-tracing-inducible-fluorescently-labeled-stem-cells-adult

Research

JoVE Journal

Neuroscience

2.9K Views.  University of Maine. Het markeren van stamcellen en hun nakomelingen met een induceerbare transgene afstammingslijn maakt ruimtelijke en temporele analyse mogelijk van activering, proliferatie, migratie en / of differentiatie van volwassen stamcellen in de hersenen in vivo. Lineage tracing kan nieuwe informatie onthullen over lineage commitment, respons op interventies en multi-potentie. Voordelen van transgene lineage tracing-benaderingen zijn onder meer specificiteit van het traceren van een bepaalde cellijn, o...

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

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Developmental Biology

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Biology

Detection and Quantitation of Label-Retaining Cells in Mouse Incisors using a 3D Reconstruction Approach after Tissue Clearing

The murine incisor is an organ that grows continuously throughout the lifespan of the mouse. The epithelial and mesenchymal stem cells residing in the proximal tissues of incisors give rise to progeny that will differentiate into ameloblasts, odontoblasts, and pulp fibroblasts. These cells are crucial in supporting the sustained turnover of incisor tissues, making the murine incisor an excellent model for studying the homeostasis of adult stem cells. Stem cells are believed to contain long-living quiescent cells that can be labeled by nucleotide analogs such as 5-ethynyl-2&#180;-deoxyuridine (EdU). The cells retain this label over time and are accordingly named&#160;label-retaining cells (LRCs). Approaches for visualizing LRCs in vivo provide a robust tool for monitoring stem cell homeostasis. In this study, we described a method for visualizing and analyzing LRCs. Our innovative approach features LRCs in mouse incisors after tissue clearing and whole-mount EdU staining followed by confocal microscopy and a 3-dimensional (3D) reconstruction with the imaging software. This method enables 3D imaging acquisition and non-biased quantitation compared to traditional LRCs analysis on sectioned slides.

The mouse incisor contains valuable label-retaining cells in its stem cell niche. We have a novel way to unbiasedly detect and quantify the label-retaining cells; our study used EdU labeling and a 3D reconstruction approach after PEGASOS tissue clearing of the mandible.

The murine incisor is an organ that grows continuously throughout the lifespan of the mouse. The epithelial and mesenchymal stem cells residing in the ...

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. ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Visualizing Neuronal and Glial Cell Dynamics in the Developing Mouse Brain

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Author Spotlight: Exploring Cell Migration and Gene Roles in the Developing Brain

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers MADM

A protocol to perform lineage tracing and functional genetic analysis of candidate genes at a single cell level using mosaic analysis with double markers (MADM) is presented. MADM clonal analysis provides a quantitative framework to measure the proliferative behavior, cellular output, and lineage relationship of individual progenitors and their daughter cells.

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying ...

Immunofluorescence Staining for Visualizing Proliferated Neural Stem Cells in Mouse Brain Sections

Source: Quintanilla, L. J., et al. <a href="https://app.jove.com/v/59237">Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells.</a> J. Vis. Exp. (2019)
This video demonstrates the procedure for staining newly proliferated neural stem cells in brain tissue sections using EdU incorporation, followed by immunofluorescence to visualize specific proteins, enhancing the understanding of cell dynamics and proliferation.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunohistochemistry

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

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