Name: isolation and characterization of the immune cells from micro-dissected mouse choroid plexuses
Uploaded: 2022-02-03T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses at JoVE.com

We thank the Institut Pasteur Animalerie Centrale and the CB-UTechS facility members for their help. This work was supported financially by Institut Pasteur.

All the procedures agreed with the guidelines of the European Commission for the handling of laboratory animals, directive 86/609/EEC. They were approved by the ethical committees No. 59, by the CETEA/CEEA No. 089, under the number dap210067 and APAFIS #32382-2021070917055505 v1.
1. Preparation of the materials
<ol>
	<li>Store all antibodies (Table of Materials) at 4 &#176;C, protected from light exposure.</li>
	<li>DAPI stock solution (1 mg/mL): Resuspend t...

<ol>
	<li>Morais, L. H., Schreiber, H. L., Mazmanian, S. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+(Kolmer's)+epiplexus+cells.">The origin of (Kolmer's) epiplexus cells.</a> Histochemistry. 44 (1), 103-104 (1975).</li><li>Baruch, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS-specific+immunity+at+the+choroid+plexus+shifts+toward+destructive+Th2+inflammation+in+brain+aging.">CNS-specific immunity at the choroid plexus shifts toward destructive Th2 inflammation in brain aging.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (6), 2264-2269 (2013).</li><li>Kunis, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN-&#947;-dependent+activation+of+the+brain's+choroid+plexus+for+CNS+immune+surveillance+and+repair.">IFN-&#947;-dependent activation of the brain's choroid plexus for CNS immune surveillance and repair.</a> Brain. 136 (11), 3427-3440 (2013).</li><li>Prinz, M., Priller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+and+brain+macrophages+in+the+molecular+age:+From+origin+to+neuropsychiatric+disease.">Microglia and brain macrophages in the molecular age: From origin to neuropsychiatric disease.</a> Nature Reviews Neuroscience. 15 (5), 300-312 (2014).</li><li>Goldmann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fate+and+dynamics+of+macrophages+at+central+nervous+system+interfaces.">fate and dynamics of macrophages at central nervous system interfaces.</a> Nature Immunology. 17 (7), 797-805 (2016).</li><li>Fung, I. 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Studies aiming to understand the immunological contributions to brain homeostasis and disease have mainly focused on cells residing within the brain parenchyma, neglecting brain borders such as CP, which are nevertheless crucial contributors to brain function2,3. The analysis of immune cell populations at CP is challenging due to the small size of CP, low numbers of resident immune cells, and complicated access to this tissue. Flow cytometry performed on total br...

Since the discovery of the blood-brain barrier by Paul Erhlich in the late 19th century, the brain has been considered virtually separated from the other organs and the bloodstream. Yet, this last decade has seen the emergence of the concept that brain function is shaped by various biological factors, such as gut microbiota and systemic immune cells and signals1,2,3,4. In parallel, other brain borders such as meninges and choroid plexuses (CP) have been identified as interfaces of active immune-brain cross talk rather than inert barrier tissues5,6,7,8.
The CP&#160;constitute the blood-cerebrospinal fluid barrier, one of the borders separating the brain and the periphery. They are located in each of the four ventricles of the brain, i.e., the third, the fourth, and both lateral ventricles, and are adjacent to areas involved in neurogenesis such as the subventricular zone and subgranular zone of the hippocampus3. Structurally, the CP are composed of a network of fenestrated blood capillaries enclosed by a monolayer of epithelial cells, which are interconnected by tight and adherens junctions9,10. Major physiological roles of the CP epithelium involve the production of cerebrospinal fluid, which flushes the brain from waste metabolites and protein aggregates, and the production and controlled blood-to-brain passage of various signaling molecules including hormones and neurotrophic factors11,12,13. Secreted molecules from CP shape brain&#39;s activity, i.e., by modulating neurogenesis and microglial function14,15,16,17,18,19, which makes CP crucial for brain homeostasis. CP also engage in various immune activities; whereas the main immune cell type in the brain parenchyma under non-pathological conditions is microglia, the diversity of CP immune cell populations is as broad as in peripheral organs3,7, suggesting that various channels of immune regulation and signaling are at work at the CP.
The space between the endothelial and epithelial cells, the CP stroma, is mainly populated by border-associated macrophages (BAM), which express pro-inflammatory cytokines and molecules related to antigen presentation in response to inflammatory signals3. Another subtype of macrophages, Kolmer&#39;s epiplexus cells, are present on the apical surface of the CP epithelium20. CP stroma is also a niche for dendritic cells, B cells, mast cells, basophils, neutrophils, innate lymphoid cells, and T cells which are mostly effector memory T cells able to recognize central nervous system antigens7,21,22,23,24. In addition, the composition and activity of immune cell populations at the CP changes upon systemic or brain perturbation, for example, during aging10,14,15,21,25, microbiota perturbation7, stress26, and disease27,28. Notably, these changes were suggested to indirectly shape brain function, i.e., a shift of CP CD4+ T cells towards Th2 inflammation occurs in brain aging and triggers immune signaling from the CP that may shape aging-associated cognitive decline14,15,21,25,29. Illuminating the properties of the CP immune cells would thus be crucial to better understand their regulatory function on CP epithelium physiology and secretion and thereby decipher their indirect impact on brain function under healthy and disease conditions.
CP are small structures that contain only a few immune cells. Their isolation requires microdissection after a preliminary step of perfusion; immune cells in the bloodstream would otherwise constitute major contaminants. This protocol aims to characterize the myeloid and T cell subsets of the CP using flow cytometry. This method identifies about 90% of the immune cell populations that compose mouse CP under non-inflammatory conditions, in accordance with recently published works using other methods to dissect immune CP heterogeneity7,10,28. This protocol could be applied to characterize changes in the CP immune cell compartment with disease and other experimental paradigms in vivo.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-mouse CD16/CD32</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Albumin, bovine</td>
			<td>MP Biomedicals</td>
			<td>160069</td>
			<td>Blocking reagent</td>
		</tr>
		<tr>
			<td>APC anti-mouse CX3CR1</td>
			<td>BioLegend</td>
			<td>149008</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC anti-mouse TCRb</td>
			<td>BioLegend</td>
			<td>109212</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC-Cy7 anti-mouse CD4</td>
			<td>BioLegend</td>
			<td>100414</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC-Cy7 anti-mouse IA-IE</td>
			<td>BioLegend</td>
			<td>107628</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>BD FACSymphony A5 Cell Analyzer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometry analyzer</td>
		</tr>
		<tr>
			<td>BV711 anti-mouse Ly6C</td>
			<td>BioLegend</td>
			<td>128037</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
			<td>Enzyme to dissociate CP tissue</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Scientific</td>
			<td>62248</td>
			<td>Live/dead marker</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td></td>
			<td></td>
			<td>Ion chelator</td>
		</tr>
		<tr>
			<td>fine scissors</td>
			<td>FST</td>
			<td>14058-11</td>
			<td>Dissection tool</td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD45</td>
			<td>BioLegend</td>
			<td>103108</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Flow controller infusion inset</td>
			<td>CareFusion</td>
			<td>RG-3-C</td>
			<td>Blood perfusion inset</td>
		</tr>
		<tr>
			<td>FlowJo software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Analysis software</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>FST</td>
			<td>11018-12</td>
			<td>Dissection tool</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3149-10KU</td>
			<td>Anticoagulant</td>
		</tr>
		<tr>
			<td>Imalgene</td>
			<td>Boehringer Ingelheim</td>
			<td></td>
			<td>Ketamine, anesthesic</td>
		</tr>
		<tr>
			<td>OneComp eBeads</td>
			<td>Invitrogen</td>
			<td>01-1111-42</td>
			<td>Control beads to realize compensation</td>
		</tr>
		<tr>
			<td>PBS-/-</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>PBS+/+</td>
			<td>Gibco</td>
			<td>14040-091</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>PE anti-mouse CD8a</td>
			<td>BioLegend</td>
			<td>100708</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>PE anti-mouse F4/80</td>
			<td>BioLegend</td>
			<td>123110</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>PE-Dazzle 594 anti-mouse CD11b</td>
			<td>BioLegend</td>
			<td>101256</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Rompun</td>
			<td>Bayer</td>
			<td></td>
			<td>Xylazine, anesthesic</td>
		</tr>
		<tr>
			<td>thin forceps</td>
			<td>Dumoxel Biology</td>
			<td>11242-40</td>
			<td>Dissection tool</td>
		</tr>
		<tr>
			<td>Vetergesic</td>
			<td>Ceva</td>
			<td></td>
			<td>Buprenorphin, analgesic</td>
		</tr>
	</tbody>
</table>

isolation and characterization of the immune cells from micro-dissected mouse choroid plexuses

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can indirectly shape brain function. At the interface between the brain and the systemic circulation, the choroid plexuses (CP), which constitute the blood-cerebrospinal fluid barrier, have been highlighted as a key site of periphery-to-brain communication. CP produce the cerebrospinal fluid, neurotrophic factors, and signaling molecules that can shape brain homeostasis. CP are also an active immunological niche. In contrast to the brain parenchyma, which is populated mainly by microglia under physiological conditions, the heterogeneity of CP immune cells recapitulates the diversity found in other peripheral organs. The CP immune cell diversity and activity change with aging, stress, and disease and modulate the activity of the CP epithelium, thereby indirectly shaping brain function. The goal of this protocol is to isolate murine CP and identify about 90% of the main immune subsets that populate them. This method is a tool to characterize CP immune cells and understand their function in orchestrating periphery-to-brain communication. The proposed protocol may help decipher how CP immune cells indirectly modulate brain function in health and across various disease conditions.

The flow cytometry analyses presented here successfully revealed the major subsets of myeloid and T cells (Figure 1 and Figure 2, respectively), and their relative total number per mouse in a highly reproducible manner (Figure 3).
The flow cytometry analysis of myeloid cells showed that CP are populated by CD11b+ CX3CR1+ F4/80high BAM, representing almost 80% of...

Watch this Scientific Journal Video about Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses at JoVE.com

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

This study uses flow cytometry and two different gating strategies on isolated perfused mice brain choroid plexuses; this protocol identifies the main immune cell subsets that populate this brain structure.

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can ...

This protocol may be useful for addressing questions about the role of the choroid plexuses and peripheral immune cells in regulation of the brain in various contexts in mouse models. This protocol allows for in-depth quantitative and qualitative analysis of the unique immune cell populations in the choroid plexuses of mice. Dissection of the choroid plexuses can be difficult at the beginning due to their small size and transparent color.We recommend practicing on non-perfused animals first. To begin, position the brain dissected from a C57 black six mouse dorsal side up in a Petri dish and place the dish below the objectives of the binocular loops. While maintaining the brain in place using forceps, insert the two ends of another pair of forceps down through the midline between the hemispheres.Using the forceps, pull the cortex with the callosum and hippocampus away from the septum, exposing the lateral ventricle and a part of the third ventricle. Identify the lateral choroid plexus as a long veil lining the lateral ventricle and flaring at both ends. Then using two ends of thin forceps, collect the lateral choroid plexus.Be careful to collect the triangular posterior part hidden by the posterior fold of the hippocampus. Next, pull the cortex with the corpus callosum and hippocampus of the contralateral hemisphere away from the septum to expose the entire third ventricle and the opposite lateral ventricle. Using fine forceps, collect the third choroid plexus identified as a short structure with a granular surface aspect.Then collect the other lateral choroid plexus. Next, insert the two ends of the forceps down between the cerebellum and midbrain and detach the cerebellum from the pons and medulla to expose the fourth ventricle. Then identify the fourth choroid plexus as a long globular structure with a granular surface aspect that lines the fourth ventricle from the lateral right end to the left end between the cerebellum and medulla.Using fine forceps, collect the fourth choroid plexus. After incubating all collected choroid plexuses with collagenase IV, gently pipette up and down around 10 times to finalize the dissociation. Then fill the tube to 1.5 milliliters with MACS buffer to stop the collagenase IV activity.Next, centrifuge the cells and discard the supernatant before washing the cells with 1.5 milliliters of MACS buffer. After adding the appropriate compensation beads and antibody solutions, incubate the samples on ice for 30 to 45 minutes, protecting them from light exposure. Then fill the tubes with 1.5 milliliters of MACS buffer and centrifuge the cells and compensation beads.After resuspending the cells and compensation beads in 500 microliters of MACS buffer, filter the cells through a 70 micron strainer. After turning on the flow cytometer and the software, select forward scatter area versus side scatter area and gate for cells based on size. Then create a daughter gate for single cells with forward scatter area versus forward scatter height.Next, create a daughter gate for live cells with DAPI versus forward scatter area to exclude the DAPI positive cells. Then create a daughter gate for CD45 positive immune cells with CD45 versus forward scatter area. Now, create a daughter gate from the CD45 positive cells and select F480 versus CD11b to identify the CD11b positive F480 positive and the CD11b positive F480 negative populations.Then create a daughter gate for CD11b positive F480 positive cells and select CX3CR1 versus IA-IE to identify both CX3CR1 positive IA-IE positive border-associated macrophages and CX3CR1 positive IA-IE negative macrophages. Next, create a daughter gate for CD11b positive F480 positive CX3CR1 positive IA-IE negative macrophages and select F480 versus CD11b to identify both the CD11b high F480 intermediate CX3CR1 positive IA-IE negative Kolmer's epiplexus macrophages and the CD11b positive F480 high CX3CR1 positive IA-IE negative border-associated macrophages. Then from the CD11b positive F480 negative population, gate for Ly6C versus IA-IE.Select both IA-IE positive Ly6C negative population and IA-IE negative Ly6C positive cells that mainly correspond to monocytes or neutrophils. Finally, create a daughter gate for CD11b positive F480 negative IA-IE positive Ly6C negative population and select CD11b versus CX3CR1. Then identify both CD11b high CX3CR1 low and CD11b positive CX3CR1 negative populations.For T-cell gating, after excluding the DAPI positive cells as described earlier, create a daughter gate for CD45 positive immune cells with CD45 versus FSC-A, then create a daughter gate from the CD45 positive cells and select TCR beta versus CD11b. Exclude the CD11b positive myeloid cells and select the TCR positive CD11b negative T-cell population. Finally, create a daughter gate for the TCR beta positive CD11b negative population and select CD4 versus CD8a.Identify both CD8 negative and CD4 positive T-cells and CD8 positive CD4 negative T-cells. The flow cytometry analyses demonstrated here successfully revealed major subsets of myeloid and T-cells and their relative total number per mouse in a highly reproducible manner. Flow cytometry analysis of the myeloid cells show that the choroid plexus is populated by CD11b positive CX3CR1 positive F480 high border-associated macrophages, representing 80%of the CD45 positive immune cells at the choroid plexus.The CD11b high F480 intermediate CX3CR1 positive IA-IE negative population of Kolmer's epiplexus macrophages was also identified. Among the CD11b positive F480 negative immune cells, two different populations of Ly6C negative IA-IE positive cells were characterized and likely correspond to dendritic cells. The analysis and gating strategy of T-cells highlighted the presence of the minor population of CD45 positive CD11b negative TCR beta positive cells.Among them, about 30-50 CD8 negative CD4 positive and CD8 positive CD4 negative T-cells per mouse populated the choroid plexus under physiological conditions. It is important to control for the quality of the perfusion since any blood contamination may blur the immune composition of the choroid plexus. This method can be followed by sorting of selected cell populations for further analysis such as bulk or single-cell RNA sequencing.

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Research

JoVE Journal

Biology

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of &#62;95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...