Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Summary

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

Abstract

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.

Protocol

1. Lung Isolation

1. Sacrifice adult mice (8-10 weeks in age; 18-20 g; C57Bl6J) mice with an overdose of isoflurane followed by exsanguination using sterile technique. Profiles will vary slightly between strains.
2. Surgically remove the diaphragm, open up the chest cavity by cutting the ribcage laterally on each side of the mouse. Flush the blood from the lungs by perfusing the right heart ventricle gently using 3-5mls of phosphate buffered saline (PBS).
3. Dissect the lung lobes removing the trachea and large bronchi and place in a petri dish filled with Hanks buffered saline (HBSS). Following collection of all lung lobes forceps are used to place tissue onto the lid of the dish. Tissue is then minced into tiny pieces using disposable scalpels with enough liquid to keep them moist but not so much that they float and move.
4. Dissect a hindlimb of one of the mice and isolate the bone marrow to use as a staining control to set the flow cytometry gates. Stain the bone marrow (BM) as previously described ¹.

2. Preparation of a Single Cell Suspension from Lung Tissue

1. Each lung is digested by using an optimized tissue weight to volume ratio of 0.2g: 5mls of pre-warmed 0.2% Worthington type 2 collagenase (Worthington Biochemicals, Lakewood, NJ; cat # LS004202) dissolved in sterile HBSS. The mixture is immersed in a 37°C water bath for 30 min ^2,3.
2. Triturate sample well until lung tissue digest flows easily through a 10ml pipette (approximately 10 repetitions). Incubate for additional 15 min at 37°C to complete the tissue digests and ensures a more uniform single cell suspension.
3. Dilute the lung cell suspension with HBSS and triturate with a 5ml pipette to disperse residual tissue fragments.
4. Filter the suspension using a 70μM cell strainer to remove undigested tissue fragments. Sample can be divided into multiple conical tubes at this point to prevent overloading one cell strainer and decrease the time.
5. Pellet the cell suspension for 10 min at 1500 rpm and decant the supernatant.
6. Resuspend cell pellet gently using room temperature RBC lysis buffer (eBiosciences, San Diego, CA; cat # 00-4333-57) and incubate at RT for 5 min. Add an equal volume of HBSS to inactivate the lysis buffer and filter the cell suspension using a 40μM cell strainer to remove debris and cell aggregates.
7. Pipette 10μl of each sample to be stained and count cell numbers for both the lung and BM samples using a hemocytometer. Note: record the total volume of the cells.
8. Pellet the single cell suspension of lung cells by centrifugation at 1500 rpm for 10 min.
9. Resuspend both lung and BM cells at 1x10⁶ cells/ml in prewarmed (37°C) DMEM+ (Dulbecco's modified Eagle's medium, high glucose (Gibco, Carlsbad, CA; cat # 11965-092) containing 2% fetal bovine serum (FBS) and 10mM HEPES.
10. Prepare a 1mg/ml stock solution of Hoechst 33342 dye (Sigma Chemical Company, St.Louis, MO; cat # B2261) in water and filter sterilize ^1,4. The Hoechst dye may be stored as aliquots at -80°C.
11. Add Hoescht 33342 dye to a final concentration of 5μg/ml (a 200x dilution of a 1mg/ml stock) to the single cell suspension.
12. Mix the cells by gently inversion and incubate in 37°C water bath for exactly 90 minutes.

3. Staining and Preparation of the Lung Cell Suspension for Flow Cytometry Analysis

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Discussion

We have adapted a method initially used to identify BM hematopoietic cells to isolate a specific population of resident lung MSC. Due to the reproducibility of isolation these cells were then well characterized as MSC. Their origin has been defined as resident in the adult mouse lung (as opposed to BM derived) and a phenotypic and molecular profile documented ². The ability to repeatedly isolate this characterized population allows the further study of the biological importance and role of the lung MSC during tissue homeostasis and disease. The recent definition of this population in vivo in both murine and human pulmonary tissue facilitates the development of a therapeutic strategy directed at the rescue of endogenous cells to facilitate lung repair during injury and disease.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phosphate buffered saline (PBS)	Sigma-Aldrich	P-5368
Hanks buffered salt solution (HBSS)	Thermo Fisher Scientific, Inc.	SH30588.01
0.2% Worthington type 2 collagenase	Worthington Biochemical	LS004202
Red blood cell lysis buffer	eBioscience	00-4333-57
DMEM	Invitrogen	11965-092
H–chst 33342 dye	Sigma-Aldrich	B2261
CD45-APC	BD Biosciences	559864
Propidium iodide	Sigma-Aldrich	81845
a-MEM	Thermo Fisher Scientific, Inc.	SH30265.01
FBS	Invitrogen	16000-069
0.5% trypsin/EDTA	Cellgro	25-053-Cl
Complete MesenCult Medium	Stem Cell Technologies	05511
0.4% w/v Giemsa staining solution	Sigma-Aldrich	GS1L
4% paraformaldehyde	Electron Microscopy Sciences	15710	16% paraformeldehyde is diluted to 4% using PBS
Carboxyfluorescein succinimidyl ester (CFSE)	Sigma-Aldrich	21888