Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Summary

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Abstract

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Introduction

Culturing of murine bone marrow cells with the cytokine Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is widely used as a method to generate monocyte-derived dendritic cells (moDC; also known as inflammatory DC) in large numbers ^1,2,3,4,5. These cells have been extremely useful in a variety of studies of dendritic cell (DC) function ^6,7,8. Typically, these murine bone marrow cells are cultured for 6-8 days and are then used for study of dendritic cell function ⁵. These cultures had long been considered mostly homogenous, consisting of a majority of differentiated moDC. More recently, it has become clear that at the end of this 6–8 day culture period, there are indeed many moDC, as well as a large subset of differentiated monocyte-derived macrophages (moMacs) ^9,10,11. Our own studies have further extended these findings demonstrating that other subsets of less developed cells, such as moDC precursors (moDP) and monocytes, remain in the cultures at low frequency even after 7 days ¹⁰. Thus, studies of dendritic cells (DC) function using cells generated by this system could reflect the responses of a broader cohort of cell types than previously appreciated.

We have learned a great deal from the study of GM-CSF-generated moDC relating to the function of these cells in the final stages of differentiation ^12,13,14. However, we understand significantly less about the developmental pathway of these cells ^2,15,16 and of how and when they exhibit specific functions such as: responsiveness to Pathogen Associated Molecular Patterns (PAMPs), phagocytosis, antigen processing and presentation ¹³, and anti-bacterial activity. A protocol for isolation of large numbers of conventional Flt3L-driven DC progenitors and precursors has been reported ¹⁷. Isolation of these distinct populations was achieved using carboxyfluorescein succinimidyl ester (CFSE)-stained bone marrow cells (to track dividing cells) and culture in Flt3L for 3 days. Cells were then depleted of linage positive cells and sorted into progenitor and precursor populations based on CD11c expression ¹⁷. Another approach by Leenen's group to identify early progenitors of DC in GM-CSF-driven culture was to sort cells based on CD31 and Ly6C ¹⁸. The initial goal was to create a similar method for obtaining progenitors and precursors of GM-CSF-driven moDC. Due to the specific cell types generated by GM-CSF, we adapted the approach and sorting strategy based on expression of molecules that were expressed at early and later stages of development. We ultimately determined that Ly6C, CD115 (CSF-1 receptor), and CD11c were the best markers for distinguishing these cell types ¹⁰.

Here, we present a method for isolation of cells at several distinct stages of development along the pathway of differentiation driven by GM-CSF: Common Myeloid Progenitor (CMP), Granulocyte-Macrophage Progenitor (GMP), monocyte, monocyte-derived Macrophage (MoMac) and monocyte-derived DC (MoDC). The moMac population can be further segregated based on level of MHC class II expression, revealing a moDC precursor population (moDP) ¹⁰. We utilize a high-speed fluorescence-activated cell sorting (FACS) strategy to isolate these 5 populations based on expression of Ly6C, CD115, and CD11c. We then demonstrate the examination of these cells in functional assays revealing their responses to PAMP stimulation.

Protocol

All animal work was approved by the Auburn University Institutional Animal Care and Use Committee in accordance with the recommendations outlined in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

1. Preparation for Bone Marrow Collection

1. Prepare 250 mL complete media by adding a solution of Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 50 µM 2-mercaptoethanol to the top of a 0.22 µm vacuum filter flask unit, and apply vacuum.
   NOTE: Complete medium can be stored at 4 °C for up to 2 months.
2. Prepare 70% ethanol solution by mixing 350 mL of 100% ethanol with 150 mL sterile H₂O in a 500 mL flask.
3. Set centrifuge to 4 °C.
4. Sterilize forceps, scalpel, and scissors in 70% ethanol.
5. Using a serological pipette, add 5 mL of complete media to three 60 mm Petri dishes.
6. Using a serological pipette, add 30 mL of complete media to a 50 mL conical tube.

2. Collection of Murine Bone Marrow Cells

1. Euthanize a C57BL/6 mouse by CO₂ narcosis in accordance with the rules established by the 2013 American Veterinary Medical Association (AVMA) Guidelines on Euthanasia.
2. Remove and strip hind legs.
   1. Saturate the hind legs and torso with 75% ethanol, and make shallow cuts through the skin around the hip joint with curved tissue scissors. Using forceps, firmly pull the skin from the hip down towards the ankle, revealing the muscle. Use scissors to remove the skin flap.
   2. Remove the whole hind leg by cutting through the bone just above the femur/hip joint.
      NOTE: In addition to sterilizing the area, the ethanol will aid in providing clean cuts and prevent hair from contaminating the samples.
   3. If the legs will be transferred to a new location for bone marrow harvesting, submerge the legs in complete media.
3. Working in a sterile biosafety cabinet, transfer the legs to one of the previously prepared Petri dishes.
4. Use scissors to cut the just below the ankle, and carefully remove as much of the muscle and elastic connective tissue as possible. Transfer the cleaned bone to the second prepared Petri dish.
   NOTE: Although it is not necessary to remove all the muscle, too much remaining tissue can make it difficult to flush out the bone marrow.
5. Separate the femur, knee, and tibia.
   1. Using forceps, hold the leg at the knee and locate the marrow.
      NOTE: Bone marrow should be visible as a faint red line inside the bone cavity at the top of the femur and toward the end of the tibia.
      1. Using scissors, make three cuts as follows.
         1. Cut the tibia just above where the marrow appears to end.
         2. Cut just below the knee joint.
         3. Cut just above the knee joint.
         4. If the hip joint is still connected to the femur, cut just below the hip joint.
      2. Return the three fragments to the Petri dish and repeat this process on other leg.
6. Flush bone marrow from the femur and tibia.
   1. Fill a 10 mL syringe with complete media from the 50 mL conical tube, and cap with 23 G needle.
   2. Holding the bone with forceps above the third prepared Petri dish, insert the needle into the bone canal and push media through, flushing out the cells (which may emerge as an intact "plug" or as several clusters). Repeat until no more color can be seen through the bone. Refill the syringe with media as necessary.
7. Crush the epiphyses.
   1. While still in the second Petri dish, hold the knee cap firmly with forceps, and mash the knees with the tip of the syringe. Continue until the epiphyses are no longer red.
8. Using the syringe, transfer the cells from the second and third Petri dish to the 50 mL tube. Breakup up clumps by gently pipetting up and down. Try not to generate bubbles. Centrifuge at 250 x g at 4 °C for 10 min.
9. Lyse red blood cells
   1. Remove supernatant with serological pipette, dislodge pellet by flicking, and lyse red blood cells by incubating in 1 mL of ACK (Ammonium-Chloride-Potassium) Lysis Buffer for 1 min at room temperature.
   2. Using a serological pipette, add 40 mL of HBSS (Hanks Balanced Salt Solution) buffer.
10. Using a serological pipette, filter the cells though a 70 µm cell strainer into a new 50 mL conical tube. Centrifuge at 250 x g at 4 °C for 10 min.
11. Using a serological pipette, remove supernatant and wash cells with 40 mL of complete media. Centrifuge at 250 x g at 4 °C for 10 min.
    NOTE: At this stage, lineage positive lymphocytes can be removed by FACS or magnetic column purification. However, lymphocytes are not maintained long term in culture. Although a large number of lineage positive cells are present in the Ly6C-CD115- population in the bone marrow ex vivo, nearly all are absent by day 5 (Figure 2).
12. Using a serological pipette, remove the supernatant, and culture the bone marrow cells in complete media with 10 ng/mL of recombinant mouse GM-CSF at a density of 1 x 10⁶ cells/mL.
    NOTE: Typically, 4 x 10⁷ total cells can be harvested after red blood cell lysis. However, expect as little as 2 x 10⁷ for beginners and up to 5 x 10⁷ cells for experienced harvesters.
13. Using a serological pipette, transfer the cells to tissue culture plates, and incubate at 37 °C in 5% CO₂. If using a 24-well plate, seed each well with 2 mL of cell suspension.
14. Every 48 h, use a serological pipette to remove half of the media and replace with fresh complete media and GM-CSF.
    NOTE: Cultures can be kept up to 9 days. However, composition changes over time. See Section 3 for more information.

3. Choosing Day of Sort

1. As cell population compositions change over time, select a day that yields the highest number of desired cells.
2. See Table 1 for the expected cell yield post sort for each of the populations after 3, 5, and 7 days of culture in GM-CSF per 1 x 10⁷ cells.

4. Staining strategy

1. Use small aliquots of cells to prepare control samples. Include an unstained control, compensation control samples stained with only one fluorescent antibody each, and fluorescence-minus-one controls in which all antibodies are added except one, to control for non-specific fluorescence in that channel. If using indirect labeling, include primary alone, secondary alone, and both primary and secondary.
   NOTE: Phycoerythrin (PE) and allophycocyanin (APC) tagged antibodies provide distinct separation with minimal bleed over when used together. However, if fluorochrome options are limited, CD115 is expressed at a relatively low level, while Ly6C is expressed at very high levels. Therefore, brighter fluorochromes are desirable for anti-CD115, and anti-Ly6C fluorochromes are chosen to prevent bleed over.
2. Prepare 100 mL of FACS Wash Buffer (FWB) by mixing 97 mL of chilled Dulbecco's phosphate buffered saline (DPBS) with 3 mL of fetal calf serum in a 50 mL conical tube, and place in an ice bath.
3. Use a pipette to gently, but thoroughly, pipette cells up and down to dislodge any loosely adherent cells.
4. Using a serological pipette, transfer cells to a 50 mL conical tube. Centrifuge at 250 x g at 4 °C for 10 min.
   1. If cell volume exceeds 50 mL, transfer cells to the necessary number of tubes and combine at the staining step.
5. Gently pour off the supernatant, and wash the pelleted cells by adding 30 mL of FWB with a serological pipette. Centrifuge at 250 x g at 4 °C for 10 min, and repeat the wash.
   NOTE: If high cell death is expected, cells can be washed with FWB with as low as 0.5% fetal calf serum (FCS). This will prevent cell clumping.
6. Suspend and stain cells per antibody manufacturer's instructions.
   1. Suspend 5 x 10⁷ cells in 1 mL of FWB and add 2 µg each of anti-Ly6C and anti-CD115 labeled with the fluorophores (of the researcher's choice). To further distinguish CMP from moDC (both are Ly6C- and CD115-), add 2 µg of anti-CD11c antibodies (CMP are CD11c^-; moDC are CD11c⁺). Incubate for 30 min on ice.
      NOTE: Figure 3 was generated using Ly6C-PE and CD115-APC.
7. Using a serological pipette, add 10 mL of FWB, and centrifuge at 250 x g at 4 °C for 10 min.
8. Gently pour off the supernatant, and wash the pelleted cells by adding 30 mL of FWB with a serological pipette. Centrifuge at 250 x g at 4 °C for 10 min, and repeat the wash.
9. Before suspending cells, flick tube thoroughly to dislodge the pellet. Use a serological pipette to suspend cells at 1 x 10⁷ cell/mL of FWB, and filter through 35-µm cell filter. Use a serological pipette to transfer filtered cells into polypropylene tube, and place on ice until ready to sort.
   NOTE: If polypropylene is unavailable, protein coated (nonfat dry milk or FCS) polystyrene tubes can be used to reduce binding.

5. Set Gates Based on Control Samples

NOTE: To prevent cell disruption due to the pressure of the high-speed flow stream, use a 100–130 µm nozzle for cell sorting.

1. Run the unstained control (see step 4.1) through the cell sorter, and apply a gate to exclude small debris (low forward scatter; FSC) and highly granular (high side scatter; SSC) particles.
   1. To analyze only the later stages (monocytes, moMac/MoDP, and MoDC), apply gating to only include larger cells (high FSC).
   2. If viability stains are being used, use these to exclude stained, non-viable events (an example is illustrated in Figure 1).
2. Run the single fluorescent control samples through the cell sorter, and adjust compensation as needed.
3. Run a sample of the multi-labeled sample. Observe four distinct populations: Ly6C+CD115- (GMPs), Ly6C+CD115+ (monocytes), Ly6C-CD115+ (moMacs/moDP), and Ly6C-CD115- (CMPs/moDCs). Apply a gate to isolate each of the four major populations.
   NOTE: CMPs and moDC share the Ly6C-CD115- phenotype. However, they can be differentiated based on CD11c expression: CMP lack CD11c, whereas MoDCs express CD11c.

6. Collection of Isolated Populations

1. Prepare collection tubes by adding enough FCS to achieve at least 20% final concentration when full. For example, if using 5 mL tubes, add 1 mL of FCS before sorting, and remove the tube when it reaches 5 mL total volume.
2. To prevent membrane turnover and antibody uptake, keep all samples (mixed and sorted) at 4 °C throughout the sort.
   1. If this is not possible, keep the tube containing the cells to be sorted on ice as much as possible and transfer aliquots to the sorter as needed.
   2. Additionally, transfer sorted samples to ice every 20–30 min.
3. After the desired number of cells have been collected, use a serological pipette to transfer the cells to a new conical tube. Centrifuge at 250 x g at 4 °C for 10 min.
   1. Confirm purity with post-sort analysis on small aliquots from each collected population.
4. Remove the supernatant, suspend in 10 mL of FWB and centrifuge at 250 x g at 4 °C for 10 min. Repeat for a total of two washes.
   NOTE: It can be difficult to remove all the supernatant without dislodging the pellet. Attaching a pipette tip to a vacuum line can help with removal. If this is not available, it is suggested that the supernatant be collected in a fresh tube in case of pellet dissociation.
5. Remove the supernatant after the second wash.
6. If the user's experimental design dictates the cells be re-cultured, follow steps 2.12–2.14. Otherwise, if cells will be used for immediate analysis, prepare cells according to desired protocol.
   NOTE: An example of typical functional analysis is included in Figure 4.

Results

In an effort to keep as many channels available for analysis as possible, viable cells were routinely selected based on forward and side scatter, excluding very small and very granular events (a typical gate is applied to all the dot plots in Figure 1A). To determine if this gating strategy reliably excluded dead cells, we stained with 7-Amino actinomycin D (7-AAD) (Figure 1B). 7AAD stains DNA in dead and dying cells due to membr...

Discussion

This protocol facilitates isolation of GM-CSF-driven progenitor and precursor cell types in numbers sufficient for several types of analyses including biochemical assays, assays of cellular function in vitro, or instillation in vivo. This method represents a significant advance in the field of monocyte-derived dendritic cell development, enabling the reliable isolation and identification of cells early in this pathway of development as well as those differentiated cell types more commonly isolated in pr...

Materials

Name	Company	Catalog Number	Comments
RPMI 1640	Corning	15-040-CV
Fetal Calf Serum (FCS)	HyClone	SV30014.04	to supplement complete medium and FWB
GlutaMAX	Gibco	35050	to supplement complete medium
2-mercaptoethanol (2-ME)	MP Biomedical	190242	to supplement complete medium
75 mM Vacuum Filter	Thermo Scientific	156-4045	to sterilize complete media
ACK Lysis Buffer	Lonza	10-548E	to lyse red blood cell
HBSS buffer	Corning	21-020-CM	to rescue leukocytes after red blood cell lysis
Phosphate Buffered Saline (PBS), Dulbecco's	Lonza	17-512F	must be endotoxin free; chilled at 4 °C
35 µm Cell filter	Falcon	352235	to break apart clumps before running through cytometer.
GM-CSF	Biosource	PMC2011	usable concentration of 10 ng/mL
Tissue cultured treated plate	VWR	10062-896	for bone marrow cells after harvest
Anti-Ly6C, Clone HK1.4	Biolegend	128018
Anti-CD115, Clone AFS98	Tonbo Bioscience	20-1152-U100
Anti-CD11c, Clone HL3	BD Biosciences	557400	to differeniate CMP and MoDCs
MoFlo XPD Flow Cytometer	Beckman Coulter	ML99030
BD Accuri C6	BD Biosciences	660517
100% Ethanol	Pharmco-Aaper	111000200CSPP
60 mm Petri Dish	Corning, Inc	353002
50 mL Conical tube	VWR	21008-242
C57BL/6 Mice	The Jackson Laboratory	000664	Female; 10-20 weeks old
Biosafety Hood	Thermo Scientific	8354-30-0011
10 mL Syringe	BD Biosciences	301604
23 G needle	BD Biosciences	305145
Centrifuge 5810 R	eppendorf	22625501
FlowJo Software v10	BD Biosciences	Version 10	flowjo.com