Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Summary

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Abstract

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of "contaminating" progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

Introduction

Monocytes, neutrophils, and dendritic cells (DCs) are myeloid cells that arise from hematopoietic progenitors, primarily in the bone marrow, by a process called myelopoiesis. Common myeloid progenitors (CMPs) have the potential to produce myeloid cells, as well as megakaryocytes and erythrocytes, but not lymphoid cells. Granulocyte-monocyte progenitors (GMPs), which are derived from CMPs, produce granulocytes and monocytes, but have lost megakaryocyte and erythrocyte potential. Monocytes and classical and plasmacytoid DCs (cDCs/pDCs) are also thought to arise from common progenitors known as monocyte-DC progenitors (MDPs), which are produced by CMPs. Gradual restricti....

Protocol

All methods described here were approved by the Institutional Animal Care and Use Committee (IACUC) of Cedars-Sinai Medical Center.

1. Isolation of Mouse Bone Marrow and Preparation of a Single Cell Suspension

1. Euthanize the mouse in accordance with institutional guidelines.
2. Place the euthanized mouse on its back and spray it with 70% ethanol (EtOH). Make a small (3 - 5 mm) incision in each hind limb at the ankle level using sharp, pointed scissors, and pull the skin up towards the body to remove it from the legs and expose the muscles and bones.
3. Remove the muscles (quadriceps, hamstrings, etc.) from ....

Results

Using the protocol described above, it is possible to obtain ~100 million cells (including red blood cells, or ~50 million nucleated cells) from both femurs and tibias (2 legs) of one C57BL/6J mouse (6 - 8 weeks old, male or female). 1 - 2 million Lin^- cells can be isolated per mouse by MACS depletion of Lin⁺ cells.

Each of the 6 myeloid progenitor subsets constitutes ~1 - 4% of the Lin^- cells. L.......

Discussion

The Weissman gating strategy for mouse myeloid progenitor identification¹ has been the gold standard for immunologists and hematologists for nearly 20 years, but it is now apparent that the "CMP" and "GMP" gates are very heterogeneous and more precise gating strategies are needed. The protocol that we have described here permits the identification of oligopotent and lineage-committed subsets in C57BL/6J mice for more precise quantification of specific myeloid progenitors and mappin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Mouse: Wild-type C57BL/6J (CD45.2)	The Jackson Laboratories	Cat#JAX:000664
Lineage Cell Depletion Kit, mouse	Miltenyi Biotec	Cat#130-090-858
Rat anti-mouse CD34 (clone RAM34) FITC	BD Biosciences	Cat#553733
Rat anti-mouse CD16/CD32 (FcγR; clone 93) APC-Cy7	BioLegend	Cat#101327
Rat anti-mouse Ly6A/E (Sca-1; clone 108113) PE-Cy7	BioLegend	Cat#108114
Rat anti-mouse CD117 (c-Kit; clone 2B8) Pacific Blue	BioLegend	Cat#105820
Rat anti-mouse Ly6C (clone HK1.4) PerCP-Cy5.5	BioLegend	Cat#128012
Rat anti-mouse CD115 (clone AFS98) PE	BioLegend	Cat#135506
Rat anti-mouse CD135 (Flt3; clone A2F10.1) APC	BD Biosciences	Cat#560718
CountBright Absolute Counting Beads	Thermo Fisher Scientific	Cat#C36950
AutoMACS Separator	Miltenyi Biotec	N/A	Use the "deplete" program
BD LSRFortessa	BD Biosciences	N/A	5 lasers, 15 colors
BD FACS Aria III cell sorter	BD Biosciences	N/A	5 lasers, 13 colors
FlowJo	FlowJo, LLC	https://www.flowjo.com	For further analysis of the .fcs files