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

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).

Analysis of the developmental stages, kinetics, and phenotypes exhibited by myeloid cells driven by GM-CSF in vitro.

The developmental progression of conventional DC has been quite well defined, yet the developmental pathway of monocyte-derived, GM-CSF-driven DC is less well understood. We addressed this issue by establishing an isolation strategy that identifies five distinct GM-CSF derived cell types. Expression of Ly6C and CD115 (Csf-1R) was used to identify and isolate four populations. One of the populations could be further separated based on CD11c expression, distinguishing five populations. We further defined these cells based on expression of transcription factors and markers of early and later stages of myeloid development. These discreet developmental stages corresponded well with previously defined populations: Common Myeloid Progenitors (CMP), Granulocyte/Macrophage Progenitors (GMP), Monocytes, as well as Monocyte-derived macrophages (moMac) and Monocyte-derived DC (moDC). Finally, within the moMac population we also identified moDC precursor activity (moDP) that could be distinguished from moMac and moDC based on their level of MHC class II expression and developmental plasticity.