Targeting Bone Marrow Mesenchymal Stromal Cells Using Cre-Recombinase Transgenes

The bone marrow microenvironment contains hematopoietic niches that regulate the proliferation, differentiation, and trafficking of hematopoietic stem/progenitors cells (HSPCs).  These hematopoietic niches are comprised of a heterogeneous population of stromal cells that include, endothelial cells, osteoblasts, CXCL12-abundant reticular (CAR) cells, mesenchymal stem cells (MSCs), arteriolar pericytes, and sympathetic nerves.  Emerging data suggest that specific stromal populations may regulate distinct types of HPSCs.  Thus, it is important to have validated approaches to interrogate and target specific stromal cell populations.  Prior studies have shown that Prx1-Cre, Osx-Cre, Lepr-Cre, and Nes-Cre broadly target mesenchymal stromal cells in the bone marrow.  Here, we rigorously define the stromal cell populations targeted by two Cre-transgenes that are commonly used to target osteolineage cells (Ocn-Cre, and Dmp1-Cre) and introduce a new Cre-transgene (Tagln-Cre) that efficiently targets bone marrow pericytes.  For each Cre-transgene, we performed lineage mapping using ROSA26^Ai9/Ai9 mice, in which cells that have undergone Cre-mediated recombination express tdTomato.  In some cases, we further crossed these mice to introduce the Cxcl12^gfp transgene, which can be used to define GFP-bright CAR cells.  Immunostaining of bone sections and flow cytometry were used to define the target stromal cell population(s) in these mice.

            Osteocalcin (Bglap, Ocn) is primarily expressed in mature osteoblasts.  Accordingly, Ocn-Cre is widely used to specifically target osteoblasts.  However, our lineage mapping studies show that Ocn-Cre targets not only all osteoblasts, but also 72 ± 4.0% of CAR cells.  Ocn-Cre also targets a subset of NG2⁺ arteriolar pericytes.  Dentin matrix acidic phosphoprotein 1 (Dmp1) is expressed primarily in osteocytes, and Dmp1-Cre has been widely used to specifically target osteocytes.  However, we show that Dmp1-Cre also efficiently targets endosteal osteoblasts and approximately 40% of CAR cells.  To target bone marrow pericytes, we tested several Cre-transgenes, ultimately focusing on Tagln-Cre.  Transgelin (Tagln, SM22a) is broadly expressed in pericytes, smooth muscle cells, and cardiomyocytes.   Lineage-mapping studies show that Tagln-Cre targets all arteriolar and venous sinusoidal pericytes in the bone marrow.  It also targets osteoblasts and 75 ± 5.2% of CAR cells.

            There are several recent studies that have ascribed specific functions to osteoblasts or osteocytes based on targeting using Ocn-Cre or Dmp1-Cre, respectively.  In light of our data, these conclusions need to be re-evaluated.  Ocn-Cre, Dmp1-Cre, and Tagln-Cre each target a subset of CAR cells.  Studies are underway to determine whether these CAR subsets have unique expression profiles and functions.  Finally, Talgn-Cre represents a new tool for investigators in the field to efficiently target bone marrow pericytes.