Isolation of murine bone marrow derived mesenchymal stem cells using Twist2 Cre transgenic mice

While human bone marrow derived mesenchymal stem cells (BMSCs) are of great interest for their potential therapeutic value, their murine equivalent remains an important basic research model that can provide critical insights into the biology of this progenitor cell population. Here we present a novel transgenic strategy that allowed for the selective identification and isolation of murine BMSCs at the early stages of stromal cell culture. This strategy involved crossing Twist2 -Cre mice with Cre reporter mice such as Z/EG or Ai9, which express EGFP or Tomato fluorescent protein, respectively, upon Cre mediated excision of a stop sequence. Using this approach, we identified an adherent fluorescent protein+cell population (T2C+) that is present during the earliest stages of colony formation and by day 5 of culture represents ~20% of the total cell population. Cell surface profiling by flow cytometry showed that T2C+cells are highly positive for SCA1 and CD29 and negative for CD45, CD117, TIE2, and TER119. Isolation of T2C+cells by FACS selected for a cell population with skeletal potential that can be directed to differentiate into osteoblasts, adipocytes, or chondrocytes. We also demonstrated in a calvarial bone defect model that T2C+cells retain a strong efficacy for osteogenic repair and can support a hematopoietic environment. Collectively, these studies provide evidence that the Twist2-Cre x Cre reporter breeding strategy can be used to positively identify and isolate multipotent murine BMSCs.