Previously published studies have identified the presence of a rare population of very small cells (< 5.0 microns in diameter) in the bone marrow of mice that are characterized by a gene expression profile consistent with pluripotent embryonic stem cells, termed ‘very small embryonic like’ (VSEL) cells (Leukemia 2006 May;20(5):857-69). Recently, a failed attempt at repeating the published protocol used to isolate VSEL cells from murine bone marrow concluded that “the existence of adult mouse VSELs in the bone marrow remains dubious.” (Stem Cell Reports 2013 Jul 24;1(2):198-208). However, close inspection of the methodology employed in this attempt reveals key missteps that may have led to this conclusion. Accordingly, we repeated the protocol as published to determine whether cells with features consistent with VSEL cells could be isolated (Stem Cells Dev. 2014 Apr 1;23(7):702-13).
Bone marrow was isolated from 5-week female C57BL/6 mice. Following lysis of red blood cells, the bone marrow preparation was incubated with antibodies targeting the following cell surface markers: stem cell antigen 1 (Sca-1), protein tyrosine phosphatase receptor type C (CD45), and lineage (Lin). The labeled sample was subjected to fluorescence activated cell sorting (FACS), using a gating strategy that employs both size (2–10µm) and fluorescence to determine the appropriate gates. Putative VSEL cells (Lin-/Sca-1+/CD45-) and hematopoietic cells (Lin-/Sca-1+/CD45+) were collected for downstream analysis.
Using the FACS gating strategy described above, Lin-/Sca-1+/CD45- cells approximately 3-5 µm in diameter were successfully identified, lending further support to the existence of VSEL cells in bone marrow of mice.