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Confirmation and Characterization of Lin‐/Sca‐1+/CD45‐ Very Small Embryonic‐Like Cells (VSELs) Purified from Bone Marrow
Author(s) -
Shin Ellie,
Truman Alisha,
Woods Dori,
Tilly Jonathan
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.559.48
Subject(s) - bone marrow , embryonic stem cell , stem cell , cell sorting , induced pluripotent stem cell , bone marrow stem cell , biology , microbiology and biotechnology , population , immunology , pathology , medicine , flow cytometry , genetics , gene , environmental health
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 ( Leukemia 2006 May;20(5):857‐69). In addition to size, these cells are characterized by a gene expression profile consistent with pluripotent embryonic stem cells and can differentiate into multiple cell lineages. While the discovery of these cells, termed 'very small embryonic like' (VSEL) cells, generated much enthusiasm in the field, it was not without controversy. Most recently, a failed attempt at repeating the published protocol that was 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 flushed from the tibiae and femurs of 10 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. Source of Research Support: Schafer Co‐op Research Scholarship