CD146 Expression in Primary Bone Marrow MSC Progenitor/Stem Cells Is Dependent On Their In Vivo Location.
Authors: Tormin Ariane , Li Ou , Walsh Stuart , Ehinger Mats , Brune Jan Claas , Scheding Stefan 
Published: October 2019

Abstract
Abstract 251
Primary MSC progenitor/stem cells (MSC-PC/SC) represent only a minute fraction of the bone marrow cells and they give rise to the well-known mesenchymal stromal cells (MSC) in culture. In vivo, MSC-PC/SC are probable constituents of the hematopoietic stem cell niche, thus playing an important role in supporting, maintaining and controlling hematopoiesis. Enrichment of primary MSC progenitors, i.e. CFU-F, has been recently reported based on expression of surface markers such as CD271, CD146, GD2, SSEA4, etc. Based on the expression of CD271 and CD146 on primary MSC (Quirici et al., Exp. Hematol. 2002; Sacchetti et al, Cell, 2007), we have recently identified two BM subpopulations (CD271+/CD146+/ CD45− and CD271+/CD146−/low/ CD45−) that highly enrich for primary MSC-PC/SC (Tormin et al, Blood 2008, 112[11]:843). These two populations contained all assayable CFU-F and both gave rise to typical cultured MSC (expression of standard surface markers, differentiation capacity into adipocytes, osteoblasts, chondrocytes). Interestingly, MSC derived from CD146−/low cells acquired CD146 expression in culture, and we therefore aimed to further investigate whether CD146 expression correlates to functional differences, e.g. stemness, or possibly differences in localization. CD271/CD146 subpopulations were FACS sorted from lineage-depleted BM-MNC. Single cell sorting of CD271/CD146/CD45− cells (n=6) confirmed the results of our prior CFU-F experiments, i.e. high enrichment of CFU-F and multipotency in the two putative stem cell populations. Six-color FACS analysis of primary BM cells showed that both populations coexpressed typical MSC markers (CD90, CD105, PDGFR-β, STRO-1), but not GD2, SSEA4, and endothelial markers. Single cell multiplex PCR on sorted primary MSC-PC/SC showed that both cell populations were negative for CD45, but did express “early” genes (Oct4, Sox2, Nanog), marker genes for the adipogenic lineage (CEBPA, LPIN1) and osteogenesis-related genes (ALPL, Runx2). Gene expression of CD146 correlated to its surface expression with some CD146 bands also detected among the CD146−/low sorted population. Next, we investigated possible differences in localization utilizing confocal microscopy of normal human BM sections. Reticular CD271/CD146 double positive and reticular CD271 single positive cells were identified. Double positive cells were mainly located adjacent to larger vessels and sinusoids but were also found in the marrow space. In contrast, CD271 single positive cells were primarily found in the endosteal space. These cells were furthermore negative for CD45 in contrast to CD45 coexpressing CD271+/low cells in the marrow space. As expected, CD146 single positive endothelial cells were found surrounding larger vessels. Thus, expression of CD146 in CD271+/CD45− cells correlated with localization (primarily endosteal versus primarily perivascular) and we therefore hypothesized that CD146 expression might be regulated by hypoxia levels. To test this, MSC were cultured under normoxic versus hypoxic conditions using deferoxamine mesylate (DFO) to mimic hypoxia. When sorted CD271+/CD146−/low/CD45− cells were cultured in normoxia, CD146 expression was lower compared with cultures initiated with CD271+/CD146+/CD45− cells in the beginning and up to the 1st passage. Thereafter, CD146 expression was comparable. However, when established MSC (CD146+) were cultured in the presence of DFO, CD146 expression was clearly downregulated, and after 7 days about 25% of cells became CD146 negative compared to 3% in normoxic controls. No changes were observed for CD90 and CD271 expression.
Taken together, CD271+/CD146+/CD45– and CD271+/CD146–/low/CD45– bone marrow cells are putative primary MSC stem/progenitor populations. Both are highly enriched for primary MSC progenitors, they have comparable functional characteristics as well as comparable surface marker and gene expression profiles. Differences in CD146 expression correlated to localization and are likely to be caused by differences in oxygen levels. We therefore conclude that CD146 expression allows to distinguish the vary rare (0.19 ± 0.09%) primary endosteal niche MSC (CD271+/CD146−/low/CD45−) from vascular niche MSC (CD271+/CD146+/CD45−, 0.31 ± 0.13% of BM cells).

Disclosures:
No relevant conflicts of interest to declare.