Primary Bone Marrow-Derived MSC Subsets with Distinct Wnt Expression Profiles Are Dynamically Distributed During Human Life.
Authors: Maijenburg Marijke , Kleijer Marion , Mul Erik , van Alphen Floris , van der Schoot Ellen , Voermans Carlijn 
Published: October 2019

Abstract
Abstract 2590
Mesenchymal stromal cells (MSC) in bone marrow (BM) consist of a heterogeneous population of cells. MSC orchestrate the BM microenvironment, and therefore have a pivotal role in hematopoietic support. Since MSC produce a large quantity and variety of Wnt proteins, it is likely that Wnts are involved in their hematopoietic support function Recent studies show enrichment for BM-derived MSC by sorting for CD271+ or CD146+ cells. However, it is unclear whether these markers are co-expressed with classical markers MSC markers CD73, CD105 and CD90. In addition mainly adult samples were studied, while it is known that MSC frequency declines with age. Most in-vitro studies with MSC as well as all therapeutic applications have been performed with a heterogeneous population of cultured cells. In this study we first characterized the different MSC subpopulations in BM from 81 donors (adult n=55 mean age 62 (19-82), pediatric n=22 mean age 4 (0-16) and fetal n=6, second trimester) by 6-color FACS analysis. Next we analyzed the subsets with respect to their distribution in different age groups, colony forming ability, and Wnt (target)-gene expression compared to cultured MSC. Three putative MSC subsets were identified in CD45−/dimCD34− cells from adult BM, based on expression of CD271 and/or CD146, CD105 and CD90. These populations were CD271brightCD146−CD105+CD90+, CD271brightCD146+CD105+CD90+ and CD271−CD146+CD105+CD90+, hereafter referred to CD271 single positive (sp) (0.011±0.001% from the mononuclear cell fraction (MNC)), double positive (0.003±0.001% from MNC) and CD146 sp (0.007±0.001%). The largest population of putative MSC in adult BM was CD271 sp (n=55, 55.9±24.3%). In contrast to adult BM, in pediatric BM the dominant population was double positive (n=22, 78.2±20.9%, 0.0115±0.003% MNC count), while the CD271 sp (0.0005±0.001% from MNC) and CD146 sp (0.0005±0.0001% from MNC) populations contained less than 25% of the CD271 and/or CD146+ cells. Remarkably, in fetal BM CD146 sp cells accounted for 35.5±8.8% (0.107±0.004% from MNC) of all presumptive MSC. Double positive cells represented 60.8±8.2% (0.1739±0.025% from MNC). CD271 sp cells, dominant in adults, were nearly absent (0.0115% ±0.004% from MNC)in fetal BM. The proportion of CD271 sp cells in BM was positively correlated with donor age (n=83, R2=0.41, p<0.000). Distribution of double positive cells was inversely correlated (n=83, R2=0.39, p<0.000). These data show that MSC subset composition changes with donor age, with CD271 sp cells dominant in adults and double positive cells dominant in pediatric and fetal BM. For verification of MSC content defined by CFU-F formation, the subpopulations were sorted. In adults CFU-F were observed in CD271 sp and double positive fraction (n=7) but not in CD146 sp cells, except for one donor. These data are in line with other reports that only describe CFU-F in the CD271bright population. In pediatric BM, only the double positive fraction contained CFU-F, whereas in fetal BM both double positive and CD146 sp cells gave rise to CFU-F. Thus not only distribution, but also CFU-F content of MSC subsets differs with donor age. To investigate whether the different subsets defined by our 6 marker FACS analysis might play different roles in hematopoietic support, we investigated by PCR-array the expression of Wnt- and Wnt-related genes. Indeed, clear differences in Wnt-(target)gene expression in the three primary adult MSC subsets were found, which were also quite different from the culture expanded MSC. Wnt5a and the Wnt target genes CCND1 and PP2CA were highly expressed in cultured MSC only. LRP6, FZD7 and Wnt11 were not detected in the CD271sp subset, but strongly expressed by the double positive cells. Thus, primary MSC subsets in adult BM may have different supportive functions through Wnt signaling. In conclusion, our data show that at least three MSC subsets are identified by expression of CD271 and/or CD146. In adults, these subsets have distinct Wnt signaling profiles. Moreover, we show for the first time that distribution of defined stromal cell subsets is significantly correlated with donor age. We hypothesize that MSC may comprise a dynamic system during human life, in which different subpopulations may have different functions during bone marrow development, homeostasis and regeneration.

Disclosures:
No relevant conflicts of interest to declare.