Hierarchical Organization of Osteoblasts and Their Impact on Hematopoietic Stem Cell Maintenance and Function.
Authors: Chitteti Brahmananda Reddy , Cheng Ying-Hua , Rodriguez-Rodriguez Sonia , Carlesso Nadia , Kacena Melissa , Srour Edward 
Published: October 2019

Abstract
Abstract 1611
We have previously demonstrated that 2-day calvariae-derived osteoblasts (OB) can significantly enhance the in vitro proliferation and functional capacity of primitive hematopoietic progenitor cells (HPC) and maintain the marrow repopulating potential of hematopoietic stem cells (HSC) thus corroborating the importance of OB in the overall competence of the hematopoietic niche (Chitteti et al, Blood, 2010). While these activities were clearly attributable to OB, the exact definition, both phenotypically and hierarchically, of OB responsible for these functions is yet to be determined. Although some of our data suggested that early stage OB maintained HSC function better than late stage OB, a more precise definition and identification of cells mediating these functions is required for a deeper understanding of the role of OB in sustaining hematopoiesis in the marrow microenvironment. Unlike HSC, the phenotypic definition of different stages of OB development is not fully described and the exact makeup of OB lineage cells responsible for the hematopoiesis enhancing activity is not well characterized. Using flow cytometric cell sorting, we recently began to fractionate calvariae-derived OB to stratify OB lineage cells based on their maturational status and to segregate the hematopoiesis enhancing activity into a phenotypically defined group of cells. Isolated cells were examined by classical OB functional assays (Ca deposition and Alkaline Phosphatase (ALP) activity) and by QRT-PCR quantification of OB-specific lineage markers (Runx-2, osteocalcin, and type I collagen) and were assessed for their hematopoiesis enhancing activity in co-cultures with marrow-derived Lin-Sca1+CD117+ (LSK) cells. LSK cells co-cultured with populations of OB cells were examined for cell proliferation, maintenance of primitive phenotype and expansion of clonogenic cells. Given that limited consensus is that OB lineage cells are Lin- (CD45, CD31, and Ter119) and Sca1-, we separated Lin- Sca1- cells based on their expression of ALCAM, CD51, and osteopontin (OPN). Most Lin-Sca1- cells expressed CD51 such that this marker was deemed dispensable in our quest to sub-fractionate osteoblastic cells. While we were able to identify Lin-Sca1-OPN+ALCAM+ as less mature OB in contrast to the more mature Lin-Sca1-OPN+ALCAM- cells, these fractionations did not compartmentalize the hematopoiesis enhancing activity and both groups of cells had comparable OB functional properties and expressed similar levels of Runx-2 and osteocalcin. We next added CD44 and CD90 to the staining panel and were able to identify four distinct groups of cells: Lin-Sca1-OPN+ALCAM-CD44+CD90- (group 1); Lin-Sca1-OPN+ALCAM-CD44+CD90+ (group 2); Lin-Sca1-OPN+ALCAM+ CD44+CD90- (group 3), and Lin-Sca1-OPN+ALCAM+CD44+CD90+ (group 4). As predicted by the expression of ALCAM, groups 1 and 2 produced the highest amounts of Ca and displayed high ALP activity illustrating that these cells are more mature OB. Interestingly, these two groups had a very low level expression of Runx-2 thus confirming their mature status. In contrast, groups 3 and 4 had very low levels of Ca deposition and ALP activity demonstrating that these cells are less mature. Most importantly, cells from group 4 had the highest level of Runx-2 expression suggesting again that these are less mature cells. Cultured cells from groups 3 and 4 gradually lost ALCAM expression with time suggesting that in vitro proliferation of less mature OB produced more mature cells and demonstrating that these markers can be used to identify classes of mature and immature OB. In co-culture experiments, OB belonging to group 4 sustained the proliferation and production of the highest number of primitive hematopoietic cells and clonogenic progenitors. Other hematopoietic studies including in-vivo repopulating potential of LSK progeny from various OB fractions are underway. These studies begin to define the hierarchical organization of osteoblastic cells and provide a more refined definition of OB that can mediate hematopoiesis enhancing activities.

Disclosures:
No relevant conflicts of interest to declare.