Recent updates on the biological basis of heterogeneity in bone marrow stromal cells/skeletal stem cells

Abstract

Based on studies over the last several decades, the self-renewing skeletal lineages derived from bone marrow stroma could be an ideal source for skeletal tissue engineering. However, the markers for osteogenic precursors; i.e., bone marrowderived skeletal stem cells (SSCs), in association with other cells of the marrow stroma (bone marrow stromal cells, BMSCs) and their heterogeneous nature both in vivo and in vitro remain to be clarified. This review aims to highlight: i) the importance of distinguishing BMSCs/SSCs from other "mesenchymal stem/stromal cells", and ii) factors that are responsible for their heterogeneity, and how these factors impact on the differentiation potential of SSCs towards bone. The prospective role of SSC enrichment, their expansion and its impact on SSC phenotype is explored. Emphasis has also been given to emerging single cell RNA sequencing approaches in scrutinizing the unique population of SSCs within the BMSC population, along with their committed progeny. Understanding the factors involved in heterogeneity may help researchers to improvise their strategies to isolate, characterize and adopt best culture practices and source identification to develop standard operating protocols for developing reproducible stem cells grafts. However, more scientific understanding of the molecular basis of heterogeneity is warranted that may be obtained from the robust high-throughput functional transcriptomics of single cells or clonal populations.

Keywords: bone marrow stromal cells; clonal analysis; heterogeneity; single cell analysis; skeletal stem cells.

Figure 1. Developmental origins of different connective tissues that have been reported to contain “MSCs”. Of note, bone originates from three different embryonic specifications (noted by a white star). Skin, muscle, tendons and ligaments, and bone arise from different specifications of the sclerotome. Bone marrow adipose tissue originates from paraxial mesoderm, lateral plate mesoderm and neural crest. While bone marrow adipose tissue arises from mesoderm and neural crest, other forms of fat originate from all three germ layers. “MSCs” are not a lineage. MSC: mesenchymal stem/stromal cell. Adapted from Bianco and Robey.

Figure 2. Gold standard assays by which to assess differentiation capacity. Many of the currently used “standard” assays of differentiation are prone to artifact or misinterpretation. However, there are assays that can faithfully report differentiation capacity: 1) the in vitro cartilage pellet assay, whereby one can see chondrocytes lying in lacunae surrounded by extracellular matrix that stains purple with toluidine blue, 2) the in vivo transplantation assay whereby donor cells are able to make bone matrix, osteocytes, osteoblasts, and in some cases, support haematopoiesis and formation of marrow adipocytes (the latter two properties are not shared by all forms of skeletal stem cells), and 3) the in vitro myogenic assay, whereby myotubes are formed in the absence of exogenous myoblasts (which will spontaneously fuse with any fibroblastic population). Adapted in part from Sacchetti et al. BM: bone marrow; DAPI: diamidino-2-phenylindole; H&E: hematoxylin and eosin; hp: haematopoiesis; MSC: mesenchymal stem/stromal cell; MU: muscle; MyHC: myosin heavy chain.

Figure 3. Differences in cell morphology (A) and colony size and habit (B) of freshly isolated bone marrow stromal cell suspensions plated at clonal density. When individual colonies with different cell shapes and colony habits are expanded ex vivo and transplanted in vivo, neither parameter correlated with the formation of a bone/marrow organ (a measure of multipotency). Adapted from Satomura et al.

Figure 4. Clonal analysis - an essential step in the determination of stem cell potency. (A) When colonies are allowed to grow beyond the 10-14 days usually used for colony forming efficiency, the individual colonies begin to spontaneously differentiate. The vast majority of the colonies are alkaline phosphatase positive (right panel), indicative of osteogenic and pre-adipogenic cells. Approximately 50% of the colonies were Alizarin Red positive (centre panel) and approximately 10% stained with oil red O (right panel, unpublished data). (B) When individual colonies were isolated, expanded ex vivo, and transplanted subcutaneously into immunocompromised mice with an hydroxyapatite/tricalcium phosphate scaffold, ∼10% of the single colony-derived strains made a complete bone/marrow organ (multipotent), whereas ∼50% formed only bone (unipotent), and the remainder formed only fibrous tissue. Adapted from Sworder et al. (C) In studies where colonies were first incubated with adipogenic medium, colonies that accumulated fat identifiable by inverted light microscopy were marked with a blue circle. When the medium was changed to an osteogenic medium, a number of the adipogenic colonies also became alizarin red positive, indicating that the original CFU-F was able to give rise to adipogenic cells, and then osteogenic cells; an indication of “flexibility” (unpublished data). CFU-F: colony forming units-fibroblast.

Figure 5. Changes in alkaline phosphatase based on position on the cell cycle. It has long been recognized that BMSC/ SSC colonies are heterogeneous with respect to alkaline phosphatase activity (left panel, slide given to PGR by Alexander Friedenstein). Later, it was determined that when cultures of osteogenic cells are synchronized using an amphidicolin protocol, cells in S phase have high levels of activity. During G2 + M phase, alkaline phosphatase is cleaved from the cell surface and released into the medium. Cell surface activity is restored during the following G1 and S phases. Adapted in part from Fedarko et al. BMSC: bone marrow stromal cells; SSC: skeletal stem cell.