Novel skeletal stem and progenitor cells and their regulation

Abstract

The development of bone and cartilage, which provide the framework of the skeletal system, is complex. In this context stem and progenitor cells play a crucial role by producing differentiated cells. Hormones are important regulators of this process, with growth hormone (GH) acting as the major endocrine regulator of longitudinal bone growth. In the present thesis, I have studied primarily the stem and progenitor cells in the skeletal system, as well as their regulation by GH.

During embryonic development, all chondrocytes and bone-forming cells originate from mesodermal or neural crest cells (NCCs). As direct descendants of the NCCs, multipotent Schwann cell precursors (SCPs) generate a variety of cell and tissue types. Employing lineage tracing, we demonstrated that during embryonic development SCPs contribute to the production of significant numbers of skeletogenic cells in bone and cartilage. These SCPs detach from the nerve fibers to become mesenchymal-type cells, which later differentiate into chondrocytes and osteocytes in the skeleton of the craniofacial region and scapula and ribs of the trunk. Our similar observations in zebrafish indicate that this process has been conserved evolutionarily. In summary, this work revealed a novel source of skeletogenic cells, as well as potential interaction between the neurological and skeletal systems during development. (Paper I)

Clonal lineage tracing with multicolor reporter mouse strains, including Confetti mice, provides a powerful tool for the study of cell behavior in vivo. However, it is challenging to maintain the fluorescent signals until they can be analyzed, especially in the case of mineralized tissues. Accordingly, we optimized a protocol that employs the Confetti model to preserve the fluorescent signals in postnatal bone tissues and visualize these directly by confocal microscopy, without additional use of antibodies. (Paper II)

During development, endochondral bone formation is driven primarily by a continuous supply of chondrocytes provided by the growth plate. Employing lineage tracing in multicolor reporter mice, we demonstrated that during fetal and neonatal development, the production of chondrocytes involves consumption of their progenitors, while at a later postnatal stage, these cells acquire the capacity for self-renewal. These findings indicate the formation of a novel stem cell niche that harbors the stem cells and facilitates their renewal. Moreover, we could show that the formation and maintenance of this niche depends to a large extent on formation of the SOC. We also found that the switch between symmetrical and asymmetrical division of these stem cells, hereafter referred to as epiphyseal stem cells (epSCs), is regulated by the mTORC1 signaling pathway. (Paper III)

Although GH is the primary endocrine regulator of longitudinal bone growth, the effects of this hormone on our newly identified epiphyseal stem cells and their niche were unknown. Using lineage tracing, we discovered that the renewal of epSCs in the growth plate involves population asymmetry and neutral competition. GH activated the JAK-STAT pathway in epSCs and the number of these cells was reduced during continuous treatment with GH. Further analysis revealed that GH stimulates epSCs to leave their niche and generate progeny. Single-cell RNA sequencing (scRNA-seq) of all cells within the growth plate further confirmed this shift of epSCs toward differentiation into transit-amplifying cells and, at the same time, revealed that several molecular pathways are involved in the regulation of epSCs by GH, including local BMP, IGF and GAS signaling. (Paper IV)

Altogether, my findings demonstrate that during development Schwann cell precursors generate chondro- and osteo-progenitors. I developed a protocol for multi-color clonal lineage tracing in mineralized tissues and applied this protocol to discover a novel stem cell niche within the epiphyseal growth plate. Finally, I characterized the dynamics and regulation of the stem cells by GH within this novel niche.