Monocytes, Macrophages, and Their Potential Niches in Synovial Joints – Therapeutic Targets in Post-Traumatic Osteoarthritis?

Synovial joints are complex structures that enable normal locomotion. Following injury, they undergo a series of changes, including a prevalent inflammatory response. This increases the risk for development of osteoarthritis (OA), the most common joint disorder. In healthy joints, macrophages are the predominant immune cells. They regulate bone turnover, constantly scavenge debris from the joint cavity and, together with synovial fibroblasts, form a protective barrier. Macrophages thus work in concert with the non-hematopoietic stroma. In turn, the stroma provides a scaffold as well as molecular signals for macrophage survival and functional imprinting: “a macrophage niche”. These intricate cellular interactions are susceptible to perturbations like those induced by joint injury. With this review, we explore how the concepts of local tissue niches apply to synovial joints. We introduce the joint micro-anatomy and cellular players, and discuss their potential interactions in healthy joints, with an emphasis on molecular cues underlying their crosstalk and relevance to joint functionality. We then consider how these interactions are perturbed by joint injury and how they may contribute to OA pathogenesis. We conclude by discussing how understanding these changes might help identify novel therapeutic avenues with the potential of restoring joint function and reducing post-traumatic OA risk.

Dissecting human embryonic skeletal stem cell ontogeny by single-cell transcriptomic and functional analyses

Human skeletal stem cells (SSCs) have been discovered in fetal and adult long bones. However, the spatiotemporal ontogeny of human embryonic SSCs during early skeletogenesis remains elusive. Here we map the transcriptional landscape of human limb buds and embryonic long bones at single-cell resolution to address this fundamental question. We found remarkable heterogeneity within human limb bud mesenchyme and epithelium, and aligned them along the proximal–distal and anterior–posterior axes using known marker genes. Osteo-chondrogenic progenitors first appeared in the core limb bud mesenchyme, which give rise to multiple populations of stem/progenitor cells in embryonic long bones undergoing endochondral ossification. Importantly, a perichondrial embryonic skeletal stem/progenitor cell (eSSPC) subset was identified, which could self-renew and generate the osteochondral lineage cells, but not adipocytes or hematopoietic stroma. eSSPCs are marked by the adhesion molecule CADM1 and highly enriched with FOXP1/2 transcriptional network. Interestingly, neural crest-derived cells with similar phenotypic markers and transcriptional networks were also found in the sagittal suture of human embryonic calvaria. Taken together, this study revealed the cellular heterogeneity and lineage hierarchy during human embryonic skeletogenesis, and identified distinct skeletal stem/progenitor cells that orchestrate endochondral and intramembranous ossification.

Dissecting human skeletal stem cell ontogeny by single-cell transcriptomic and functional analyses

Human skeletal stem cells (SSCs) have been discovered in fetal and adult bones. However, the spatiotemporal ontogeny of human SSCs during embryogenesis has been elusive. Here we map the transcriptional landscape of human embryonic skeletogenesis at single-cell resolution to address this fundamental question. We found remarkable heterogeneity within human limb bud mesenchyme and epithelium, as well as the earliest osteo-chondrogenic progenitors. Importantly, embryonic SSCs (eSSCs) were found in the perichondrium of human long bones, which self-renew and generate osteochondral lineage cells, but not adipocytes or hematopoietic stroma. eSSCs are marked by the adhesion molecule CADM1 and highly enrich FOXP1/2 transcriptional network. Interestingly, neural crest-derived cells with similar phenotypic markers and transcriptional network were also found in the sagittal suture of human embryonic calvaria. Taken together, this study revealed the cellular heterogeneity and lineage hierarchy during human embryonic skeletogenesis, and identified distinct skeletal stem/progenitor cells that orchestrate endochondral and intramembranous ossification.

Early Growth Response (EGR)-1 Expression Regulates Colony Forming Capacity and Hematopoietic Support Function in Human Primary Bone Marrow Stromal Stem Cells

Bone marrow stromal stem cells (BMSCs) are essential components of the hematopoietic environment. BMSCs play a key role in regulating hematopoiesis and, furthermore, as skeletal stem cells they give rise to osteoblasts, adipocytes, and chondrocytes. However, despite this important role of BMSCs in bone and bone marrow physiology, little is known about how proliferation, differentiation and hematopoietic support functions of BMSCs are regulated. We hypothesized that primary human BMSCs have a distinct transcriptional regulatory system, which control BMSC stem cell properties and biological functions. We have previously reported gene expression profiling of highly-enriched human primary BMSCs (Li et al, Stem Cell Reports, 3(6):965-74, 2014), which demonstrated a substantially higher expression of early growth response 1 (EGR1) in primary cells compared to the non-colony-forming cells and cultured stromal cells, respectively. EGR1 is a member of the immediate early response transcription factor family, which has a function in cell growth, development, and stress responses in many tissues. EGR1 has been previously reported to be important for hematopoietic stem cell (HSC) proliferation and localization (Min et al. Cell Stem Cell, 2(4):380-91, 2008), but its role in non-hematopoietic bone marrow cells has thus far not been investigated. Therefore, we aimed to study the possible role of EGR1 in stroma stem cell proliferation and hematopoietic supporting function. Our data demonstrate that the expression of EGR1 as measured by qPCR was 126 ± 9-fold higher in highly fibroblast colony-forming cells (CFU-F) enriched human primary linneg/CD45neg/CD271pos/CD140aneg bone marrow cells compared to the non-colony forming CD45neg/CD271neg cell population. Furthermore, EGR1 expression in CD271posCD140aneg cells was 3 ± 0.2 -fold higher than in the CD271posCD140apos cell population, which has only minimal CFU-F activity. EGR1 expression decreased dramatically during culture with a more than 30-fold difference between primary and passage one and six cells. Down-regulation of EGR1 expression by shRNA did not affect the multi-differentiation capacity (adipogenic, osteogenic) and surface marker expression profile of BMSCs in vitro compared to controls. However, colony-forming capacity and proliferation was considerably increased in EGR1 knockdown cells, i.e. shRNA- transduced stromal cells produced up to 1.8 ± 0.1-fold more CFU-F compared to controls, whereas CFU-F were virtually absent when assaying EGR1 overexpressing cells. Furthermore, population doubling times were decreased in EGR1 knockdown cells but significantly increased (2.4 ± 0.3-fold) in EGR1 overexpressing cells. These data indicate that EGR1 expression negatively regulates BMSC proliferation and colony-forming capacity. On the other hand, hematopoietic support function was decreased in EGR1 knockdown cells as measured by the production of transplantable CD34posCD90pos HSC in stroma co-culture experiments (4-day serum-free culture supplemented with SCF 25 ng/ml, TPO 25 ng/ml, and FLT3L 25 ng/ml, 1×104 CD34pos seeded cord blood cells). Here, the positive effect of the supporting stroma was neutralized by knockdown of EGR1. Numbers of CD34posCD90pos HSC produced in co-cultures with EGR1 knockdown stroma cells were as low as 1,053 ± 316 compared to 6,100 ± 840 in control co-cultures (scamble control). Without stroma, 840 ± 210 CD34posCD90pos cells were generated from 1×104 seeded CD34pos cells. Furthermore, expansion of CD34posCD90posCB cells was increased in co-cultures with EGR1 overexpressing cells, indicating that EGR1is a positive regulator of hematopoietic stroma support in human BMSC, and confirmatory in-vivo xenotransplantation studies are ongoing. In summary, EGR1 is highly expressed by primary BM-MSC compared with non-colony forming cells and is downregulated during culture. EGR1 expression negatively regulates BMSC proliferation and colony formation while positively regulating hematopoietic stroma support function. Our data thus indicate that EGR1 may act as an important BMSC regulator coordinating the specific functions of BMSC in their different biological contexts. Disclosures No relevant conflicts of interest to declare.