Fibrous periosteum repairs bone fracture and maintains the healed bone throughout mouse adulthood.

Bone is regarded as one of few tissues that heals without fibrous scar. The outer layer of the periosteum is covered with fibrous tissue, whose function in bone formation is unknown. We herein developed a system to distinguish the fate of fibrous-layer periosteal cells (FL-PCs) from the skeletal stem/progenitor cells (SSPCs) in the cambium-layer periosteum and bone marrow in mice. We showed that FL-PCs did not participate in steady-state osteogenesis, but formed the main body of fibrocartilaginous callus during fracture healing. Moreover, FL-PCs invaded the cambium-layer periosteum and bone marrow after fracture, forming neo-SSPCs that continued to maintain the healed bones throughout adulthood. The FL-PC-derived neo-SSPCs expressed lower levels of osteogenic signature genes and displayed lower osteogenic differentiation activity than the preexisting SSPCs. Consistent with this, healed bones were thinner and formed more slowly than normal bones. Thus, the fibrous periosteum becomes the cellular origin of bones after fracture and alters bone properties permanently.

Sertoli cells are the source of stem cell factor for spermatogenesis.

Several cell types have been proposed to create the required microenvironment for spermatogenesis. However, expression patterns of the key growth factors produced by these somatic cells have not been systematically studied and no such factor has been conditionally deleted from its primary source(s), raising the question of which cell type(s) are the physiological sources of these growth factors. Here, using single-cell RNA sequencing and a series of fluorescent reporter mice, we found that stem cell factor (Scf), one of the essential growth factors for spermatogenesis, was broadly expressed in testicular stromal cells, including Sertoli, endothelial, Leydig, smooth muscle and Tcf21-CreER+ stromal cells. Both undifferentiated and differentiating spermatogonia were associated with Scf-expressing Sertoli cells in the seminiferous tubule. Conditional deletion of Scf from Sertoli cells, but not any other Scf-expressing cells, blocked the differentiation of spermatogonia, leading to complete male infertility. Conditional overexpression of Scf in Sertoli cells, but not endothelial cells, significantly increased spermatogenesis. Our data reveal the importance of anatomical localization for Sertoli cells in regulating spermatogenesis and that SCF produced specifically by Sertoli cells is essential for spermatogenesis.

Uncovering the emergence of HSCs in the human fetal bone marrow by single-cell RNA-seq analysis.

During fetal development, human hematopoietic stem cells (HSCs) colonize the bone marrow (BM), where they self-renew and sustain hematopoiesis throughout life; however, the precise timepoint at which HSCs seed the BM is unclear. We used single-cell RNA-sequencing to map the transcriptomic landscape of human fetal BM and spleen hematopoietic stem/progenitor cells (HSPCs) and their microenvironment from 10 to 14 post-conception weeks (PCWs). We further demonstrated that functional HSCs capable of reconstituting long-term multi-lineage hematopoiesis in adult NOG mice do not emerge in the BM until 12 PCWs. In contrast, functional HSCs were not detected in the spleen by 14 PCWs. By comparing the niche-HSPC interactions between BM and spleen, we identified ligand-receptor pairs likely to be involved in fetal HSC migration and maintenance. Our work paves the way for research into the mechanisms underlying HSC colonization in human fetal BM and provides invaluable resources for future studies on HSC development.

Tracing the skeletal progenitor transition during postnatal bone formation.

Multiple distinct types of skeletal progenitors have been shown to contribute to endochondral bone development and maintenance. However, the division of labor and hierarchical relationship between different progenitor populations remain undetermined. Here we developed dual-recombinase fate-mapping systems to capture the skeletal progenitor transition during postnatal bone formation. We showed that postnatal osteoblasts arose primarily from chondrocytes before adolescence and from Lepr+ bone marrow stromal cells (BMSCs) after adolescence. This transition occurred in the diaphysis during adolescence and progressively spread to the metaphysis. The osteoblast-forming Lepr+ BMSCs derived primarily from fetal Col2+ cells. Conditional deletion of Runx2 from perinatal chondrocytes and adult Lepr+ BMSCs impaired bone lengthening and thickening, respectively. Forced running increased osteoblast formation by perinatal chondrocytes but not by adult Lepr+ BMSCs. Thus, the short-term developmental skeletal progenitors generated the long-term adult skeletal progenitors. They sequentially control the growth and maintenance of endochondral bones.

Perivenous Stellate Cells Are the Main Source of Myofibroblasts and Cancer-Associated Fibroblasts Formed After Chronic Liver Injuries.

BACKGROUND AND AIMS: Studies of the identity and pathophysiology of fibrogenic HSCs have been hampered by a lack of genetic tools that permit specific and inducible fate-mapping of these cells in vivo. Here, by single-cell RNA sequencing of nonparenchymal cells from mouse liver, we identified transcription factor 21 (Tcf21) as a unique marker that restricted its expression to quiescent HSCs. APPROACH AND RESULTS: Tracing Tcf21+ cells by Tcf21-CreER (Cre-Estrogen Receptor fusion protein under the control of Tcf21 gene promoter) targeted ~10% of all HSCs, most of which were located at periportal and pericentral zones. These HSCs were quiescent under steady state but became activated on injuries, generating 62%-67% of all myofibroblasts in fibrotic livers and ~85% of all cancer-associated fibroblasts (CAFs) in liver tumors. Conditional deletion of Transforming Growth Factor Beta Receptor 2 (Tgfbr2) by Tcf21-CreER blocked HSC activation, compromised liver fibrosis, and inhibited liver tumor progression. CONCLUSIONS: In conclusion, Tcf21-CreER-targeted perivenous stellate cells are the main source of myofibroblasts and CAFs in chronically injured livers. TGF-ß signaling links HSC activation to liver fibrosis and tumorigenesis.

C-KIT Expression Distinguishes Fetal from Postnatal Skeletal Progenitors.

Hematopoietic stem cells (HSCs) and skeletal stem cells (SSCs) cohabit in the bone marrow. KITL (C-KIT ligand) from LEPR+ adult bone marrow stromal cells is pivotal for HSC maintenance. In contrast, it remains unclear whether KITL/C-KIT signaling also regulates SSCs. Here, we lineage traced C-KIT+ cells and found that C-KIT was expressed by fetal, but not postnatal skeletal progenitors. Fetal C-KIT+ cells gave rise to 20% of LEPR+ stromal cells in adult bone marrow, forming nearly half of all osteoblasts. Disruption of mTOR signaling in fetal C-KIT+ cells impaired bone formation. Notably, conditional deletion of Kitl from PRX1+ fetal bone marrow stromal cells, but not LEPR+ adult bone marrow stromal cells, significantly increased bone formation. Thus, our work identified C-KIT+ skeletal progenitors as an important source of bones formed during development.