Loss of Notch signaling in skeletal stem cells enhances bone formation with aging.

Skeletal stem and progenitor cells (SSPCs) perform bone maintenance and repair. With age, they produce fewer osteoblasts and more adipocytes leading to a loss of skeletal integrity. The molecular mechanisms that underlie this detrimental transformation are largely unknown. Single-cell RNA sequencing revealed that Notch signaling becomes elevated in SSPCs during aging. To examine the role of increased Notch activity, we deleted Nicastrin, an essential Notch pathway component, in SSPCs in vivo. Middle-aged conditional knockout mice displayed elevated SSPC osteo-lineage gene expression, increased trabecular bone mass, reduced bone marrow adiposity, and enhanced bone repair. Thus, Notch regulates SSPC cell fate decisions, and moderating Notch signaling ameliorates the skeletal aging phenotype, increasing bone mass even beyond that of young mice. Finally, we identified the transcription factor Ebf3 as a downstream mediator of Notch signaling in SSPCs that is dysregulated with aging, highlighting it as a promising therapeutic target to rejuvenate the aged skeleton.

Hox genes are crucial regulators of periosteal stem cell identity.

Periosteal stem and progenitor cells (PSPCs) are major contributors to bone maintenance and repair. Deciphering the molecular mechanisms that regulate their function is crucial for the successful generation and application of future therapeutics. Here, we pinpoint Hox transcription factors as necessary and sufficient for periosteal stem cell function. Hox genes are transcriptionally enriched in periosteal stem cells and their overexpression in more committed progenitors drives reprogramming to a naïve, self-renewing stem cell-like state. Crucially, individual Hox family members are expressed in a location-specific manner and their stem cell-promoting activity is only observed when the Hox gene is matched to the anatomical origin of the PSPC, demonstrating a role for the embryonic Hox code in adult stem cells. Finally, we demonstrate that Hoxa10 overexpression partially restores the age-related decline in fracture repair. Together, our data highlight the importance of Hox genes as key regulators of PSPC identity in skeletal homeostasis and repair.

Rosiglitazone induces adipogenesis of both marrow and periosteum derived mesenchymal stem cells during endochondral fracture healing.

BACKGROUND: Type 2 diabetes mellitus (T2DM) afflicts about six percent of the global population, and these patients suffer from a two-fold increased fracture risk. Thiazolidinediones (TZDs), including rosiglitazone, are commonly used medications in T2DM because they have a low incidence of monotherapy failure. It is known that rosiglitazone is associated with secondary osteoporosis, further increasing the fracture risk in an already susceptible population. However, it is not yet understood how rosiglitazone impacts endochondral bone healing after fracture. The aim of this study is to elucidate how rosiglitazone treatment impacts endochondral fracture healing, and how rosiglitazone influences the differentiation of skeletal stem and progenitor cells from the bone marrow and the periosteum. METHODS: An in-vivo mouse femur fracture model was employed to evaluate differences in fracture healing between mice treated with and without rosiglitazone chow. Fracture healing was assessed with histology and micro computed tomography (µCT). In-vitro assays utilized isolated mouse bone marrow stromal cells and periosteal cells to investigate how rosiglitazone impacts the osteogenic capability and adipogenicity of these cells. RESULTS: The in-vivo mouse femur fracture model showed that fracture callus in mice treated with rosiglitazone had significantly more adipose content than those of control mice that did not receive rosiglitazone. In addition, µCT analysis showed that rosiglitazone treated mice had significantly greater bone volume, but overall greater porosity when compared to control mice. In-vitro experimentation showed significantly less osteogenesis and more adipogenesis in bone marrow derived progenitor cells that were cultured in osteogenic media. In addition, rosiglitazone treatment alone caused significant increases in adipogenesis in both bone marrow and periosteum derived cells. CONCLUSION: Rosiglitazone impairs endochondral fracture healing in mice by increasing adipogenesis and decreasing osteogenesis of both bone marrow and periosteum derived skeletal progenitor cells.

Modulating the systemic and local adaptive immune response after fracture improves bone regeneration during aging.

Tissue injury leads to the well-orchestrated mobilization of systemic and local innate and adaptive immune cells. During aging, immune cell recruitment is dysregulated, resulting in an aberrant inflammatory response that is detrimental for successful healing. Here, we precisely define the systemic and local immune cell response after femur fracture in young and aging mice and identify increased toll-like receptor signaling as a potential culprit for the abnormal immune cell recruitment observed in aging animals. Myd88, an upstream regulator of TLR-signaling lies at the core of this aging phenotype, and local treatment of femur fractures with a Myd88 antagonist in middle-aged mice reverses the aging phenotype of impaired fracture healing, thus offering a promising therapeutic target that could overcome the negative impact of aging on bone regeneration.

Systemic NF-κB-mediated inflammation promotes an aging phenotype in skeletal stem/progenitor cells.

Aging tissues undergo a progressive decline in regenerative potential. This decline in regenerative responsiveness has been attributed to changes in tissue-specific stem cells and their niches. In bone, aged skeletal stem/progenitor cell dysfunction is characterized by decreased frequency and impaired osteogenic differentiation potential. This aging phenotype ultimately results in compromised regenerative responsiveness to injury. The age-associated increase of inflammatory mediators, known as inflamm-aging, has been identified as the main culprit driving skeletal stem cell dysfunction. Here, we utilized a mouse model of parabiosis to decouple aging from inflammation. Using the Nfkb1-/- mouse as a model of inflamm-aging, we demonstrate that a shared systemic circulation between a wild-type and Nfkb1-/- mouse results in an aging phenotype of the wild-type skeletal stem and progenitor cells, shown by CFU-fs and osteogenic and adipogenic differentiation assays. Our findings demonstrate that exposure to an inflammatory secretome results in a phenotype similar to the one observed in aging.

Author Correction: Hox gene expression determines cell fate of adult periosteal stem/progenitor cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Propranolol Reverses Impaired Fracture Healing Response Observed With Selective Serotonin Reuptake Inhibitor Treatment.

Selective serotonin reuptake inhibitors (SSRIs) are one of the most commonly prescribed antidepressants worldwide and recent data show significant impairment of fracture healing after treatment with the SSRI fluoxetine in mice. Here, we provide evidence that the negative effects of SSRIs can be overcome by administration of the beta-blocker propranolol at the time of fracture. First, in vitro experiments established that propranolol does not affect osteogenic differentiation. We then used a murine model of intramembranous ossification to study the potential rescue effect of propranolol on SSRI-induced impaired fracture healing. Micro-CT analysis revealed that fluoxetine treatment resulted in a smaller bony regenerate and that this decrease in bone formation can be overcome by co-treatment with propranolol. We then tested this in a clinically relevant model of endochondral ossification. Fluoxetine-treated mice with a femur fracture were treated with propranolol initiated at the time of fracture, and a battery of analyses demonstrated a reversal of the detrimental effect of fluoxetine on fracture healing in response to propranolol treatment. These experiments show for the first time to our knowledge that the negative effects of SSRIs on fracture healing can be overcome by co-treatment with a beta-blocker. © 2019 American Society for Bone and Mineral Research.

Age-related inflammation triggers skeletal stem/progenitor cell dysfunction.

Aging is associated with impaired tissue regeneration. Stem cell number and function have been identified as potential culprits. We first demonstrate a direct correlation between stem cell number and time to bone fracture union in a human patient cohort. We then devised an animal model recapitulating this age-associated decline in bone healing and identified increased cellular senescence caused by a systemic and local proinflammatory environment as the major contributor to the decline in skeletal stem/progenitor cell (SSPC) number and function. Decoupling age-associated systemic inflammation from chronological aging by using transgenic Nfkb1KO mice, we determined that the elevated inflammatory environment, and not chronological age, was responsible for the decrease in SSPC number and function. By using a pharmacological approach inhibiting NF-κB activation, we demonstrate a functional rejuvenation of aged SSPCs with decreased senescence, increased SSPC number, and increased osteogenic function. Unbiased, whole-genome RNA sequencing confirmed the reversal of the aging phenotype. Finally, in an ectopic model of bone healing, we demonstrate a functional restoration of regenerative potential in aged SSPCs. These data identify aging-associated inflammation as the cause of SSPC dysfunction and provide mechanistic insights into its reversal.

Hox gene expression determines cell fate of adult periosteal stem/progenitor cells.

Hox genes are evolutionarily conserved transcription factors that during embryonic development function as master regulators of positional identity. In postnatal life, the function of Hox proteins is less clear: Hox genes are expressed during tissue repair, but in this context their function(s) are largely unknown. Here we show that Hox genes are expressed in periosteal stem/progenitor cells in a distribution similar to that during embryonic development. Using unbiased sequencing, we established that periosteal stem/progenitor cells from distinct anatomic sites within the skeleton significantly differ in their transcriptome, and that Hox expression status best defines these differences. Lastly, we provide evidence that Hox gene expression is one potential mechanism that maintains periosteal stem/progenitor cells in a more primitive, tripotent state, while suppression of Hox genes leads to fate changes with loss of tripotency. Together, our data describe an adult role of Hox genes other than positional identity, and the modulatory role of Hox genes in fate decisions may offer potential druggable targets for the treatment of fractures, non-unions and bone defects.

Ecto-5'-nucleotidase (CD73) regulates bone formation and remodeling during intramembranous bone repair in aging mice.

Ecto-5'-nucleotidase (CD73) generates adenosine, an osteoblast activator and key regulator of skeletal growth. It is unknown, however, if CD73 regulates osteogenic differentiation during fracture healing in adulthood, and in particular how CD73 activity regulates intramembranous bone repair in the elderly. Monocortical tibial defects were created in 46-52-week-old wild type (WT) and CD73 knock-out mice (CD73-/-) mice. Injury repair was analyzed at post-operative days 5, 7, 14 and 21 by micro-computed tomography (micro-CT), histomorphometry, proliferating cell nuclear antigen (PCNA) immunostaining, alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) histochemistry. Middle-aged CD73 knock-out mice exhibited delayed bone regeneration and significantly reduced bone matrix deposition detected by histomorphometry and micro-CT. Cell proliferation, ALP activity and osteoclast number were reduced in the CD73-/- mice, suggesting a combined defect in bone formation and resorption due the absence of CD73 activity in this model of intramembranous bone repair. Results from this study demonstrate that osteoblast activation through CD73 activity is essential during bone repair in aging mice, and it may present a drugable target for future biomimetic therapeutic approaches that aim at enhancing bone formation in the elderly patients.

Mosaic analysis of cell rearrangements during ureteric bud branching in dissociated/reaggregated kidney cultures and in vivo.

BACKGROUND: Cell rearrangements mediated by GDNF/Ret signaling underlie the formation of the ureteric bud (UB) tip domain during kidney development. Whether FGF signaling also influences these rearrangements is unknown. Chimeric embryos are a powerful tool for examining the genetic controls of cellular behaviors, but generating chimeras by traditional methods is expensive and laborious. Dissociated fetal kidney cells can reorganize to form complex structures including branching UB tubules, providing an easier method to generate renal chimeras. RESULTS: Cell behaviors in normal or chimeric kidney cultures were investigated using time-lapse imaging. In Spry1(-/-) ↔ wild-type chimeras, cells lacking Spry1 (a negative regulator of Ret and FGF receptor signaling) preferentially occupied the UB tips, as previously observed in traditional chimeras, thus validating this experimental system. In Fgfr2(UB-/-) ↔ wild-type chimeras, the wild-type cells preferentially occupied the tips. Independent evidence for a role of Fgfr2 in UB tip formation was obtained using Mosaic mutant Analysis with Spatial and Temporal control of Recombination (MASTR). CONCLUSIONS: Dissociation and reaggregation of fetal kidney cells of different genotypes, with suitable fluorescent markers, provides an efficient way to analyze cell behaviors in chimeric cultures. FGF/Fgfr2 signaling promotes UB cell rearrangements that form the tip domain, similarly to GDNF/Ret signaling.

Generation of Nkx2.2:lacZ mice using recombination-mediated cassette exchange technology.

Nkx2.2 encodes a homeodomain transcription factor required for the correct specification and/or differentiation of cells in the pancreas, intestine, and central nervous system (CNS). To follow the fate of cells deleted for Nkx2.2 within these tissues, we generated Nkx2.2:lacZ knockin mice using a recombination-mediated cassette exchange (RMCE) approach. Expression analysis of lacZ and/or ß-galactosidase in Nkx2.2(lacZ/+) heterozygote embryos and adults demonstrates that lacZ faithfully recapitulates endogenous Nkx2.2 expression. Furthermore, the Nkx2.2(lacZ/lacZ) homozygous embryos display phenotypes indistinguishable from the previously characterized Nkx2.2(-/-) strain. LacZ expression analyses in the Nkx2.2(lacZ/lacZ) homozygous embryos indicate that Nkx2.2-expressing progenitor cells within the pancreas are generated in their normal numbers and are not mislocalized within the pancreatic ductal epithelium or developing islets. In the CNS of Nkx2.2(lacZ/lacZ) embryos, LacZ-expressing cells within the ventral P3 progenitor domain display different migration properties depending on the developmental stage and their respective differentiation potential.