Molars to Medicine: A Focused Review on the Pre-Clinical Investigation and Treatment of Secondary Degeneration following Spinal Cord Injury Using Dental Stem Cells.

Spinal cord injury (SCI) can result in the permanent loss of mobility, sensation, and autonomic function. Secondary degeneration after SCI both initiates and propagates a hostile microenvironment that is resistant to natural repair mechanisms. Consequently, exogenous stem cells have been investigated as a potential therapy for repairing and recovering damaged cells after SCI and other CNS disorders. This focused review highlights the contributions of mesenchymal (MSCs) and dental stem cells (DSCs) in attenuating various secondary injury sequelae through paracrine and cell-to-cell communication mechanisms following SCI and other types of neurotrauma. These mechanistic events include vascular dysfunction, oxidative stress, excitotoxicity, apoptosis and cell loss, neuroinflammation, and structural deficits. The review of studies that directly compare MSC and DSC capabilities also reveals the superior capabilities of DSC in reducing the effects of secondary injury and promoting a favorable microenvironment conducive to repair and regeneration. This review concludes with a discussion of the current limitations and proposes improvements in the future assessment of stem cell therapy through the reporting of the effects of DSC viability and DSC efficacy in attenuating secondary damage after SCI.

Impact of Environmental and Epigenetic Changes on Mesenchymal Stem Cells during Aging.

Many crucial epigenetic changes occur during early skeletal development and throughout life due to aging, disease and are heavily influenced by an individual's lifestyle. Epigenetics is the study of heritable changes in gene expression as the result of changes in the environment without any mutation in the underlying DNA sequence. The epigenetic profiles of cells are dynamic and mediated by different mechanisms, including histone modifications, non-coding RNA-associated gene silencing and DNA methylation. Given the underlining role of dysfunctional mesenchymal tissues in common age-related skeletal diseases such as osteoporosis and osteoarthritis, investigations into skeletal stem cells or mesenchymal stem cells (MSC) and their functional deregulation during aging has been of great interest and how this is mediated by an evolving epigenetic landscape. The present review describes the recent findings in epigenetic changes of MSCs that effect growth and cell fate determination in the context of aging, diet, exercise and bone-related diseases.

A senescence stress secretome is a hallmark of therapy-related myeloid neoplasm stromal tissue occurring soon after cytotoxic exposure.

Therapy-related myeloid neoplasm (tMN) is considered a direct consequence of DNA damage in hematopoietic stem cells. Despite increasing recognition that altered stroma can also drive leukemogenesis, the functional biology of the tMN microenvironment remains unknown. We performed multiomic (transcriptome, DNA damage response, cytokine secretome and functional profiling) characterization of bone marrow stromal cells from tMN patients. Critically, we also compared (i) patients with myeloid neoplasm and another cancer but without cytotoxic exposure, (ii) typical primary myeloid neoplasm, and (iii) age-matched controls to decipher the microenvironmental changes induced by cytotoxics vs. neoplasia. Strikingly, tMN exhibited a profoundly senescent phenotype with induction of CDKN1A and ß-Galactosidase, defective phenotype, and proliferation. Moreover, tMN stroma showed delayed DNA repair and defective adipogenesis. Despite their dormant state, tMN stromal cells were metabolically highly active with a switch toward glycolysis and secreted multiple pro-inflammatory cytokines indicative of a senescent-secretory phenotype that inhibited adipogenesis. Critically, senolytics not only eliminated dormant cells, but also restored adipogenesis. Finally, sequential patient sampling showed senescence phenotypes are induced within months of cytotoxic exposure, well prior to the onset of secondary cancer. Our data underscores a role of senescence in the pathogenesis of tMN and provide a valuable resource for future therapeutics.

Craniomaxillofacial morphology in a murine model of ephrinB1 conditional deletion in osteoprogenitor cells.

OBJECTIVE: EFNB1 mutation causes craniofrontonasal dysplasia (CFND), a congenital syndrome associated with craniomaxillofacial anomalies characterised by coronal craniosynostosis, orbital hypertelorism, and midface dysplasia. The aim of this murine study was to investigate the effect of the EfnB1 conditional gene deletion in osteoprogenitor cells on the craniomaxillofacial skeletal morphology. DESIGN: The skulls of male and female mice, in which EfnB1 was deleted by Cre (a site-specific DNA recombinase) under the control of the Osterix (Osx) promoter (EfnB1OB-/-), were compared to those without EfnB1 deletion (Osx:Cre control) at two ages (4 and 8 weeks; n = 6 per group). The three-dimensional micro-computed tomography reconstructions were prepared to calculate 17 linear measurements in the cranial vault (brain box), midface and mandible. Coronal and sagittal sutures from the 8-week-old mice were also subjected to histological examination. RESULTS: EfnB1OB-/- mice displayed significantly larger cranial height, larger interorbital and nasal widths, smaller maxillary width than controls by 8 weeks (p < 0.05), but mandibular size was not significantly different (p > 0.05). Binomial testing showed significantly smaller EfnB1OB-/- skulls at 4 weeks but larger at 8 weeks (p < 0.05). Histological examination revealed increased bony fusion and fibrous connective tissue deposition at the coronal suture of EfnB1OB-/- mice compared with controls. CONCLUSIONS: Craniofacial phenotype of the murine model of EfnB1 deletion in osteoprogenitor cells partially represents the human CFND phenotype, with implications for better understanding mechanisms involved in skeletal morphogenesis and malocclusion.

Generation of two multipotent mesenchymal progenitor cell lines capable of osteogenic, mature osteocyte, adipogenic, and chondrogenic differentiation.

Mesenchymal progenitors differentiate into several tissues including bone, cartilage, and adipose. Targeting these cells in vivo is challenging, making mesenchymal progenitor cell lines valuable tools to study tissue development. Mesenchymal stem cells (MSCs) can be isolated from humans and animals; however, obtaining homogenous, responsive cells in a reproducible fashion is challenging. As such, we developed two mesenchymal progenitor cell (MPC) lines, MPC1 and MPC2, generated from bone marrow of male C57BL/6 mice. These cells were immortalized using the temperature sensitive large T-antigen, allowing for thermal control of proliferation and differentiation. Both MPC1 and MPC2 cells are capable of osteogenic, adipogenic, and chondrogenic differentiation. Under osteogenic conditions, both lines formed mineralized nodules, and stained for alizarin red and alkaline phosphatase, while expressing osteogenic genes including Sost, Fgf23, and Dmp1. Sost and Dmp1 mRNA levels were drastically reduced with addition of parathyroid hormone, thus recapitulating in vivo responses. MPC cells secreted intact (iFGF23) and C-terminal (cFGF23) forms of the endocrine hormone FGF23, which was upregulated by 1,25 dihydroxy vitamin D (1,25D). Both lines also rapidly entered the adipogenic lineage, expressing adipose markers after 4 days in adipogenic media. MPC cells were also capable of chondrogenic differentiation, displaying increased expression of cartilaginous genes including aggrecan, Sox9, and Comp. With the ability to differentiate into multiple mesenchymal lineages and mimic in vivo responses of key regulatory genes/proteins, MPC cells are a valuable model to study factors that regulate mesenchymal lineage allocation as well as the mechanisms that dictate transcription, protein modification, and secretion of these factors.

Eph-Ephrin Signaling Mediates Cross-Talk Within the Bone Microenvironment.

Skeletal integrity is maintained through the tightly regulated bone remodeling process that occurs continuously throughout postnatal life to replace old bone and to repair skeletal damage. This is maintained primarily through complex interactions between bone resorbing osteoclasts and bone forming osteoblasts. Other elements within the bone microenvironment, including stromal, osteogenic, hematopoietic, endothelial and neural cells, also contribute to maintaining skeletal integrity. Disruption of the dynamic interactions between these diverse cellular systems can lead to poor bone health and an increased susceptibility to skeletal diseases including osteopenia, osteoporosis, osteoarthritis, osteomalacia, and major fractures. Recent reports have implicated a direct role for the Eph tyrosine kinase receptors and their ephrin ligands during bone development, homeostasis and skeletal repair. These membrane-bound molecules mediate contact-dependent signaling through both the Eph receptors, termed forward signaling, and through the ephrin ligands, referred to as reverse signaling. This review will focus on Eph/ ephrin cross-talk as mediators of hematopoietic and stromal cell communication, and how these interactions contribute to blood/ bone marrow function and skeletal integrity during normal steady state or pathological conditions.

Ageing human bone marrow mesenchymal stem cells have depleted NAD(P)H and distinct multispectral autofluorescence.

Stem cell exhaustion plays a major role in the ageing of different tissues. Similarly, in vitro cell ageing during expansion prior to their use in regenerative medicine can severely compromise stem cell quality through progressive declines in differentiation and growth capacity. We utilized non-destructive multispectral assessment of native cell autofluorescence to investigate the metabolic mechanisms of in vitro mesenchymal stem cell (MSC) ageing in human bone marrow MSCs over serial passages (P2-P10). The spectral signals for NAD(P)H, flavins and protein-bound NAD(P)H were successfully isolated using Robust Dependent Component Analysis (RoDECA). NAD(P)H decreased over the course of hMSC ageing in absolute terms as well as relative to flavins (optical redox ratio). Relative changes in other fluorophore levels (flavins, protein-bound NAD(P)H) suggested that this reduction was due to nicotinamide adenine dinucleotide depletion rather than a metabolic shift from glycolysis to oxidative phosphorylation. Using multispectral features, which are determined without cell fixation or fluorescent labelling, we developed and externally validated a reliable, linear model which could accurately categorize the age of culture-expanded hMSCs. The largest shift in spectral characteristics occurs early in hMSC ageing. These findings demonstrate the feasibility of applying multispectral technology for the non-invasive monitoring of MSC health in vitro.

Clinical Application of Bone Marrow Mesenchymal Stem/Stromal Cells to Repair Skeletal Tissue.

There has been an escalation in reports over the last decade examining the efficacy of bone marrow derived mesenchymal stem/stromal cells (BMSC) in bone tissue engineering and regenerative medicine-based applications. The multipotent differentiation potential, myelosupportive capacity, anti-inflammatory and immune-modulatory properties of BMSC underpins their versatile nature as therapeutic agents. This review addresses the current limitations and challenges of exogenous autologous and allogeneic BMSC based regenerative skeletal therapies in combination with bioactive molecules, cellular derivatives, genetic manipulation, biocompatible hydrogels, solid and composite scaffolds. The review highlights the current approaches and recent developments in utilizing endogenous BMSC activation or exogenous BMSC for the repair of long bone and vertebrae fractures due to osteoporosis or trauma. Current advances employing BMSC based therapies for bone regeneration of craniofacial defects is also discussed. Moreover, this review discusses the latest developments utilizing BMSC therapies in the preclinical and clinical settings, including the treatment of bone related diseases such as Osteogenesis Imperfecta.

Pharmacological targeting of KDM6A and KDM6B, as a novel therapeutic strategy for treating craniosynostosis in Saethre-Chotzen syndrome.

BACKGROUND: During development, excessive osteogenic differentiation of mesenchymal progenitor cells (MPC) within the cranial sutures can lead to premature suture fusion or craniosynostosis, leading to craniofacial and cognitive issues. Saethre-Chotzen syndrome (SCS) is a common form of craniosynostosis, caused by TWIST-1 gene mutations. Currently, the only treatment option for craniosynostosis involves multiple invasive cranial surgeries, which can lead to serious complications. METHODS: The present study utilized Twist-1 haploinsufficient (Twist-1del/+) mice as SCS mouse model to investigate the inhibition of Kdm6a and Kdm6b activity using the pharmacological inhibitor, GSK-J4, on calvarial cell osteogenic potential. RESULTS: This study showed that the histone methyltransferase EZH2, an osteogenesis inhibitor, is downregulated in calvarial cells derived from Twist-1del/+ mice, whereas the counter histone demethylases, Kdm6a and Kdm6b, known promoters of osteogenesis, were upregulated. In vitro studies confirmed that siRNA-mediated inhibition of Kdm6a and Kdm6b expression suppressed osteogenic differentiation of Twist-1del/+ calvarial cells. Moreover, pharmacological targeting of Kdm6a and Kdm6b activity, with the inhibitor, GSK-J4, caused a dose-dependent suppression of osteogenic differentiation by Twist-1del/+ calvarial cells in vitro and reduced mineralized bone formation in Twist-1del/+ calvarial explant cultures. Chromatin immunoprecipitation and Western blot analyses found that GSK-J4 treatment elevated the levels of the Kdm6a and Kdm6b epigenetic target, the repressive mark of tri-methylated lysine 27 on histone 3, on osteogenic genes leading to repression of Runx2 and Alkaline Phosphatase expression. Pre-clinical in vivo studies showed that local administration of GSK-J4 to the calvaria of Twist-1del/+ mice prevented premature suture fusion and kept the sutures open up to postnatal day 20. CONCLUSION: The inhibition of Kdm6a and Kdm6b activity by GSK-J4 could be used as a potential non-invasive therapeutic strategy for preventing craniosynostosis in children with SCS. Pharmacological targeting of Kdm6a/b activity can alleviate craniosynostosis in Saethre-Chotzen syndrome. Aberrant osteogenesis by Twist-1 mutant cranial suture mesenchymal progenitor cells occurs via deregulation of epigenetic modifiers Ezh2 and Kdm6a/Kdm6b. Suppression of Kdm6a- and Kdm6b-mediated osteogenesis with GSK-J4 inhibitor can prevent prefusion of cranial sutures.

Epigenetic Regulators of Mesenchymal Stem/Stromal Cell Lineage Determination.

PURPOSE OF REVIEW: Although many signalling pathways have been discovered to be essential in mesenchymal stem/stromal (MSC) differentiation, it has become increasingly clear in recent years that epigenetic regulation of gene transcription is a vital component of lineage determination, encompassing diet, lifestyle and parental influences on bone, fat and cartilage development. RECENT FINDINGS: This review discusses how specific enzymes that modify histone methylation and acetylation or DNA methylation orchestrate the differentiation programs in lineage determination of MSC and the epigenetic changes that facilitate development of bone related diseases such as osteoporosis. The review also describes how environmental factors such as mechanical loading influence the epigenetic signatures of MSC, and how the use of chemical agents or small peptides can regulate epigenetic drift in MSC populations during ageing and disease. Epigenetic regulation of MSC lineage commitment is controlled through changes in enzyme activity, which modifies DNA and histone residues leading to alterations in chromatin structure. The co-ordinated epigenetic regulation of transcriptional activation and repression act to mediate skeletal tissue homeostasis, where deregulation of this process can lead to bone loss during ageing or osteoporosis.

HOPX regulates bone marrow-derived mesenchymal stromal cell fate determination via suppression of adipogenic gene pathways.

Previous studies of global binding patterns identified the epigenetic factor, EZH2, as a regulator of the homeodomain-only protein homeobox (HOPX) gene expression during bone marrow stromal cell (BMSC) differentiation, suggesting a potential role for HOPX in regulating BMSC lineage specification. In the present study, we confirmed that EZH2 direct binds to the HOPX promoter region, during normal growth and osteogenic differentiation but not under adipogenic inductive conditions. HOPX gene knockdown and overexpression studies demonstrated that HOPX is a promoter of BMSC proliferation and an inhibitor of adipogenesis. However, functional studies failed to observe any affect by HOPX on BMSC osteogenic differentiation. RNA-seq analysis of HOPX overexpressing BMSC during adipogenesis, found HOPX function to be acting through suppression of adipogenic pathways associated genes such as ADIPOQ, FABP4, PLIN1 and PLIN4. These findings suggest that HOPX gene target pathways are critical factors in the regulation of fat metabolism.

The changing epigenetic landscape of Mesenchymal Stem/Stromal Cells during aging.

There is mounting evidence in the literature that mesenchymal stromal/stem cell (MSC) like populations derived from different tissues, undergo epigenetic changes during aging, leading to compromised connective tissue integrity and function. This body of work has linked the biological aging of MSC to changes in their epigenetic signatures affecting growth, lifespan, self-renewal and multi-potential, due to deregulation of processes such as cellular senescence, oxidative stress, DNA damage, telomere shortening and DNA damage. This review addresses recent findings examining DNA methylation, histone modifications and miRNA changes in aging MSC populations. Moreover, we explore how epigenetic factors alter cellular pathways and associated biological networks, contributing to the MSC aging phenotype. Finally we discuss the crucial areas requiring a greater understanding of these processes, in order to piece together a global picture of the changing epigenetic landscape in MSC during aging.

CMTM8 Is a Suppressor of Human Mesenchymal Stem Cell Osteogenic Differentiation and Promoter of Proliferation Via EGFR Signaling.

Multipotent bone marrow-derived mesenchymal stem/stromal cells (BMSCs) exhibit a finite life span after ex vivo expansion leading to cellular senescence. Many factors can contribute to this. Recently, our group has identified for the first time expression of the chemokine-like factor superfamily 8 (CMTM8) gene in cultured human BMSCs. In this study, we examine the role of CMTM8 in BMSC proliferation, migration, and differentiation. Functional studies using siRNA-mediated knockdown of CMTM8 in human BMSCs resulted in decreased capacity to undergo proliferation and migration and an increased capacity for osteogenic differentiation in vitro. Furthermore, reduced CMTM8 levels led to a decrease in the epidermal growth factor receptor (EGFR) signaling pathway during BMSC proliferation and migration, respectively. Supportive studies using retroviral mediated enforced expression of CMTM8 in BMSC resulted in an increased capacity for proliferation and migration but a decreased osteogenic differentiation potential. Collectively, these data suggest that CMTM8 promotes BMSC proliferation and BMSC migration through the EGFR/ERK1/2 pathway. This study provides insight into novel regulatory mechanisms of human BMSC growth and cell fate determination.

Twist-1 is upregulated by NSD2 and contributes to tumour dissemination and an epithelial-mesenchymal transition-like gene expression signature in t(4;14)-positive multiple myeloma.

Approximately 15% of patients with multiple myeloma (MM) harbour the t(4;14) chromosomal translocation, leading to the overexpression of the histone methyltransferase NSD2. Patients with this translocation display increased tumour dissemination, accelerated disease progression and rapid relapse. Using publicly available gene expression profile data from NSD2high (n = 135) and NSD2low (n = 878) MM patients, we identified 39 epithelial-mesenchymal transition (EMT)-associated genes which are overexpressed in NSD2high MM plasma cells. In addition, our analyses identified Twist-1 as a key transcription factor upregulated in NSD2high MM patients and t(4;14)-positive cell lines. Overexpression and knockdown studies confirmed that Twist-1 is involved in driving the expression of EMT-associated genes in the human MM cell line KMS11 and promoted the migration of myeloma cell lines in vitro. Notably, Twist-1 overexpression in the mouse MM cell line 5TGM1 significantly increased tumour dissemination in an intratibial tumour model. These findings demonstrate that Twist-1, downstream of NSD2, contributes to the induction of an EMT-like signature in t(4;14)-positive MM and enhances the dissemination of MM plasma cells in vivo, which may, in part, explain the aggressive disease features associated with t(4;14)-positive MM.

Stem cell-directed therapies for osteoarthritis: The promise and the practice.

Osteoarthritis (OA) is a disease of an entire synovial joint characterized by clinical symptoms and distortion of joint tissues, including cartilage, muscles, ligaments, and bone. Although OA is a disease of all joint tissues, it is a defined accessible compartment and is thus amenable to topical surgical and regenerative therapies, including stem cells. All tissues arise from stem progenitor cells, and the relative capacity of different cellular compartments, and different individuals, to renew tissues into adulthood may be important in the onset of many different degenerative diseases. OA is driven by both mechanical and inflammatory factors, but how these factors affect the proliferation and differentiation of cells into cartilage in vivo is largely unknown. Indeed, our very basic understanding of the physiological cellular kinetics and biology of the stem-progenitor cell unit of the articular cartilage, and how this is influenced by mechano-inflammatory injury, is largely unknown. OA seems, rather deceptively, to be the low-hanging fruit for stem cell therapy. Without the basic understanding of the stem cell and progenitor unit that generate and maintain articular cartilage in vivo, we will continue to waste opportunities to both prevent and manage this disease. In this review, we discuss the biology of chondrogenesis, the stem cell populations that support articular cartilage in health and disease, and future opportunities afforded through the translation of basic articular chondrocyte stem cell biology into new clinical therapies.

Non-destructive, label free identification of cell cycle phase in cancer cells by multispectral microscopy of autofluorescence.

BACKGROUND: Cell cycle analysis is important for cancer research. However, available methodologies have drawbacks including limited categorisation and reliance on fixation, staining or transformation. Multispectral analysis of endogenous cell autofluorescence has been shown to be sensitive to changes in cell status and could be applied to the discrimination of cell cycle without these steps. METHODS: Cells from the MIA-PaCa-2, PANC-1, and HeLa cell lines were plated on gridded dishes and imaged using a multispectral fluorescence microscope. They were then stained for proliferating cell nuclear antigen (PCNA) and DNA intensity as a reference standard for their cell cycle position (G1, S, G2, M). The multispectral data was split into training and testing datasets and models were generated to discriminate between G1, S, and G2 + M phase cells. A standard decision tree classification approach was taken, and a two-step system was generated for each line. RESULTS: Across cancer cell lines accuracy ranged from 68.3% (MIA-PaCa-2) to 73.3% (HeLa) for distinguishing G1 from S and G2 + M, and 69.0% (MIA-PaCa-2) to 78.0% (PANC1) for distinguishing S from G2 + M. Unmixing the multispectral data showed that the autofluorophores NADH, FAD, and PPIX had significant differences between phases. Similarly, the redox ratio and the ratio of protein bound to free NADH were significantly affected. CONCLUSIONS: These results demonstrate that multispectral microscopy could be used for the non-destructive, label free discrimination of cell cycle phase in cancer cells. They provide novel information on the mechanisms of cell-cycle progression and control, and have practical implications for oncology research.

Epigenetic Regulation of Bone Marrow Stem Cell Aging: Revealing Epigenetic Signatures associated with Hematopoietic and Mesenchymal Stem Cell Aging.

In this review we explore the importance of epigenetics as a contributing factor for aging adult stem cells. We summarize the latest findings of epigenetic factors deregulated as adult stem cells age and the consequence on stem cell self-renewal and differentiation, with a focus on adult stem cells in the bone marrow. With the latest whole genome bisulphite sequencing and chromatin immunoprecipitations we are able to decipher an emerging pattern common for adult stem cells in the bone marrow niche and how this might correlate to epigenetic enzymes deregulated during aging. We begin by briefly discussing the initial observations in yeast, drosophila and Caenorhabditis elegans (C. elegans) that led to the breakthrough research that identified the role of epigenetic changes associated with lifespan and aging. We then focus on adult stem cells, specifically in the bone marrow, which lends strong support for the deregulation of DNA methyltransferases, histone deacetylases, acetylates, methyltransferases and demethylases in aging stem cells, and how their corresponding epigenetic modifications influence gene expression and the aging phenotype. Given the reversible nature of epigenetic modifications we envisage "epi" targeted therapy as a means to reprogram aged stem cells into their younger counterparts.

Mesenchymal stem cells and biologic factors leading to bone formation.

BACKGROUND: Physiological bone formation and bone regeneration occurring during bone repair can be considered distinct but similar processes. Mesenchymal stem cells (MSC) and associated biologic factors are crucial to both bone formation and bone regeneration. AIM: To perform a narrative review of the current literature regarding the role of MSC and biologic factors in bone formation with the aim of discussing the clinical relevance of in vitro and in vivo animal studies. METHODS: The literature was searched for studies on MSC and biologic factors associated with the formation of bone in the mandible and maxilla. The search specifically targeted studies on key aspects of how stem cells and biologic factors are important in bone formation and how this might be relevant to bone regeneration. The results are summarized in a narrative review format. RESULTS: Different types of MSC and many biologic factors are associated with bone formation in the maxilla and mandible. CONCLUSION: Bone formation and regeneration involve very complex and highly regulated cellular and molecular processes. By studying these processes, new clinical opportunities will arise for therapeutic bone regenerative treatments.

Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination.

BACKGROUND: The 5 hydroxymethylation (5hmC) mark and TET DNA dioxygenases play a pivotal role in embryonic stem cell differentiation and animal development. However, very little is known about TET enzymes in lineage determination of human bone marrow-derived mesenchymal stem/stromal cells (BMSC). We examined the function of all three TET DNA dioxygenases, responsible for DNA hydroxymethylation, in human BMSC cell osteogenic and adipogenic differentiation. RESULTS: We used siRNA knockdown and retroviral mediated enforced expression of TET molecules and discovered TET1 to be a repressor of both osteogenesis and adipogenesis. TET1 was found to recruit the co-repressor proteins, SIN3A and the histone lysine methyltransferase, EZH2 to osteogenic genes. Conversely, TET2 was found to be a promoter of both osteogenesis and adipogenesis. The data showed that TET2 was directly responsible for 5hmC levels on osteogenic and adipogenic lineage-associated genes, whereas TET1 also played a role in this process. Interestingly, TET3 showed no functional effect in BMSC osteo-/adipogenic differentiation. Finally, in a mouse model of ovariectomy-induced osteoporosis, the numbers of clonogenic BMSC were dramatically diminished corresponding to lower trabecular bone volume and reduced levels of TET1, TET2 and 5hmC. CONCLUSION: The present study has discovered an epigenetic mechanism mediated through changes in DNA hydroxymethylation status regulating the activation of key genes involved in the lineage determination of skeletal stem cells, which may have implications in BMSC function during normal bone regulation. Targeting TET molecules or their downstream targets may offer new therapeutic strategies to help prevent bone loss and repair following trauma or disease.

Loss of EfnB1 in the osteogenic lineage compromises their capacity to support hematopoietic stem/progenitor cell maintenance.

The bone marrow stromal microenvironment contributes to the maintenance and function of hematopoietic stem/progenitor cells (HSPCs). The Eph receptor tyrosine kinase family members have been implicated in bone homeostasis and stromal support of HSPCs. The present study examined the influence of EfnB1-expressing osteogenic lineage on HSPC function. Mice with conditional deletion of EfnB1 in the osteogenic lineage (EfnB1OB-/-), driven by the Osterix promoter, exhibited a reduced prevalence of osteogenic progenitors and osteoblasts, correlating to lower numbers of HSPCs compared with Osx:Cre mice. Long-term culture-initiating cell (LTC-IC) assays confirmed that the loss of EfnB1 within bone cells hindered HSPC function, with a significant reduction in colony formation in EfnB1OB-/- mice compared with Osx:Cre mice. Human studies confirmed that activation of EPHB2 on CD34+ HSPCs via EFNB1-Fc stimulation enhanced myeloid/erythroid colony formation, whereas functional blocking of either EPHB1 or EPHB2 inhibited the maintenance of LTC-ICs. Moreover, EFNB1 reverse signaling in human and mouse stromal cells was found to be required for the activation of the HSPC-promoting factor CXCL12. Collectively, the results of this study confirm that EfnB1 contributes to the stromal support of HSPC function and maintenance and may be an important factor in regulating the HSPC niche.