Phenotype Of Progenitor Cells Research Articles

Sa1367 Hepatocyte Reprogramming to a Progenitor-Cell Phenotype Is a Potential Mechanism of Hepatocarcinogenesis - The Role of Reprogramming Factors: OCT4, SOX2 and NANOG

Sa1367 Hepatocyte Reprogramming to a Progenitor-Cell Phenotype Is a Potential Mechanism of Hepatocarcinogenesis - The Role of Reprogramming Factors: OCT4, SOX2 and NANOG

Effect of hyperglycemia on the number of CD117+ progenitor cells and their differentiation toward endothelial progenitor cells in young and old ages

Dysfunction of endothelial progenitor cells (EPCs) has been reported either in aging or diabetes, though the influence of an “old” environment on numerical and functional changes of diabetes associated EPCs is not known. We evaluated the effect of both aging and early stage of streptozotocin-induced diabetes on the number of bone marrow-derived CD117+ progenitor cells, and on their differentiation in vitro toward EPCs. The phenotype of progenitor cells and the uptake of acetylated-low density lipoprotein (Ac-LDL) were evaluated after cell culture in VEGF, FGF-1, and IGF-1 supplemented medium. Hyperglycemia similarly reduced the number of CD117+ cells both in young and old mice. CD117+ cells from young mice differentiated better than those from old animals “in vitro”, with a greater reduction of CD117+ cells and an higher increase of CD184+VEGFR-2+ cells. In diabetic mice, in vitro CD117+ cells differentiation was significantly reduced in young animals. Diabetes did not impact on the scarce differentiation of CD117+ cells from old mice. Hyperglycemia reduced the uptake of acLDL by EPCs greatly in young than in old mice. These findings indicate that part of the EPCs functional alterations induced by hyperglicemia in young mice are observed in normal aged mice.

Protein profile of basal prostate epithelial progenitor cells--stage-specific embryonal antigen 4 expressing cells have enhanced regenerative potential in vivo.

The long‐term propagation of basal prostate progenitor cells ex vivo has been very difficult in the past. The development of novel methods to expand prostate progenitor cells in vitro allows determining their cell surface phenotype in greater detail. Mouse (Lin−Sca‐1+ CD49f+ Trop2high‐phenotype) and human (Lin− CD49f+ TROP2high) basal prostate progenitor cells were expanded in vitro. Human and mouse cells were screened using 242 anti‐human or 176 antimouse monoclonal antibodies recognizing the cell surface protein profile. Quantitative expression was evaluated at the single‐cell level using flow cytometry. Differentially expressed cell surface proteins were evaluated in conjunction with the known CD49f+/TROP2high phenotype of basal prostate progenitor cells and characterized by in vivo sandwich‐transplantation experiments using nude mice. The phenotype of basal prostate progenitor cells was determined as CD9+/CD24+/CD29+/CD44+/CD47+/CD49f+/CD104+/CD147+/CD326+/Trop2high of mouse as well as human origin. Our analysis revealed several proteins, such as CD13, Syndecan‐1 and stage‐specific embryonal antigens (SSEAs), as being differentially expressed on murine and human CD49f+ TROP2+ basal prostate progenitor cells. Transplantation experiments suggest that CD49f+ TROP2high SSEA‐4high human prostate basal progenitor cells to be more potent to regenerate prostate tubules in vivo as compared with CD49f+ TROP2high or CD49f+ TROP2high SSEA‐4low cells. Determination of the cell surface protein profile of functionally defined murine and human basal prostate progenitor cells reveals differentially expressed proteins that may change the potency and regenerative function of epithelial progenitor cells within the prostate. SSEA‐4 is a candidate cell surface marker that putatively enables a more accurate identification of the basal PESC lineage.

Valproic acid and 5-azacytidine promote an increase of stemness phenotype in human osteosarcomas

Osteosarcoma (OS) is the most common pediatric tumour of bone in the world. It leads to local invasion and early metastasis to lung. The prognosis is poor with the 5-year survival rate of 65% for patients with primary tumour and 20% for patients with metastasis. OS contain a small cell population with stem cell like properties, referred to as cancer stem cells characterized by CD133 expression [1,2]. Recent studies have implicated epigenetic alterations, including DNA methylation and histone modifications, as prominent factors that contribute to the phenotype of cancer progenitor or stem cells. Here, we examined the epigenetic effects of the HDAC inhibitor valproic acid (VPA) and demethylating agent, 5’azacytidine (DAC), on the stemness phenotype in osteosarcoma cell lines. Saos-2 and MG63 cell lines were treated with 0,5 mM VPA and 3µM DAC for 48 hours alone, and in combination. CD133 expression as well as stemness markers expression including OCT4, Sox2 and Nanog was analyzed by flow cytometry and real-time PCR. Vimentin and osteocalcin levels were also tested. Sarcospheres formation rate was assessed as spheres number/seed single cells number. Specific histone modifications including H3-trymethyl-k9, H3-acetyl-k9, H3-trymethil-k27 and global methylation were analysed by flow cytometry and immunofluorescence. Moreover, soft agar and invasion assays were performed. Our findings indicated that DAC or VPA and their combination induced an increase of stemness characteristics of OS cells in terms of high expression of CD133, OCT4, Sox2 and Nanog and high sarcospheres-forming efficiency. Interestingly, combined treatment with DAC and VPA induced an increase of CD133 expression in a synergistic manner in all cell lines. Vimentin resulted up-regulated after treatment, whereas, the level of osteocalcin remained similar before and after treatment. Furthermore, soft agar assay revealed major colony-forming efficiency in treated cells compared to untreated cells, whereas invasion potential decreased after drug treatment. Interestingly, histone modification analyses correlated with those typical of embryonic stemness with an increase of H3-acetyl-k9 and decrease of H3-trymethyl-k9, and H3-trymethil-k27 after drug treatment as well as global methylation decreased in treated cells. In conclusion, DAC and VPA induced an increase of stemness associated to an decrease of global methylation, increase of acetylation and decrease of H3-trymethyl-k9, and H3-trymethil-k27. This study was supported by MIUR-EU funds (PON 01_02834 and PRIN 2011/13)

232: Excessive fetal growth associated with premature aging phenotype of hematopoietic progenitor cells

232: Excessive fetal growth associated with premature aging phenotype of hematopoietic progenitor cells

Localization and characterization of hematopoietic cells in the mouse embryonic head

The midgestation mouse head harbors hematopoietic stem cells (HSCs) at the same time HSCs are detected in the aorta, gonads, mesonephros region (AGM). Within the complex structure of the embryonic head, the localization and phenotype of HSCs and hematopoietic progenitor cells (HPCs) remains unknown. Since the Ly6a(Sca1)GFP transgene is a reporter for all AGM HSCs and some hemogenic endothelial cells (HEC), we examined these embryos for head HSC, HEC and HPC. Wholemount staining reveals some GFP+ cells in E10.5 head vasculature. By FACS analysis, 0.6% of E11.5 head cells are GFP+. To examine whether head HPC/HSC are enriched in the Ly6aGFP+ population, we sorted E11.5-E12.5 head cells and performed functional assays. Preliminary data show that head HSCs are GFP+. When we subdissected the head into forebrain (FB), midbrain (MB), and hindbrain with brachial arches (HBA) to localize HPCs, we found erythro-myeloid progenitor cells in GFP+ and GFP- fractions of all three brain regions at E10-11.5. Interestingly, in OP9 co-cutures, B cell potential was mostly observed in CD31+GFP+ of the three subregions. These results suggest that head HSCs (Ly6aGFP) are phenotypically and functionally similar to AGM HSCs. We are continuing to identify the precise head subregion(s) in which HSCs occur, examine the hemogenic potential of the head vasculature, and also the pathways affecting their development.

Maintenance of hematopoietic stem and progenitor cell phenotype of intra-aortic cell clusters in the AGM region through the Sox17-Notch1-Hes1 axis

Maintenance of hematopoietic stem and progenitor cell phenotype of intra-aortic cell clusters in the AGM region through the Sox17-Notch1-Hes1 axis

Hypoxic metabolism in human hematopoietic stem cells.

BackgroundAdult hematopoietic stem cells (HSCs) are maintained in a microenvironment, known as niche in the endosteal regions of the bone marrow. This stem cell niche with low oxygen tension requires HSCs to adopt a unique metabolic profile. We have recently demonstrated that mouse long-term hematopoietic stem cells (LT-HSCs) utilize glycolysis instead of mitochondrial oxidative phosphorylation as their main energy source. However, the metabolic phenotype of human hematopoietic progenitor and stem cells (HPSCs) remains unknown.ResultsWe show that HPSCs have a similar metabolic phenotype, as shown by high rates of glycolysis, and low rates of oxygen consumption. Fractionation of human mobilized peripheral blood cells based on their metabolic footprint shows that cells with a low mitochondrial potential are highly enriched for HPSCs. Remarkably, low MP cells had much better repopulation ability as compared to high MP cells. Moreover, similar to their murine counterparts, we show that Hif-1α is upregulated in human HPSCs, where it is transcriptionally regulated by Meis1. Finally, we show that Meis1 and its cofactors Pbx1 and HoxA9 play an important role in transcriptional activation of Hif-1α in a cooperative manner.ConclusionsThese findings highlight the unique metabolic properties of human HPSCs and the transcriptional network that regulates their metabolic phenotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-015-0020-3) contains supplementary material, which is available to authorized users.

Altered gut–liver axis and hepatic adiponectin expression in OSAS: novel mediators of liver injury in paediatric non-alcoholic fatty liver

BackgroundMechanism(s) connecting obstructive sleep apnoea syndrome (OSAS) to liver injury in paediatric non-alcoholic fatty liver disease (NAFLD) are unknown. We hypothesised alterations in gut–liver axis and in the pool and...

Characterization of Reversibly Immortalized Calvarial Mesenchymal Progenitor Cells

Bone morphogenetic proteins (BMPs) play a sentinel role in osteoblastic differentiation, and their implementation into clinical practice can revolutionize cranial reconstruction. Preliminary data suggest a therapeutic role of adenoviral gene delivery of BMPs in murine calvarial defect healing. Poor transgene expression inherent in direct adenoviral therapy prompted investigation of cell-based strategies. To isolate and immortalize calvarial cells as a potential progenitor source for osseous tissue engineering. Cells were isolated from murine skulls, cultured, and transduced with a retroviral vector bearing the loxP-flanked SV40 large T antigen. Immortalized calvarial cells (iCALs) were evaluated via light microscopy, immunohistochemistry, and flow cytometry to determine whether the immortalization process altered cell morphology or progenitor cell profile. Immortalized calvarial cells were then infected with adenoviral vectors encoding BMP-2 or GFP and assessed for early and late stages of osteogenic differentiation. Immortalization of calvarial cells did not alter cell morphology as demonstrated by phase contrast microscopy. Mesenchymal progenitor cell markers CD166, CD73, CD44, and CD105 were detected at varying levels in both primary cells and iCALs. Significant elevations in alkaline phosphatase activity, osteocalcin mRNA transcription, and matrix mineralization were detected in BMP-2 treated iCALs compared with GFP-treated cells. Gross and histological analyses revealed ectopic bone production from treated cells compared with controls in an in vivo stem cell implantation assay. We have established an immortalized osteoprogenitor cell line from juvenile calvarial cells that retain a progenitor cell phenotype and can successfully undergo osteogenic differentiation upon BMP-2 stimulation. These cells provide a valuable platform to investigate the molecular mechanisms underlying intramembranous bone formation and to screen for factors/small molecules that can facilitate the healing of osseous defects in the craniofacial skeleton.

Hepatocellular carcinoma: from hepatocyte to liver cancer stem cell.

Hepatocellular carcinoma: from hepatocyte to liver cancer stem cell.

Endothelial progenitor cell phenotype and function are impaired in childhood-onset systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by increased cardiovascular risk in adult-onset and childhood-onset SLE. Type I interferons (IFNs) appear to play a prominent role in premature vascular damage in adult-onset SLE, at least in part, by inducing impairments in the phenotype and function of endothelial progenitor cells (EPCs), thereby hampering vascular repair. It is not clear whether EPC dysfunction is present in childhood-onset SLE in association with a type I IFN signature. The phenotype and numbers of EPCs were quantified in patients with childhood-onset SLE, patients with juvenile idiopathic arthritis (JIA), and matched healthy control subjects. In a separate cohort of patients with childhood-onset SLE, markers of subclinical atherosclerosis and endothelial dysfunction were quantified using standardized protocols and analyzed for associations with serum type I IFN activity. EPC numbers and function were significantly decreased in patients with childhood-onset SLE compared with patients with JIA and healthy control subjects. Serum from patients with childhood-onset SLE impaired differentiation of EPCs into mature endothelial cells in healthy controls, and this effect was blocked by inhibition of the type I IFN pathway. Type I IFN activity in serum was not significantly associated with subclinical atherosclerosis and endothelial function in patients with childhood-onset SLE. As in adult-onset SLE, childhood-onset SLE is characterized by phenotypic and functional EPC abnormalities, which are likely triggered by type I IFNs. Although cross-sectional analysis revealed no global association between type I IFN signatures and vascular measures of subclinical atherosclerosis, longitudinal assessments are needed to evaluate whether progression of vascular damage in patients with childhood-onset SLE is associated with type I IFNs, as observed in patients with adult-onset SLE.

Treatment of antibody-mediated rejection including immunoadsorption during first year after renal transplantation – Clinical results and regulation of endothelial progenitor cells

ObjectiveAntibody-mediated rejection (AMR) is associated with poor allograft survival. Therefore, effective treatment strategies are required. Extracorporeal strategies are increasingly included in treatment of antibody-mediated rejection to eliminate the detrimental alloantibodies. Yet, other mechanisms contributing to the beneficial effect of apheresis besides the removal of antibodies are under consideration. MethodsWe retrospectively analyzed data of 427 transplant patients from 2006 to 2013 with special focus on occurrence, treatment – always including immunoadsorption – and 12-months outcome of antibody-mediated rejection.Besides, we prospectively monitored how the number and phenotype of endothelial progenitor cells in four patients experiencing antibody-mediated rejection changed during the treatment course of 6–20 sessions of immunoadsorption in comparison to seven patients subjected to immunoadsorption because of preparation for ABO-incompatible transplantation. Results24 patients were diagnosed with acute AMR and treated with immunoadsorption resulting in patient and allograft survival of 100% and 87.5%, respectively. In patients with antibody-mediated rejection, the endothelial progenitor cell number after successful immunoadsorption therapy was always transiently decreased and the adhesive and migratory ability improved. This regulation of circulating endothelial precursor cells was not seen in patients undergoing repetitive immunoadsorptions before ABO-incompatible transplantation. ConclusionCombined therapy with immunoadsorption allows a successful treatment of AMR. Treatment seems to be associated with a transient regulation of circulating endothelial precursor cells.

Hypoxia after transarterial chemoembolization may trigger a progenitor cell phenotype in hepatocellular carcinoma.

To test the hypothesis that the hypoxia marker carbonic anhydrase IX (CAIX), and the cholangiocytic/progenitor markers cytokeratin (CK) 19 and epithelial cell adhesion molecule (EpCAM), may be expressed in areas of hypoxia in hepatocellular carcinoma (HCC) after transarterial chemoembolization (TACE). Immunohistochemistry for CAIX, CK19 and EpCAM (BerEP4) was performed in 57 HCCs, including 40 residual/recurrent tumours adjacent to the TACE treatment site and 17 untreated tumours from the same 40 patients. CAIX was exxpressed in 19 of 40 residual/recurrent HCCs and in two of 17 untreated HCCs. The rate of CAIX immunoreactivity in the treated tumours was significantly higher than that in the non-treated tumours (47.5% versus 11.8%, P = 0.015). CK19 and EpCAM were expressed in six of 19 and in seven of 19 CAIX-positive TACE-treated HCCs, respectively, but were not expressed in CAIX-negative tumours or untreated tumours. There were significant associations between CK19 and CAIX immunoreactivity, and between EpCAM and CAIX immunoreactivity (P = 0.007 and P = 0.003, respectively). Double staining of CAIX and CK19 showed co-localization of both proteins in five of six cases. Three of seven EpCAM-positive tumours were also positive for CK19. Hypoxia may trigger the expression of proteins that are normally associated with cholangiocytic/progenitor cell differentiation, suggesting that TACE paradoxically causes an aggressive tumour phenotype.

Bone marrow-derived stem cell therapy for metastatic brain cancers.

We propose that stem cell therapy may be a potent treatment for metastatic melanoma in the brain. Here we discuss the key role of a leaky blood-brain barrier (BBB) that accompanies the development of brain metastases. We review the need to characterize the immunological and inflammatory responses associated with tumor-derived BBB damage in order to reveal the contribution of this brain pathological alteration to the formation and growth of brain metastatic cancers. Next, we discuss the potential repair of the BBB and attenuation of brain metastasis through transplantation of bone marrow-derived mesenchymal stem cells with the endothelial progenitor cell phenotype. In particular, we review the need for evaluation of the efficacy of stem cell therapy in repairing a disrupted BBB in an effort to reduce neuroinflammation, eventually attenuating brain metastatic cancers. The demonstration of BBB repair through augmented angiogenesis and vasculogenesis will be critical to establishing the potential of stem cell therapy for the treatment/prevention of metastatic brain tumors. The overarching hypothesis we advanced here is that BBB breakdown is closely associated with brain metastatic cancers of melanoma, exacerbating the inflammatory response of the brain during metastasis, and ultimately worsening the outcome of metastatic brain cancers. Abrogating this leaky BBB-mediated inflammation via stem cell therapy represents a paradigm-shifting approach to treating brain cancer. This review article discusses the pros and cons of cell therapy for melanoma brain metastases.

Immunohistochemical Analysis of LGR5 Expression in Pediatric Liver Disease

BACKGROUNDIn regenerating liver, hepatic progenitor cells (HPCs) are recruited in response to liver injury; however, there is a lack of highly specific human HPC markers for the hepatocyte lineage. LGR5, a Wnt‐associated adult stem cell marker, has been extensively studied in intestinal stem cells. Although LGR5 appears to be exclusively expressed by HPCs in mice, little is known about its expression in human liver. We hypothesized that LGR5+ cells are induced in the regenerative response to liver injury to potentially rescue dysfunctional hepatocytes.METHODSUsing immunohistochemistry, we characterized LGR5 expression in pediatric liver diseases, using mainly liver explants (n=36 cases total).RESULTSWe found cytoplasmic LGR5 over‐expression in all 36 pediatric cases, including acute hepatic necrosis and cholestatic liver diseases. In 44% of cases, >50% of hepatocytes were LGR5+, signifying a robust regenerative response. In contrast, there was only weak staining in bile ducts, suggesting hepatocyte‐specific expression. Marked peri‐portal expression was prominent in cholestatic diseases, consistent with known patterns of regeneration. Comparison to other regeneration markers will further characterize this hepatocyte progenitor cell phenotype.CONCLUSIONSAlthough we observed some degree of regenerative response in all cases examined, LGR5 was highly expressed in cholestatic liver diseases, possibly due to alternate regeneration pathways. These studies are the first to support a unique role for LGR5 in human hepatocyte regeneration and as a potential predictive marker for recovery of liver function in children. LGR5 INTENSITY TOTAL CASES WEAK + 1+ 2+ 3+ A1AT Deficiency (ATD) 15 0 2 5 8 Biliary Atresia (BA) 5 0 0 2 3 PFIC 10 1 1 5 3 Acute Hepatic Necrosis (ALF) 6 1 4 1 0 TOTAL CASES 36 2 7 13 14 imageimage

Umbilical Cord Blood-Derived Mononuclear Cells Exhibit Pericyte-Like Phenotype and Support Network Formation of Endothelial Progenitor Cells In Vitro.

Umbilical cord blood represents a promising cell source for pro-angiogenic therapies. The present study examined the potential of mononuclear cells (MNCs) from umbilical cord blood to support endothelial progenitor cell (EPC) microvessel formation. MNCs were isolated from the cord blood of 20 separate donors and selected for further characterization based upon their proliferation potential and morphological resemblance to human vascular pericytes (HVPs). MNCs were screened for their ability to support EPC network formation using an in vitro assay (Matrigel™) as well as a reductionist, coculture system consisting of no additional angiogenic cytokines beyond those present in serum. In less than 15% of the isolations, we identified a population of highly proliferative MNCs that phenotypically resembled HVPs as assessed by expression of PDGFR-β, NG2, α-SMA, and ephrin-B2. Within a Matrigel™ system, MNCs demonstrated pericyte-like function through colocalization to EPC networks and similar effects as HVPs upon total EPC tubule length (p=0.95) and number of branch points (p=0.93). In a reductionist coculture system, MNCs served as pro-angiogenic mural cells by supporting EPC network formation to a significantly greater extent than HVP cocultures, by day 14 of coculture, as evidenced through EPCtotal tubule length (p<0.0001) and number of branch points (p<0.0001). Our findings are significant as we demonstrate mural cell progenitors can be isolated from umbilical cord blood and develop culture conditions to support their use in microvascular tissue engineering applications.

Coronary stents seeded with human trophoblastic endovascular progenitor cells show accelerated strut coverage without excessive neointimal proliferation in a porcine model.

The success of percutaneous coronary intervention (PCI) has been limited by restenosis and stent thrombosis. Delayed or incomplete endothelial regeneration is believed to be a key factor responsible for these events. Developing a stent with an accelerated healing profile may be of benefit. We aimed to evaluate the feasibility and safety of seeding a bare metal stent (BMS) with human trophoblastic endovascular progenitor cells (hTEC) derived from human embryonic stem cells. A porcine coronary artery model was used to compare the rate and extent of endothelial regeneration and the degree of neointimal proliferation. Characterisation of hTEC confirmed a mixed progenitor and endothelial cell phenotype. The biodistribution and fate of hTEC were studied using radiolabelled 111Indium oxine and fluorescent in situ hybridisation. Scanning electron microscopy showed earlier endothelial coverage in hTEC-seeded stents as compared to similar BMS. hTEC-seeded BMS achieved complete stent coverage in three days. Quantitative coronary angiography, intravascular ultrasound assessment and histomorphometry showed no difference in neointimal hyperplasia between hTEC-seeded and control BMS. hTEC seeding of coronary stents is a novel and safe approach to accelerate endothelial regeneration without increasing neointimal proliferation.

Serial explant culture provides novel insights into the potential location and phenotype of corneal endothelial progenitor cells

The routine cultivation of human corneal endothelial cells, with the view to treating patients with endothelial dysfunction, remains a challenging task. While progress in this field has been buoyed by the proposed existence of progenitor cells for the corneal endothelium at the corneal limbus, strategies for exploiting this concept remain unclear. In the course of evaluating methods for growing corneal endothelial cells, we have noted a case where remarkable growth was achieved using a serial explant culture technique. Over the course of 7 months, a single explant of corneal endothelium, acquired from cadaveric human tissue, was sequentially seeded into 7 culture plates and on each occasion produced a confluent cell monolayer. Sample cultures were confirmed as endothelial in origin by positive staining for glypican-4. On each occasion, small cells, closest to the tissue explant, developed into a highly compact layer with an almost homogenous structure. This layer was resistant to removal with trypsin and produced continuous cell outgrowth during multiple culture periods. The small cells gave rise to larger cells with phase-bright cell boundaries and prominent immunostaining for both nestin and telomerase. Nestin and telomerase were also strongly expressed in small cells immediately adjacent to the wound site, following transfer of the explant to another culture plate. These findings are consistent with the theory that progenitor cells for the corneal endothelium reside within the limbus and provide new insights into expected expression patterns for nestin and telomerase within the differentiation pathway.

Resolving Salmonella infection reveals dynamic and persisting changes in murine bone marrow progenitor cell phenotype and function.

The generation of immune cells from BM precursors is a carefully regulated process. This is essential to limit the potential for oncogenesis and autoimmunity yet protect against infection. How infection modulates this is unclear. Salmonella can colonize systemic sites including the BM and spleen. This resolving infection has multiple IFN-γ-mediated acute and chronic effects on BM progenitors, and during the first week of infection IFN-γ is produced by myeloid, NK, NKT, CD4+ T cells, and some lineage-negative cells. After infection, the phenotype of BM progenitors rapidly but reversibly alters, with a peak ∼30-fold increase in Sca-1hi progenitors and a corresponding loss of Sca-1lo/int subsets. Most strikingly, the capacity of donor Sca-1hi cells to reconstitute an irradiated host is reduced; the longer donor mice are exposed to infection, and Sca-1hic-kitint cells have an increased potential to generate B1a-like cells. Thus, Salmonella can have a prolonged influence on BM progenitor functionality not directly related to bacterial persistence. These results reflect changes observed in leucopoiesis during aging and suggest that BM functionality can be modulated by life-long, periodic exposure to infection. Better understanding of this process could offer novel therapeutic opportunities to modulate BM functionality and promote healthy aging.