Cellular senescence mediates the detrimental effect of prenatal dexamethasone exposure on postnatal long bone growth in mice offspring

Cellular senescence mediates the detrimental effect of prenatal dexamethasone exposure on postnatal long bone growth in mice offspring | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Cellular senescence mediates the detrimental effect of prenatal dexamethasone exposure on postnatal long bone growth in mice offspring Jianwen Su, Yu Chai, Zhiguo Ji, Yongheng Xie, Bin Yu, Xianrong Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-17277/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jul, 2020 Read the published version in Stem Cell Research & Therapy → Version 2 posted 11 You are reading this latest preprint version Show more versions Abstract Background : Prenatal dexamethasone exposure (PDE) induces low birth weight and retardation of fetal bone development which are associated with lower peak bone mass in adult offspring. Here we evaluated whether and how PDE affects postnatal long bone growth in mice offspring. Methods : Pregnant mice were injected subcutaneously with dexamethasone (1.2 mg/kg/day) every morning from gestational day (GD) 12-14. Femurs and tibias of 2-, 4-, 6-, and 12-week-old female offspring were harvested for histological, immunofluorescence, flow cytometric analysis, or microcomputed tomography (μCT) measurement. Results : PDE leads to impaired bone remodeling as well as decreased bone mass in the long bone of female mice offspring. During postnatal bone growth, significant decrease of CD45 - CD29 + CD105 + Sca-1 + bone marrow mesenchymal stem cells (BMSCs) and CD45 - Nestin + cells, loss of type H vessels as well as increment of cellular senescence were found in metaphysis of long bone in mice offspring after PDE. We further show that eliminating the excessive senescent cells with dasatinib (5 mg/kg/day) and quercetin (50 mg/kg/day) during GD 12-14 rescues the above toxic effect of PDE on the postnatal long bone growth in female mice offspring. Conclusion : Cellular senescence mediates the toxic effect of PDE on postnatal long bone growth in mice offspring, and inhibition of cellular senescence may be proposed for treating the retardation of bone growth caused by PDE. Stem Cell & Developmental Cell Biology Dexamethasone bone development bone mesenchymal stem cells cellular senescence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Prenatal corticosteroid use in clinic has been shown to be effective in accelerating lung maturation and reducing the incidence of respiratory complications in infant [1]. However, clinical studies provide evidence that these short-term benefits are associated with reduction in birth size for infants born preterm, near term, or at term [2], and suppression of fetal bone turnover in infants at birth [3]. In addition, clinical trial data show that lower birth weight is associated with lower peak bone mass in adult offspring [4, 5]. Evidence from experimental animal models by our group and others demonstrate that prenatal dexamethasone exposure (PDE) impairs long bone development in fetal animals [6-9], reduces bone mass in adult offspring [7, 10]. These data imply that developmental overexposure to glucocorticoid alters bone programming and results in less bone mass in offspring. Although studies have found the detrimental effect of PDE on osteogenesis [11, 12], little is known about the most critical cellular target of PDE in the developing bone. Mesenchymal stem/stromal cells from bone marrow are multipotent cells that play crucial roles in bone development, maintenance and regeneration due to their multilineage differentiation and self-renewal capacity [13, 14]. During childhood and puberty, skeletal bone grows quickly owing to rapid self-renewal and differentiation of bone marrow mesenchymal stem or stromal cells (BMSCs) and osteoprogenitors [15, 16]. The activities of stem cells are controlled by the local stem cell microenvironment, which is composed of cellular components such as stromal cells, immune cells, endothelial cells, osteoblasts and a non-cellular compartment which includes extracellular matrix components and signal molecules [17]. This local environment plays important role in regulating stem cell survival, function and fate [18], and alterations of the microenvironment may impair skeletal stem cells functions, leading to decreased osteogenesis and bone formation [19-21]. Cellular senescence is a state of irreversible growth arrest and occurs throughout life. Senescent cells can secrete numerous biologically active factors, termed the senescence-associated secretory phenotype (SASP), leading to pathological consequences in the tissue microenvironment [22]. It has been demonstrated that senescence associated bone microenvironment contribute to age-related bone loss [23]. Recent evidence suggests that cellular senescence is also a key regulator during bone development [24]. Senescence can be induced by various intrinsic and extrinsic triggers. Extensive use of dexamethasone has been associated with cellular senescence and aging-related pathological process in vivo and in vitro [25, 26]. However, the potential relationship between cellular senescence and development retardation in bone induced by PDE remains unclear. Our recent study found that PDE during gestational day 12-14 (GD12-14) retards bone development in fetal mice [6]. Here we show that young and adult female mice offspring by PDE have impaired bone formation and significant lower bone mass. We have found significant reduced amount of CD45 - Nestin + and CD45 - CD29 + CD105 + Sca-1 + cells as well as impaired angiogenesis in metaphysis of long bone in young offspring mice after PDE. We also have found that PDE promotes cellular senescence and suppresses cells proliferation in trabecular area of long bone. Interestingly, eliminating senescent cells using senolytics dasatinib and quercetin (D+Q) rescues significantly the decreased BMSCs and osteoprogenitors by PDE and prevents developmental retardation of long bone in young mice offspring. 2. Materials And Methods 2.1 Animals This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals of Nanfang Hospital Southern Medical University. The protocol was approved by the Animal Care and Use Committee of Nanfang Hospital. Pathogen-free mice were maintained under standard conditions in a 12h light and 12h dark cycle, at 25 ± 3°C, with a relative humidity of 40-60%, and with food and tap water available ad libitum. PDE during GD 12-14 was applied according to the procedure previously described [6]. Briefly, virgin C57BL/6 female mice at 10-12 weeks old were mate with male mice overnight. The day on which the presence of a vaginal plug was set as GD 0, the pregnant mice were randomly assigned to the PDE group or vehicle treatment (control) group. To construct PDE mice model, dexamethasone sodium phosphate (Cat. 2392-39-4, Tianxin, China) was injected subcutaneously (1.2 mg/kg/day) during GD 12-14. To construct the vehicle control of PDE model, pregnant mice were treated with the same amount of vehicle (normal saline) daily during GD 12-14. The pregnant mice were housed individually in cages with freely available food and water. Two female offspring were selected randomly from each litter for postnatal bone development investigation. Femurs and tibias from mice offspring at 2-, 4-, 6-, and 12-week-old were dissected for further analysis. To evaluate the effect of dasatinib (S1021, Selleck Chemicals，Houston，TX, USA) and quercetin (S2391, Selleck Chemicals，Houston，TX, USA), PDE pregnant mice during GD 12-14 were treated with vehicle (200 μl 1% methyl cellulose) or dasatinib (5 mg/kg/day) plus quercetin (50 mg/kg/day) by oral gavage, respectively. 2.2 Microcomputed Tomography (μCT) analysis Tibias from 12-week-old mice offspring were dissected free of soft tissue, fixed and stored in 70% ethanol, and imaged using a μCT specimen scanner (Scanco Medical, AG, Switzerland). The scan was performed using an X-ray energy of 55 kV and current of 145 mA, with a voxel size of 12μm and an integration time of 400 msec. Trabecular bone measurements consisting of 250 slices (3mm) were performed from 0.215 mm (18 image slices) below the growth plate. Bone volume (BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th) and trabecular separation (Tb. Sp) were determined. Quantitative analyses were carried out using IPL software (Image Processing Language V5.15, Scanco Medical AG, Switzerland). 2.3 Histochemistry To study the morphology of postnatal long bone growth in offspring after PDE, femurs and tibias of mice offspring at 2-, 4-, and 6-week-old were fixed in 4% paraformaldehyde, decalcified in 0.5M ethylenediamine-tetraacetic acid (EDTA, pH 7.4), followed by paraffine embedding or frozen embedding. Hematoxylin-eosin (H&E) staining, Goldner’s trychrome staining, and tartrate-resistant acid phosphatase (TRAP) staining were performed on 4 μm paraffin sections according to standard procedures. The number of osteoblasts per square millimeter of metaphyseal area (N. per mm 2 ) were quantified in the area from 0-0.5mm below growth plate. For detecting the osteoclastic activity in bone, TRAP staining was performed on the deparaffinized and rehydrated sections using a Leukocyte Acid Phosphatase kit (Cat. 387A-1KT, Sigma-Aldrich, USA). The TRAP + multinucleated cells containing at least three nuclei were identified as osteoclasts under light microscope (Olympus, BX53). The number of TRAP + cells per square millimeter of metaphyseal area (N. per mm 2 ) in the area from 0-0.5mm below growth plate was quantified. For detecting senescence associated β-galactosidase (SA-β-Gal) activity, frozen sections were stained using SA-β-Gal staining kit (Cat. 9860, Cell Signaling Technology, USA) according to manufacturer’s instructions. Senescent cells were identified as blue-stained cells under light microscope (Olympus, BX53). The number of SA-β-Gal + cells per square millimeter of metaphyseal area (N. per mm 2 ) in the area from 0-0.5mm below growth plate was quantified. 2.4 Immunofluorescence For immunofluorescence staining, frozen sections were incubated in blocking buffer (3% BSA in PBS with Tween (PBST)) for 1 hour at room temperature, incubated with primary antibodies overnight at 4°C. The primary antibodies for immunostaining include: Nestin (ab134017, Abcam, Cambridge, MA, USA), CD31 (FAB3629G-100, R&D Systems, Minneapolis, MN, USA), Endomucin (Emcn, SC-65495，Santa Cruz, Dallas, TX USA), Ki67 (ab15580, Abcam, Cambridge, MA, USA). Sections were washed 3 times in PBS and then incubated with secondary antibodies at room temperature for 1 hour. The secondary antibodies for immunostaining include: 488-conjugated secondary antibody (703-546-155, Jackson ImmunoResearch, West Grove, PA, USA), 594-conjugated secondary antibody (712-586-153, Jackson ImmunoResearch, West Grove, PA, USA), 488-conjugated secondary antibody (A21206, ThermoFish Scientific, USA). Nuclei were counterstained with DAPI (S2110，Solarbio, China). Images were captured using a fluorescence microscrope (Olympus, BX53, Japan). Positive-stained area or the number of positive-stained cells per square millimeter of the metaphyseal area was measured from 0-0.5mm below growth plate. 2.5 Flow cytometric analysis Bone marrow cells were collected from femurs and tibias of mice offspring at 4-week-old. Cell numbers were determined after removal of red blood cells with ACK Lysis Buffer (CS0001, Leagene, China). After washed with PBS twice, pellets were resuspended and blocked in 1% BSA on ice for 15min. Cells were then washed twice with PBS and incubated with primary antibody (for cell surface marker) solution diluted by 0.5% BSA for 30min on ice in the dark. After being fixed by 4% paraformaldehyde and permeabilized by PBST and washed with PBS, cells were incubated with primary antibody (for intracellular antigen) solution diluted by 0.5% BSA for 30min on ice in the dark. The primary antibody used were PE-conjugated Nestin Antibody (MA5-23574, ThermoFish Scientific, Rockford, IL, USA), and BV421−conjugated anti-mouse CD45 (563890, BD Biosciences, San Jose, CA, USA). Cells were then washed once and re-suspended in 300 μl PBS and transferred to flow tubes. For BMSCs, CD45 - CD29 + CD105 + Sca-1 + bone marrow cells were detected using a Mouse mesenchymal stem cell Multi-color Flow kit (FMC003, R&D systems, Canada) following manufactures protocol. Flow cytometric analysis was performed on a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (BD Life Sciences San Jose, CA, USA). 2.6 Statistics The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology. All quantitative data were presented as mean ± S.E.M. For comparisons between two groups, independent Student’s t -test was performed. For multiple comparisons, one-way analysis of variance (ANOVA) with Bonferroni post hoc test was used. Statistical analysis was performed using SPSS, version 20 software (International Business Machines Corporation, IBM Corp.). Significant level was defined as P < 0.05. 3. Results 3.1 Adult mice offspring from PDE has low bone mass in the long bone Our previous work has shown the adverse effect of PDE on the long bone development in fetal mice [6]. We then tested whether long bone development retardation during prenatal period results in bone mass deficit in adult offspring. Tibias were harvested from 12-week-old mice offspring of PDE and measured by μCT. A significant reduction in the mass of trabecular bone was observed in female offspring relative to control mice offspring (Fig. 1). There was a significant decrease of bone volume fraction (BV/TV) in female adult mice offspring from PDE, which was attributed to a large decrease in trabecular number (Tb. N) along with a notable increase in trabecular separation (Tb. Sp), with no change observed in trabecular thickness (Tb.Th) (Fig. 1b-e). But, we didn’t observe change of the above microstructural parameters in male mice offspring (Fig. 1f-i). Together, these data revealed that PDE has long-term detrimental effect on bone mass in female adult mice offspring. 3.2 Postnatal long bone development is retarded in female mice offspring from PDE Bone mass in adulthood has been closely correlated with fetal and postnatal bone growth, which is an important process for bone mineral accrual [27]. To investigate how PDE induces low bone mass in female adult offspring, we evaluated the morphology of long bone of female offspring at 2-, 4-, and 6-week-old. H&E staining and Goldner’s trychrome staining results showed reduced amount of trabecular bones in female mice offspring after PDE (Fig. 2a). Similarly, histomorphometric analysis revealed significantly less osteoblast counts in offspring from PDE, compared with that in control (Fig. 2b). As the reduced trabecular bone could be due to either lower bone formation or higher bone resorption or both, we examined the changes of osteoclastogenesis in mice offspring after PDE. TRAP staining and quantitative analysis revealed that osteoclast number was significantly lower in PDE mice offspring compared to controls (Fig. 2c). Therefore, PDE impedes both bone formation and bone absorption during skeletal bone development. 3.3 PDE induces loss of Nestin expressing cells and blood vessels in long bone of female mice offspring Nestin expressing (Nestin + cells) in postnatal bones are heterogeneous populations mainly in endothelial and osteoblast lineage [24, 28]. These cells are highly proliferative and critical for osteoblast replenishment for bone formation during postnatal bone development [24]. We thus assessed whether PDE affects Nestin + cells in postnatal long bone of female mice offspring. Results showed that the number of Nestin + cells in femoral metaphysis was significantly reduced as assessed by immunofluorescence staining (Fig. 3a, b). A specific subtype of vessels, termed H-type vessels which characterized by high expression of the endothelial markers CD31 and Emcn (CD31 hi Emcn hi ), generate distinct microenvironments for maintaining perivascular osteoprogenitors and coupling angiogenesis to osteoprogenitors [29]. We then examined the effect of PDE on type-H vessels in postnatal long bone of offspring. Double immunofluorescence staining for CD31 and Emcn showed a significant lower proportion of type-H vessels in the femoral metaphysis of mice offspring after PDE compared to that of control offspring (Fig. 3c, d). Therefore, PDE reduces type-H vessels, which are closely associated with impaired bone formation in mice offspring. 3.4 Cellular senescence is increased in postnatal long bone of mice offspring after PDE A recent report showed that premature cellular senescence in the long bone of young mice leads to reduction of osteoprogenitors, impairing of blood vessel formation and bone formation [24]. We then tested whether the progression of the cellular senescence in long bone of postnatal offspring is altered by PDE. SA-β-Gal staining was conducted in femoral bones of mice offspring at 2-, 4-, and 6-week-old. We found a significant increase in the number of SA-β-Gal + cells in femoral metaphysis of PDE offspring at 2-, 4-, and 6-week-old compared to those in control offspring, respectively (Fig. 4a, b). Consistently, immunofluorescence staining for Ki67, the proliferative marker, showed reduced staining in the trabecular bone adjacent to the growth plate, the same region that SA-β-Gal + cells located (Fig. 4c). Quantitative results confirmed dramatically reduction of Ki67 + cells in the femoral metaphysis of mice offspring after PDE (Fig. 4d). The above results strongly suggest that PDE induces growth arrest and senescence in certain cell types. To identify whether PDE induced senescence in osteoprogenitors, we performed co-staining of SA-β-Gal with osteoprogenitor marker Nestin and Osterix, and vessel marker Emcn, respectively. However, we didn’t find overlapping staining of SA-β-Gal with any of those three markers (Supplementary Fig. 1, 2). The above data suggest that PDE might not stimulate cellular senescence in osteoprogenitors and cells of type H vessels directly, but suppress those cells indirectly by stimulating senescence of other cell populations in bone. 3.5 Targeting cellular senescence prevents PDE-induced bone development retardation in mice offspring It was reported that clearance of senescent cells using D+Q can improve bone mass in aged mice [23]. To evaluate the role of increased cellular senescence in PDE-induced bone growth retardation in mice offspring, pregnant mice by PDE were treated with D+Q or vehicle during GD 12-14 once every day. Consistent with the effect of D+Q on aged mice, SA-β-Gal + cells number in femoral metaphysis was significantly suppressed in D+Q-treated PDE mice offspring as compared to vehicle-treated ones (Fig. 5a, b). As anticipated, D+Q treatment significantly improved trabecular bone amount in femoral metaphysis in 2-week-old PDE mice offspring relative to vehicle-treated ones, as assessed by H&E staining and Goldner’s trychrome staining results (Fig. 5a). Trabecular bone histomorphometry demonstrated the rescued osteoblast numbers and increased Nestin + cells in PDE mice offspring treated by D+Q than in vehicle-treated ones (Fig. 5c, d). Concomitant with reduced burden of cellular senescence by D+Q and markedly rescued bone phenotype in mice offspring after PDE, we further found that the percentage of CD45 - Nestin + (Fig. 6a, b) and CD45 - CD29 + CD105 + Sca-1 + (Fig. 6c, d) was significantly decreased in bone marrow of mice offspring after PDE. It is noteworthy that D+Q treatment only partially rescued the loss of CD45 - Nestin + cells in mice offspring after PDE, but completely restored the ratio of CD45 - CD29 + CD105 + Sca-1 + cells (Fig. 6). Compared with control mice, mice offspring after PDE and D+Q treatment had significantly lower percentage of CD45 - Nestin + cells (Fig. 6a, b), but had a considerable increment in the percentage of CD45 - CD29 + CD105 + Sca-1 + cells (Fig. 6b, d). Additionally, D+Q treatment also dramatically restored the osteoblast number (Fig. 7a, b) and angiogenesis potential in mice offspring after PDE (Fig. 7c, d). All the data indicate that cellular senescence mediates the detrimental effect of PDE on BMSCs and osteoprogenitors, leading to retardation of postnatal long bone growth in mice offspring. 4. Discussion Here we provide unique insights into the mechanisms by which PDE leads to retardation of postnatal bone development. Our data show that PDE induces cellular senescence, reduction of BMSCs amount and decrease of bone modeling and remodeling during postnatal bone growth in female mice offspring. Furthermore, elimination of excessive senescent-cells can rescue the toxic effect of PDE on postnatal bone growth in female mice offspring. The present study has identified that PDE induces cellular senescence which may contribute to the toxic effect of PDE on BMSCs and type H vessels in the metaphysis of long bone, thereby impeding bone formation in mice offspring. We believe that these results have opened a door to elucidating poorly understood aspects of osteoporosis with developmental origin and highlighted D+Q as a therapeutic approach for treating PDE-induced retardation of bone growth in offspring. Our previous work demonstrated that continuous PDE during GD 9-20 retards long bone development in fetal rats and leads to low bone mass in adult rat offspring [7], and further delineated that PDE at dose higher than 0.8 mg/kg/day during GD 12-14 developed severe retardation of long bone development in fetal mice [6]. In the present study, the data clearly indicate that PDE at 1.2 mg/kg/day during GD 12-14 induces low bone mass in 12-week-old mice offspring. Our finding is supported by a recent report that PDE at GD 16-17 induces decreased bone mass and biomechanical strength in adult mice offspring [30]. Furthermore, we observed a notable decrease in both osteoblasts and osteoclasts in femoral metaphysis of female mice offspring after PDE, indicating a decreased bone modeling and remodeling induced by PDE. Overall, the works from our lab and others have highlighted that PDE has long-term effect on bone mass of offspring in later life, which might be consequences of PDE programming on intrauterine development of fetal tissue [31, 32]. During bone modeling and remodeling, bone formation requires continuous production of osteoblasts from osteoprogenitors and BMSCs in mammals. Nestin + cells in postnatal bone encompass osteoblastic and endothelial progenitors, and several subsets of stem cells with self-renewal capacity [28, 33]. Additionally, type H vessels are important for maintaining osteoprogenitors and coupling angiogenesis to osteoprogenitors [29]. Our studies provide the first evidence of decreased amount of CD45 - CD29 + CD105 + Sca-1 + BMSCs and Nestin + cells and impaired type H vessels in the long bone of female mice offspring after PDE. It is possible that PDE has extensive toxic effect on osteoprogenitors, BMSCs and type H vessels, leading to impaired osteogenesis and diminished bone formation. Interestingly, we found the dramatically increased cellular senescence and diminished cellular proliferation in femoral metaphysis of young female mice offspring after PDE. Senescent cells are known to instigate SASP which may exert detrimental paracrine, thereby contributing to senescence-related inflammation and stem cell dysfunction [34]. Indeed, senescent cells are found to be associated with embryonic and postnatal development [24, 35, 36]. In the present study, we didn’t find much overlapping of osteoprogenitor markers with SA-β-Gal staining, however, our data revealed that reducing senescent cell burden using senolytics D+Q have dramatically restored the percentage of BMSCs, the numbers of osteoblasts and type H vessels in trabecular bone of female mice offspring after PDE. These data support our conclusion that excessive senescent-cells mediate the detrimental effect of PDE on postnatal long bone development in female mice offspring. As Nestin + cells has been found to undergo normal programmed senescence in trabecular bone during late puberty, and prednisolone injection during puberty further stimulate senescence of Nestin + cells in mice [24], it is possible that loss of Nestin + cells in PDE mice offspring might be at least partially due to the senescence of Nestin + cells. Interestingly, we found that senolytics D+Q treatment only partially rescued the loss of Nestin + cells in mice offspring after PDE. One reason could be that PDE suppresses Nestin + cells by a mechanism of action that is distinct from that of D+Q, which is known to reduce senescent cell burden by reducing pro-inflammatory cytokine secretion [37]. Bone is a dynamic organ composed of various bone marrow-derived cell types including hematopoietic, mesenchymal, endothelial cells [38]. As dexamethasone has far-ranging effects on various cells and tissues [31], it might trigger the senescence on a heterogeneous population of cells in bone. Considering that almost all of SA-β-Gal-positive stained cells locate in endosteal bone marrow close to the trabecular bone surface, but not inside the trabecular bone where osteocytes locate, it is reasonable to rule out the senescence of osteocytes in young mice offspring after PDE. Nevertheless, a limitation of our study cannot be neglected that we failed (despite extensive effort) to identify the cell types and molecular mechanism of cellular senescence induced by PDE. Growing evidences indicate that prenatal glucocorticoid may exert long-term programming of fetal tissue by epigenetic modifications of genome [31, 32], therefore, further study is necessary to analyze the transcriptome and epigenome of SA-β-Gal + cells, to unravel the cellular and molecular mechanisms involved in long-term programming of cellular senescence as a result of PDE, and how a deregulation of cellular senescence retards long bone development in female mice offspring. 5. Conclusions Although the precise mechanisms by which PDE enhances cellular senescence will require further experimentation, our studies have significant advancements by identifying cellular senescence as a key regulator linking PDE with postnatal bone development retardation in female mice offspring and, further, targeting cellular senescence may be a promising avenue for prevention of bone development retardation in female mice offspring after PDE. 5. Declarations 5.1 Author contributions X.Z. designed the research, drafted and revised the article; J.S., Y.C., Z.J. and Y.X. acquired the data; J.S., Y.C., B.Y. and X.Z. analyzed the data. 5.2 Funding This study was supported by grants from the National Natural Science Foundation of China (No.81573515, to X.Z.) and The Major Program of National Natural Science Foundation of China (No.81830079, to B.Y.), Key Research Project of Science and Technology Planning Program of Guangdong Province (No.2019B020201013, to X.Z.). 5.3 Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (No.81573515, to X.Z.), The Major Program of National Natural Science Foundation of China (No.81830079, to B.Y.), Key Research Project of Science and Technology Planning Program of Guangdong Province (No.2019B020201013, to X.Z.). 5.4 Conflicts of interest statement The authors declare no conflicts of interest. 5.5 Ethics approval and consent to participate All experiments were conducted in accordance with the guidelines set by the Institutional Animal Care and Use Committee of Nanfang Hospital, Southern Medical University. 5.6 Availability of Data and Materials Please contact the corresponding author for data requests. 5.7 Consent for publication Not applicable 6. References Kemp MW, Schmidt AF, Jobe AH. Optimizing antenatal corticosteroid therapy. Semin Fetal Neonatal Med 2019; 24: 176-181. Rodriguez A, Wang Y, Ali Khan A, et al. Antenatal corticosteroid therapy (ACT) and size at birth: A population-based analysis using the Finnish Medical Birth Register. PLoS Med 2019; 16: e1002746. Korakaki E, Damilakis J, Gourgiotis D, et al. Quantitative ultrasound measurements in premature infants at 1 year of age: the effects of antenatal administered corticosteroids. Calcif Tissue Int 2011; 88: 215-222. Dalziel SR, Fenwick S, Cundy T, et al. Peak bone mass after exposure to antenatal betamethasone and prematurity: follow-up of a randomized controlled trial. J Bone Miner Res 2006; 21:1175-1186. Xie LF, Alos N, Cloutier A, et al. The long-term impact of very preterm birth on adult bone mineral density. Bone Rep 2018; 10: 100189. Chen Z, Zhao X, Li Y, et al. Course-, dose-, and stage-dependent toxic effects of prenatal dexamethasone exposure on long bone development in fetal mice. Toxicol Appl Pharmacol 2018; 351:12-20. Zhang X, Shang-Guan Y, Ma J, et al. Mitogen-inducible gene-6 partly mediates the inhibitory effects of prenatal dexamethasone exposure on endochondral ossification in long bones of fetal rats. Br J Pharmacol 2016; 173: 2250-2262. Cheng X, Wang G, Lee KK, Yang X. Dexamethasone use during pregnancy: potential adverse effects on embryonic skeletogenesis. Curr Pharm Des 2014; 20:5430-5437. Sliwa E, Dobrowolski P, Piersiak T. Bone development of suckling piglets after prenatal, neonatal or perinatal treatment with dexamethasone. J Anim Physiol Anim Nutr (Berl) 2010; 94: 293-306. Tomaszewska E, Dobrowolski P, Bieńko M, et al. Effects of 2-oxoglutaric acid on bone morphometry, densitometry, mechanics, and immunohistochemistry in 9-month-old boars with prenatal dexamethasone-induced osteopenia. Connect Tissue Res 2015; 56: 483-492. Xiao H, Wen Y, Pan Z, et al. Increased H3K27ac level of ACE mediates the intergenerational effect of low peak bone mass induced by prenatal dexamethasone exposure in male offspring rats. Cell Death Dis 2018; 9: 638. Cheng X, Wang G, Lee KK, et al. Dexamethasone use during pregnancy: potential adverse effects on embryonic skeletogenesis. Curr Pharm Des 2014; 20: 5430-5437. Grayson WL, Bunnell BA, Martin E, et al. Stromal cells and stem cells in clinical bone regeneration. Nat Rev Endocrinol 2015;11: 140-150. Ono N, Balani DH, Kronenberg HM. Stem and progenitor cells in skeletal development. Curr Top Dev Biol 2019; 133: 1-24. Li P, Deng Q, Liu J, et al. Roles for HB-EGF in mesenchymal stromal cell proliferation and differentiation during skeletal growth. J Bone Miner Res 2019; 34: 295-309. Papaioannou G, Mirzamohammadi F, Kobayashi T. Ras signaling regulates osteoprogenitor cell proliferation and bone formation. Cell Death Dis 2016; 7: e2405. Moore KA, Lemischka IR. Stem cells and their niches. Science 2006; 311: 1880-1885. Ferreira SA, Motwani MS, Faull PA, et al. Bi-directional cell-pericellular matrix interactions direct stem cell fate. Nat Commun 2018; 9: 4049. Zhang B, Liu N, Shi H, et al. High glucose microenvironments inhibit the proliferation and migration of bone mesenchymal stem cells by activating GSK3β. J Bone Miner Metab 2016; 34: 140-150. Pierce JL, Begun DL, Westendorf JJ, et al. Defining osteoblast and adipocyte lineages in the bone marrow. Bone 2019; 118: 2-7. Tencerova M, Figeac F, Ditzel N, et al. High-Fat Diet-Induced Obesity Promotes Expansion of bone marrow adipose tissue and impairs skeletal stem cell functions in Mice. J Bone Miner Res 2018; 33: 1154-1165. He S, Sharpless NE. Senescence in health and disease. Cell 2017; 169: 1000-1011. Farr JN, Xu M, Weivoda MM, et al. Targeting cellular senescence prevents age-related bone loss in mice. Nat Med 2017; 23:1072-1079. Li C, Chai Y, Wang L, et al. Programmed cell senescence in skeleton during late puberty. Nat Commun 2017; 8: 1312. Martin LF, Richardson LS, da Silva MG, et al. Dexamethasone induces primary amnion epithelial cell senescence through telomere-P21 associated pathway. Biol Reprod 2019; 100: 1605-1616. van Olst L, Bielefeld P, Fitzsimons CP, et al. Glucocorticoid-mediated modulation of morphological changes associated with aging in microglia. Aging Cell 2018; 17: e12790. Dennison EM, Cooper C, Cole ZA. Early development and osteoporosis and bone health. J Dev Orig Health Dis 2010; 1: 142-149. Ono N, Ono W, Mizoguchi T, et al. Vasculature-associated cells expressing nestin in developing bones encompass early cells in the osteoblast and endothelial lineage. Dev Cell 2014; 29: 330–339. Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 2014; 507: 323-328. Beier EE, Sheu TJ, Resseguie EA, et al. Sclerostin activity plays a key role in the negative effect of glucocorticoid signaling on osteoblast function in mice. Bone Res 2017; 5: 17013. Moisiadis VG, Matthews SG. Glucocorticoids and fetal programming part 2: Mechanisms. Nat Rev Endocrinol 2014; 10:403-411. Fowden AL, Forhead AJ. Glucocorticoids as regulatory signals during intrauterine development. Exp Physiol 2015;100: 1477-1487. Bernal A, Arranz L. Nestin-expressing progenitor cells: function, identity and therapeutic implications. Cell Mol Life Sci 2018; 75: 2177-2195. Kirkland JL, Tchkonia T. Cellular Senescence: A translational perspective. EBioMedicine 2017; 21: 21-28. Muñoz-Espín D, Cañamero M, Maraver A, et al. Programmed cell senescence during mammalian embryonic development. Cell 2013; 155: 1104-1118. Storer M, Mas A, Robert-Moreno A, et al. Senescence is a developmental mechanism that contributes to embryonic growth and patterning. Cell 2013; 155: 1119-1130. Xu M, Pirtskhalava T, Farr JN, et al. Senolytics improve physical function and increase lifespan in old age. Nat Med 2018; 24: 1246-1256. Tikhonova AN, Dolgalev I, Hu H, et al. The bone marrow microenvironment at single-cell resolution. Nature 2019; 569: 222-228. Abbreviations PDE, prenatal dexamethasone exposure; GD, gestational day; BMSCs, bone marrow mesenchymal stem cells; SASP, senescence-associated secretory phenotype; μCT, microcomputed tomography; BV/TV, bone volume; Tb. N, trabecular number; Tb. Th, trabecular thickness; Tb. Sp, trabecular separation; H&E, hematoxylin-eosin; TRAP, tartrate-resistant acid phosphatase; SA-β-Gal, senescence associated β-galactosidase; PBST, PBS with Tween; Emcn, Endomucin; ANOVA, analysis of variance; D+Q, dasatinib and quercetin. Supplementary Files SupplementaryFigure2.tif SupplementaryFigure1.tif SupplementaryLegends.docx Cite Share Download PDF Status: Published Journal Publication published 06 Jul, 2020 Read the published version in Stem Cell Research & Therapy → Version 2 posted Editorial decision: Accept 06 Jun, 2020 Review # 3 received at journal 20 May, 2020 Review # 2 received at journal 20 May, 2020 Review # 1 received at journal 20 May, 2020 Reviewer # 3 agreed at journal 15 May, 2020 Reviewer # 2 agreed at journal 13 May, 2020 Reviewers invited by journal 12 May, 2020 Reviewer # 1 agreed at journal 12 May, 2020 Editor assigned by journal 10 May, 2020 Submission checks completed at journal 09 May, 2020 Editor invited by journal 09 May, 2020 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-17277","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":669971,"identity":"5078b2b9-6341-43ba-8662-1449517a0971","order_by":0,"name":"Jianwen Su","email":"","orcid":"","institution":"Southern Medical University Nanfang Hospital Department of Spinal Surgery","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jianwen","middleName":"","lastName":"Su","suffix":""},{"id":669972,"identity":"c85c89a6-46f5-4812-a52a-eaaed2d476bd","order_by":1,"name":"Yu Chai","email":"","orcid":"","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Chai","suffix":""},{"id":669973,"identity":"51261a6c-c2eb-4ea9-9817-eb1d7ded8527","order_by":2,"name":"Zhiguo Ji","email":"","orcid":"","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhiguo","middleName":"","lastName":"Ji","suffix":""},{"id":669974,"identity":"22999c71-4020-431a-98c8-5772667f6885","order_by":3,"name":"Yongheng Xie","email":"","orcid":"","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yongheng","middleName":"","lastName":"Xie","suffix":""},{"id":669975,"identity":"a8349128-22b7-403d-b312-5f99ec294443","order_by":4,"name":"Bin Yu","email":"","orcid":"","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Yu","suffix":""},{"id":669976,"identity":"c0d20baa-069c-437f-b5ed-be11f4fb4ee2","order_by":5,"name":"Xianrong Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYDACZhhDgvkAhHGAeC1sCURqgQMJHgPitBgcZ372uKLijt382T2fP91sY5Dju5HA+LkAjxbJZjZzwzNnniU3zjm7TTq3jcFY8kYCs/QMPFr4mRnMJBvbDiczS+RuYwZqSdxwI4GNmQePFjZm9m9gLWwSOY8/A7XUE9TCz8wDtsWORyKHAeSwBANCWiSbecokG84cTpCQSDOTzjknYTjzzMNmaXxaDM4f3ybZUHHYXn5G8uPPOWU28nzHkw9+xqcFBhIbILQEEDM2EKGBgcGeKFWjYBSMglEwMgEAdupG/ujBt8QAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0992-5013","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xianrong","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2020-03-11 16:36:56","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-17277/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-17277/v2","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13287-020-01790-9","type":"published","date":"2020-07-06T12:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":1346446,"identity":"1ad39352-e527-4b2c-a3b3-532ec372f970","added_by":"auto","created_at":"2020-06-16 23:09:50","extension":"tif","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":804639,"visible":true,"origin":"","legend":"Prenatal dexamethasone exposure (PDE) induces bone loss in later life of female mice offspring. (a) Representative μCT images of proximal tibia in 12-week-old female and male mice offspring. Scale bar, 1 mm. Quantitative analyses of bone microstructural parameters of female mice offspring including trabecular bone volume fraction (BV/TV) (b), trabecular number (Tb. N) (c), trabecular thickness (Tb. Th) (d), and trabecular separation (Tb. Sp) (e). Quantitative analysis of BV/TV (f), Tb.N (g), Tb.Th (h) and Tb.Sp (i) in male mice offspring. Data are represented as mean ± S.E.M. * P \u003c 0.05 versus control (n = 6 per group, Student’s t test).","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-17277/v2/Figure1.tif"},{"id":1346447,"identity":"9790982f-f7a5-4bbe-9b5c-597fc8a8d43c","added_by":"auto","created_at":"2020-06-16 23:09:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2304950,"visible":true,"origin":"","legend":"Prenatal dexamethasone exposure (PDE) suppresses postnatal long bone development in female mice offspring. (a) Representative images of hematoxylin-eosin (H\u0026E) staining, Goldner’s trychrome staining, and tartrate-resistant acid phosphatase (TRAP) staining in femoral sections from 2-, 4-, and 6-week-old female mice offspring. 2W, 4W and 6W represent 2-, 4-, and 6-week-old mice, respectively. Scale bar, 100 μm. Quantitative analysis of the numbers of osteoblast per tissue area in metaphyseal bone below growth plate (N. Ob per mm2) (b) and the numbers of TRAP+ cells per tissue area in metaphyseal bone below growth plate (N. TRAP+ cells per mm2) (c). Data are represented as mean ± S.E.M. * P \u003c 0.05 versus control (n = 5 per group, Student’s t test).","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-17277/v2/Figure2.jpg"},{"id":1346448,"identity":"ee2c0082-9b9d-4551-8701-d743f10325ec","added_by":"auto","created_at":"2020-06-16 23:09:51","extension":"tif","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6451270,"visible":true,"origin":"","legend":"Prenatal dexamethasone exposure (PDE) induces loss of Nestin+ cells and type H vessels in the long bone of female mice offspring. (a) Representative images of immunofluorescence staining for Nestin (green) and (b) quantitative analysis of Nestin+ cells in femoral metaphysis from 2-, 4-, and 6-week-old female offspring. 2W, 4W and 6W represent 2-, 4-, and 6-week-old mice, respectively. DAPI stains nuclei blue. Scale bar, 30 μm. (c) Representative images of double-immunofluorescence staining for CD31 (green) and Endomucin (Emcn, red) in femoral metaphysis from 2-, 4-, and 6-week-old female offspring. DAPI stains nuclei blue. Scale bar, 40 μm. (d) Quantification of the relative fluorescence area of CD31+Emcn+ cells per tissue area in femoral metaphysis (CD31+Emcn+ area per mm2). Data are represented as mean ± S.E.M. * P \u003c 0.05 versus control (n = 5 per group, Student’s t test).","description":"","filename":"Figure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-17277/v2/Figure3.tif"},{"id":1346449,"identity":"413cc304-03d5-465a-8374-413535ed06f4","added_by":"auto","created_at":"2020-06-16 23:09:53","extension":"tif","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6088126,"visible":true,"origin":"","legend":"Prenatal dexamethasone exposure (PDE) enhances cellular senescence in trabecular bone of female mice offspring. (a) Representative images of senescence associated β-galactosidase (SA-β-Gal) staining in femoral metaphysis from 2-, 4-, and 6-week-old female offspring. 2W, 4W and 6W represent 2-, 4-, and 6-week-old mice, respectively. Sections were counterstained with eosin (pink color). Scale bar, 30 μm. (b) Quantitative analysis of the number of SA-β-Gal+ cells per tissue area in metaphyseal bone below growth plate. (c) Representative images of immunofluorescence staining for Ki67 (green) and (d) quantitative analysis of the number of Ki67+ cells in femoral metaphysis from 2-, 4-, and 6-week-old female offspring. DAPI stains nuclei blue. Scale bar, 50 μm. Data are represented as mean ± S.E.M. * P \u003c 0.05 versus control (n = 7 per group, Student’s t test).","description":"","filename":"Figure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-17277/v2/Figure4.tif"},{"id":1346450,"identity":"e59d5b9c-a500-4b64-b365-f4bf1abb8866","added_by":"auto","created_at":"2020-06-16 23:09:53","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2066493,"visible":true,"origin":"","legend":"Clearance of senescent-cell by treatment with senolytics dasatinib and quercetin (D+Q) prevents the suppressed osteogenesis in female mice offspring after PDE. (a) Representative images of senescence associated β-galactosidase (SA-β-Gal) staining, hematoxylin-eosin (H\u0026E) staining, Goldner’s trychrome staining, and Nestin immunofluorescence (green) in femoral metaphysis from 2-week-old female offspring. Pregnant mice were injected subcutaneously with normal saline (control) or dexamethasone (1.2 mg/kg/day) (PDE group) during gestational day (GD) 12-14. Additionally, control pregnant mice were treated with 1% methyl cellulose, and PDE pregnant mice were treated with 1% methyl cellulose (vehicle) or dasatinib (5 mg/kg/day) and quercetin (50 mg/kg/day) by oral gavage during GD 12-14. Femurs of 2-week-old female mice offspring were harvested for analysis. DAPI stains nuclei blue. Scale bar, 100 μm. (b) Quantitative analysis of the numbers of SA-β-Gal+ cells per tissue area in femoral metaphysis. n = 6 per group. (c) Quantitative analysis of the osteoblast numbers per tissue area in femoral metaphysis. n = 6 per group. (d) Quantitative analysis of the Nestin+ cells per tissue area. n = 5 per group. * P \u003c 0.05, one-way analysis of variance (ANOVA) with Bonferroni post hoc test.","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-17277/v2/Figure5.jpg"},{"id":1346451,"identity":"2e4d3f84-14a8-4afd-9192-224108688115","added_by":"auto","created_at":"2020-06-16 23:09:53","extension":"tif","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1100461,"visible":true,"origin":"","legend":"Clearance of senescent-cell by treatment with senolytics dasatinib and quercetin (D+Q) rescues the loss of osteoprogenitors and BMSCs in female mice offspring after PDE. (a) Representative images of the flow cytometry analysis and (b) the percentage of Nestin+ cells in femoral bone from 4-week-old female mice offspring. Pregnant mice were injected subcutaneously with normal saline (control) or dexamethasone (1.2 mg/kg/day) (PDE group) during gestational day (GD) 12-14. Additionally, control pregnant mice were treated with 1% methyl cellulose, and PDE pregnant mice were treated with 1% methyl cellulose (vehicle) or dasatinib (5 mg/kg/day) and quercetin (50 mg/kg/day) by oral gavage during GD 12-14. Femoral bone marrow of 4-week-old female mice offspring were harvested for analysis. (c) Representative images of the flow cytometry analysis and (b) the percentage of CD45-CD29+CD105+Sca-1+ cells in femoral bone from 4-week old female mice offspring. n = 5 per group. * P \u003c 0.05, one-way analysis of variance (ANOVA) with Bonferroni post hoc test.","description":"","filename":"Figure6.tif","url":"https://assets-eu.researchsquare.com/files/rs-17277/v2/Figure6.tif"},{"id":1346452,"identity":"cc6296b9-aec4-4ac0-be8e-1c6d84a7dffe","added_by":"auto","created_at":"2020-06-16 23:09:53","extension":"tif","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5047471,"visible":true,"origin":"","legend":"Senolytics dasatinib and quercetin (D+Q) rescues the loss of osteoblasts and H type vessels in female mice offspring after PDE. (a) Representative images of the immunohistochemistry staining for osteocalcin (Ocn, dark brown stained cells on the surface of trabecular bone) and (b) the number of Ocn+ cells per mm of bone surface in femoral bone from 4-week-old female mice offspring. Scale bar, 50 μm. (c) Representative images of double-immunofluorescence staining for CD31 (green) and Endomucin (Emcn, red) in femoral metaphysis from 4-week-old female offspring. DAPI stains nuclei blue. Scale bar, 30 μm. (d) Quantification of the relative fluorescence area of CD31+Emcn+ cells per tissue area in femoral metaphysis (CD31+Emcn+ area per mm2). Data are represented as mean ± S.E.M. n = 5 per group, *P \u003c 0.05, one-way analysis of variance (ANOVA) with Bonferroni post hoc test.","description":"","filename":"Figure7.tif","url":"https://assets-eu.researchsquare.com/files/rs-17277/v2/Figure7.tif"},{"id":13542115,"identity":"ebe85e12-b782-4c9e-ba22-cc43448001a7","added_by":"auto","created_at":"2021-09-17 01:53:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":31506419,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-17277/v2/9d0abd62-44d5-4f55-bf7e-d77ea4fff1e2.pdf"},{"id":1346455,"identity":"b946b16b-86e5-4fa3-aa28-3fb75e395f9c","added_by":"auto","created_at":"2020-06-16 23:09:57","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5650100,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-17277/v2/SupplementaryFigure2.tif"},{"id":1346456,"identity":"93d6e9fc-1580-4223-bfbd-df5de85e4ad9","added_by":"auto","created_at":"2020-06-16 23:09:57","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13095228,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-17277/v2/SupplementaryFigure1.tif"},{"id":1346457,"identity":"994e1167-5a11-4205-a1dc-00f55f0a7c5d","added_by":"auto","created_at":"2020-06-16 23:09:57","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13744,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-17277/v2/SupplementaryLegends.docx"}],"financialInterests":"","formattedTitle":"Cellular senescence mediates the detrimental effect of prenatal dexamethasone exposure on postnatal long bone growth in mice offspring","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePrenatal corticosteroid use in clinic has been shown to be effective in accelerating lung maturation and reducing the incidence of respiratory complications in infant [1]. However, clinical studies provide evidence that these short-term benefits are associated with reduction in birth size for infants born preterm, near term, or at term [2], and suppression of fetal bone turnover in infants at birth [3]. In addition, clinical trial data show that lower birth weight is associated with lower peak bone mass in adult offspring [4, 5]. Evidence from experimental animal models by our group and others demonstrate that prenatal dexamethasone exposure (PDE) impairs long bone development in fetal animals [6-9], reduces bone mass in adult offspring [7, 10]. These data imply that developmental overexposure to glucocorticoid alters bone programming and results in less bone mass in offspring. Although studies have found the detrimental effect of PDE on osteogenesis [11, 12], little is known about the most critical cellular target of PDE in the developing bone.\u003c/p\u003e\n\u003cp\u003eMesenchymal stem/stromal cells from bone marrow are multipotent cells that play crucial roles in bone development, maintenance and regeneration due to their multilineage differentiation and self-renewal capacity [13, 14]. During childhood and puberty, skeletal bone grows quickly owing to rapid self-renewal and differentiation of bone marrow mesenchymal stem or stromal cells (BMSCs) and osteoprogenitors [15, 16]. The activities of stem cells are controlled by the local stem cell microenvironment, which is composed of cellular components such as stromal cells, immune cells, endothelial cells, osteoblasts and a non-cellular compartment which includes extracellular matrix components and signal molecules [17]. This local environment plays important role in regulating stem cell survival, function and fate [18], and alterations of the microenvironment may impair skeletal stem cells functions, leading to decreased osteogenesis and bone formation [19-21].\u003c/p\u003e\n\u003cp\u003eCellular senescence is a state of irreversible growth arrest and occurs throughout life. Senescent cells can secrete numerous biologically active factors, termed the senescence-associated secretory phenotype (SASP), leading to pathological consequences in the tissue microenvironment [22]. It has been demonstrated that senescence associated bone microenvironment contribute to age-related bone loss [23]. Recent evidence suggests that cellular senescence is also a key regulator during bone development [24]. Senescence can be induced by various intrinsic and extrinsic triggers. Extensive use of dexamethasone has been associated with cellular senescence and aging-related pathological process \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro \u003c/em\u003e[25, 26]. However, the potential relationship between cellular senescence and development retardation in bone induced by PDE remains unclear.\u003c/p\u003e\n\u003cp\u003eOur recent study found that PDE during gestational day 12-14 (GD12-14) retards bone development in fetal mice [6]. Here we show that young and adult female mice offspring by PDE have impaired bone formation and significant lower bone mass. We have found significant reduced amount of CD45\u003csup\u003e-\u003c/sup\u003eNestin\u003csup\u003e+\u003c/sup\u003e and CD45\u003csup\u003e-\u003c/sup\u003eCD29\u003csup\u003e+\u003c/sup\u003eCD105\u003csup\u003e+\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003e cells as well as impaired angiogenesis in metaphysis of long bone in young offspring mice after PDE. We also have found that PDE promotes cellular senescence and suppresses cells proliferation in trabecular area of long bone. Interestingly, eliminating senescent cells using senolytics dasatinib and quercetin (D+Q) rescues significantly the decreased BMSCs and osteoprogenitors by PDE and prevents developmental retardation of long bone in young mice offspring.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals of Nanfang Hospital Southern Medical University. The protocol was approved by the Animal Care and Use Committee of Nanfang Hospital. Pathogen-free mice were maintained under standard conditions in a 12h light and 12h dark cycle, at 25 \u0026plusmn; 3\u0026deg;C, with a relative humidity of 40-60%, and with food and tap water available ad libitum. PDE during GD 12-14 was applied according to the procedure previously described [6]. Briefly, virgin C57BL/6 female mice at 10-12 weeks old were mate with male mice overnight. The day on which the presence of a vaginal plug was set as GD 0, the pregnant mice were randomly assigned to the PDE group or vehicle treatment (control) group. To construct PDE mice model, dexamethasone sodium phosphate (Cat. 2392-39-4, Tianxin, China) was injected subcutaneously (1.2 mg/kg/day) during GD 12-14. To construct the vehicle control of PDE model, pregnant mice were treated with the same amount of vehicle (normal saline) daily during GD 12-14. The pregnant mice were housed individually in cages with freely available food and water. Two female offspring were selected randomly from each litter for postnatal bone development investigation. Femurs and tibias from mice offspring at 2-, 4-, 6-, and 12-week-old were dissected for further analysis. To evaluate the effect of dasatinib (S1021, Selleck Chemicals，Houston，TX, USA) and quercetin (S2391, Selleck Chemicals，Houston，TX, USA), PDE pregnant mice during GD 12-14 were treated with vehicle (200 \u0026mu;l 1% methyl cellulose) or dasatinib (5 mg/kg/day) plus quercetin (50 mg/kg/day) by oral gavage, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Microcomputed Tomography (\u0026mu;CT) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTibias from 12-week-old mice offspring were dissected free of soft tissue, fixed and stored in 70% ethanol, and imaged using a \u0026mu;CT specimen scanner (Scanco Medical, AG, Switzerland). The scan was performed using an X-ray energy of 55 kV and current of 145 mA, with a voxel size of 12\u0026mu;m and an integration time of 400 msec. Trabecular bone measurements consisting of 250 slices (3mm) were performed from 0.215 mm (18 image slices) below the growth plate. Bone volume (BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th) and trabecular separation (Tb. Sp) were determined. Quantitative analyses were carried out using IPL software (Image Processing Language V5.15, Scanco Medical AG, Switzerland).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Histochemistry \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the morphology of postnatal long bone growth in offspring after PDE, femurs and tibias of mice offspring at 2-, 4-, and 6-week-old were fixed in 4% paraformaldehyde, decalcified in 0.5M ethylenediamine-tetraacetic acid (EDTA, pH 7.4), followed by paraffine embedding or frozen embedding. Hematoxylin-eosin (H\u0026amp;E) staining, Goldner\u0026rsquo;s trychrome staining, and tartrate-resistant acid phosphatase (TRAP) staining were performed on 4 \u0026mu;m paraffin sections according to standard procedures. The number of osteoblasts per square millimeter of metaphyseal area (N. per mm\u003csup\u003e2\u003c/sup\u003e) were quantified in the area from 0-0.5mm below growth plate.\u003c/p\u003e\n\u003cp\u003eFor detecting the osteoclastic activity in bone, TRAP staining was performed on the deparaffinized and rehydrated sections using a Leukocyte Acid Phosphatase kit (Cat. 387A-1KT, Sigma-Aldrich, USA). The TRAP\u003csup\u003e+\u003c/sup\u003e multinucleated cells containing at least three nuclei were identified as osteoclasts under light microscope (Olympus, BX53). The number of TRAP\u003csup\u003e+\u003c/sup\u003e cells per square millimeter of metaphyseal area (N. per mm\u003csup\u003e2\u003c/sup\u003e) in the area from 0-0.5mm below growth plate was quantified.\u003c/p\u003e\n\u003cp\u003eFor detecting senescence associated \u0026beta;-galactosidase (SA-\u0026beta;-Gal) activity, frozen sections were stained using SA-\u0026beta;-Gal staining kit (Cat. 9860, Cell Signaling Technology, USA) according to manufacturer\u0026rsquo;s instructions. Senescent cells were identified as blue-stained cells under light microscope (Olympus, BX53). The number of SA-\u0026beta;-Gal\u003csup\u003e+\u003c/sup\u003e cells per square millimeter of metaphyseal area (N. per mm\u003csup\u003e2\u003c/sup\u003e) in the area from 0-0.5mm below growth plate was quantified.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Immunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor immunofluorescence staining, frozen sections were incubated in blocking buffer (3% BSA in PBS with Tween (PBST)) for 1 hour at room temperature, incubated with primary antibodies overnight at 4\u0026deg;C. The primary antibodies for immunostaining include: Nestin (ab134017, Abcam, Cambridge, MA, USA), CD31 (FAB3629G-100, R\u0026amp;D Systems, Minneapolis, MN, USA), Endomucin (Emcn, SC-65495，Santa Cruz, Dallas, TX USA), Ki67 (ab15580, Abcam, Cambridge, MA, USA). Sections were washed 3 times in PBS and then incubated with secondary antibodies at room temperature for 1 hour. The secondary antibodies for immunostaining include: 488-conjugated secondary antibody (703-546-155, Jackson ImmunoResearch, West Grove, PA, USA), 594-conjugated secondary antibody (712-586-153, Jackson ImmunoResearch, West Grove, PA, USA), 488-conjugated secondary antibody (A21206, ThermoFish Scientific, USA). Nuclei were counterstained with DAPI (S2110，Solarbio, China). Images were captured using a fluorescence microscrope (Olympus, BX53, Japan). Positive-stained area or the number of positive-stained cells per square millimeter of the metaphyseal area was measured from 0-0.5mm below growth plate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Flow cytometric analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBone marrow cells were collected from femurs and tibias of mice offspring at 4-week-old. Cell numbers were determined after removal of red blood cells with ACK Lysis Buffer (CS0001, Leagene, China). After washed with PBS twice, pellets were resuspended and blocked in 1% BSA on ice for 15min. Cells were then washed twice with PBS and incubated with primary antibody (for cell surface marker) solution diluted by 0.5% BSA for 30min on ice in the dark. After being fixed by 4% paraformaldehyde and permeabilized by PBST and washed with PBS, cells were incubated with primary antibody (for intracellular antigen) solution diluted by 0.5% BSA for 30min on ice in the dark. The primary antibody used were PE-conjugated Nestin Antibody (MA5-23574, ThermoFish Scientific, Rockford, IL, USA), and BV421\u0026minus;conjugated anti-mouse CD45 (563890, BD Biosciences, San Jose, CA, USA). Cells were then washed once and re-suspended in 300 \u0026mu;l PBS and transferred to flow tubes. For BMSCs, CD45\u003csup\u003e-\u003c/sup\u003eCD29\u003csup\u003e+\u003c/sup\u003eCD105\u003csup\u003e+\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003e bone marrow cells were detected using a Mouse mesenchymal stem cell Multi-color Flow kit (FMC003, R\u0026amp;D systems, Canada) following manufactures protocol. Flow cytometric analysis was performed on a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (BD Life Sciences San Jose, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Statistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology. All quantitative data were presented as mean \u0026plusmn; S.E.M. For comparisons between two groups, independent Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was performed. For multiple comparisons, one-way analysis of variance (ANOVA) with Bonferroni post hoc test was used. Statistical analysis was performed using SPSS, version 20 software (International Business Machines Corporation, IBM Corp.). Significant level was defined as \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Adult mice offspring from PDE has low bone mass in the long bone \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur previous work has shown the adverse effect of PDE on the long bone development in fetal mice [6]. We then tested whether long bone development retardation during prenatal period results in bone mass deficit in adult offspring. Tibias were harvested from 12-week-old mice offspring of PDE and measured by \u0026mu;CT. A significant reduction in the mass of trabecular bone was observed in female offspring relative to control mice offspring (Fig. 1). There was a significant decrease of bone volume fraction (BV/TV) in female adult mice offspring from PDE, which was attributed to a large decrease in trabecular number (Tb. N) along with a notable increase in trabecular separation (Tb. Sp), with no change observed in trabecular thickness (Tb.Th) (Fig. 1b-e). But, we didn\u0026rsquo;t observe change of the above microstructural parameters in male mice offspring (Fig. 1f-i). Together, these data revealed that PDE has long-term detrimental effect on bone mass in female adult mice offspring.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Postnatal long bone development is retarded in female mice offspring from PDE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBone mass in adulthood has been closely correlated with fetal and postnatal bone growth, which is an important process for bone mineral accrual [27]. To investigate how PDE induces low bone mass in female adult offspring, we evaluated the morphology of long bone of female offspring at 2-, 4-, and 6-week-old. H\u0026amp;E staining and Goldner\u0026rsquo;s trychrome staining results showed reduced amount of trabecular bones in female mice offspring after PDE (Fig. 2a). Similarly, histomorphometric analysis revealed significantly less osteoblast counts in offspring from PDE, compared with that in control (Fig. 2b). As the reduced trabecular bone could be due to either lower bone formation or higher bone resorption or both, we examined the changes of osteoclastogenesis in mice offspring after PDE. TRAP staining and quantitative analysis revealed that osteoclast number was significantly lower in PDE mice offspring compared to controls (Fig. 2c). Therefore, PDE impedes both bone formation and bone absorption during skeletal bone development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 PDE induces loss of Nestin expressing cells and blood vessels in long bone of female mice offspring\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNestin expressing (Nestin\u003csup\u003e+\u003c/sup\u003e cells) in postnatal bones are heterogeneous populations mainly in endothelial and osteoblast lineage [24, 28]. These cells are highly proliferative and critical for osteoblast replenishment for bone formation during postnatal bone development [24]. We thus assessed whether PDE affects Nestin\u003csup\u003e+\u003c/sup\u003e cells in postnatal long bone of female mice offspring. Results showed that the number of Nestin\u003csup\u003e+\u003c/sup\u003e cells in femoral metaphysis was significantly reduced as assessed by immunofluorescence staining (Fig. 3a, b).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; A specific subtype of vessels, termed H-type vessels which characterized by high expression of the endothelial markers CD31 and Emcn (CD31\u003csup\u003ehi\u003c/sup\u003eEmcn\u003csup\u003ehi\u003c/sup\u003e), generate distinct microenvironments for maintaining perivascular osteoprogenitors and coupling angiogenesis to osteoprogenitors [29]. We then examined the effect of PDE on type-H vessels in postnatal long bone of offspring. Double immunofluorescence staining for CD31 and Emcn showed a significant lower proportion of type-H vessels in the femoral metaphysis of mice offspring after PDE compared to that of control offspring (Fig. 3c, d). Therefore, PDE reduces type-H vessels, which are closely associated with impaired bone formation in mice offspring.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Cellular senescence is increased in postnatal long bone of mice offspring after PDE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA recent report showed that premature cellular senescence in the long bone of young mice leads to reduction of osteoprogenitors, impairing of blood vessel formation and bone formation [24]. We then tested whether the progression of the cellular senescence in long bone of postnatal offspring is altered by PDE. SA-\u0026beta;-Gal staining was conducted in femoral bones of mice offspring at 2-, 4-, and 6-week-old. We found a significant increase in the number of SA-\u0026beta;-Gal\u003csup\u003e+\u003c/sup\u003e cells in femoral metaphysis of PDE offspring at 2-, 4-, and 6-week-old compared to those in control offspring, respectively (Fig. 4a, b). Consistently, immunofluorescence staining for Ki67, the proliferative marker, showed reduced staining in the trabecular bone adjacent to the growth plate, the same region that SA-\u0026beta;-Gal\u003csup\u003e+\u003c/sup\u003e cells located (Fig. 4c). Quantitative results confirmed dramatically reduction of Ki67\u003csup\u003e+\u003c/sup\u003e cells in the femoral metaphysis of mice offspring after PDE (Fig. 4d). The above results strongly suggest that PDE induces growth arrest and senescence in certain cell types. To identify whether PDE induced senescence in osteoprogenitors, we performed co-staining of SA-\u0026beta;-Gal with osteoprogenitor marker Nestin and Osterix, and vessel marker Emcn, respectively. However, we didn\u0026rsquo;t find overlapping staining of SA-\u0026beta;-Gal with any of those three markers (Supplementary Fig. 1, 2). The above data suggest that PDE might not stimulate cellular senescence in osteoprogenitors and cells of type H vessels directly, but suppress those cells indirectly by stimulating senescence of other cell populations in bone.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Targeting cellular senescence prevents PDE-induced bone development retardation in mice offspring\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; It was reported that clearance of senescent cells using D+Q can improve bone mass in aged mice [23]. To evaluate the role of increased cellular senescence in PDE-induced bone growth retardation in mice offspring, pregnant mice by PDE were treated with D+Q or vehicle during GD 12-14 once every day. Consistent with the effect of D+Q on aged mice, SA-\u0026beta;-Gal\u003csup\u003e+\u003c/sup\u003e cells number in femoral metaphysis was significantly suppressed in D+Q-treated PDE mice offspring as compared to vehicle-treated ones (Fig. 5a, b). As anticipated, D+Q treatment significantly improved trabecular bone amount in femoral metaphysis in 2-week-old PDE mice offspring relative to vehicle-treated ones, as assessed by H\u0026amp;E staining and Goldner\u0026rsquo;s trychrome staining results (Fig. 5a). Trabecular bone histomorphometry demonstrated the rescued osteoblast numbers and increased Nestin\u003csup\u003e+\u003c/sup\u003e cells in PDE mice offspring treated by D+Q than in vehicle-treated ones (Fig. 5c, d).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; Concomitant with reduced burden of cellular senescence by D+Q and markedly rescued bone phenotype in mice offspring after PDE, we further found that the percentage of CD45\u003csup\u003e-\u003c/sup\u003eNestin\u003csup\u003e+\u003c/sup\u003e (Fig. 6a, b) and CD45\u003csup\u003e-\u003c/sup\u003eCD29\u003csup\u003e+\u003c/sup\u003eCD105\u003csup\u003e+\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003e (Fig. 6c, d) was significantly decreased in bone marrow of mice offspring after PDE. It is noteworthy that D+Q treatment only partially rescued the loss of CD45\u003csup\u003e-\u003c/sup\u003eNestin\u003csup\u003e+\u003c/sup\u003e cells in mice offspring after PDE, but completely restored the ratio of CD45\u003csup\u003e-\u003c/sup\u003eCD29\u003csup\u003e+\u003c/sup\u003eCD105\u003csup\u003e+\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003e cells (Fig. 6). Compared with control mice, mice offspring after PDE and D+Q treatment had significantly lower percentage of CD45\u003csup\u003e-\u003c/sup\u003eNestin\u003csup\u003e+\u003c/sup\u003e cells (Fig. 6a, b), but had a considerable increment in the percentage of CD45\u003csup\u003e-\u003c/sup\u003eCD29\u003csup\u003e+\u003c/sup\u003eCD105\u003csup\u003e+\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003e cells (Fig. 6b, d). Additionally, D+Q treatment also dramatically restored the osteoblast number (Fig. 7a, b) and angiogenesis potential in mice offspring after PDE (Fig. 7c, d). All the data indicate that cellular senescence mediates the detrimental effect of PDE on BMSCs and osteoprogenitors, leading to retardation of postnatal long bone growth in mice offspring.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eHere we provide unique insights into the mechanisms by which PDE leads to retardation of postnatal bone development. Our data show that PDE induces cellular senescence, reduction of BMSCs amount and decrease of bone modeling and remodeling during postnatal bone growth in female mice offspring. Furthermore, elimination of excessive senescent-cells can rescue the toxic effect of PDE on postnatal bone growth in female mice offspring. The present study has identified that PDE induces cellular senescence which may contribute to the toxic effect of PDE on BMSCs and type H vessels in the metaphysis of long bone, thereby impeding bone formation in mice offspring. We believe that these results have opened a door to elucidating poorly understood aspects of osteoporosis with developmental origin and highlighted D+Q as a therapeutic approach for treating PDE-induced retardation of bone growth in offspring.\u003c/p\u003e\n\u003cp\u003eOur previous work demonstrated that continuous PDE during GD 9-20 retards long bone development in fetal rats and leads to low bone mass in adult rat offspring [7], and further delineated that PDE at dose higher than 0.8 mg/kg/day during GD 12-14 developed severe retardation of long bone development in fetal mice [6]. In the present study, the data clearly indicate that PDE at 1.2 mg/kg/day during GD 12-14 induces low bone mass in 12-week-old mice offspring. Our finding is supported by a recent report that PDE at GD 16-17 induces decreased bone mass and biomechanical strength in adult mice offspring [30]. Furthermore, we observed a notable decrease in both osteoblasts and osteoclasts in femoral metaphysis of female mice offspring after PDE, indicating a decreased bone modeling and remodeling induced by PDE. Overall, the works from our lab and others have highlighted that PDE has long-term effect on bone mass of offspring in later life, which might be consequences of PDE programming on intrauterine development of fetal tissue [31, 32].\u003c/p\u003e\n\u003cp\u003eDuring bone modeling and remodeling, bone formation requires continuous production of osteoblasts from osteoprogenitors and BMSCs in mammals. Nestin\u003csup\u003e+\u003c/sup\u003e cells in postnatal bone encompass osteoblastic and endothelial progenitors, and several subsets of stem cells with self-renewal capacity [28, 33]. Additionally, type H vessels are important for maintaining osteoprogenitors and coupling angiogenesis to osteoprogenitors [29]. Our studies provide the first evidence of decreased amount of CD45\u003csup\u003e-\u003c/sup\u003eCD29\u003csup\u003e+\u003c/sup\u003eCD105\u003csup\u003e+\u003c/sup\u003eSca-1\u003csup\u003e+ \u003c/sup\u003eBMSCs and Nestin\u003csup\u003e+\u003c/sup\u003e cells and impaired type H vessels in the long bone of female mice offspring after PDE. It is possible that PDE has extensive toxic effect on osteoprogenitors, BMSCs and type H vessels, leading to impaired osteogenesis and diminished bone formation.\u003c/p\u003e\n\u003cp\u003eInterestingly, we found the dramatically increased cellular senescence and diminished cellular proliferation in femoral metaphysis of young female mice offspring after PDE. Senescent cells are known to instigate SASP which may exert detrimental paracrine, thereby contributing to senescence-related inflammation and stem cell dysfunction [34]. Indeed, senescent cells are found to be associated with embryonic and postnatal development [24, 35, 36]. In the present study, we didn\u0026rsquo;t find much overlapping of osteoprogenitor markers with SA-\u0026beta;-Gal staining, however, our data revealed that reducing senescent cell burden using senolytics D+Q have dramatically restored the percentage of BMSCs, the numbers of osteoblasts and type H vessels in trabecular bone of female mice offspring after PDE. These data support our conclusion that excessive senescent-cells mediate the detrimental effect of PDE on postnatal long bone development in female mice offspring.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; As Nestin\u003csup\u003e+\u003c/sup\u003e cells has been found to undergo normal programmed senescence in trabecular bone during late puberty, and prednisolone injection during puberty further stimulate senescence of Nestin\u003csup\u003e+\u003c/sup\u003e cells in mice [24], it is possible that loss of Nestin\u003csup\u003e+\u003c/sup\u003e cells in PDE mice offspring might be at least partially due to the senescence of Nestin\u003csup\u003e+\u003c/sup\u003e cells. Interestingly, we found that senolytics D+Q treatment only partially rescued the loss of Nestin\u003csup\u003e+\u003c/sup\u003e cells in mice offspring after PDE. One reason could be that PDE suppresses Nestin\u003csup\u003e+\u003c/sup\u003e cells by a mechanism of action that is distinct from that of D+Q, which is known to reduce senescent cell burden by reducing pro-inflammatory cytokine secretion [37].\u003c/p\u003e\n\u003cp\u003eBone is a dynamic organ composed of various bone marrow-derived cell types including hematopoietic, mesenchymal, endothelial cells [38]. As dexamethasone has far-ranging effects on various cells and tissues [31], it might trigger the senescence on a heterogeneous population of cells in bone. Considering that almost all of SA-\u0026beta;-Gal-positive stained cells locate in endosteal bone marrow close to the trabecular bone surface, but not inside the trabecular bone where osteocytes locate, it is reasonable to rule out the senescence of osteocytes in young mice offspring after PDE. Nevertheless, a limitation of our study cannot be neglected that we failed (despite extensive effort) to identify the cell types and molecular mechanism of cellular senescence induced by PDE. Growing evidences indicate that prenatal glucocorticoid may exert long-term programming of fetal tissue by epigenetic modifications of genome [31, 32], therefore, further study is necessary to analyze the transcriptome and epigenome of SA-\u0026beta;-Gal\u003csup\u003e+\u003c/sup\u003e cells, to unravel the cellular and molecular mechanisms involved in long-term programming of cellular senescence as a result of PDE, and how a deregulation of cellular senescence retards long bone development in female mice offspring.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eAlthough the precise mechanisms by which PDE enhances cellular senescence will require further experimentation, our studies have significant advancements by identifying cellular senescence as a key regulator linking PDE with postnatal bone development retardation in female mice offspring and, further, targeting cellular senescence may be a promising avenue for prevention of bone development retardation in female mice offspring after PDE.\u003c/p\u003e\n"},{"header":"5. Declarations","content":"\u003cp\u003e\u003cstrong\u003e5.1 Author contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.Z. designed the research, drafted and revised the article; J.S., Y.C., Z.J. and Y.X. acquired the data; J.S., Y.C., B.Y. and X.Z. analyzed the data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2 Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the National Natural Science Foundation of China (No.81573515, to X.Z.) and The Major Program of National Natural Science Foundation of China (No.81830079, to B.Y.), Key Research Project of Science and Technology Planning Program of Guangdong Province (No.2019B020201013, to X.Z.).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.3 Acknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u003c/strong\u003e\u0026nbsp;This study was supported by grants from the National Natural Science Foundation of China (No.81573515, to X.Z.), The Major Program of National Natural Science Foundation of China (No.81830079, to B.Y.), Key Research Project of Science and Technology Planning Program of Guangdong Province (No.2019B020201013, to X.Z.).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.4 Conflicts of interest statement \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.5 Ethics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were conducted in accordance with the guidelines set by the Institutional Animal Care and Use Committee of Nanfang Hospital, Southern Medical University.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.6 Availability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlease contact the corresponding author for data requests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.7 Consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"6. References","content":"\u003col\u003e\n\u003cli\u003eKemp MW, Schmidt AF, Jobe AH. Optimizing antenatal corticosteroid therapy. \u003cem\u003eSemin Fetal Neonatal Med \u003c/em\u003e2019; 24: 176-181.\u003c/li\u003e\n\u003cli\u003eRodriguez A, Wang Y, Ali Khan A, et al. Antenatal corticosteroid therapy (ACT) and size at birth: A population-based analysis using the Finnish Medical Birth Register. \u003cem\u003ePLoS Med \u003c/em\u003e2019; 16: e1002746.\u003c/li\u003e\n\u003cli\u003eKorakaki E, Damilakis J, Gourgiotis D, et al. Quantitative ultrasound measurements in premature infants at 1 year of age: the effects of antenatal administered corticosteroids. \u003cem\u003eCalcif Tissue Int \u003c/em\u003e2011; 88: 215-222.\u003c/li\u003e\n\u003cli\u003eDalziel SR, Fenwick S, Cundy T, et al. Peak bone mass after exposure to antenatal betamethasone and prematurity: follow-up of a randomized controlled trial. \u003cem\u003eJ Bone Miner Res\u003c/em\u003e 2006; 21:1175-1186.\u003c/li\u003e\n\u003cli\u003eXie LF, Alos N, Cloutier A, et al. The long-term impact of very preterm birth on adult\u0026nbsp;bone\u0026nbsp;mineral density. \u003cem\u003eBone Rep\u003c/em\u003e 2018; 10: 100189.\u003c/li\u003e\n\u003cli\u003eChen Z, Zhao X, Li Y, et al. Course-, dose-, and stage-dependent toxic effects of prenatal dexamethasone exposure on long bone development in fetal mice. \u003cem\u003eToxicol Appl Pharmacol\u003c/em\u003e 2018; 351:12-20.\u003c/li\u003e\n\u003cli\u003eZhang X, Shang-Guan Y, Ma J, et al. Mitogen-inducible gene-6 partly mediates the inhibitory effects of prenatal dexamethasone exposure on endochondral ossification in long bones of fetal rats. \u003cem\u003eBr J Pharmacol\u003c/em\u003e 2016; 173: 2250-2262.\u003c/li\u003e\n\u003cli\u003eCheng X, Wang G, Lee KK, Yang X. Dexamethasone use during pregnancy: potential adverse effects on embryonic skeletogenesis.\u003cem\u003e Curr Pharm Des\u003c/em\u003e 2014; 20:5430-5437.\u003c/li\u003e\n\u003cli\u003eSliwa E, Dobrowolski P, Piersiak T. Bone development of suckling piglets after prenatal, neonatal or perinatal treatment with dexamethasone.\u003cem\u003e J Anim Physiol Anim Nutr (Berl) \u003c/em\u003e2010; 94: 293-306.\u003c/li\u003e\n\u003cli\u003eTomaszewska E, Dobrowolski P, Bieńko M, et al. Effects of 2-oxoglutaric acid on bone morphometry, densitometry, mechanics, and immunohistochemistry in 9-month-old boars with prenatal dexamethasone-induced osteopenia. \u003cem\u003eConnect Tissue Res\u003c/em\u003e 2015; 56: 483-492.\u003c/li\u003e\n\u003cli\u003eXiao H, Wen Y, Pan Z, et al. Increased H3K27ac level of ACE mediates the intergenerational effect of low peak bone mass induced by prenatal dexamethasone exposure in male offspring rats. \u003cem\u003eCell Death Dis\u003c/em\u003e 2018; 9: 638.\u003c/li\u003e\n\u003cli\u003eCheng X, Wang G, Lee KK, et al. Dexamethasone use during pregnancy: potential adverse effects on embryonic skeletogenesis. \u003cem\u003eCurr Pharm Des\u003c/em\u003e 2014; 20: 5430-5437.\u003c/li\u003e\n\u003cli\u003eGrayson WL, Bunnell BA, Martin E, et al. Stromal cells and stem cells in clinical bone regeneration.\u003cem\u003e Nat Rev Endocrinol \u003c/em\u003e2015;11: 140-150.\u003c/li\u003e\n\u003cli\u003eOno N, Balani DH, Kronenberg HM. Stem and progenitor cells in skeletal development. \u003cem\u003eCurr Top Dev Biol \u003c/em\u003e2019; 133: 1-24.\u003c/li\u003e\n\u003cli\u003eLi P, Deng Q, Liu J, et al. Roles for HB-EGF in mesenchymal stromal cell proliferation and differentiation during skeletal growth. \u003cem\u003eJ Bone Miner Res\u003c/em\u003e 2019; 34: 295-309.\u003c/li\u003e\n\u003cli\u003ePapaioannou G, Mirzamohammadi F, Kobayashi T. Ras signaling regulates osteoprogenitor cell proliferation and bone formation. \u003cem\u003eCell Death Dis \u003c/em\u003e2016; 7: e2405.\u003c/li\u003e\n\u003cli\u003eMoore KA, Lemischka IR. Stem cells\u0026nbsp;and their niches. \u003cem\u003eScience\u003c/em\u003e 2006; 311: 1880-1885.\u003c/li\u003e\n\u003cli\u003eFerreira SA, Motwani MS, Faull PA, et al. Bi-directional cell-pericellular matrix interactions direct stem cell fate.\u003cem\u003e Nat Commun\u003c/em\u003e 2018; 9: 4049.\u003c/li\u003e\n\u003cli\u003eZhang B, Liu N, Shi H, et al. High glucose microenvironments inhibit the proliferation and migration of bone mesenchymal stem cells by activating GSK3\u0026beta;. \u003cem\u003eJ Bone Miner Metab \u003c/em\u003e2016; 34: 140-150.\u003c/li\u003e\n\u003cli\u003ePierce JL, Begun DL, Westendorf JJ, et al. Defining osteoblast and adipocyte lineages in the bone marrow. \u003cem\u003eBone\u003c/em\u003e 2019; 118: 2-7.\u003c/li\u003e\n\u003cli\u003eTencerova M, Figeac F, Ditzel N, et al. High-Fat Diet-Induced Obesity Promotes Expansion of bone marrow adipose tissue and impairs skeletal stem cell functions in Mice. \u003cem\u003eJ Bone Miner Res\u003c/em\u003e 2018; 33: 1154-1165.\u003c/li\u003e\n\u003cli\u003eHe S, Sharpless NE. Senescence in health and disease. \u003cem\u003eCell \u003c/em\u003e2017; 169: 1000-1011.\u003c/li\u003e\n\u003cli\u003eFarr JN, Xu M, Weivoda MM, et al. Targeting cellular senescence prevents age-related bone loss in mice. \u003cem\u003eNat Med\u003c/em\u003e 2017; 23:1072-1079.\u003c/li\u003e\n\u003cli\u003eLi C, Chai Y, Wang L, et al. Programmed cell senescence in skeleton during late puberty. \u003cem\u003eNat Commun \u003c/em\u003e2017; 8: 1312.\u003c/li\u003e\n\u003cli\u003eMartin LF, Richardson LS, da Silva MG, et al. Dexamethasone induces primary amnion epithelial cell senescence through telomere-P21 associated pathway. \u003cem\u003eBiol Reprod\u003c/em\u003e 2019; 100: 1605-1616.\u003c/li\u003e\n\u003cli\u003evan Olst L, Bielefeld P, Fitzsimons CP, et al. Glucocorticoid-mediated modulation of morphological changes associated with aging in microglia.\u003cem\u003e Aging Cell \u003c/em\u003e2018; 17: e12790.\u003c/li\u003e\n\u003cli\u003eDennison EM, Cooper C, Cole ZA. Early development and osteoporosis and bone health. \u003cem\u003eJ Dev Orig Health Dis \u003c/em\u003e2010; 1: 142-149.\u003c/li\u003e\n\u003cli\u003eOno N, Ono W, Mizoguchi T, et al. Vasculature-associated cells expressing nestin in developing bones encompass early cells in the osteoblast and endothelial lineage. \u003cem\u003eDev Cell\u003c/em\u003e 2014; 29: 330\u0026ndash;339.\u003c/li\u003e\n\u003cli\u003eKusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. \u003cem\u003eNature \u003c/em\u003e2014; 507: 323-328.\u003c/li\u003e\n\u003cli\u003eBeier EE, Sheu TJ, Resseguie EA, et al. Sclerostin activity plays a key role in the negative effect of glucocorticoid signaling on osteoblast function in mice. \u003cem\u003eBone Res \u003c/em\u003e2017; 5: 17013.\u003c/li\u003e\n\u003cli\u003eMoisiadis VG, Matthews SG. \u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/24863383/?from_term=Glucocorticoids+and+fetal+programming+part+2%3A+mechanisms\u0026amp;from_pos=1\"\u003eGlucocorticoids and fetal programming part 2: Mechanisms.\u003c/a\u003e \u003cem\u003eNat Rev Endocrinol\u003c/em\u003e 2014; 10:403-411.\u003c/li\u003e\n\u003cli\u003eFowden AL, Forhead AJ. Glucocorticoids as regulatory signals during intrauterine development. \u003cem\u003eExp Physiol\u003c/em\u003e 2015;100: 1477-1487.\u003c/li\u003e\n\u003cli\u003eBernal A, Arranz L. Nestin-expressing progenitor cells: function, identity and therapeutic implications. \u003cem\u003eCell Mol Life Sci \u003c/em\u003e2018; 75: 2177-2195.\u003c/li\u003e\n\u003cli\u003eKirkland JL, Tchkonia T. Cellular Senescence: A translational perspective. \u003cem\u003eEBioMedicine\u003c/em\u003e 2017; 21: 21-28.\u003c/li\u003e\n\u003cli\u003eMu\u0026ntilde;oz-Esp\u0026iacute;n D, Ca\u0026ntilde;amero M, Maraver A, et al. Programmed cell senescence during mammalian embryonic development. \u003cem\u003eCell \u003c/em\u003e2013; 155: 1104-1118.\u003c/li\u003e\n\u003cli\u003eStorer M, Mas A, Robert-Moreno A, et al. Senescence is a developmental mechanism that contributes to embryonic growth and patterning. \u003cem\u003eCell \u003c/em\u003e2013; 155: 1119-1130.\u003c/li\u003e\n\u003cli\u003eXu M, Pirtskhalava T, Farr JN, et al. Senolytics improve physical function and increase lifespan in old age. \u003cem\u003eNat Med\u003c/em\u003e 2018; 24: 1246-1256.\u003c/li\u003e\n\u003cli\u003eTikhonova AN, Dolgalev I, Hu H, et al. The bone marrow microenvironment at single-cell resolution. \u003cem\u003eNature\u003c/em\u003e 2019; 569: 222-228.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations ","content":"\u003cp\u003ePDE, prenatal dexamethasone exposure; GD, gestational day; BMSCs, bone marrow mesenchymal stem cells; SASP, senescence-associated secretory phenotype; \u0026mu;CT, microcomputed tomography; BV/TV, bone volume; Tb. N, trabecular number; Tb. Th, trabecular thickness; Tb. Sp, trabecular separation; H\u0026amp;E, hematoxylin-eosin; TRAP, tartrate-resistant acid phosphatase; SA-\u0026beta;-Gal, senescence associated \u0026beta;-galactosidase; PBST, PBS with Tween; Emcn, Endomucin; ANOVA, analysis of variance; D+Q, dasatinib and quercetin.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Dexamethasone, bone development, bone mesenchymal stem cells, cellular senescence ","lastPublishedDoi":"10.21203/rs.3.rs-17277/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-17277/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Prenatal dexamethasone exposure (PDE) induces low birth weight and retardation of fetal bone development which are associated with lower peak bone mass in adult offspring. Here we evaluated whether and how PDE affects postnatal long bone growth in mice offspring. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Pregnant mice were injected subcutaneously with dexamethasone (1.2 mg/kg/day) every morning from gestational day (GD) 12-14. Femurs and tibias of 2-, 4-, 6-, and 12-week-old female offspring were harvested for histological, immunofluorescence, flow cytometric analysis, or microcomputed tomography (μCT) measurement. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: PDE leads to impaired bone remodeling as well as decreased bone mass in the long bone of female mice offspring. During postnatal bone growth, significant decrease of CD45\u003csup\u003e-\u003c/sup\u003eCD29\u003csup\u003e+\u003c/sup\u003eCD105\u003csup\u003e+\u003c/sup\u003eSca-1\u003csup\u003e+ \u003c/sup\u003ebone marrow mesenchymal stem cells (BMSCs) and CD45\u003csup\u003e-\u003c/sup\u003eNestin\u003csup\u003e+\u003c/sup\u003e cells, loss of type H vessels as well as increment of cellular senescence were found in metaphysis of long bone in mice offspring after PDE. We further show that eliminating the excessive senescent cells with dasatinib (5 mg/kg/day) and quercetin (50 mg/kg/day) during GD 12-14 rescues the above toxic effect of PDE on the postnatal long bone growth in female mice offspring. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: Cellular senescence mediates the toxic effect of PDE on postnatal long bone growth in mice offspring, and inhibition of cellular senescence may be proposed for treating the retardation of bone growth caused by PDE.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","manuscriptTitle":"Cellular senescence mediates the detrimental effect of prenatal dexamethasone exposure on postnatal long bone growth in mice offspring","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2020-06-16 23:09:49","doi":"10.21203/rs.3.rs-17277/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2020-06-06T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-05-20T12:00:00+00:00","index":3,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2020-05-20T12:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2020-05-20T12:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2020-05-15T12:00:00+00:00","index":3,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-05-13T12:00:00+00:00","index":2,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-05-12T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-05-12T12:00:00+00:00","index":1,"fulltext":""},{"type":"editorAssigned","content":"","date":"2020-05-10T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-05-09T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-05-09T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2020-03-15 20:50:22","doi":"10.21203/rs.3.rs-17277/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2020-04-02T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-03-31T12:00:00+00:00","index":3,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2020-03-29T12:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2020-03-25T12:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewersInvited","content":"","date":"2020-03-17T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-03-17T12:00:00+00:00","index":1,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-03-17T12:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-03-17T12:00:00+00:00","index":3,"fulltext":""},{"type":"editorAssigned","content":"","date":"2020-03-13T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-03-12T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-03-11T12:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-03-10T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"22bb682b-bd67-43e0-91fd-443b238cc469","owner":[],"postedDate":"June 16th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":104039,"name":"Stem Cell \u0026 Developmental Cell Biology"}],"tags":[],"updatedAt":"2020-07-12T15:02:28+00:00","versionOfRecord":{"articleIdentity":"rs-17277","link":"https://doi.org/10.1186/s13287-020-01790-9","journal":{"identity":"stem-cell-research-and-therapy","isVorOnly":false,"title":"Stem Cell Research \u0026 Therapy"},"publishedOn":"2020-07-06 12:00:00","publishedOnDateReadable":"July 6th, 2020"},"versionCreatedAt":"2020-06-16 23:09:49","video":"","vorDoi":"10.1186/s13287-020-01790-9","vorDoiUrl":"https://doi.org/10.1186/s13287-020-01790-9","workflowStages":[]},"version":"v2","identity":"rs-17277","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-17277","identity":"rs-17277","version":["v2"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}