Isolation of Human Bone Marrow Non-hematopoietic Cells for Single-cell RNA Sequencing.

The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME's intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche. Key features • Isolation of high-quality human non-hematopoietic bone marrow cells for scRNAseq • Targeted strategy for enriching low-frequency stromal cells.

Targeting histone deacetylase 1 (HDAC1) in the bone marrow stromal cells revers imatinib resistance by modulating IL-6 in Ph + acute lymphoblastic leukemia.

Bone marrow stromal cells (BMSCs) can promote the growth of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). Histone deacetylases (HDACs) play essential roles in the proliferation and apoptosis resistance of Ph + ALL cells. In our previous study, inhibiting histone deacetylase 1 (HDAC1) decreases the proliferation of Ph + ALL cells. However, little is known regarding how HDAC1 in BMSCs of Ph + ALL patients affects the imatinib (IM) resistance. Therefore, the present work examined the roles of HDAC1 in BMSCs. Overexpression of HDAC1 was found in BMSCs of Ph + ALL patients with IM resistance. In addition, the Ph + ALL cell line SUP-B15 was co-cultured with BMSCs after lentivirus transfection for regulating HDAC1 expression. Knockdown of HDAC1 within BMSCs elevated the IM-mediated SUP-B15 cell apoptosis, while increasing HDAC1 expression had an opposite effect. IL-6 in BMSCs, which is an important factor for the microenvironment-associated chemoresistance, showed evident up-regulation in HDAC1-upregulated BMSCs and down-regulation in HDAC1-downregulated BMSCs. While recombinant IL-6 (rIL-6) can reversed the sensitivity of SUP-B15 cells to IM induced by downregulating HDAC1 expression in BMSCs. HDAC1 showed positive regulation on IL-6 transcription and secretion. Moreover, IL-6 secretion induced by HDAC1 in BMSCs might enhance IM resistance in Ph + ALL cells. With regard to the underlying molecular mechanism, NF-κB, an important signal responsible for IL-6 transcription in BMSCs, mediated the HDAC1-regulated IL-6 expression. Collectively, this study facilitated to develop HDAC1 inhibitors based not only the corresponding direct anti-Ph + ALL activity but also the regulation of bone marrow microenvironment.

CRIP1 regulates osteogenic differentiation of bone marrow stromal cells and pre-osteoblasts via the Wnt signaling pathway.

With the aging of the global demographic, the prevention and treatment of osteoporosis are becoming crucial issues. The gradual loss of self-renewal and osteogenic differentiation capabilities in bone marrow stromal cells (BMSCs) is one of the key factors contributing to osteoporosis. To explore the regulatory mechanisms of BMSCs differentiation, we collected bone marrow cells of femoral heads from patients undergoing total hip arthroplasty for single-cell RNA sequencing analysis. Single-cell RNA sequencing revealed significantly reduced CRIP1 (Cysteine-Rich Intestinal Protein 1) expression and osteogenic capacity in the BMSCs of osteoporosis patients compared to non-osteoporosis group. CRIP1 is a gene that encodes a member of the LIM/double zinc finger protein family, which is involved in the regulation of various cellular processes including cell growth, development, and differentiation. CRIP1 knockdown resulted in decreased alkaline phosphatase activity, mineralization and expression of osteogenic markers, indicating impaired osteogenic differentiation. Conversely, CRIP1 overexpression, both in vitro and in vivo, enhanced osteogenic differentiation and rescued bone mass reduction in ovariectomy-induced osteoporosis mice model. The study further established CRIP1's modulation of osteogenesis through the Wnt signaling pathway, suggesting that targeting CRIP1 could offer a novel approach for osteoporosis treatment by promoting bone formation and preventing bone loss.

Skeletal stem and progenitor cells in bone development and repair.

Bone development, growth, and repair are complex processes involving various cell types and interactions, with central roles played by skeletal stem and progenitor cells. Recent research brought new insights into the skeletal precursor populations that mediate intramembranous and endochondral bone development. Later in life, many of the cellular and molecular mechanisms determining development are reactivated upon fracture, with powerful trauma-induced signaling cues triggering a variety of postnatal skeletal stem/progenitor cells (SSPCs) residing near the bone defect. Interestingly, in this injury context, the current evidence suggests that the fates of both SSPCs and differentiated skeletal cells can be considerably flexible and dynamic, and that multiple cell sources can be activated to operate as functional progenitors generating chondrocytes and/or osteoblasts. The combined implementation of in vivo lineage tracing, cell surface marker-based selection, single-cell molecular analyses, and high-resolution in situ imaging has strongly improved our insights into the diversity and roles of developmental and reparative stem/progenitor subsets, while also unveiling the complexity of their dynamics, hierarchies, and relationships. Albeit incompletely understood at present, findings supporting lineage flexibility and possibly plasticity among sources of osteogenic cells challenge the classical dogma of a single primitive, self-renewing, multipotent stem cell driving bone tissue formation and regeneration from the apex of a hierarchical and strictly unidirectional differentiation tree. We here review the state of the field and the newest discoveries in the origin, identity, and fates of skeletal progenitor cells during bone development and growth, discuss the contributions of adult SSPC populations to fracture repair, and reflect on the dynamism and relationships among skeletal precursors and differentiated cell lineages. Further research directed at unraveling the heterogeneity and capacities of SSPCs, as well as the regulatory cues determining their fate and functioning, will offer vital new options for clinical translation toward compromised fracture healing and bone regenerative medicine.

Skeletal progenitor cells are crucial for bone development and growth, as they provide the cellular building blocks (chondrocytes and osteoblasts) that form the cartilage and bone tissues that the skeleton is composed of. In adult life, the occurrence of a bone fracture reactivates similar tissue-forming mechanisms, starting with the trauma triggering various postnatal skeletal stem/progenitor cells (SSPCs) residing near the bone defect to divide and migrate. These cells subsequently generate functional fracture-repairing cells by differentiating into mature chondrocytes and/or osteoblasts. In recent years, the combined use of various advanced research approaches and new techniques has strongly improved our insights into the origin, identity, fates, and roles of developmental and reparative skeletal stem cells and progenitor subsets. Concomitantly, this research also unveiled considerable complexity in their dynamics, diversity, hierarchies, and relationships, which is incompletely understood at present. In this review, we discuss the state of the field and the newest discoveries in the identity and roles of skeletal stem and progenitor cells mediating bone development, growth, and repair. Further research on these cell populations, including determining their exact nature, fate, and functioning, and how they can be harvested and regulated, is critical to develop new treatments for non-healing fractures.

Exploring miR-21 as a key regulator in two distinct approaches of bone marrow stromal cells differentiation into Schwann-like cells.

The differentiation of bone marrow stromal cells (BMSCs) into Schwann-like cells (SCLCs) has the potential to promote the structural and functional restoration of injured axons. However, the optimal induction protocol and its underlying mechanisms remain unclear. This study aimed to compare the effectiveness of different induction protocols in promoting the differentiation of rat BMSCs into SCLCs and to explore their potential mechanisms. BMSCs were induced using two distinct methods: a composite factor induction approach (Protocol-1) and a conditioned culture medium induction approach (Protocol-2). The expression of Schwann cells (SCs) marker proteins and neurotrophic factors (NTFs) in the differentiated cells was assessed. Cell proliferation and apoptosis were also measured. During induction, changes in miR-21 and Sprouty RTK signaling antagonist 2 (SPRY2) mRNA were analyzed. Following the transfection of BMSCs with miR-21 agomir or miR-21 antagomir, induction was carried out using both protocols, and the expression of SPRY2, ERK1/2, and SCs marker proteins was examined. The results revealed that NTFs expression was higher in Protocol-1, whereas SCs marker proteins expression did not significantly differ between the two groups. Compared to Protocol-1, Protocol-2 exhibited enhanced cell proliferation and fewer apoptotic and necrotic cells. Both protocols showed a negative correlation between miR-21 and SPRY2 expression throughout the induction stages. After induction, the miR-21 agomir group exhibited reduced SPRY2 expression, increased ERK1/2 expression, and significantly elevated expression of SCs marker proteins. This study demonstrates that Protocol-1 yields higher NTFs expression, whereas Protocol-2 results in stronger SCLCs proliferation. Upregulating miR-21 suppresses SPRY2 expression, activates the ERK1/2 signaling pathway, and promotes BMSC differentiation into SCLCs.

Interplay Between Skeletal and Hematopoietic Cells in the Bone Marrow Microenvironment in Homeostasis and Aging.

PURPOSE OF THE REVIEW: In this review, we discuss the most recent scientific advances on the reciprocal regulatory interactions between the skeletal and hematopoietic stem cell niche, focusing on immunomodulation and its interplay with the cell's mitochondrial function, and how this impacts osteoimmune health during aging and disease. RECENT FINDINGS: Osteoimmunology investigates interactions between cells that make up the skeletal stem cell niche and immune system. Much work has investigated the complexity of the bone marrow microenvironment with respect to the skeletal and hematopoietic stem cells that regulate skeletal formation and immune health respectively. It has now become clear that these cellular components cooperate to maintain homeostasis and that dysfunction in their interaction can lead to aging and disease. Having a deeper, mechanistic appreciation for osteoimmune regulation will lead to better research perspective and therapeutics with the potential to improve the aging process, skeletal and hematologic regeneration, and disease targeting.

Therapeutic effects of ginsenosides on osteoporosis for novel drug applications.

Osteoporosis (OP) is a metabolic bone disease with a high incidence rate worldwide. Its main features are decreased bone mass, increased bone fragility and deterioration of bone microstructure. It is caused by an imbalance between bone formation and bone resorption. Ginsenoside is a safe and effective traditional Chinese medicine (TCM) usually extracted from ginseng plants, having various therapeutic effects, of which the effect against osteoporosis has been extensively studied. We searched a total of 44 relevant articles with using keywords including osteoporosis, ginsenosides, bone mesenchymal cells, osteoblasts, osteoclasts and bone remodeling, all of which investigated the cellular mechanisms of different types of ginsenosides affecting the activity of bone remodeling by mesenchymal stem cells, osteoblasts and osteoclasts to counteract osteoporosis. This review describes the different types of ginsenosides used to treat osteoporosis from different perspectives, providing a solid theoretical basis for future clinical applications.

Wnt16 Increases Bone-to-Implant Contact in an Osteopenic Rat Model by Increasing Proliferation and Regulating the Differentiation of Bone Marrow Stromal Cells.

Osseointegration is a complex biological cascade that regulates bone regeneration after implant placement. Implants possessing complex multiscale surface topographies augment this regenerative process through the regulation of bone marrow stromal cells (MSCs) that are in contact with the implant surface. One pathway regulating osteoblastic differentiation is Wnt signaling, and upregulation of non-canonical Wnts increases differentiation of MSCs on these titanium substrates. Wnt16 is a non-canonical Wnt shown to regulate bone morphology in mouse models. This study evaluated the role of Wnt16 during surface-mediated osteoblastic differentiation of MSCs in vitro and osseointegration in vivo. MSCs were cultured on Ti substrates with different surface properties and non-canonical Wnt expression was determined. Subsequently, MSCs were cultured on Ti substrates +/-Wnt16 (100 ng/mL) and anti-Wnt16 antibodies (2 µg/mL). Wnt16 expression was increased in cells grown on microrough surfaces that were processed to be hydrophilic and have nanoscale roughness. However, treatment MSCs on these surfaces with exogenous rhWnt16b increased total DNA content and osteoprotegerin production, but reduced osteoblastic differentiation and production of local factors necessary for osteogenesis. Addition of anti-Wnt16 antibodies blocked the inhibitor effects of Wnt16. The response to Wnt16 was likely independent of other osteogenic pathways like Wnt11-Wnt5a signaling and semaphorin 3a signaling. We used an established rat model of cortical and trabecular femoral bone impairment following botox injections (2 injections of 8 units/leg each, starting and maintenance doses) to assess Wnt16 effects on whole bone morphology and implant osseointegration. Wnt16 injections did not alter whole bone morphology significantly (BV/TV, cortical thickness, restoration of trabecular bone) but were effective at increasing cortical bone-to-implant contact during impaired osseointegration in the botox model. The mechanical quality of the increased bone was not sufficient to rescue the deleterious effects of botox. Clinically, these results are important to understand the interaction of cortical and trabecular bone during implant integration. They suggest a role for Wnt16 in modulating bone remodeling by reducing osteoclastic activity. Targeted strategies to temporally regulate Wnt16 after implant placement could be used to improve osseointegration by increasing the net pool of osteoprogenitor cells.

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

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

Comparison between stem cell therapy and stem cell derived exosomes on induced multiple sclerosis in dogs.

BACKGROUND: Multiple sclerosis (MS) is a chronic condition that primarily manifests as demyelination of neuronal axons in the central nervous system, due to the loss or attack of oligodendroglia cells that form myelin. Stem cell therapy has shown promising results for the treatment of MS due to its capability to halt the immune attack, stop apoptosis and axonal degeneration, and differentiate into oligodendrocytes. Stem cell-derived Exosomes (Exosomes) have shown great capabilities for neuronal diseases as they have growth factors, complex sets of miRNA, enzymes, proteins, major peptides, lipids, and macromolecules with anti-inflammatory, angiogenesis, and neurogenesis activities. METHODS: This study aimed to compare the healing properties of stem cells, against Exosomes for the treatment of an experimentally induced MS dog model. Dog models of MS received either a single treatment of stem cells or a single treatment of Exosomes intrathecally and the treatment process was evaluated clinically, radiologically, histopathologically, and electron microscopy and cerebrospinal fluid analysis. RESULTS: showed marked amelioration of the clinical signs in both treated groups compared to the control one, magnetic resonance scans showed the resolution of the hyperintense lesions at the end of the study period, the histopathology and electron microscopy showed marked healing properties and remyelination in treated groups with superiority of the stem cells compared to Exosomes. CONCLUSIONS: Although stem cell results were superior to Exosomes therapy; Exosomes have proven to be effective and safe important actors in myelin regeneration, and their use in diseases like MS helps to stimulate remyelination.

Erratum: Maternal GNAS contributes to the extra-large G protein α-subunit (XLαs) expression in a cell type-specific manner.

[This corrects the article DOI: 10.3389/fgene.2021.680537.].

Young minds, deeper insights: a recap of the BMAS Summer School 2023, ranging from basic research to clinical implications of bone marrow adipose tissue.

Bone marrow adiposity (BMA) is a rapidly growing yet very young research field that is receiving worldwide attention based on its intimate relationship with skeletal and metabolic diseases, as well as hematology and cancer. Moreover, increasing numbers of young scientists and students are currently and actively working on BMA within their research projects. These developments led to the foundation of the International Bone Marrow Adiposity Society (BMAS), with the goal to promote BMA knowledge worldwide, and to train new generations of researchers interested in studying this field. Among the many initiatives supported by BMAS, there is the BMAS Summer School, inaugurated in 2021 and now at its second edition. The aim of the BMAS Summer School 2023 was to educate and train students by disseminating the latest advancement on BMA. Moreover, Summer School 2023 provided suggestions on how to write grants, deal with negative results in science, and start a laboratory, along with illustrations of alternative paths to academia. The event was animated by constructive and interactive discussions between early-career researchers and more senior scientists. In this report, we highlight key moments and lessons learned from the event.

The unique role of bone marrow adipose tissue in ovariectomy-induced bone loss in mice.

BACKGROUND: Accumulation of bone marrow adipose tissue (BMAT) is always seen in osteoporosis induced by estrogen deficiency. Herein, we aimed to investigate the mechanisms and consequences of this phenomenon by establishing a mouse model of osteoporosis caused by ovariectomy (OVX)-mimicked estrogen deficiency. METHODS: Micro-CT, osmium tetroxide staining, and histological analyses were performed to examine the changes in bone microstructure, BMAT and white adipose tissue (WAT) in OVX mice compared to sham mice. The osteogenesis and adipogenesis of primary bone marrow stromal cells (BMSCs) isolated from sham and OVX mice were compared in vitro. The molecular phenotypes of BMAT and WAT were determined and compared by quantitative PCR (qPCR). Bone marrow adipocyte-conditioned medium (BMA CM) was prepared from sham or OVX mice for coculture assays, and BMSCs or bone marrow monocytes/macrophages (BMMs) were isolated and subjected to osteoblast and osteoclast differentiation, respectively. Cell staining and qPCR were used to assess the effects of BMAT on bone metabolism. RESULTS: OVX-induced estrogen deficiency induced reductions in both cortical and trabecular bone mass along with an expansion of BMAT volume. At the cellular level, loss of estrogen inhibited BMSC osteogenesis and promoted BMSC adipogenesis, whereas addition of estradiol exerted the opposite effects. In response to estrogen deficiency, despite the common proinflammatory molecular phenotype observed in both fat depots, BMAT, unlike WAT, unexpectedly exhibited an increase in adipocyte differentiation and lipolytic activity as well as the maintenance of insulin sensitivity. Importantly, BMAT, but not WAT, presented increased mRNA levels of both BMP receptor inhibitors (Grem1, Chrdl1) and Rankl following OVX. In addition, treatment with BMA CM, especially from OVX mice, suppressed the osteoblast differentiation of BMSCs while favoring the osteoclast differentiation of BMMs. CONCLUSION: Our study illustrates that OVX-induced estrogen deficiency results in bone loss and BMAT expansion by triggering imbalance between the osteogenesis and adipogenesis of BMSCs. Furthermore, expanded BMAT, unlike typical WAT, may negatively regulate bone homeostasis through paracrine inhibition of osteoblast-mediated bone formation and promotion of osteoclast-mediated bone resorption.

The Effect of Lanthanum (III) Nitrate on the Osteogenic Differentiation of Mice Bone Marrow Stromal Cells.

To study the species of lanthanum (III) nitrate (La[NO3]3) dispersed in cell media and the effect on the osteoblast differentiation of bone marrow stroma cells (BMSCs). Different La-containing precipitations were obtained by adding various concentrations of La(NO3)3 solutions to Dulbecco's modified Eagle medium (DMEM) or DMEM with fetal bovine serum (FBS). A series of characterisation methods, including dynamic light scattering, scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, and protein quantification were employed to clarify the species of the different La-containing precipitations. The primary BMSCs were isolated, and the cell viability, alkaline phosphatase activity, and the formation of a mineralised nodule of BMSCs were tested when treated with different La-containing precipitations. The La(NO3)3 solutions in DMEM could form LaPO4, which exits in the particle formation, while the La(NO3)3 solutions in DMEM with FBS could form a La-PO4-protein compound. When treated with La(NO3)3 solutions in DMEM, the cell viability of the BMSCs was inhibited at the concentrations of 1, 10, and 100 µM at 1 day and 3 days. Meanwhile, the supernatant derived from the La(NO3)3 solutions in DMEM did not affect the cell viability of the BMSCs. In addition, the precipitate derived from the La(NO3)3 solutions in DMEM added to the complete medium inhibited the cell viability of the BMSCs at concentrations of 10 µM and 100 µM. When treated with La(NO3)3 solutions in DMEM with FBS, the derived precipitate and supernatant did not affect the cell viability of the BMSCs, except for the concentration of 100 µM La(NO3)3. The La-PO4-protein formed from the La(NO3)3 solutions in DMEM with FBS inhibited the osteoblast differentiation of BMSCs at the concentration of 1 µM La(NO3)3 (P < 0.05) but had no effect on either the osteoblast differentiation at the concentrations of 0.001 and 0.1 µM or on the formation of a mineralised nodule at all tested concentrations of La(NO3)3. Overall, La(NO3)3 solutions in different cell culture media could form different La-containing compounds: La-PO4 particles (in DMEM) and a La-PO4-protein compound (in DMEM with FBS). The different La-containing compounds caused different effects on the cell viability, osteoblast differentiation, and the formation of a mineralised nodule of the BMSCs. The La-containing precipitation inhibited the osteoblast differentiation by inhibiting the expression of osteoblast-related genes and proteins, providing a theoretical basis for clinical doctors to apply phosphorus-lowering drugs such as lanthanum carbon.

Total flavonoids of Rhizoma drynariae improves tendon-bone healing for anterior cruciate ligament reconstruction in mice and promotes the osteogenic differentiation of bone mesenchymal stem cells by the ERR1/2-Gga1-TGF-ß/MAPK pathway.

BACKGROUND: Total flavonoids of Rhizoma drynariae (TFRD) is broadly used in the treatment of orthopedic diseases. Nevertheless, the effects and underlying mechanism of TFRD on tendon-bone healing after anterior cruciate ligament reconstruction (ACLR) remain unclear. METHODS: The ACLR mouse model was established. Hematoxylin and Eosin (HE) staining was used for histological analysis of tendon-bone healing. Western blot was utilized to detect the levels of osteogenic related factors (ALP, OCN, RUNX2). The viability and alkaline phosphatase (ALP) activity of bone mesenchymal stem cells (BMSCs) were determined by Cell Counting Kit-8 (CCK-8) and ALP assays. The interaction of estrogen related receptor alpha (ESRRA), estrogen related receptor beta (ESRRB), and golgi-localized γ-ear containing ADP ribosylation factor-binding protein 1 (Gga1) was detected by luciferase reporter assays. The levels of important proteins on the TGF-ß/MAPK pathway were measured by western blot. RESULTS: TFRD improved tendon-bone healing, restored biomechanics of ACLR mice and activated the TGF-ß/MAPK pathway. TFRD treatment also enhanced the viability and osteogenic differentiation of BMSCs in vitro. Then, we demonstrated that TFRD targeted ESRRA and ESRRB to transcriptionally activate Gga1 expression. Knockdown of ESRRA, ESRRB, or Gga1 suppressed the viability and osteogenic differentiation of TFRD-induced BMSCs, which was revealed to be restored by Gga1 overexpression. The overexpression of ESRRA, ESRRB, or Gga1 was demonstrated to promote the BMSC viability and osteogenic differentiation. TGF-ß1 treatment can reverse the impact of Gga1 inhibition on osteogenic differentiation in TFRD-induced BMSCs. CONCLUSION: TFRD improves tendon-bone healing in ACLR mouse models and facilitates the osteogenic differentiation of BMSCs through the ERR1/2-Gga1-TGF-ß/MAPK pathway, which might deepen our understanding of the underlying mechanism of TFRD in tendon-bone healing.

Circular RNA circ-3626 promotes bone formation by modulating the miR-338-3p/Runx2 axis.

OBJECTIVE: Disorders of bone homeostasis are the key factors leading to metabolic bone disease, such as senile osteoporosis, which is characterized by age-related bone loss. Bone marrow stromal cells (BMSCs) possess high osteogenic capacity which has been regarded as a practical approach to preventing bone loss. Previous studies have shown that the osteogenic differentiation ability of BMSCs is significantly decreased in senile osteoporosis. Recently, circular RNAs (circRNAs) have been regarded as critical regulators in controlling the osteogenic differentiation of BMSCs by sponging microRNAs (miRNAs). Our study aimed to discover new and critical osteogenesis-related circRNAs that can promote bone formation in senile osteoporosis. METHODS: We detected the dysregulated circRNAs of BMSCs upon osteogenic differentiation induction and identified the critical osteogenic circRNA (circ-3626). The relationship between circ-3626 and osteoporosis was further verified in clinical bone samples and aged mice by qPCR. Moreover, circ-3626 AAV was constructed to examine the osteogenic effect of circ-3626 on bone formation via using Micro-CT, double calcein labeling, and the three-point bending tests. Bioinformatics analysis, Luciferase report gene assays, FISH, RNA pull-down, qPCR, Western Blots, and alizarin red staining assay explore the effects and mechanisms of circ-3626 on osteogenic differentiation of BMSCs. RESULTS: Circ-3626 was identified as a pivotal osteogenesis-related circRNA via RNA sequencing. The results of alizarin red staining, Western blots, and qPCR assays suggest that overexpressing circ-3626 dramatically accelerates the osteogenic capability of BMSCs. Furthermore, the bone repair capability of aging mice could be significantly improved by circ-3626 AAV treatment. Micro RNA miR-338-3p was identified as the downstream target of circ-3626. Overexpression of circ-3626 increases the expression of Runx2 by sponging miR-338-3p, thereby promoting the osteogenic differentiation of BMSCs by upregulating the expression of osteogenic genes. In addition, Western blots, and qPCR assays suggest circ-3626 AAV treatment promote the expression of Runx2 and osteogenic marker genes. CONCLUSION: Thus, we demonstrate that circ-3626 plays a pivotal role in promoting bone formation through the miR-338-3p/Runx2 axis and may provide new strategies for preventing and treating the bone loss of senile osteoporosis.

DNA methylation of promoter region inhibits galectin-1 expression in BMSCs of aged mice.

Senile osteoporosis increases fracture risks. Bone marrow stromal cells (BMSCs) are sensitive to aging. Deep insights into BMSCs aging are vital to elucidate the mechanisms underlying age-related bone loss. Recent advances showed that osteoporosis is associated with aberrant DNA methylation of many susceptible genes. Galectin-1 (Gal-1) has been proposed as a mediator of BMSCs functions. In our previous study, we showed that Gal-1 was downregulated in aged BMSCs and global deletion of Gal-1 in mice caused bone loss via impaired osteogenesis potential of BMSCs. Gal-1 promoter is featured by CpG islands. However, there are no reports concerning the DNA methylation status in Gal-1 promoter during osteoporosis. In the current study, we sought to investigate the role of DNA methylation in Gal-1 downregulation in aged BMSCs. The potential for anti-bone loss therapy based on modulating DNA methylation is explored. Our results showed that Dnmt3b-mediated Gal-1 promoter DNA hypermethylation plays an important role in Gal-1 downregulation in aged BMSCs, which inhibited ß-catenin binding on Gal-1 promoter. Bone loss of aged mice was alleviated in response to in vivo deletion of Dnmt3b from BMSCs. Finally, when bone marrow of young wild-type (WT) mice or young Dnmt3bPrx1-Cre mice was transplanted into aged WT mice, Gal-1 level in serum and trabecular bone mass were elevated in recipient aged WT mice. Our study will benefit for deeper insights into the regulation mechanisms of Gal-1 expression in BMSCs during osteoporosis development, and for the discovery of new therapeutic targets for osteoporosis via modulating DNA methylation status.NEW & NOTEWORTHY There is Dnmt3b-mediated DNA methylation in Gal-1 promoter in aged bone marrow stromal cell (BMSC). DNA methylation causes Gal-1 downregulation and osteogenesis attenuation of aged BMSC. DNA methylation blocks ß-catenin binding on Gal-1 promoter. Bone loss of aged mice is alleviated by in vivo deletion of Dnmt3b from BMSC.

Influence of quercetin on aging of bone marrow mesenchymal stem cells induced by microgravity / 中国药理学通报

Aim To investigate the effect of quercetin on the aging model of bone marrow mesenchymal stem cells established under microgravity. Methods Using 3D gyroscope, a aging model of bone marrow mesenchymal stem cells was constructed, and after receiving quercetin and microgravity treatment, the anti-aging effect of the quercetin was evaluated by detecting related proteins and oxidation indexes. Results Compared to the control group, the expressions of age-related proteins p21, pi6, p53 and RB in the microgravity group significantly increased, while the expressions of cyclin D1 and lamin B1 significantly decreased, with statistical significance (P<0.05). In the microgravity group, mitochondrial membrane potential significantly decreased (P<0.05), ROS accumulation significantly increased (P <0.05), SOD content significantly decreased and MDA content significantly increased (P<0.05). Compared to the microgravity group, the expressions of age-related proteins p21, pi6, p53 and RB in the quercetin group significantly decreased, while the expressions of cyclin D1 and lamin B1 significantly increased, with statistical significance (P<0.05). In the quercetin group, mitochondrial membrane potential significantly increased (P<0.05), ROS accumulation significantly decreased (P<0.05), SOD content significantly increased and MDA content significantly decreased (P<0.05). Conclusions Quercetin can resist oxidation, protect mitochondrial function and normal cell cycle, thus delaying the aging of bone marrow mesenchymal stem cells induced by microgravity.

HIF-1α is an important regulator of IL-8 expression in human bone marrow stromal cells under hypoxic microenvironment.

The secretion of IL-8 has been found increasing for different reasons in human bone marrow stromal cells (BMSCs), resulting in poor prognosis in patients with hematologic neoplasms. Hypoxia, a typical feature of numerous hematologic neoplasms microenvironment, often produces hypoxia inducible factor-1α (HIF-1α) which stabilizes and promotes tumor progression. Besides, hypoxic conditions also induce IL-8 production in BMSCs. However, very little is known about the mechanism of increased IL-8 expression in BMSCs caused by hypoxia. In the present study, HIF-1α and IL-8 were found highly expressed in BMSC lines under hypoxic conditions. In addition, the expression and secretion of IL-8 were significantly inhibited by the knockdown of HIF-1α under hypoxic conditions. Furthermore, HIF-1α was found to transcriptionally regulate IL-8 by binding to the region of IL-8 promoter at - 147 to - 140. Collectively, these results demonstrate that IL-8's increase is partly due to the hypoxic microenvironment in hematologic neoplasms, and activation of HIF-1α in BMSCs contributes to the induction and transcriptional regulation of IL-8 expression.

Preliminary delivery efficiency prediction of nanotherapeutics into crucial cell populations in bone marrow niche.

Several crucial stromal cell populations regulate hematopoiesis and malignant diseases in bone marrow niches. Precise regulation of these cell types can remodel niches and develop new therapeutics. Multiple nanocarriers have been developed to transport drugs into the bone marrow selectively. However, the delivery efficiency of these nanotherapeutics into crucial niche cells is still unknown, and there is no method available for predicting delivery efficiency in these cell types. Here, we constructed a three-dimensional bone marrow niche composed of three crucial cell populations: endothelial cells (ECs), mesenchymal stromal cells (MSCs), and osteoblasts (OBs). Mimetic niches were used to detect the cellular uptake of three typical drug nanocarriers into ECs/MSCs/OBs in vitro. Less than 5% of nanocarriers were taken up by three stromal cell types, and most of them were located in the extracellular matrix. Delivery efficiency in sinusoidal ECs, arteriole ECs, MSCs, and OBs in vivo was analyzed. The correlation analysis showed that the cellular uptake of three nanocarriers in crucial cell types in vitro is positively linear correlated with its delivery efficiency in vivo. The delivery efficiency into MSCs was remarkably higher than that into ECs and OBs, no matter what kind of nanocarrier. The overall efficiency into sinusoidal ECs was greatly lower than that into arteriole ECs. All nanocarriers were hard to be delivered into OBs (<1%). Our findings revealed that cell tropisms of nanocarriers with different compositions and ligand attachments in vivo could be predicted via detecting their cellular uptake in bone marrow niches in vitro. This study provided the methodology for niche-directed nanotherapeutics development.