Bone Marrow Stromal Cell Function Research Articles

METTL7A-mediated m6A modification of corin reverses bisphosphonates-impaired osteogenic differentiation of orofacial BMSCs

Bisphosphonate-related osteonecrosis of jaw (BRONJ) is characterized by impaired osteogenic differentiation of orofacial bone marrow stromal cells (BMSCs). Corin has recently been demonstrated to act as a key regulator in bone development and orthopedic disorders. However, the role of corin in BRONJ-related BMSCs dysfunction remains unclarified. A m6A epitranscriptomic microarray study from our group shows that the CORIN gene is significantly upregulated and m6A hypermethylated during orofacial BMSCs osteogenic differentiation. Corin knockdown inhibits BMSCs osteogenic differentiation, whereas corin overexpression or soluble corin (sCorin) exerts a promotion effect. Furthermore, corin expression is negatively regulated by bisphosphonates (BPs). Corin overexpression or sCorin reverses BPs-impaired BMSCs differentiation ability. Mechanistically, we find altered expression of phos-ERK in corin knockdown/overexpression BMSCs and BMSCs under sCorin stimulation. PD98059 (a selective ERK inhibitor) blocks the corin-mediated promotion effect. With regard to the high methylation level of corin during osteogenic differentiation, we apply a non-selective m6A methylase inhibitor, Cycloleucine, which also blocks the corin-mediated promotion effect. Furthermore, we demonstrate that METTL7A modulates corin m6A modification and reverses BPs-impaired BMSCs function, indicating that METTL7A regulates corin expression and thus contributes to orofacial BMSCs differentiation ability. To conclude, our study reveals that corin reverses BPs-induced BMSCs dysfunction, and METTL7A-mediated corin m6A modification underlies corin promotion of osteogenic differentiation via the ERK pathway. We hope this brings new insights into future clinical treatments for BRONJ.

Batch Effects during Human Bone Marrow Stromal Cell Propagation Prevail Donor Variation and Culture Duration: Impact on Genotype, Phenotype and Function.

Donor variation is a prominent critical issue limiting the applicability of cell-based therapies. We hypothesized that batch effects during propagation of bone marrow stromal cells (BMSCs) in human platelet lysate (hPL), replacing fetal bovine serum (FBS), can affect phenotypic and functional variability. We therefore investigated the impact of donor variation, hPL- vs. FBS-driven propagation and exhaustive proliferation, on BMSC epigenome, transcriptome, phenotype, coagulation risk and osteochondral regenerative function. Notably, propagation in hPL significantly increased BMSC proliferation, created significantly different gene expression trajectories and distinct surface marker signatures, already after just one passage. We confirmed significantly declining proliferative potential in FBS-expanded BMSC after proliferative challenge. Flow cytometry verified the canonical fibroblastic phenotype in culture-expanded BMSCs. We observed limited effects on DNA methylation, preferentially in FBS-driven cultures, irrespective of culture duration. The clotting risk increased over culture time. Moreover, expansion in xenogenic serum resulted in significant loss of function during 3D cartilage disk formation and significantly increased clotting risk. Superior chondrogenic function under hPL-conditions was maintained over culture. The platelet blood group and isoagglutinins had minor impact on BMSC function. These data demonstrate pronounced batch effects on BMSC transcriptome, phenotype and function due to serum factors, partly outcompeting donor variation after just one culture passage.

Research on Action of Bone Marrow Stromal Cells (BMSC) Modified by Toll-Like Receptor for Myocardial Function of Rats with Myocardial Infarction (MI)

MI could affect myocardial function seriously. The action and mechanism of BSMC modified by the transplantation of Toll-like receptor on myocardial function of rats with MI was studied. The sixty SD rats were divided into sham-operation group and model group randomly. The isolated and cultivated BMSC was divided into three groups such as group with added by TLR4 agonist, group with added by TLR-4 antagonist as TAK-242, group with only BMSC. The model of rats with MT was established in model group. BMSC in different group was transplanted into rats respectively. The cardiac function of rats in sham-operation group and model group was observed. The distribution condition of surface molecule in BMSC was detected by FCM. There was expression CD44 (+), CD54 (+), CD90 (+), no expression CD34 (−) from results. It could indicated that BMSCs with high purity was cultivated successfully. The high MT expression was affected by TLR-4 generally which could also illustrate the degree of deformation and necrosis of cardiac muscle cell, could be enhanced in established rat model because of negative regulatory action. The zone of MI in rats could be amplified by TLR-4. The cytobiological function of BMSC could be adjusted by TLR-4 through the transplantation of TLR-4 after there was MI in rats. The cytobiological function of BMSC could be adjusted by TLR-4 through modification of the transplantation of TLT-4 in rats after there was MI in rats. The BMSCs modified by high TLR-4 expression had negative regulatory action on the treatment on MI.

Extracellular vesicles derived from T-cell acute lymphoblastic leukemia inhibit osteogenic differentiation of bone marrow mesenchymal stem cells via miR-34a-5p.

Reduced bone formation in patients with T-cell acute lymphoblastic leukemia (T-ALL) may be related to the interaction between tumour cells and bone marrow stromal cells (BMSCs). The miRNAs in extracellular vesicles derived from leukemia cells play an essential role in regulating the function of BMSCs; however, the regulatory mechanisms remain unclear. The expression of miR-34a-5p in T-ALL patients and cells was measured by quantitative real-time PCR. BMSCs were co-cultured with extracellular vesicles isolated from T-ALL cells in mineralization medium. The osteogenic differentiation of BMSCs was evaluated by Alizarin Red S staining, alkaline phosphatase (ALP) staining, and detection of osteogenic differentiation markers. A dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-34a-5p and Wnt family member 1 (WNT1). MiR-34a-5p expression was upregulated in T-ALL patients and Jurkat cells. After BMSCs were co-cultured with extracellular vesicles derived from T-ALL cells, osteogenic differentiation of BMSCs was inhibited, and bone mineralization and ALP activity were decreased compared to those of control cells. MiR-34a-5p knockdown in T-ALL cells restored osteogenic differentiation of BMSCs co-cultured with extracellular vesicles. In addition, miR-34a-5p targets and negatively regulates WNT1 expression. In conclusion, our results demonstrated that knockdown of miR-34a-5p in extracellular vesicles derived from T-ALL cells promoted osteogenic differentiation of BMSCs by regulating WNT1.

Osteoblastic Wntless deletion differentially regulates the fate and functions of bone marrow-derived stem cells in relation to age.

Although functional association between Wnt signaling and bone homeostasis has been well described through genetic ablation of Wntless (Wls), the mechanisms of how osteoblastic Wls regulates the fate of bone marrow stromal cells (BMSCs) and hematopoietic stem cells (HSCs) in relation to age are not yet understood. Here, we generated Col2.3-Cre;Wlsfl/fl mice that were free from premature lethality and investigated age-related impacts of osteoblastic Wls deficiency on hematopoiesis, BM microenvironment, and maintenance of BMSCs (also known as BM-derived mesenchymal stem/stromal cells) and HSCs. Ablation of osteoblastic Wls deteriorated BM microenvironment and bone mass accrual along with age-independent effects on functions of BMSCs. Osteoblastic Wls deletion impaired HSC repopulation and progeny with skewing toward myeloid lineage cells only at old stage. As proven by hallmarks of stem cell senescence, osteoblastic Wls ablation differentially induced senescence of BMSCs and HSCs in relation to age without alteration in their BM frequency. Our findings support that deletion of Wls in Col2.3-expressing cells induces senescence of BMSCs and impairs BM microenvironment in age-independent manner. Overall, long-term deterioration in BM microenvironment contributes to age-related HSC senescence with impaired progeny and hematopoiesis, which also suggests possible roles of osteoblastic Wls on the maintenance of BM HSCs.

Accumulation of kynurenine elevates oxidative stress and alters microRNA profile in human bone marrow stromal cells.

Kynurenine, a metabolite of tryptophan breakdown, has been shown to increase with age, and plays a vital role in a number of age-related pathophysiological changes, including bone loss. Accumulation of kynurenine in bone marrow stromal cells (BMSCs) has been associated with a decrease in cell proliferation and differentiation, though the exact mechanism by which kynurenine mediates these changes is poorly understood. MiRNAs have been shown to regulate BMSC function, and accumulation of kynurenine may alter the miRNA expression profile of BMSCs. The aim of this study was to identify differentially expressed miRNAs in human BMSCs in response to treatment with kynurenine, and correlate miRNAs function in BMSCs biology through bioinformatics analysis. Human BMSCs were cultured and treated with and without kynurenine, and subsequent miRNA isolation was performed. MiRNA array was performed to identify differentially expressed miRNA. Microarray analysis identified 50 up-regulated, and 36 down-regulated miRNAs in kynurenine-treated BMSC cultures. Differentially expressed miRNA included miR-1281, miR-330-3p, let-7f-5p, and miR-493-5p, which are important for BMSC proliferation and differentiation. KEGG analysis found up-regulated miRNA targeting glutathione metabolism, a pathway critical for removing oxidative species. Our data support that the kynurenine dependent degenerative effect is partially due to changes in the miRNA profile of BMSCs.

Long-Term Clearance of Senescent Cells Prevents the Hematopoietic Stem Cell Aging in Naturally Aged Mice

Senescent cells (SnCs) accumulate with age. Genetic or pharmacological clearance of SnCs delays several age-associated disorders and extends the healthspan of both progeroid and wild-type (WT) mice (Baker DJ et al. Nature, 479: 232, 2011; Baker DJ et al. Nature, 530: 184, 2016). In addition, our previous study showed that transient depletion of SnCs could rejuvenated aged hematopoietic stem cells (HSCs) and muscle stem cells in normally aged mice (Chang J et al. Nat Med, 22:78, 2016). These findings suggest that SnCs play a causative role in aging and certain aging-related pathologies, and that selective clearance of SnCs with a senolytic agent that can selectively kill SnCs can be beneficial in part by rejuvenating aged tissue stem cells. Thus, senolytic drugs are potential novel anti-aging agents. However, SnCs can re-accumulate within a few weeks after depletion in mice, suggesting that long-term and intermittent clearance of SnCs is required to extend healthspan, as shown in previous studies (Baker DJ et al. Nature, 479: 232, 2011; Baker DJ et al. Nature, 530: 184, 2016). It is not known how long-term and intermittent clearance of SnCs affects tissue stem cells such as HSCs because HSCs are mostly quiescent in order to self-renew and prevent exhaustion. We used p16-3MR transgenic mice to evaluate the effect on HSCs of long-term and intermittent clearance of SnCs by ganciclovir (GCV), which selectively kills SnCs in these mice. We treated WT and p16-3MR mice with GCV (25 mg/kg/day x 5 days/per cycle with a 2-week interval between the cycles by ip injection) or saline (vehicle control) from 12 to 24 months of age. GCV effectively prevented SnC accumulation in p16-3MR mice but had no effect in WT mice. In general, aged p16-3MR mice treated with GCV looked healthier than old vehicle-treated p16-3MR mice and old WT mice treated with vehicle or GCV as expected (Baker DJ et al. Nature, 530: 184, 2016). Importantly, GCV abrogated aging-induced increases in p16 expression, p38MAPK activation, and accumulation of DNA double-strand breaks in HSCs in p16-3MR mice. This abrogation was associated with a significant improvement in HSC clonogenicity, suggesting that long-term and intermittent clearance of SnCs prevents HSC aging in naturally aged mice. This suggestion is confirmed by competitive serial and limited dilution HSC transplantation assays, which revealed that HSCs from GCV-treated old p16-3MR mice were functionally indistinguishable from those of young mice. To elucidate the mechanism by which SnC clearance prevents HSC aging, we evaluated the clonogenicity of Lin−Sca1+c-Kit+ (LSK) cells from young or vehicle- or GCV-treated old p16-3MR mice in co-cultures with bone marrow stromal cells (BMSCs) from these mice. We found that clearance of SnCs not only prevented the decline in HSC clonogenic function with aging but also preserved the hematopoietic supporting function of BMSCs. This preservation was associated with reduced levels of Cdkn2a and Cdkn1a mRNAs and senescence-associated secretory phenotype (SASP) factors (such as Il1β, Tnfα and Ccl5), which can adversely affect HSC self-renewal, and decrease of adipogenic and osteoclastic differentiation. In addition, the levels of the HSC self-renewal promoting factor Spp1 mRNA was higher in BMSCs from GCV-treated old p16-3MR mice than in the cells from vehicle-treated old p16-3MR mice. These findings suggest that long-term and intermittent clearance of SnCs can also inhibit BMSC senescence, which contributes to the prevention of HSC aging. Disclosures He: University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Chang:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Kim:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Almeida:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Campisi:Unity Biotechnology: Equity Ownership, Other: co-founder of Unity Biotechnology which develops small-molecule senolytic drugs. Zhou:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents; Unity Biotechnology: Equity Ownership, Other: Co-founder of Unity Biotechnology which develops small-molecule senolytic drugs; Dialectic Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents.

Early growth response 1 regulates hematopoietic support and proliferation in human primary bone marrow stromal cells.

Human bone marrow stromal cells (BMSC) are key elements of the hematopoietic environment and they play a central role in bone and bone marrow physiology. However, how key stromal cell functions are regulated is largely unknown. We analyzed the role of the immediate early response transcription factor EGR1 as key stromal cell regulator and found that EGR1 was highly expressed in prospectively-isolated primary BMSC, down-regulated upon culture, and low in non-colony-forming CD45neg stromal cells. Furthermore, EGR1 expression was lower in proliferative regenerating adult and fetal primary cells compared to adult steady-state BMSC. Overexpression of EGR1 in stromal cells induced potent hematopoietic stroma support as indicated by an increased production of transplantable CD34+CD90+ hematopoietic stem cells in expansion co-cultures. The improvement in bone marrow stroma support function was mediated by increased expression of hematopoietic supporting genes, such as VCAM1 and CCL28. Furthermore, EGR1 overexpression markedly decreased stromal cell proliferation whereas EGR1 knockdown caused the opposite effects. These findings thus show that EGR1 is a key stromal transcription factor with a dual role in regulating proliferation and hematopoietic stroma support function that is controlling a genetic program to co-ordinate the specific functions of BMSC in their different biological contexts.

Immunosuppression in Experimental Chagas Disease Is Mediated by an Alteration of Bone Marrow Stromal Cell Function During the Acute Phase of Infection.

After infection with Trypanosoma cruzi, the etiologic agent of Chagas disease, immunosuppression, and apoptosis of mature lymphocytes contribute to the establishment of the parasite in the host and thereby to persistence and pathology in the chronic stage of infection. In a systemic mouse model of experimental Chagas disease, we have demonstrated a strong depletion of mature B cells in the spleen during the first 2 weeks of infection. Remarkably, the decrease in this cell population commenced already in the bone marrow from infected mice and was a concomitant of an increased apoptosis in pro- and pre-B cell populations. Pro- and pre-B cells in the bone marrow showed a significant reduction accompanied by a functional disturbance of bone marrow-derived stromal cells resulting in diminished levels of IL-7, an essential factor for the development of B cell precursors. Ex vivo, stromal cells isolated from the bone marrow of infected mice had a strikingly impaired capacity to maintain the development of pro- and pre-B cells obtained from uninfected animals. Together, the reduction of an active humoral immune response during acute Chagas disease suggests to be an initial immune evasion mechanism of the parasite to establish persistent infection. Therefore, prevention of B cell depletion by rescuing the stromal cells during this early phase, could give rise to new therapeutic approaches.

Lanthanum-containing bioparticles are associated with the influence of lanthanum on high phosphate mediated bone marrow stromal cells viability.

Lanthanum (La)-based binder appears effective in reducing serum inorganic phosphate (Pi) levels among chronic dialysis patients, yet concern remains about La accumulation in bone during long-term oral administration. In this study, the effect of lanthanum chloride (LaCl3) on bone marrow stromal cells (BMSCs) viability was investigated under high Pi situation. We found low concentration (10-9M) of LaCl3 increased BMSCs viability, while high concentration (10-5M) of LaCl3 not only inhibited BMSCs viability but also stimulated high Pi induced cell apoptosis. In addition, La-containing calcium phosphate (CaP) particles can be detected on cell surfaces and inside cells. We also found that inhibition of CaP formation by adding sodium pyrophosphate, a recognized inhibitor of hydroxyapatite formation, abrogated LaCl3 induced the BMSCs viability. For isolated La-containing CaP particles, the particle size increased and crystal phase switched with elevated concentration of LaCl3. These results demonstrated that La-containing CaP particles were associated with the process of LaCl3 mediated BMSCs viability and the physicochemical properties of these particles played an important role in modulating BMSCs function.

Epigenetic Modification of the CCL5/CCR1/ERK Axis Enhances Glioma Targeting in Dedifferentiation-Reprogrammed BMSCs

SummaryThe success of stem cell-mediated gene therapy in cancer treatment largely depends on the specific homing ability of stem cells. We have previously demonstrated that after in vitro induction of neuronal differentiation and dedifferentiation, bone marrow stromal cells (BMSCs) revert to a primitive stem cell population (De-neu-BMSCs) distinct from naive BMSCs. We report here that De-neu-BMSCs express significantly higher levels of chemokines, and display enhanced homing abilities to glioma, the effect of which is mediated by the activated CCL5/CCR1/ERK axis. Intriguingly, we find that the activated chemokine axis in De-neu-BMSCs is epigenetically regulated by histone modifications. On the therapeutic front, we show that De-neu-BMSCs elicit stronger homing and glioma-killing effects together with cytosine deaminase/5-fluorocytosine compared with unmanipulated BMSCs in vivo. Altogether, the current study provides an insight into chemokine regulation in BMSCs, which may have more profound effects on BMSC function and their application in regenerative medicine and cancer targeting.

Organic Nanoparticles with Aggregation‐Induced Emission for Bone Marrow Stromal Cell Tracking in a Rat PTI Model

Stem-cell based therapy is an emerging therapeutic approach for ischemic stroke treatment. Bone marrow stromal cells (BMSCs) are in common use as a cell source for stem cell therapy and show promising therapeutic outcomes for stroke treatment. One challenge is to develop a reliable tracking strategy to monitor the fate of BMSCs and assess their therapeutic effects in order to improve the success rate of such treatment. Herein, TPEEP, a fluorogen with aggregation-induced emission characteristics and near-infrared emission are designed and synthesized and further fabricated into organic nanoparticles (NPs). The obtained NPs show high fluorescence quantum yield, low cytotoxicity with good physical and photostability, which display excellent tracking performance of BMSCs in vitro and in vivo. Using a rat photothrombotic ischemia model as an example, the NP-labeled BMSCs are able to migrate to the stroke lesion site to yield bright red fluorescence. Immunofluorescence staining shows that the NP labeling does not affect the normal function of BMSCs, proving their good biocompatibility in vivo. These merits make TPEEP NP a potential cell tracker to evaluate the fate of BMSCs in cell therapy.

Bone and Stem Cells. Cellular network in bone micro-environment - histological and ultrastructural aspects -

Bone micro-environment appears to reflect bone turnover, i.e., frequency of bone remodeling. There are many bone-synthesizing mature osteoblasts, bone-resorbing osteoclasts, and a thick cell layer of preosteoblasts overlying mature osteoblasts in the region which shows active bone remodeling. Bone lining cells, - flattened, resting form of osteoblasts cover the quiescent bone surface, in which, however, osteocyte-lacunar canalicular system tend to be geometrically well-arranged. Thus, bone micro-environment seems to be regulated by preosteoblasts, bone marrow stromal cells and vascular endothelial cells, as well as osteoblasts and osteoclasts. But, precious biological function of preosteoblasts and bone marrow stromal cells are still under the investigation, e.g., due to many phenotypes of preosteoblasts. In this review, we will introduce histological and ultrastructural aspects on cellular involvement in bone micro-environment.

RhPDGF-BB Promotes Proliferation and Osteogenic Differentiation of Bone Marrow Stromal Cells from Streptozotocin-Induced Diabetic Rats through ERK Pathway

Management of nonunion fracture and massive segmental bone defects in diabetes remains a challenging clinical problem. Bone marrow stromal cells (BMSCs) are crucial for bone remodeling and hold promise for bone regeneration. However, we have showed previously that diabetes can affect the proliferation and osteogenic potential of BMSCs adversely and a strategy to attenuate the impaired functions of BMSCs is required. Platelet-derived growth factor-BB (PDGF-BB) plays an important role in bone formation. However, little information is available about its effect on diabetic BMSCs. In this study, BMSCs were isolated from streptozotocin-induced diabetic rats. After treatment with recombinant human PDGF-BB (rhPDGF-BB), diabetic BMSCs demonstrated enhanced cell proliferation and osteogenic differentiation based on increased expressions of osteogenic genes (Runx2, alkaline phosphatase, and osteocalcin) and Runx2 protein, as well as upregulated alkaline phosphatase activity and mineralization. Furthermore, blocking extracellular signal regulated kinase (ERK) pathway by inhibitor PD98059 repressed the enhanced proliferation and osteogenic differentiation in diabetic BMSCs induced by rhPDGF-BB. Together, these results indicated that rhPDGF-BB stimulates proliferation and osteogenic differentiation partially through ERK pathway in diabetic BMSCs. Therefore, modulation of diabetic BMSCs could augment BMSCs function affected by diabetes and holds significance for future strategies to treat diabetic bone complications.

Osteogenic potential of bone marrow stromal cells derived from streptozotocin-induced diabetic rats

Type1 diabetes mellitus (T1DM) is associated with a series of bone complications, which are still a great challenge in the clinic. Bone marrow stromal cells (BMSCs) are crucial to bone remodeling and are attractive candidates for tissue engineering. Hence, we aimed to investigate whether impaired functions of BMSCs play a role in the pathogenesis of bone complications associated with T1DM. BMSCs were isolated from normal and streptozotocin-induced diabetic rats, and their proliferation and osteogenic differentiation ability were analyzed. Diabetic BMSCs demonstrated reduced proliferation ability, osteoblast gene expression, alkaline phosphatase activity and mineralization. Nude mice transplanted with diabetic BMSCs in a calcium phosphate cement scaffold exhibited reduced new bone formation, as detected by hematoxylin and eosin staining and immunohistochemistry. These changes may be partially related to impaired insulin and insulin-like growth factor1 (IGF-1) signaling. Weak gene expression of insulin receptor (IR), IGF-1, insulin-like growth factor1 receptor (IGF-1R), and insulin receptor substrate-1 (IRS-1) was observed in the diabetic BMSCs compared with normal BMSCs, together with decreased protein level of IGF-1, IGF-1R, IRS-1 and phosphorylated extracellular signal-regulated kinase. Therefore, impaired proliferation and osteogenic potential of BMSCs may be responsible for bone complications related to T1DM, mediated partially by impaired insulin and IGF-1 signaling. These findings may provide a new target with which to devise strategies for therapy.

Stromal Cells Expressing Hedgehog-Interacting Protein Are One of the Main Regulators of the Hedgehog Signaling System in Myeloid Neoplasms

Abstract 3840Aberrant reactivation of Hedgehog (Hh) signaling has been described in a wide variety of human cancers including cancer stem cells. However, involvement of the Hh signaling system in the bone marrow (BM) microenvironment during the development of myeloid neoplasm is unclear. In this study we assessed the expression of Hh-related proteins in normal human CD34+ cells, CD34+ leukemic/dysplastic cells and BM stromal cells. Both Indian hedgehog (Ihh) and its signal transducer, SMO, were expressed in CD34+ acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) derived cells, suggesting that Ihh could be affected in an autocrine manner. Remarkably, expression of the endogenous Hh signaling inhibitor, human hedgehog-interacting protein (HHIP), in AML/MDS-associated stromal cells was significantly lower than in healthy donor-derived stromal cells. Moreover, HHIP expression level in BM stromal cells highly correlated with their tumor-supporting activity of SMO+ leukemic cells. Demethylating agent 5-aza-dC rescued HHIP expression via demethylation of HHIP gene and reduced the leukemia-supporting activity of AML/MDS-associated stromal cells. This effect of 5-aza-dC was negated by HHIP shRNA transfer into stromal cells. These results indicate that suppression of stromal HHIP expression could be involved in the progression of AML/MDS and 5-aza-dC may improve the protective function of BM stromal cells for AML/MDS. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Silencing HIF-1α reduces the adhesion and secretion functions of acute leukemia hBMSCs

Hypoxia inducible factor-1α (HIF-1α) is an important transcription factor, which plays a critical role in the formation of solid tumor and its microenviroment. The objective of the present study was to evaluate the expression and function of HIF-1α in human leukemia bone marrow stromal cells (BMSCs) and to identify the downstream targets of HIF-1α. HIF-1α expression was detected at both the RNA and protein levels using real-time PCR and immunohistochemistry, respectively. Vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) were detected in stromal cells by enzyme-linked immunosorbent assay. HIF-1α was blocked by constructing the lentiviral RNAi vector system and infecting the BMSCs. The Jurkat cell/BMSC co-cultured system was constructed by putting the two cells into the same suitable cultured media and conditions. Cell adhesion and secretion functions of stromal cells were evaluated after transfection with the lentiviral RNAi vector of HIF-1α. Increased HIF-1α mRNA and protein was detected in the nucleus of the acute myeloblastic and acute lymphoblastic leukemia compared with normal BMSCs. The lentiviral RANi vector for HIF-1α was successfully constructed and was applied to block the expression of HIF-1α. When HIF-1α of BMSCs was blocked, the expression of VEGF and SDF-1 secreted by stromal cells were decreased. When HIF-1α was blocked, the co-cultured Jurkat cell’s adhesion and migration functions were also decreased. Taken together, these results suggest that HIF-1α acts as an important transcription factor and can significantly affect the secretion and adhesion functions of leukemia BMSCs.

The influence of prefreezing temperature on pore structure in freeze-dried β-TCP scaffolds

A combined method of tricalcium phosphate (TCP) scaffold production, which comprised negative mold and scaffold fabrication, was reported in this study. The negative mold structure was designed by computer and fabricated by fused deposition modeling (FDM) technology, while the TCP scaffold was produced by freeze-drying technology under different prefreezing temperatures of -10 degrees C, -30 degrees C, and -86 degrees C and thermal treatment to get beta-TCP. The scaffold structure was evaluated with X-ray, scanning electron microscopy (SEM), compressive mechanical testing, and micro-computerized tomography (micro-CT). The cell-scaffold interaction was studied by culturing dog bone marrow stromal cells (BMSCs) on the scaffolds and assessing differentiated BMSC function by measuring cellular alkaline phosphatase (ALP) activity. The results showed good interconnectivity and good pore distribution with the pore size ranging from 50 to 250 microm and compressive modulus of 1.18 MPa at a prefreezing temperatures of -10 degrees C. In vitro cell culture results indicated that the porous scaffolds were not toxic to bone cells. These results demonstrate that rapid prototyping and freeze-drying technologies for creating beta-TCP scaffolds are promising for bone tissue engineering.

Silencing HIF-1α reduces the adhesion and secretion functions of acute leukemia hBMSCs

Hypoxia inducible factor-1α (HIF-1α) is an important transcription factor, which plays a critical role in the formation of solid tumor and its microenvironment. The objective of the present study was to evaluate the expression and function of HIF-1α in human leukemia bone marrow stromal cells (BMSCs) and to identify the downstream targets of HIF-1α. HIF-1α expression was detected at both the RNA and protein levels using real-time PCR and immunohistochemistry, respectively. Vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) were detected in stromal cells by enzyme-linked immunosorbent assay. HIF-1α was blocked by constructing the lentiviral RNAi vector system and infecting the BMSCs. The Jurkat cell/BMSC co-cultured system was constructed by putting the two cells into the same suitable cultured media and conditions. Cell adhesion and secretion functions of stromal cells were evaluated after transfection with the lentiviral RNAi vector of HIF-1α. Increased HIF-1α mRNA and protein was detected in the nucleus of the acute myeloblastic and acute lymphoblastic leukemia compared with normal BMSCs. The lentiviral RANi vector for HIF-1α was successfully constructed and was applied to block the expression of HIF-1α. When HIF-1α of BMSCs was blocked, the expression of VEGF and SDF-1α secreted by stromal cells was decreased. When HIF-1α was blocked, the co-cultured Jurkat cell's adhesion and migration functions were also decreased. Taken together, these results suggest that HIF-1α acts as an important transcription factor and can significantly affect the secretion and adhesion functions of leukemia BMSCs.

Neurotrophins regulate bone marrow stromal cell IL-6 expression through the MAPK pathway.

BackgroundThe host's response to infection is characterized by altered levels of neurotrophins and an influx of inflammatory cells to sites of injured tissue. Progenitor cells that give rise to the differentiated cellular mediators of inflammation are derived from bone marrow progenitor cells where their development is regulated, in part, by cues from bone marrow stromal cells (BMSC). As such, alteration of BMSC function in response to elevated systemic mediators has the potential to alter their function in biologically relevant ways, including downstream alteration of cytokine production that influences hematopoietic development.Methodology/Principal FindingsIn the current study we investigated BMSC neurotrophin receptor expression by flow cytometric analysis to determine differences in expression as well as potential to respond to NGF or BDNF. Intracellular signaling subsequent to neurotrophin stimulation of BMSC was analyzed by western blot, microarray analysis, confocal microscopy and real-time PCR. Analysis of BMSC Interleukin-6 (IL-6) expression was completed using ELISA and real-time PCR.ConclusionBMSC established from different individuals had distinct expression profiles of the neurotrophin receptors, TrkA, TrkB, TrkC, and p75NTR. These receptors were functional, demonstrated by an increase in Akt-phosphorylation following BMSC exposure to recombinant NGF or BDNF. Neurotrophin stimulation of BMSC resulted in increased IL-6 gene and protein expression which required activation of ERK and p38 MAPK signaling, but was not mediated by the NFκB pathway. BMSC response to neurotrophins, including the up-regulation of IL-6, may alter their support of hematopoiesis and regulate the availability of inflammatory cells for migration to sites of injury or infection. As such, these studies are relevant to the growing appreciation of the interplay between neurotropic mediators and the regulation of hematopoiesis.