Oxytocin preprotein and oxytocin receptor mRNA expression is altered in semen samples with abnormal semen parameters.

RESEARCH QUESTION: Are oxytocin preprotein and the oxytocin receptor expressed in human spermatozoa and is their mRNA expression different between normal semen samples and samples with at least one abnormal parameter? DESIGN: An in-vitro prospective study of 175 semen samples from Greek men, according to World Health Organization criteria, 2010. mRNA expression levels were compared between different categories of semen samples, classified according to their concentration, total number, motility and morphology. Immunohistochemistry was used to detect oxytocin preprotein and its receptor on spermatozoa smears. RESULTS: Compared with normal samples (normal motility and normal concentration), samples with at least one abnormal sperm parameter had statistically significantly lower oxytocin preprotein mRNA expression (P = 0.019) and higher oxytocin receptor mRNA expression levels (P < 0.001). Oligozoospermic samples had statistically significantly higher oxytocin preprotein mRNA expression levels (P = 0.002) and lower oxytocin receptor mRNA expression levels (P = 0.047). Asthenozoospermic samples had statistically significantly lower oxytocin preprotein mRNA expression levels (P < 0.001). Teratozoospermic samples had statistically significantly lower oxytocin preprotein mRNA expression levels (P = 0.049) and higher oxytocin receptor mRNA expression levels (P < 0.001). Oxytocin preprotein mRNA expression was positively associated with total progressive motility (P < 0.001) and negatively associated with the percentage of immotile spermatozoa (P = 0.001). Oxytocin receptor mRNA expression was negatively associated with the percentage of normal forms (P < 0.001). CONCLUSION: Oxytocin preprotein and oxytocin receptor mRNA expression in spermatozoa could be used as a novel and unbiased diagnostic tool for male infertility.

Expression of genes that regulate follicle development and maturation during ovarian stimulation in poor responders.

RESEARCH QUESTION: Sex hormone-binding globulin (SHBG), androgen receptor (AR), LH beta polypeptide (LHB), progesterone receptor membrane component 1 (PGRMC1) and progesterone receptor membrane component 2 (PGRMC2) regulate follicle development and maturation. Their mRNA expression was assessed in peripheral blood mononuclear cells (PBMC) of normal and poor responders, during ovarian stimulation. DESIGN: Fifty-two normal responders and 15 poor responders according to the Bologna criteria were enrolled for IVF and intracytoplasmic sperm injection and stimulated with 200 IU of follitrophin alpha and gonadotrophin-releasing hormone antagonist. HCG was administered for final oocyte maturation. On days 1, 6 and 10 of stimulation, blood samples were obtained, serum hormone levels were measured, RNA was extracted from PBMC and real-time polymerase chain reaction was carried out to identify the mRNA levels. Relative mRNA expression of each gene was calculated by the comparative 2-DDCt method. RESULTS: Differences between mRNA levels of each gene on the same time point between the two groups were not significant. PGRMC1 and PGRMC2 mRNA levels were downregulated, adjusted for ovarian response and age. Positive correlations between PGRMC1 and AR (standardized beta = 0.890, P < 0.001) from day 1 to 6 and PGRMC1 and LHB (standardized beta = 0.806, P < 0.001) from day 1 to 10 were found in poor responders. PGRMC1 and PGRMC2 were positively correlated on days 6 and 10 in normal responders. CONCLUSIONS: PGRMC1 and PGRMC2 mRNA are significantly decreased during ovarian stimulation, with some potential differences between normal and poor responders.

mRNA overexpression of prolyl hydroxylase PHD3 is inversely related to nuclear grade in renal cell carcinoma.

The aim of the present study was to evaluate the relative mRNA expression levels of genes involved in the hypoxia inducible factor (HIF) signalling pathway in renal cell carcinoma (RCC) and to analyse their associations with clinicopathological parameters and survival outcomes. Reverse transcription-quantitative PCR was used to quantify the mRNA expression levels of HIF-1α, HIF-2α, prolyl hydroxylase (PHD)1, PHD2 and PHD3 in formalin-fixed paraffin-embedded (FFPE) tumour tissue samples from 41 patients with RCC, including 33 cases of clear cell RCC (ccRCC). FFPE samples of corresponding adjacent normal kidney tissues were used as a comparison. mRNA expression levels were analysed in regard to clinical parameters, histological type, stage, nuclear grade, cancer specific survival and overall survival. Compared with adjacent normal kidney tissue, HIF-1α levels were lower in 16/33 ccRCC samples (48.48%), while HIF-2α, PHD1 and PHD2 levels did not exhibit a specific expression pattern. By contrast, the PHD3 mRNA level was higher in 29/33 (87.87%) of the tumour samples. HIF-1α was positively associated with HIF-2α, PHD1 and PHD2. HIF-2α levels were associated with PHD1, PHD2 and PHD3, while PHD3 was strongly associated with PHD2. PHD3 mRNA levels were inversely associated with nuclear grade (P=0.015). However, in univariate analysis, PHD3 was not associated with cancer-specific or overall survival rates. The present findings suggest an important involvement of PHD3 in ccRCC, since PHD3 mRNA expression was inversely associated with nuclear grade. However, PHD3 mRNA levels did not have an independent prognostic value. Further studies are required to investigate whether PHD3 could be used as either a therapeutic target or prognostic marker.

The effects of postnatal exposure of endocrine disruptors on testicular function: a systematic review and a meta-analysis.

BACKGROUND: Despite many epidemiological studies having been conducted, the impact of postnatal exposure of endocrine disruptors (EDs) on testicular function remains a controversial issue. AIM: To systematically review the literature and perform a quantitative synthesis to evaluate the effect of EDs on testicular function. MATERIALS AND METHODS: A comprehensive search was conducted in the MEDLINE, Scopus, and CENTRAL databases. Eligible for the systematic review were observational (cross-sectional and cohort) studies with (i) adult men who had a high probability of postnatal exposure to EDs ("exposed"), (ii) adult men who had a low probability of postnatal exposure to EDs ("non-exposed"), and (iii) an outcome of interest [seminal parameters and reproductive hormone concentrations]. The continuous outcomes in each of the studies were synthesized by the random effects model and expressed as standardized mean difference (SMD) with 95% confidence interval (CI). RESULTS: Thirteen studies, including 959 exposed and 907 non-exposed men, fulfilled the inclusion criteria. Exposure to EDs was associated with decreased LH [SMD - 0.17, 95% CI - 0.33 to - 0.02, 10 studies (616 exposed, 563 non-exposed), I2 40%, p = 0.09], progressive motility [SMD - 0.45, 95% CI - 0.77 to - 0.13, three studies (133 cases, 153 controls), I2 38%, p = 0.20], and normal morphology [SMD - 0.50, 95% CI - 0.85 to - 0.14, eight studies (562 cases, 540 controls), I2 87%, p < 0.01] compared with non-exposure. No difference was observed between the other study groups. CONCLUSIONS: Postnatal exposure to EDs is associated with decreased semen quality. Nevertheless, there is no evidence that a disruption of testicular function mediates the deterioration in semen quality.

Addition of procyanidine to semen preserves progressive sperm motility up to three hours of incubation.

Several studies on semen physiology and sperm fertilizing capacity have shown a beneficial effect of antioxidants. Procyanidine is a natural antioxidant, more efficient compared with vitamin C and E, with many applications in the food, agriculture, pharmaceutical and cosmetic industry. Thus, we tested whether the addition of procyanidine to the semen of infertile men has a beneficial effect on spermatozoa during their in vitro incubation and during the cryopreservation process. Semen samples of 25 infertile men were divided in to two aliquots, in which procyanidine was added or not. Semen analysis, measurement of sperm DNA fragmentation index (DFI) and measurement of reactive oxygen species (ROS) were performed 3 h after incubation at 37 °C and after sperm cryopreservation and thawing. In-vitro addition of procyanidine to semen of infertile men resulted in a lesser decrease in progressive motility [-4 (-31:+6) vs. -6 (-31:+5), p < 0.001] and total motility [-5 (-29:+3) vs. -9 (-32:+2), p < 0.001] after 3 h of incubation compared with no addition of procyanidine. Sperm morphology was decreased only in the control group after 3 h of incubation [2 (0:+6) vs. 1 (0:+4), p = 0.009]. Furthermore, a larger increase in sperm DFI was observed in the control compared with the procyanidine group [9 (-7:+27) vs. 3 (-3:+18), p = 0.005] after thawing of cryopreserved semen samples. In conclusion, in-vitro addition of procyanidine to the semen of infertile men exerts a protective effect on progressive motility during handling and after 3 h of incubation as well as on sperm DFI during the process of cryopreservation.

Endocrine disruptors and testicular function.

Despite concerns of the scientific community regarding the adverse effects of human exposure to exogenous man-made chemical substances or mixtures that interfere with normal hormonal balance, the so called "endocrine disruptors (EDs)", their production has been increased during the last few decades. EDs' extensive use has been implicated in the increasing incidence of male reproductive disorders including poor semen quality, testicular malignancies and congenital developmental defects such as hypospadias and cryptorchidism. Several animal studies have demonstrated that exposure to EDs during fetal, neonatal and adult life has deleterious consequences on male reproductive system; however, the evidence on humans remains ambiguous. The complexity of their mode of action, the differential effect according to the developmental stage that exposure occurs, the latency from exposure and the influence of the genetic background in the manifestation of their toxic effects are all responsible factors for the contradictory outcomes. Furthermore, the heterogeneity in the published human studies has hampered agreement in the field. Interventional studies to establish causality would be desirable, but unfortunately the nature of the field excludes this possibility. Therefore, future studies based on standardized guidelines are necessary, in order to estimate human health risks and implement policies to limit public exposure.

A High-Resolution Proteomic Landscaping of Primary Human Dental Stem Cells: Identification of SHED- and PDLSC-Specific Biomarkers.

Dental stem cells (DSCs) have emerged as a promising tool for basic research and clinical practice. A variety of adult stem cell (ASC) populations can be isolated from different areas within the dental tissue, which, due to their cellular and molecular characteristics, could give rise to different outcomes when used in potential applications. In this study, we performed a high-throughput molecular comparison of two primary human adult dental stem cell (hADSC) sub-populations: Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) and Periodontal Ligament Stem Cells (PDLSCs). A detailed proteomic mapping of SHEDs and PDLSCs, via employment of nano-LC tandem-mass spectrometry (MS/MS) revealed 2032 identified proteins in SHEDs and 3235 in PDLSCs. In total, 1516 proteins were expressed in both populations, while 517 were unique for SHEDs and 1721 were exclusively expressed in PDLSCs. Further analysis of the recorded proteins suggested that SHEDs predominantly expressed molecules that are involved in organizing the cytoskeletal network, cellular migration and adhesion, whereas PDLSCs are highly energy-producing cells, vastly expressing proteins that are implicated in various aspects of cell metabolism and proliferation. Applying the Rho-GDI signaling pathway as a paradigm, we propose potential biomarkers for SHEDs and for PDLSCs, reflecting their unique features, properties and engaged molecular pathways.

Should we expand the indications for varicocele treatment?

Current guidelines suggest that treatment of varicocele should be considered in patients with clinically palpable disease and abnormal semen parameters. However, the clinicians are often challenged with the decision whether to treat varicocele in patients with testicular pain or low testosterone levels. Moreover, varicocele is highly associated with DNA fragmentation due to the oxidative stress and it has been demonstrated that surgical repair of varicocele ameliorates oxidative stress markers and consequently the sperm DNA integrity. These new markers could have an adjunctive role to standard semen parameters especially when normal semen analysis is found in adult men with conventional methods. This review presents a contemporary overview of the rationale for varicocele treatment, as well as of the relationship between varicocele and other novel parameters such as DNA fragmentation index and reactive oxygen species. We will also discuss data from several recent series demonstrating that surgical treatment and especially microsurgical approach could resolve testicular pain, increase testosterone levels and fertility rate both in patients with non-obstructive azoospermia as well as in normozoospermia men. The correlation with progressive testicular failure will be also examined. We hope that this overview will provide clinicians with an evidence-based approach to managing these unanswered and conflicting topics.

Interaction of dental pulp stem cells with Biodentine and MTA after exposure to different environments.

OBJECTIVE:: The aim of the present study was to evaluate and compare the cytotoxic effects of Biodentine and MTA on dental pulp stem cells (DPSCs) and to assess cell viability and adherence after material exposure to an acidic environment. MATERIAL AND METHODS:: DPSCs were cultured either alone or in contact with either: Biodentine; MTA set for 1 hour; or MTA set for 24 hours. After 4 and 7 days, cell viability was measured using the MTT assay. Biodentine and MTA were also prepared and packed into standardized bovine dentin disks and divided into three groups according to the storage media (n=6/group): freshly mixed materials without storage medium (Group A); materials stored in saline (Group B); materials stored in citric acid buffered at pH 5.4 (Group C). After 24 hours, DPSCs were introduced in the wells and cell adherence, viability, and cellular morphology were observed via confocal microscopy after three days of culture. Cell viability was analyzed using repeated-measures analysis of variance test with Tukey's post hoc tests (α=0.05). RESULTS:: Biodentine expressed significantly higher cell viability compared with all other groups after 4 days, with no differences after 7 days. Notably, cell viability was significantly greater in 24-hour set MTA compared with 1-hour set MTA and control groups after 7 days. Material exposure to an acidic environment showed an increase in cell adherence and viability in both groups. CONCLUSIONS:: Biodentine induced a significantly accelerated cell proliferation compared with MTA. Setting of these materials in the presence of citric acid enhanced DPSC viability and adherence.

Interaction of dental pulp stem cells with Biodentine and MTA after exposure to different environments

ABSTRACT Objective: The aim of the present study was to evaluate and compare the cytotoxic effects of Biodentine and MTA on dental pulp stem cells (DPSCs) and to assess cell viability and adherence after material exposure to an acidic environment. Material and Methods: DPSCs were cultured either alone or in contact with either: Biodentine; MTA set for 1 hour; or MTA set for 24 hours. After 4 and 7 days, cell viability was measured using the MTT assay. Biodentine and MTA were also prepared and packed into standardized bovine dentin disks and divided into three groups according to the storage media (n=6/group): freshly mixed materials without storage medium (Group A); materials stored in saline (Group B); materials stored in citric acid buffered at pH 5.4 (Group C). After 24 hours, DPSCs were introduced in the wells and cell adherence, viability, and cellular morphology were observed via confocal microscopy after three days of culture. Cell viability was analyzed using repeated-measures analysis of variance test with Tukey's post hoc tests (α=0.05). Results: Biodentine expressed significantly higher cell viability compared with all other groups after 4 days, with no differences after 7 days. Notably, cell viability was significantly greater in 24-hour set MTA compared with 1-hour set MTA and control groups after 7 days. Material exposure to an acidic environment showed an increase in cell adherence and viability in both groups. Conclusions: Biodentine induced a significantly accelerated cell proliferation compared with MTA. Setting of these materials in the presence of citric acid enhanced DPSC viability and adherence.

Distinctive Mesenchymal-Parenchymal Cell Pairings Govern B Cell Differentiation in the Bone Marrow.

Bone marrow niches for hematopoietic progenitor cells are not well defined despite their critical role in blood homeostasis. We previously found that cells expressing osteocalcin, a marker of mature osteolineage cells, regulate the production of thymic-seeding T lymphoid progenitors. Here, using a selective cell deletion strategy, we demonstrate that a subset of mesenchymal cells expressing osterix, a marker of bone precursors in the adult, serve to regulate the maturation of early B lymphoid precursors by promoting pro-B to pre-B cell transition through insulin-like growth factor 1 (IGF-1) production. Loss of Osx(+) cells or Osx-specific deletion of IGF-1 led to a failure of B cell maturation and the impaired adaptive immune response. These data highlight the notion that bone marrow is a composite of specialized niches formed by pairings of specific mesenchymal cells with parenchymal stem or lineage committed progenitor cells, thereby providing distinctive functional units to regulate hematopoiesis.

Transcriptome comparison of distinct osteolineage subsets in the hematopoietic stem cell niche using a triple fluorescent transgenic mouse model.

The bone marrow niche is recognized as a central player in maintaining and regulating the behavior of hematopoietic stem and progenitor cells. Specific gain-of and loss-of function experiments perturbing a range of osteolineage cells or their secreted proteins had been shown to affect stem cell maintenance (Calvi et al, 2003 [1]; Stier et al., 2005 [2]; Zhang et al., 2003 [3]; Nilsson et al., 2005 [4]; Greenbaum et al., 2013 [5]) and engraftment (Adam et al., 2006, 2009 [6,7]). We used specific in vivo cell deletion approaches to dissect the niche cell-parenchymal cell dependency in a complex bone marrow microenvironment. Endogenous deletion of osteocalcin-expressing (Ocn(+)) cells led to a loss of T immune cells (Yu et al., 2015 [8]. Ocn(+) cells express the Notch ligand DLL4 to communicate with T-competent progenitors, and thereby ensuring T precursor production and expression of chemotactic molecules on their cell surface for subsequent thymic seeding. In contrast, depletion of osterix-expressing (Osx(+)) osteoprogenitors led to reduced B immune cells. These distinct hematopoietic phenotypes suggest specific pairing of mesenchymal niche cells and parenchymal hematopoietic cells in the bone marrow to create unique functional units to support hematopoiesis. Here, we present the global gene expression profiles of these osteolineage subtypes utilizing a triple fluorescent transgenic mouse model (OsxCre(+);Rosa-mCh(+);Ocn:Topaz(+)) that labels Osx(+) cells red, Ocn(+) cells green, and Osx(+) Ocn(+) cells yellow. This system allows isolation of distinct osteolineage subsets within the same animal by flow cytometry. Array data that have been described in our study [8] are also publically available from NCBI Gene Expression Omnibus (GEO) with the accession number GSE66042. Differences in gene expression may correlate with functional difference in supporting hematopoiesis.

Dental Stem Cell Migration on Pulp Ceiling Cavities Filled with MTA, Dentin Chips, or Bio-Oss.

MTA, Bio-Oss, and dentin chips have been successfully used in endodontics. The aim of this study was to assess the adhesion and migration of dental stem cells on human pulp ceiling cavities filled with these endodontic materials in an experimental model, which mimics the clinical conditions of regenerative endodontics. Cavities were formed, by a homemade mold, on untouched third molars, filled with endodontic materials, and observed with electron microscopy. Cells were seeded on cavities' surface and their morphology and number were analysed. The phenomenon of tropism was assessed in a migration assay. All three materials demonstrated appropriate microstructures for cell attachment. Cells grew on all reagents, but they showed a differential morphology. Moreover, variations were observed when comparing cells numbers on cavity's filling versus the surrounding dentine disc. The highest number of cells was recorded on dentin chips whereas the opposite was true for Bio-Oss. This was confirmed in the migration assay where a statistically significant lower number of cells migrated towards Bio-Oss as compared to MTA and dentin chips. This study highlights that MTA and dentin chips have a greater potential compared to Bio-Oss regarding the attraction of dental stem cells and are good candidates for bioengineered pulp regeneration.

Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow.

Production of the cells that ultimately populate the thymus to generate α/ß T cells has been controversial, and their molecular drivers remain undefined. Here, we report that specific deletion of bone-producing osteocalcin (Ocn)-expressing cells in vivo markedly reduces T-competent progenitors and thymus-homing receptor expression among bone marrow hematopoietic cells. Decreased intrathymic T cell precursors and decreased generation of mature T cells occurred despite normal thymic function. The Notch ligand DLL4 is abundantly expressed on bone marrow Ocn(+) cells, and selective depletion of DLL4 from these cells recapitulated the thymopoietic abnormality. These data indicate that specific mesenchymal cells in bone marrow provide key molecular drivers enforcing thymus-seeding progenitor generation and thereby directly link skeletal biology to the production of T cell-based adaptive immunity.

Inhibiting stromal cell heparan sulfate synthesis improves stem cell mobilization and enables engraftment without cytotoxic conditioning.

The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions.

Diabetes impairs hematopoietic stem cell mobilization by altering niche function.

Success with transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) in patients depends on adequate collection of these cells after mobilization from the bone marrow niche by the cytokine granulocyte colony-stimulating factor (G-CSF). However, some patients fail to achieve sufficient HSPC mobilization. Retrospective analysis of bone marrow transplant patient records revealed that diabetes correlated with poor mobilization of CD34+ HSPCs. In mouse models of type 1 and type 2 diabetes (streptozotocin-induced and db/db mice, respectively), we found impaired egress of murine HSPCs from the bone marrow after G-CSF treatment. Furthermore, HSPCs were aberrantly localized in the marrow niche of the diabetic mice, and abnormalities in the number and function of sympathetic nerve termini were associated with this mislocalization. Aberrant responses to ß-adrenergic stimulation of the bone marrow included an inability of marrow mesenchymal stem cells expressing the marker nestin to down-modulate the chemokine CXCL12 in response to G-CSF treatment (mesenchymal stem cells are reported to be critical for HSPC mobilization). The HSPC mobilization defect was rescued by direct pharmacological inhibition of the interaction of CXCL12 with its receptor CXCR4 using the drug AMD3100. These data suggest that there are diabetes-induced changes in bone marrow physiology and microanatomy and point to a potential intervention to overcome poor HSPC mobilization in diabetic patients.

Inhibition of osteoclast function reduces hematopoietic stem cell numbers in vivo.

Osteoblasts play a crucial role in the hematopoietic stem cell (HSC) niche; however, an overall increase in their number does not necessarily promote hematopoiesis. Because the activity of osteoblasts and osteoclasts is coordinately regulated, we hypothesized that active bone-resorbing osteoclasts would participate in HSC niche maintenance. Mice treated with bisphosphonates exhibited a decrease in proportion and absolute number of Lin(-)cKit(+)Sca1(+) Flk2(-) (LKS Flk2(-)) and long-term culture-initiating cells in bone marrow (BM). In competitive transplantation assays, the engraftment of treated BM cells was inferior to that of controls, confirming a decrease in HSC numbers. Accordingly, bisphosphonates abolished the HSC increment produced by parathyroid hormone. In contrast, the number of colony-forming-unit cells in BM was increased. Because a larger fraction of LKS in the BM of treated mice was found in the S/M phase of the cell cycle, osteoclast impairment makes a proportion of HSCs enter the cell cycle and differentiate. To prove that HSC impairment was a consequence of niche manipulation, a group of mice was treated with bisphosphonates and then subjected to BM transplantation from untreated donors. Treated recipient mice experienced a delayed hematopoietic recovery compared with untreated controls. Our findings demonstrate that osteoclast function is fundamental in the HSC niche.

The HSC niche concept has turned 31. Has our knowledge matured?

The hematopoietic stem cell (HSC) niche is currently defined as the specific microenvironment in the bone marrow (BM) which anatomically harbors HSCs and governs their fate. It plays a pivotal role in regulating the survival and self-renewal ability of HSCs, protecting them from exhaustion while preventing their excessive proliferation. Many different stromal cell types have been proposed as putative constituents of the niche, but their integrated function is still unrevealed. Mechanisms by which stem/progenitor cell behavior is regulated in the niche include cell-to-cell interaction and the production of growth factors, cytokines, and extracellular matrix proteins. The HSC niche is a dynamic entity reflecting and responding to the needs of the organism. An understanding of how the niche participates in the maintenance of tissue homeostasis and repair offers new opportunities for the development of novel therapeutic tools.

Bone marrow mesenchymal stromal cells non-selectively protect chronic myeloid leukemia cells from imatinib-induced apoptosis via the CXCR4/CXCL12 axis.

BACKGROUND: Residual chronic myeloid leukemia disease following imatinib treatment has been attributed to the presence of quiescent leukemic stem cells intrinsically resistant to imatinib. Mesenchymal stromal cells in the bone marrow may favor the persistence and progression of leukemia by preserving the proliferation and self-renewal capacities of the malignant progenitor cells. DESIGN AND METHODS: BV173 or primary chronic myeloid leukemia cells were co-cultured with human mesenchymal stromal cells and imatinib-induced cell death was then measured. The roles of pro-and anti-apoptotic proteins and chemokine CXCL12 in this context were evaluated. We also studied the ability of BV173 cells to repopulate NOD/SCID mice following in vitro exposure to imatinib and mesenchymal stromal cells. RESULTS: Whilst imatinib induced dose-dependent apoptosis of BV173 cells and primary chronic myeloid leukemia cells, co-culture with mesenchymal stromal cells protected both types of chronic myeloid leukemia cells. Molecular analysis indicated that mesenchymal stromal cells reduced caspase-3 activation and modulated the expression of the anti-apoptotic protein Bcl-XL. Furthermore, chronic myeloid leukemia cells exposed to imatinib in the presence of mesenchymal stromal cells retained the ability to engraft into NOD/SCID mice. We observed that chronic myeloid leukemia cells and mesenchymal stromal cells express functional levels of CXCR4 and CXCL12, respectively. Finally, the CXCR4 antagonist, AMD3100 restored apoptosis by imatinib and the susceptibility of the SCID leukemia repopulating cells to the tyrosine kinase inhibitor. CONCLUSIONS: Human mesenchymal stromal cells mediate protection of chronic myeloid leukemia cells from imatinib-induced apoptosis. Disruption of the CXCL12/CXCR4 axis restores, at least in part, the leukemic cells' sensitivity to imatinib. The combination of anti-CXCR4 antagonists with tyrosine kinase inhibitors may represent a powerful approach to the treatment of chronic myeloid leukemia.