Adult stem cells in endometrial regeneration: Molecular insights and clinical applications.

Endometrial damage is an important cause of female reproductive problems, manifested as menstrual abnormalities, infertility, recurrent pregnancy loss, and other complications. These conditions are collectively termed "Asherman syndrome" (AS) and are typically associated with recurrent induced pregnancy terminations, repeated diagnostic curettage and intrauterine infections. Cancer treatment also has unexpected detrimental side effects on endometrial function in survivors independently of ovarian effects. Endometrial stem cells act in the regeneration of the endometrium and in repair through direct differentiation or paracrine effects. Nonendometrial adult stem cells, such as bone marrow-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells, with autologous and allogenic applications, can also repair injured endometrial tissue in animal models of AS and in human studies. However, there remains a lack of research on the repair of the damaged endometrium after the reversal of tumors, especially endometrial cancers. Here, we review the biological mechanisms of endometrial regeneration, and research progress and challenges for adult stem cell therapy for damaged endometrium, and discuss the potential applications of their use for endometrial repair after cancer remission, especially in endometrial cancers. Successful application of such cells will improve reproductive parameters in patients with AS or cancer. Significance: The endometrium is the fertile ground for embryos, but damage to the endometrium will greatly impair female fertility. Adult stem cells combined with tissue engineering scaffold materials or not have made great progress in repairing the injured endometrium due to benign lesions. However, due to the lack of research on the repair of the damaged endometrium caused by malignant tumors or tumor therapies, the safety and effectiveness of such stem cell-based therapies need to be further explored. This review focuses on the molecular insights and clinical application potential of adult stem cells in endometrial regeneration and discusses the possible challenges or difficulties that need to be overcome in stem cell-based therapies for tumor survivors. The development of adult stem cell-related new programs will help repair damaged endometrium safely and effectively and meet fertility needs in tumor survivors.

Abcg2-expressing side population cells contribute to cardiomyocyte renewal through fusion.

The adult mammalian heart has a limited regenerative capacity. Therefore, identification of endogenous cells and mechanisms that contribute to cardiac regeneration is essential for the development of targeted therapies. The side population (SP) phenotype has been used to enrich for stem cells throughout the body; however, SP cells isolated from the heart have been studied exclusively in cell culture or after transplantation, limiting our understanding of their function in vivo. We generated a new Abcg2-driven lineage-tracing mouse model with efficient labeling of SP cells. Labeled SP cells give rise to terminally differentiated cells in bone marrow and intestines. In the heart, labeled SP cells give rise to lineage-traced cardiomyocytes under homeostatic conditions with an increase in this contribution following cardiac injury. Instead of differentiating into cardiomyocytes like proposed cardiac progenitor cells, cardiac SP cells fuse with preexisting cardiomyocytes to stimulate cardiomyocyte cell cycle reentry. Our study is the first to show that fusion between cardiomyocytes and non-cardiomyocytes, identified by the SP phenotype, contribute to endogenous cardiac regeneration by triggering cardiomyocyte cell cycle reentry in the adult mammalian heart.

Cellular stress induces cancer stem-like cells through expression of DNAJB8 by activation of heat shock factor 1.

In a previous study, we found that DNAJB8, a heat shock protein (HSP) 40 family member is expressed in kidney cancer stem-like cells (CSC)/cancer-initiating cells (CIC) and that it has a role in the maintenance of kidney CSC/CIC. Heat shock factor (HSF) 1 is a key transcription factor for responses to stress including heat shock, and it induces HSP family expression through activation by phosphorylation. In the present study, we therefore examined whether heat shock (HS) induces CSC/CIC. We treated the human kidney cancer cell line ACHN with HS, and found that HS increased side population (SP) cells. Western blot analysis and qRT-PCR showed that HS increased the expression of DNAJB8 and SOX2. Gene knockdown experiments using siRNAs showed that the increase in SOX2 expression and SP cell ratio depends on DNAJB8 and that the increase in DNAJB8 and SOX2 depend on HSF1. Furthermore, treatment with a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, decreased the expression of DNAJB8 and SOX2 and the ratio of SP cells. Taken together, the results indicate that heat shock induces DNAJB8 by activation of HSF1 and induces cancer stem-like cells.

A Synthetic Polymer Scaffold Reveals the Self-Maintenance Strategies of Rat Glioma Stem Cells by Organization of the Advantageous Niche.

Cancer stem cells (CSCs) are believed to be maintained within a microenvironmental niche. Here we used polymer microarrays for the rapid and efficient identification of glioma CSC (GSC) niche mimicries and identified a urethane-based synthetic polymer, upon which two groups of niche components, namely extracellular matrices (ECMs) and iron are revealed. In cultures, side population (SP) cells, defined as GSCs in the rat C6 glioma cell line, are more efficiently sustained in the presence of their differentiated progenies expressing higher levels of ECMs and transferrin, while in xenografts, ECMs are supplied by the vascular endothelial cells (VECs), including SP cell-derived ones with distinctively greater ability to retain xenobiotics than host VECs. Iron is stored in tumor infiltrating host macrophages (Mφs), whose protumoral activity is potently enhanced by SP cell-secreted soluble factor(s). Finally, coexpression of ECM-, iron-, and Mφ-related genes is found to be predictive of glioma patients' outcome. Our polymer-based approach reveals the intrinsic capacities of GSCs, to adapt the environment to organize a self-advantageous microenvironment niche, for their maintenance and expansion, which redefines the current concept of anti-CSC niche therapy and has the potential to accelerate cancer therapy development. Stem Cells 2016;34:1151-1162.

Kinetic Modeling of ABCG2 Transporter Heterogeneity: A Quantitative, Single-Cell Analysis of the Side Population Assay.

The side population (SP) assay, a technique used in cancer and stem cell research, assesses the activity of ABC transporters on Hoechst staining in the presence and absence of transporter inhibition, identifying SP and non-SP cell (NSP) subpopulations by differential staining intensity. The interpretation of the assay is complicated because the transporter-mediated mechanisms fail to account for cell-to-cell variability within a population or adequately control the direct role of transporter activity on staining intensity. We hypothesized that differences in dye kinetics at the single-cell level, such as ABCG2 transporter-mediated efflux and DNA binding, are responsible for the differential cell staining that demarcates SP/NSP identity. We report changes in A549 phenotype during time in culture and with TGFß treatment that correlate with SP size. Clonal expansion of individually sorted cells re-established both SP and NSPs, indicating that SP membership is dynamic. To assess the validity of a purely kinetics-based interpretation of SP/NSP identity, we developed a computational approach that simulated cell staining within a heterogeneous cell population; this exercise allowed for the direct inference of the role of transporter activity and inhibition on cell staining. Our simulated SP assay yielded appropriate SP responses for kinetic scenarios in which high transporter activity existed in a portion of the cells and little differential staining occurred in the majority of the population. With our approach for single-cell analysis, we observed SP and NSP cells at both ends of a transporter activity continuum, demonstrating that features of transporter activity as well as DNA content are determinants of SP/NSP identity.

[Differentiation of Cultivated CD45-/CD31+ Mouse Lung Side Population Cells into Vascular Smooth Muscle Cells in vitro].

OBJECTIVES: To investigate the characteristics of differentiation of lung side population cells (LSP cells)in vitro. METHODS: CD45-/CD31+ LSP cells sorted by flow cytometry were taken from mouse lung tissues and cultured for 14 d. The cultured LSP cells were observed with colony formation assay and flow cytometryin vitro. The mRNA expressions of ATP-binding cassette transporter G2 (ABCG2), smooth muscle actin (SMA) and α-smooth muscle tropomyosin (α-SMT) in both freshly isolated LSP cells and cultured LSP cells were examined. The expressions of ABCG2 and stem cell antigen 1 (Sca1) in LSP cells were detected using immunofluorescence. RT-PCR tests were performed to detect the expressions of ABCG2, SMA and α-SMT in LSP cells. RESULTS: The isolated CD45-/CD31+ lung side population cells expressed ABCG2, SMA and Sca1, but not α-SMT. A large number of LSP in aggregated state were observed after 14 d of culture. Before induction of differentiation, the CD45-/CD31+ LSP cells expressed ABCG2 and SMA, but not α-SMT. After induction of differentiation, the CD45-/CD31+ lung side population cells expressed α-SMT and SMA, but not ABCG2. CONCLUSIONS: CD45-/CD31+ LSP cells might be progenitor cells of vascular smooth muscle cells, possessing the characteristics of stem cell differentiations.

Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR.

Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells.

Toxicity of Hoechst 33342: implication in side population analysis.

In the current study, we discovered that Hoechst 33342 (H342) could produce significant toxicity to the human neuroblastoma SK-N-SH cells at concentrations typically used to identify side population (SP) cells. At 3.75 and 5 µg/ml, H342 induced cell death and apoptosis. Flow cytometry analysis revealed cell cycle arrest at S-phase. Our findings caution against using H342 for SP analysis without considering differential susceptibility of different cells to H342. Specifically, concentration of H342 needs to be adjusted carefully to minimize toxicity for each cell line.

Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK-LRP6 signalling.

Cardiac side population cells (CSPs) are promising cell resource for the regeneration in diseased heart as intrinsic cardiac stem cells. However, the relative low ratio of CSPs in the heart limited the ability of CSPs to repair heart and improve cardiac function effectively under pathophysiological condition. Which factors limiting the proliferation of CSPs in diseased heart are unclear. Here, we show that urotensin II (UII) regulates the proliferation of CSPs by c-Jun N-terminal kinase (JNK) and low density lipoprotein receptor-related protein 6 (LRP6) signalling during pressure overload. Pressure overload greatly upregulated UII level in plasma, UII receptor (UT) antagonist, urantide, promoted CSPs proliferation and improved cardiac dysfunction during chronic pressure overload. In cultured CSPs subjected to mechanical stretch (MS), UII significantly inhibited the proliferation by UT. Nanofluidic proteomic immunoassay showed that it is the JNK activation, but not the extracellular signal-regulated kinase signalling, that involved in the UII-inhibited- proliferation of CSPs during pressure overload. Further analysis in vitro indicated UII-induced-phospho-JNK regulates phosphorylation of LRP6 in cultured CSPs after MS, which is important in the inhibitory effect of UII on the CSPs during pressure overload. In conclusion, UII inhibited the proliferation of CSPs by JNK/LRP6 signalling during pressure overload. Pharmacological inhibition of UII promotes CSPs proliferation in mice, offering a possible therapeutic approach for cardiac failure induced by pressure overload.

Cardiac side population cells: moving toward the center stage in cardiac regeneration.

Over the past decade, extensive work in animal models and humans has identified the presence of adult cardiac progenitor cells, capable of cardiomyogenic differentiation and likely contributors to cardiomyocyte turnover during normal development and disease. Among cardiac progenitor cells, there is a distinct subpopulation, termed "side population" (SP) progenitor cells, identified by their unique ability to efflux DNA binding dyes through an ATP-binding cassette transporter. This review highlights the literature on the isolation, characterization, and functional relevance of cardiac SP cells. We review the initial discovery of cardiac SP cells in adult myocardium as well as their capacity for functional cardiomyogenic differentiation and role in cardiac regeneration after myocardial injury. Finally, we discuss recent advances in understanding the molecular regulators of cardiac SP cell proliferation and differentiation, as well as likely future areas of investigation required to realize the goal of effective cardiac regeneration.

Effective Detection of Hoechst Side Population Cells by Flow Cytometry.

The side population (SP) cells are identified through Hoechst 33342 staining and analyzed using flow cytometry (FCM). The Hoechst SP method is utilized for the isolation of stem cells based on the dye efflux properties of ATP-binding cassette (ABC) transporters. The method was initially employed for the identification and isolation of hematopoietic stem cells (HSCs), but it has now evolved to primarily focus on the identification and isolation of cancer stem cells (CSCs). The traditional detection method of FCM uses a 355 nm laser to excite the dye to detect SP cells. Through this study, we have successfully identified alternative approaches for dye excitation that can effectively replace the detection of SP cells using a 355 nm laser. This is achieved through the utilization of high-power 375 nm or 405 nm lasers. This allows us to exercise enhanced selectivity in the detection of SP cells rather than being solely limited to the 355 nm laser flow cytometry.

Ex vivo-expanded DCs induce donor-specific central and peripheral tolerance and prolong the acceptance of donor skin grafts.

Dendritic cells (DCs) are known to regulate immune responses by inducing both central and peripheral tolerance. DCs play a vital role in negative selection of developing thymocytes by deleting T cells with high-affinity for self-peptide-major histocompatibility complexes. In the periphery, DCs mediate peripheral tolerance by promoting regulatory T-cell development, induction of T-cell unresponsiveness, and deletion of activated T cells. We studied whether allogeneic DCs, obtained from bone marrow cultured with either Flt3L (FLDCs) or granulocyte-macrophage colony-stimulating factor (GMDCs), could induce allospecific central and peripheral tolerance after IV injection; B cells were used as a control. The results showed that only FLDCs reached the thymus after injection and that these cells induced both central and peripheral tolerance to donor major histocompatibility complexes. For central tolerance, injection of FLDCs induced antigen-specific clonal deletion of both CD8 and CD4 single-positive thymocytes. For peripheral tolerance, injection of FLDCs induced donor-specific T-cell unresponsiveness and prolonged survival of donor-derived skin grafts. Tolerance induction by adoptive transfer of FLDCs could be a useful approach for promoting graft acceptance after organ transplantation.

Isolation of side population cells in B-cell non-Hodgkin's lymphomas.

BACKGROUND: Side population (SP) cells are characterized by the ability to exclude Hoechst 33342 dye due to high expression of the ATP-binding cassette transporter. This ability is associated with drug-resistant characteristics of cancer stem cells. METHODS: We analyzed SP cells from human B-cell non-Hodgkin's lymphoma cell lines and primary cells derived from patients and compared them with non-SP (NSP) cells. RESULTS: SP cells comprised a minor fraction of all cells ranging from 1.5 ± 1.8 to 8.3 ± 5.7% in cell lines and had higher ABCG2 expression than NSP cells. SP cells had better cell viability, colony-forming ability and drug resistance than NSP cells. The SP cells also showed stem cell-like characteristics, including elevated telomerase activity and higher expression of OCT4 and NANOG. A cDNA microarray demonstrated that SP cells had decreased expression of genes associated with apoptosis and cell death compared to NSP cells. CONCLUSIONS: The presence of SP cells might imply the possibility of lymphoma stem cells and be associated with a malignant potential of B-cell lymphoma.

Not all side population cells contain cancer stem-like cells in human gastric cancer cell lines.

INTRODUCTION: Side population (SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer. Many kinds of cell lines and tissue have demonstrated presence of SP cells including different gastric cancer cell lines. However, is that true all SP cells contain cancer stem-like cells in gastric cancer cell lines? MATERIALS AND METHODS: MKN-45 and BGC-823 cells labeled with Hoechst 33342 were chosen to obtain SP cells, then characterized the cancer stem-like properties of SP cells both in vitro and in vivo. Five stemness-related genes expression profiles, including OCT-4, SOX-2, NANOG, CD44 and ATP-binding cassette transporters gene ABCG-2, were tested in SP and MP cells using quantitative real-time RT-PCR. Western blot was chosen to show the difference of protein expression between SP and MP cells. When inoculated into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, SP cells from MKN-45 showed higher tumorigenesis tendency than MP cells, but SP cells from BGC-823 showed same tumorgenesis tendency as MP cells. CONCLUSION: SP cells from MKN-45 possess cancer stem cell properties and proved that they were gastric cancer stem-like cells. SP cells from BGC-823 didn't possess cancer stem cell properties and proved that not all SP cells contain cancer stem-like cells in gastric cancer cell lines.

[Epithelial-mesenchymal transition of prostate cancer: cancer stem cells or bulk cancer cells].

OBJECTIVE: To test the hypothesis that epithelial-mesenchymal transition (EMT) of prostate cancer is most likely to occur in cancer stem cells (CSC). METHODS: The isolation of CSC from LNCaP cell line was performed by flow cytometry based on side-population (SP) phenotype. After SP sorting, LNCaP/SP and LNCaP/NSP were used for further transfection of hypoxia-inducible factor-1α (HIF-1α). Subsequently, EMT-associated proteins were detected by Western blotting. And the assays of Transwell and methyl thiazolyl tetrazolium (MTT) were used to compare invasive and proliferative potency between LNCaP/SP and LNCaP/NSP after HIF-1α induction. Eventually, xenograft experiments were performed with LNCaP/HIF-1α/SP and LNCaP/HIF-1α/NSP cells for further analysis of in vivo tumorigenesis and distant metastasis. RESULTS: Through HIF-1α-induced EMT, LNCaP/HIF-1α/SP exhibited such remarkable EMT characteristics as a positive expression of epithelial markers (E-cadherin and CK18) and a negative expression of mesenchymal markers (vimentin, N-cadherin, fibronectin, cathepsin D, MMP-2 and uPAR). And LNCaP/HIF-1α/NSP underwent partial EMT with an abnormal expression of some mesenchymal proteins (vimentin and cathepsin D) and loss of epithelial protein (CK18) despite reservation of another important epithelial marker (E-cadherin). Further Transwell and MTT assays indicated that LNCaP/HIF-1α/SP exhibited stronger in vitro invasive and proliferative potency than LNCaP/HIF-1α/NSP cells. In animal models, the volume of subcutaneous tumor by LNCaP/HIF-1α/SP cells was much greater than that by LNCaP/HIF-1α/NSP counterparts ((1008 ± 230) vs (288 ± 145) mm(3), P < 0.01). Moreover, LNCaP/HIF-1α/SP cells also had a significantly higher rate of subcutaneous tumor incidence (80% vs 53%, P < 0.05) and bone metastasis (40% vs 0, P < 0.01) as compared with LNCaP/HIF-1α/NSP counterparts. CONCLUSION: As the main target cells of prostatic EMT, CSCs may develop a more malignant phenotype after EMT.

Left atrium of the human adult heart contains a population of side population cells.

Cardiac "side population" (SP) cells have previously been found to differentiate into both endothelial cells and cardiomyocytes in mice and rats, but there are no data on SP cells in the human adult heart. Therefore, human cardiac atrial biopsies were dissociated, stained for SP cells and analyzed with FACS. Identified cell populations were analyzed for gene expression by quantitative real-time PCR and subjected to in vitro differentiation. Only biopsies from the left atrium contained a clearly distinguishable population of SP cells (0.22 ± 0.08%). The SP population was reduced by co-incubation with MDR1 inhibitor Verapamil, while the ABCG2 inhibitor FTC failed to decrease the number of SP cells. When the gene expression was analyzed, SP cells were found to express significantly more MDR1 than non-SP cells. For ABCG2, there was no detectable difference. SP cells also expressed more of the stem cell-associated markers C-KIT and OCT-4 than non-SP cells. On the other hand, no significant difference in the expression of endothelial and cardiac genes could be detected. SP cells were further subdivided based on CD45 expression. The CD45-SP population showed evidence of endothelial commitment at gene expression level. In conclusion, the results show that a SP population of cells is present also in the human adult heart.

Quantitative gene expression profiling of CD45+ and CD45- skeletal muscle-derived side population cells.

The skeletal muscle-derived side population (mSP) which highly excludes Hoechst 33342 is composed of CD45(+) and CD45(-) subpopulations; yet, rareness of mSP cells in general has complicated extensive quantitative analysis of gene expression profiles in primarily isolated mSP cells. Here, we describe the isolation of adult mouse normal skeletal muscle residing SPCD45(+) and SPCD45(-) cells from a parent mononuclear muscle-derived cell (MDC) population. Relative quantitative real time PCR (RT-PCR) of 64 genes revealed that mSPCD45(-) compared with mSPCD45(+) was enriched for cells expressing transcripts associated with endothelial cells, Notch signaling and myogenic precursors. By comparing the mRNA signatures of mSPs with those of adipose tissue-derived SP populations, a common endothelial component seemed to reside in both muscle and fat-derived SPCD45(-) entities. However, each SP subset was clearly specified by the tissue from which the cells originated suggesting that muscle SPs compared with adipose tissue SPs are predisposed towards differentiation into the myogenic lineage. Thus, our data support the previously suggested hypothesis that satellite cell precursors (or alternatively a satellite cell subpopulation) remain in the mSPCD45(-) fraction, and we show that these cells express high levels of many of the known myogenic precursor/stem cell related markers, including Pax7 and Myf5.

[Sorting and identification of cancer stem cells in human prostate cancer cell lines].

OBJECTIVE: To sort and identify side population (SP) cancer stem cells (CSC) in human prostate cancer (PCa) cell lines. METHODS: Stem-like cells were isolated from five PCa cell lines Du145, IA8, LNCaP, TSU-Pr and PC-3 using FACS based on CD133+ CD44+ immunophenotype and SP in Hoechst staining. The in vitro growth pattern and tumorigenicity of SP stem cells were verified by soft agar colony-formation trial. LNCaP/SP cells were selected for further identification of stem cell properties using immunostaining, proliferation and invasion assay. Eventually, tumorigenicity and metastasis ability of LNCaP/SP were confirmed by xenograft experiments. RESULTS: The percentages of CSCs of the CD133 CD44 + immunophenotype were extremely low in the five PCa cell lines. On the contrary, the percentages of the isolated SP cells were significantly higher in Du145 ([0.15 +/- 0.02]%), IA8 ([0.60 +/- 0.07 ]%), LNCaP ([0.8 +/- 0.1]%) and TSU-PrL ([2.0 +/- 0.4]%), but none was detected in PC-3. Besides, IA8/SP, LNCaP/SP and TSU-PrL/SP cells showed a significantly greater colony-forming efficiency than non-side population (NSP) cells (P < 0.05). Compared with LNCaP/NSP cells, LNCaP/SP cells exhibited high expressions of integrin alpha2, Nanog, CD44, OCT4 and ABCG2, remarkably enhanced invasive and proliferative potentials in vitro, and markedly increased tumorigenicity and metastasis (P < 0.01). CONCLUSION: SP sorting is more suitable than CD133+ CD44+ selection for enriching CSCs from PCa cell lines, and LNCaP/ SP represents a typical CSC population.

Fms-like tyrosine kinase 3 is a regulator of the cardiac side population in mice.

Fms-like tyrosine kinase 3 (Flt3) is a regulator of hematopoietic progenitor cells and a target of tyrosine kinase inhibitors. Flt3-targeting tyrosine kinase inhibitors can have cardiovascular side effects. Flt3 and its ligand (Flt3L) are expressed in the heart, but little is known about their physiological functions. Here, we show that cardiac side population progenitor cells (SP-CPCs) from mice produce and are responsive to Flt3L. Compared with wild-type, flt3L-/- mice have less SP-CPCs with less contribution of CD45-CD34+ cells and lower expression of genes related to epithelial-to-mesenchymal transition, cardiovascular development and stem cell differentiation. Upon culturing, flt3L-/- SP-CPCs show increased proliferation and less vasculogenic commitment, whereas Akt phosphorylation is lower. Notably, proliferation and differentiation can be partially restored towards wild-type levels in the presence of alternative receptor tyrosine kinase-activating growth factors signaling through Akt. The lower vasculogenic potential of flt3L-/- SP-CPCs reflects in decreased microvascularisation and lower systolic function of flt3L-/- hearts. Thus, Flt3 regulates phenotype and function of murine SP-CPCs and contributes to cellular and molecular properties that are relevant for their cardiovasculogenic potential.

Endocannabinoids are expressed in bone marrow stromal niches and play a role in interactions of hematopoietic stem and progenitor cells with the bone marrow microenvironment.

Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB(1) and CB(2). The endocannabinoid system is capable of activation of distinct signaling pathways on demand in response to pathogenic events or stimuli, hereby enhancing cell survival and promoting tissue repair. However, the role of endocannabinoids in hematopoietic stem and progenitor cells (HSPCs) and their interaction with hematopoietic stem cells (HSC) niches is not known. HSPCs are maintained in the quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signaling. We report that HSPCs express the CB(1) receptors and that BM stromal cells secrete endocannabinoids, anandamide (AEA) (35 pg/10(7) cells), and 2-AG (75.2 ng/10(7) cells). In response to the endotoxin lipopolysaccharide (LPS), elevated levels of AEA (75.6 pg/10(7) cells) and 2-AG (98.8 ng/10(7) cells) were secreted from BM stromal cells, resulting in migration and trafficking of HSPCs from the BM niches to the peripheral blood. Furthermore, administration of exogenous cannabinoid CB(1) agonists in vivo induced chemotaxis, migration, and mobilization of human and murine HSPCs. Cannabinoid receptor knock-out mice Cnr1(-/-) showed a decrease in side population (SP) cells, whereas fatty acid amide hydrolase (FAAH)(-/-) mice, which have elevated levels of AEA, yielded increased colony formation as compared with WT mice. In addition, G-CSF-induced mobilization in vivo was modulated by endocannabinoids and was inhibited by specific cannabinoid antagonists as well as impaired in cannabinoid receptor knock-out mice Cnr1(-/-), as compared with WT mice. Thus, we propose a novel function of the endocannabinoid system, as a regulator of HSPC interactions with their BM niches, where endocannabinoids are expressed in HSC niches and under stress conditions, endocannabinoid expression levels are enhanced to induce HSPC migration for proper hematopoiesis.