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Hematopoietic stem cells (HSCs) develop from a specialized subpopulation of endothelial cells known as hemogenic endothelium (HE). Although the HE origin of HSCs is now well established in different species,the signaling pathways that control this transition remain poorly understood. Here,we show that activation of retinoic acid (RA) signaling in aorta-gonad-mesonephros-derived HE ex vivo dramatically enhanced its HSC potential,whereas conditional inactivation of the RA metabolizing enzyme retinal dehydrogenase 2 in VE-cadherin expressing endothelial cells in vivo abrogated HSC development. Wnt signaling completely blocked the HSC inductive effects of RA modulators,whereas inhibition of the pathway promoted the development of HSCs in the absence of RA signaling. Collectively,these findings position RA and Wnt signaling as key regulators of HSC development and in doing so provide molecular insights that will aid in developing strategies for their generation from pluripotent stem cells. View Publication

The neural differentiation of human embryonic stem cells (ESCs) is a potential tool for elucidating the key mechanisms involved in human neurogenesis. Nestin and ??-III-tubulin,which are cytoskeleton proteins,are marker proteins of neural stem cells (NSCs) and neurons,respectively. However,the expression patterns of nestin and ??-III-tubulin in neural derivatives from human ESCs remain unclear. In this study,we found that neural progenitor cells (NPCs) derived from H9 cells express high levels of nestin and musashi-1. In contrast,??-III-tubulin was weakly expressed in a few NPCs. Moreover,in these cells,nestin formed filament networks,whereas ??-III-tubulin was distributed randomly as small particles. As the differentiation proceeded,the nestin filament networks and the ??-III-tubulin particles were found in both the cell soma and the cellular processes. Moreover,the colocalization of nestin and ??-III-tubulin was found mainly in the cell processes and neurite-like structures and not in the cell soma. These results may aid our understanding of the expression patterns of nestin and ??-III-tubulin during the neural differentiation of H9 cells. ?? 2013 Elsevier Inc. View Publication

Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes,for drug discovery,and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimensional,defined and highly efficient protocol that avoids the use of feeder cells,serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells. View Publication

An important risk in the clinical application of human pluripotent stem cells (hPSCs),including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs),is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs,designated anti-stage-specific embryonic antigen (SSEA)-5,which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells,we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9,CD30,CD50,CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures. View Publication

Embryoid bodies (EBs) can be generated by culturing human pluripotent stem cells in ultra-low attachment culture vessels,under conditions that are adverse to pluripotency and proliferation. EBs generated in suspension cultures are capable of differentiating into cells of the ectoderm,mesoderm,and endoderm. In this chapter,we describe techniques for generation of EBs from human pluripotent stem cells. Once formed,the EBs can then be dissociated using specific enzymes to acquire a single cell population that has the potential to differentiate into cells of all three germ layers. This population can then be cultured in specialized conditions to obtain progenitor cells of specific lineages. Pure populations of progenitor cells generated on a large scale basis can be used for research,drug discovery/development,and cellular transplantation therapy. View Publication

Granulocyte colony-stimulating factor (G-CSF),the prototypical mobilizing cytokine,induces hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow in a cell-nonautonomous fashion. This process is mediated,in part,through suppression of osteoblasts and disruption of CXCR4/CXCL12 signaling. The cellular targets of G-CSF that initiate the mobilization cascade have not been identified. We use mixed G-CSF receptor (G-CSFR)-deficient bone marrow chimeras to show that G-CSF-induced mobilization of HSPCs correlates poorly with the number of wild-type neutrophils. We generated transgenic mice in which expression of the G-CSFR is restricted to cells of the monocytic lineage. G-CSF-induced HSPC mobilization,osteoblast suppression,and inhibition of CXCL12 expression in the bone marrow of these transgenic mice are intact,demonstrating that G-CSFR signals in monocytic cells are sufficient to induce HSPC mobilization. Moreover,G-CSF treatment of wild-type mice is associated with marked loss of monocytic cells in the bone marrow. Finally,we show that bone marrow macrophages produce factors that support the growth and/or survival of osteoblasts in vitro. Together,these data suggest a model in which G-CSFR signals in bone marrow monocytic cells inhibit the production of trophic factors required for osteoblast lineage cell maintenance,ultimately leading to HSPC mobilization. View Publication

Circulating cancer cells (CCCs) are closely related to the process of distant metastasis. In early step of the metastasis cascade,CCCs must evade the detachment-induced cell death (anoikis) for their survival. Here,we examined whether Na + /K + -ATPase α3-isoform (α3NaK) in CCCs contributes to avoidance of anoikis. In CCCs isolated from gastric cancer patients,α3NaK was predominantly localized in the plasma membrane (PM),but it moved to the cytoplasm when the CCCs were attached to culture dishes. The CCCs showed significant expression of integrin α5 but not fibronectin,one of components of the extracellular matrix (ECM). In human gastric cancer MKN45 cells,digoxin (20 and 50 nM),a cardiac glycoside,significantly inhibited the enzyme activity and translocation (from cytoplasm to PM) of α3NaK,while they had no significant effect on ubiquitous Na + /K + -ATPase α1-isoform (α1NaK) in the PM. The translocation of α3NaK required the loss of ECM components from the cells. Additionally,digoxin significantly enhanced caspase 3/7 activity,as well as the expression of cleaved caspase 3,while reducing the viability of detached (floating) cells. In the MKN45 xenograft mouse model,intraperitoneal administration of digoxin (2 mg/kg/day) significantly decreased the number of CCCs and suppressed their liver metastasis. Our results suggest that α3NaK plays an essential role in the survival of CCCs in gastric cancer,and that digoxin enhances anoikis in detached (metastatic) gastric cancer cells by inhibiting the α3NaK translocation from cytoplasm to PM,thereby reducing CCCs. Targeting α3NaK may be a promising therapeutic strategy against CCC survival. Subject terms: Metastasis,Gastric cancer,Apoptosis View Publication

High telomerase activity is a characteristic of human embryonic stem cells (hESCs),however,the regulation and maintenance of correct telomere length in hESCs is unclear. In this study we investigated telomere elongation in hESCs in vitro and found that telomeres lengthened from their derivation in blastocysts through early expansion,but stabilized at later passages. We report that the core unit of telomerase,hTERT,was highly expressed in hESCs in blastocysts and throughout long-term culture; furthermore,this was regulated in a Wnt-β-catenin-signaling-dependent manner. Our observations that the alternative lengthening of telomeres (ALT) pathway was suppressed in hESCs and that hTERT knockdown partially inhibited telomere elongation,demonstrated that high telomerase activity was required for telomere elongation. We observed that chromatin modification through trimethylation of H3K9 and H4K20 at telomeric regions decreased during early culture. This was concurrent with telomere elongation,suggesting that epigenetic regulation of telomeric chromatin may influence telomerase function. By measuring telomere length in 96 hESC lines,we were able to establish that telomere length remained relatively stable at 12.02±1.01 kb during later passages (15-95). In contrast,telomere length varied in hESCs with genomic instability and hESC-derived teratomas. In summary,we propose that correct,stable telomere length may serve as a potential biomarker for genetically stable hESCs. View Publication

Recent evidence points to extra-telomeric,noncanonical roles for telomerase in regulating stem cell function. In this study,human embryonic stem cells (hESCs) were cultured in 20% or 2% O2 microenvironments for up to 5 days and evaluated for telomerase reverse transcriptase (TERT) expression and telomerase activity. Results showed increased cell survival and maintenance of the undifferentiated state with elevated levels of nuclear TERT in 2% O2-cultured hESCs despite no significant difference in telomerase activity compared with their high-O2-cultured counterparts. Pharmacological inhibition of telomerase activity using a synthetic tea catechin resulted in spontaneous hESC differentiation,while telomerase inhibition with a phosphorothioate oligonucleotide telomere mimic did not. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed variations in transcript levels of full-length and alternate splice variants of TERT in hESCs cultured under varying O2 atmospheres. Steric-blocking of Δα and Δβ hTERT splicing using morpholino oligonucleotides altered the hTERT splicing pattern and rapidly induced spontaneous hESC differentiation that appeared biased toward endomesodermal and neuroectodermal cell fates,respectively. Together,these results suggest that post-transcriptional regulation of TERT under varying O2 microenvironments may help regulate hESC survival,self-renewal,and differentiation capabilities through expression of extra-telomeric telomerase isoforms. View Publication

The role of autophagy,a lysosomal degradation pathway which prevents cellular damage,in the maintenance of adult mouse hematopoietic stem cells (HSCs) remains unknown. Although normal HSCs sustain life-long hematopoiesis,malignant transformation of HSCs leads to leukemia. Therefore,mechanisms protecting HSCs from cellular damage are essential to prevent hematopoietic malignancies. In this study,we crippled autophagy in HSCs by conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system. This resulted in the loss of normal HSC functions,a severe myeloproliferation,and death of the mice within weeks. The hematopoietic stem and progenitor cell compartment displayed an accumulation of mitochondria and reactive oxygen species,as well as increased proliferation and DNA damage. HSCs within the Lin(-)Sca-1(+)c-Kit(+) (LSK) compartment were significantly reduced. Although the overall LSK compartment was expanded,Atg7-deficient LSK cells failed to reconstitute the hematopoietic system of lethally irradiated mice. Consistent with loss of HSC functions,the production of both lymphoid and myeloid progenitors was impaired in the absence of Atg7. Collectively,these data show that Atg7 is an essential regulator of adult HSC maintenance. View Publication

Hematopoietic cells (HCs) promote blood vessel formation by producing various proangiogenic cytokines and chemokines and matrix metalloproteinases. We injected mouse colon26 colon cancer cells or human PC3 prostate adenocarcinoma cells into mice and studied the localization of HCs during tumor development. HCs were distributed in the inner tumor mass in all of the tumor tissues examined; however,the localization of HCs in the tumor tissue differed depending on the tumor cell type. In the case of colon26 tumors,as the tumor grew,many mature HCs migrated into the tumor mass before fine capillary formation was observed. On the other hand,although very few HCs migrated into PC3 tumor tissue,c-Kit+ hematopoietic stem/progenitor cells accumulated around the edge of the tumor. Bone marrow suppression induced by injection of anti-c-Kit neutralizing antibody suppressed tumor angiogenesis by different mechanisms according to the tumor cell type: bone marrow suppression inhibited the initiation of sprouting angiogenesis in colon26 tumors,while it suppressed an increase in the caliber of newly developed blood vessels at the tumor edge in PC3 tumors. Our findings suggest that HCs are involved in tumor angiogenesis and regulate the angiogenic switch during tumorigenesis. View Publication