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Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties,yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME,we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway,possibly by modulating availability of hyaluronic acid (HA),the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships,and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions,respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction,whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo,targeting CD44 decreased PI3K-4EBP1-SOX2 signaling,tumor growth,and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally,a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes,respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44,TAM,and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery. View Publication

Bone marrow-derived mesenchymal stem cells (BMMSCs) are multipotent postnatal stem cells that have been used for the treatment of bone defects and graft-versus-host diseases in clinics. In this study,we found that subcutaneously transplanted human BMMSCs are capable of organizing hematopoietic progenitors of recipient origin. These hematopoietic cells expressed multiple lineages of hematopoietic cell associated markers and were able to rescue lethally irradiated mice,with successful engraftment in the recipient,suggesting a potential bone marrow (BM) resource for stem cell therapies. Furthermore,we found that platelet-derived growth factor (PDGF) promotes the formation of BMMSC-generated BM niches through upregulation of beta-catenin,implying that the PDGF pathway contributes to the formation of ectopic BM. These results indicate that the BMMSC-organized BM niche system represents a unique hematopoietic progenitor resource possessing potential clinical value. View Publication

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that increases the expression of detoxifying enzymes upon ligand stimulation. Recent studies now suggest that novel endogenous roles of the AHR exist throughout development. In an effort to create an optimized model system for the study of AHR signaling in several cellular lineages,we have employed a CRISPR/CAS9 genome editing strategy in induced pluripotent stem cells (iPSCs) to incorporate a reporter cassette at the transcription start site of one of its canonical targets,cytochrome P450 1A1 (CYP1A1). This cell line faithfully reports on CYP1A1 expression,with luciferase levels as its functional readout,when treated with an endogenous AHR ligand (FICZ) at escalating doses. iPSC-derived fibroblast-like cells respond to acute exposure to environmental and endogenous AHR ligands,and iPSC-derived hepatocytes increase CYP1A1 in a similar manner to primary hepatocytes. This cell line is an important innovation that can be used to map AHR activity in discrete cellular subsets throughout developmental ontogeny. As further endogenous ligands are proposed,this line can be used to screen for safety and efficacy and can report on the ability of small molecules to regulate critical cellular processes by modulating the activity of the AHR. View Publication

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from bone marrow into peripheral blood by the cytokine granulocyte colony-stimulating factor (G-CSF) has become the preferred source of HSPCs for stem cell transplants. However,G-CSF fails to mobilize sufficient numbers of stem cells in up to 10% of donors,precluding autologous transplantation in those donors or substantially delaying transplant recovery time. Consequently,new regimens are needed to increase the number of stem cells in peripheral blood upon mobilization. Using a forward genetic approach in mice,we mapped the gene encoding the epidermal growth factor receptor (Egfr) to a genetic region modifying G-CSF-mediated HSPC mobilization. Amounts of EGFR in HSPCs inversely correlated with the cells' ability to be mobilized by G-CSF,implying a negative role for EGFR signaling in mobilization. In combination with G-CSF treatment,genetic reduction of EGFR activity in HSPCs (in waved-2 mutant mice) or treatment with the EGFR inhibitor erlotinib increased mobilization. Increased mobilization due to suppression of EGFR activity correlated with reduced activity of cell division control protein-42 (Cdc42),and genetic Cdc42 deficiency in vivo also enhanced G-CSF-induced mobilization. Our findings reveal a previously unknown signaling pathway regulating stem cell mobilization and provide a new pharmacological approach for improving HSPC mobilization and thereby transplantation outcomes. View Publication

The t(12;21)(p13;q22) translocation is the most common chromosomal abnormality yet identified in any pediatric leukemia and gives rise to the TEL-AML1 fusion product. To investigate the effects of TEL-AML1 on hematopoiesis,fetal liver hematopoietic progenitor cells (HPCs) were transduced with retroviral vectors expressing this fusion protein. We show that TEL-AML1 dramatically alters differentiation of HPCs in vitro,preferentially promoting B-lymphocyte development,enhancing self-renewal of B-cell precursors,and leading to the establishment of long-term growth factor-dependent pre-B-cell lines. However,it had no effect on myeloid development in vitro. Further experiments were performed to determine whether TEL-AML1 also demonstrates lineage-specific activity in vivo. TEL-AML1-expressing HPCs displayed a competitive advantage in reconstituting both B-cell and myeloid lineages in vivo but had no effect on reconstitution of the T-cell lineage. Despite promoting these alterations in hematopoiesis,TEL-AML1 did not induce leukemia in transplanted mice. Our study provides a unique insight into the role of TEL-AML1 in leukemia predisposition and a potential model to study the mechanism of leukemogenesis associated with this fusion. View Publication

Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid,beta-glycerophosphate,and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after,but not before,14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair. View Publication

Anterior foregut endoderm (AFE) gives rise to therapeutically relevant cell types in tissues such as the esophagus,salivary glands,lung,thymus,parathyroid and thyroid. Despite its importance,reports describing the generation of AFE from pluripotent stem cells (PSCs) by directed differentiation have mainly focused on the Nkx2.1(+) lung and thyroid lineages. Here,we describe a novel protocol to derive a subdomain of AFE,identified by expression of Pax9,from PSCs using small molecules and defined media conditions. We generated a reporter PSC line for isolation and characterization of Pax9(+) AFE cells,which when transplanted in vivo,can form several distinct complex AFE-derived epithelia,including mucosal glands and stratified squamous epithelium. Finally,we show that the directed differentiation protocol can be used to generate AFE from human PSCs. Thus,this work both broadens the range of PSC-derived AFE tissues and creates a platform enabling the study of AFE disorders. View Publication

SummaryMutations in the DMD gene lead to Duchenne muscular dystrophy (DMD),a severe neuromuscular disorder affecting young boys as they acquire motor functions. DMD is typically diagnosed at 2–4 years of age,but the absence of dystrophin has negative impacts on skeletal muscles before overt symptoms appear in patients,which poses a serious challenge in current standards of care. Here,we investigated the consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Dystrophin deficiency was linked to marked dysregulations of cell junction proteins involved in the cell state transitions characteristic of embryonic somitogenesis. Altogether,this work demonstrates that in vitro,dystrophin deficiency has deleterious effects on cell-cell communication during myogenic development,which should be considered in future therapeutic strategies for DMD. Graphical abstract Highlights•Myogenic differentiation of DMD hiPSCs diverges at the somite stage•Cell junction formation is dysregulated in DMD somite cells•Somite cells from DMD hiPSCs have impaired epithelialization properties•Migration velocity of DMD-mutant somite progenitors is upregulated Natural sciences; Biological sciences; Biochemistry; Cell biology; Stem cells research; Developmental biology. View Publication

Purine nucleoside phosphorylase (PNP) deficiency in humans results in T lymphocytopenia. Forodesine,a potent inhibitor of PNP,was designed based on the transition-state structure stabilized by the enzyme. Previous studies established that forodesine in the presence of deoxyguanosine (dGuo) inhibits the proliferation of T lymphocytes. A phase 1 clinical trial of forodesine in T-cell malignancies demonstrated significant antileukemic activity with an increase in intracellular dGuo triphosphate (dGTP). High accumulation of dGTP in T cells may be dependent on the levels of deoxynucleoside kinases. Because B-cell chronic lymphocytic leukemia (B-CLL) cells have high activity of deoxycytidine kinase (dCK),we hypothesized that these lymphocytes would respond to forodesine. This postulate was tested in primary lymphocytes during in vitro investigations. Lymphocytes from 12 patients with CLL were incubated with forodesine and dGuo. These CLL cells showed a wide variation in the accumulation of intracellular dGTP without any effect on other deoxynucleotides. This was associated with DNA damage-induced p53 stabilization,phosphorylation of p53 at Ser15,and activation of p21. The dGTP accumulation was related to induction of apoptosis measured by caspase activation,changes in mitochondrial membrane potential,and PARP cleavage. Based on these data,a phase 2 clinical trial of forodesine has been initiated for CLL patients. View Publication

Polycomb-repressive complex 2 (PRC2)-mediated histone methylation plays an important role in aberrant cancer gene silencing and is a potential target for cancer therapy. Here we show that S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) induces efficient apoptotic cell death in cancer cells but not in normal cells. We found that DZNep effectively depleted cellular levels of PRC2 components EZH2,SUZ12,and EED and inhibited associated histone H3 Lys 27 methylation (but not H3 Lys 9 methylation). By integrating RNA interference (RNAi),genome-wide expression analysis,and chromatin immunoprecipitation (ChIP) studies,we have identified a prominent set of genes selectively repressed by PRC2 in breast cancer that can be reactivated by DZNep. We further demonstrate that the preferential reactivation of a set of these genes by DZNep,including a novel apoptosis affector,FBXO32,contributes to DZNep-induced apoptosis in breast cancer cells. Our results demonstrate the unique feature of DZNep as a novel chromatin remodeling compound and suggest that pharmacologic reversal of PRC2-mediated gene repression by DZNep may constitute a novel approach for cancer therapy. View Publication

The identification of neural stem cells (NSCs) in situ has been prevented by the inability to identify a marker consistently expressed in all adult NSCs and is thus generally accomplished using the in vitro neurosphere-forming assay. The high-mobility group transcription factor Sox2 is expressed in embryonic neural epithelial stem cells; because these cells are thought to give rise to the adult NSC population,we hypothesized that Sox2 may continue to be expressed in adult NSCs. Using Sox2:EGFP transgenic mice,we show that Sox2 is expressed in neurogenic regions along the rostral-caudal axis of the central nervous system throughout life. Furthermore,all neurospheres derived from these neurogenic regions express Sox2,suggesting that Sox2 is indeed expressed in adult NSCs. We demonstrate that NSCs are heterogeneous within the adult brain,with differing capacities for cell production. In vitro,all neurospheres express Sox2,but the expression of markers common to early progenitor cells within individual neurospheres varies; this heterogeneity of NSCs is mirrored in vivo. For example,both glial fibrillary acidic protein and NG2 are expressed within individual neurospheres,but their expression is mutually exclusive; likewise,these two markers show distinct staining patterns within the Sox2+ regions of the brain's neurogenic regions. Thus,we propose that the expression of Sox2 is a unifying characteristic of NSCs in the adult brain,but that not all NSCs maintain the ability to form all neural cell types in vivo. View Publication

Summary Human embryonic stem cells (hESCs) regularly acquire nonrandom genomic aberrations during culture,raising concerns about their safe therapeutic application. The International Stem Cell Initiative identified a copy number variant (CNV) amplification of chromosome 20q11.21 in 25% of hESC lines displaying a normal karyotype. By comparing four cell lines paired for the presence or absence of this CNV,we show that those containing this amplicon have higher population doubling rates,attributable to enhanced cell survival through resistance to apoptosis. Of the three genes encoded within the minimal amplicon and expressed in hESCs,only overexpression of BCL2L1 (BCL-XL isoform) provides control cells with growth characteristics similar to those of CNV-containing cells,whereas inhibition of BCL-XL suppresses the growth advantage of CNV cells,establishing BCL2L1 as a driver mutation. Amplification of the 20q11.21 region is also detectable in human embryonal carcinoma cell lines and some teratocarcinomas,linking this mutation with malignant transformation. View Publication