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ETV6-RUNX1 gene fusion is usually an early,prenatal event in childhood acute lymphoblastic leukemia (ALL). Transformation results in the generation of a persistent (> 14 years) preleukemic clone,which postnatally converts to ALL after the acquisition of necessary secondary genetic alterations. Many cancer cells show some expression of the erythropoietin receptor (EPOR) gene,although the functionality" of any EPOR complexes and their relevant signaling pathways in nonerythroid cells has not been validated. EPOR mRNA is selectively and ectopically expressed in ETV6-RUNX1(+) ALL but the presence of a functional EPOR on the cell surface and its role in leukemogenesis driven by ETV6-RUNX1 remains to be identified. Here we show that ETV6-RUNX1 directly binds the EPOR promoter and that expression of ETV6-RUNX1 alone in normal pre-B cells is sufficient to activate EPOR transcription. We further reveal that murine and human ETV6-RUNX1(+) cells expressing EPOR mRNA have EPO ligand binding activity that correlates with an increased cell survival through activation of the JAK2-STAT5 pathway and up-regulation of antiapoptotic BCL-XL. These data support the contention that ETV6-RUNX1 directly activates ectopic expression of a functional EPOR and provides cell survival signals that may contribute critically to persistence of covert premalignant clones in children. View Publication

HIV-1 Tat-interacting protein of 110 kDa [Tip110; p110(nrb)/SART3/p110] is an RNA binding nuclear protein implicated in regulation of HIV-1 gene and host gene transcription,pre-mRNA splicing,and cancer immunology. Recently,we demonstrated a role for Tip110 in regulation of hematopoiesis. Here,we show that TIP110 is also expressed in human embryonic stem cells (hESCs) and expression was decreased with differentiation of these ESCs. TIP110 was found,through up- and down-modulation of expression of Tip110,to be important in maintaining pluripotent factor (NANOG,OCT4,and SOX2) expression in and pluripotency of hESCs,although the mechanisms involved and whether the Tip110 effects are direct remain to be determined. View Publication

PURPOSE:
Human embryonic stem cells (hESC) hold a great potential for regenerative medicine because,in principle,they can differentiate into any cell type found in the human body. In addition,studying the effect of ionizing radiation (IR) on hESC may provide valuable information about the response of human cells to IR exposure in their most naive state,as well as the consequences of IR exposure on the development of organisms. However,the effect of IR,in particular radionuclide uptake,on the pluripotency,proliferation and survival of hESC has not been extensively studied.
METHODS:
In this study we treated cultured hESC with 5-[(125)I]iodo-2'-deoxyuridine ((125)IdU),a precursor of DNA synthesis. Then we measured the expansion of colonies and expression of pluripotency markers in hESC.
RESULTS:
We found that uptake of (125)IdU was similar in both hESC and HT1080 human fibrosarcoma cells. However,treatment with 0.1 μCi/ml (125)IdU for 24 hours resulted in complete death of the hESC population; whereas HT1080 cancer cells continued to grow. Treatment with a 10-fold lower dose (125)IdU (0.01 μCi/ml) resulted in colonies of hESC becoming less defined with numerous cells growing in monolayer outside of the colonies showing signs of differentiation. Then we analyzed the expression of pluripotency markers (octamer-binding transcription factor 4 [Oct-4] and stage-specific embryonic antigen-4 [SSEA4]) in the surviving hESC. We found that hESC in the surviving colonies expressed pluripotency markers at levels comparable with those in the non-treated controls.
CONCLUSIONS:
Our results provide important initial insights into the sensitivity of hESC to IR,and especially that produced by the decay of an internalized radionuclide. View Publication

Cellular reprogramming from adult somatic cells into an embryonic cell-like state,termed induced pluripotency,has been achieved in several cell types. However,the ability to reprogram human amniotic epithelial cells (hAECs),an abundant cell source derived from discarded placental tissue,has only recently been investigated. Here we show that not only are hAECs easily reprogrammed into induced pluripotent stem cells (AE-iPSCs),but hAECs reprogram faster and more efficiently than adult and neonatal somatic dermal fibroblasts. Furthermore,AE-iPSCs express higher levels of NANOG and OCT4 compared to human foreskin fibroblast iPSCs (HFF1-iPSCs) and express decreased levels of genes associated with differentiation,including NEUROD1 and SOX17,markers of neuronal differentiation. To elucidate the mechanism behind the higher reprogramming efficiency of hAECs,we analyzed global DNA methylation,global histone acetylation,and the mitochondrial DNA A3243G point mutation. Whereas hAECs show no differences in global histone acetylation or mitochondrial point mutation accumulation compared to adult and neonatal dermal fibroblasts,hAECs demonstrate a decreased global DNA methylation compared to dermal fibroblasts. Likewise,quantitative gene expression analyses show that hAECs endogenously express OCT4,SOX2,KLF4,and c-MYC,all four factors used in cellular reprogramming. Thus,hAECs represent an ideal cell type for testing novel approaches for generating clinically viable iPSCs and offer significant advantages over postnatal cells that more likely may be contaminated by environmental exposures and infectious agents. View Publication

Considerable progress has been made in identifying signaling pathways that direct the differentiation of human pluripotent stem cells (hPSCs) into specialized cell types,including neurons. However,differentiation of hPSCs with extrinsic factors is a slow,step-wise process,mimicking the protracted timing of human development. Using a small-molecule screen,we identified a combination of five small-molecule pathway inhibitors that yield hPSC-derived neurons at textgreater75% efficiency within 10 d of differentiation. The resulting neurons express canonical markers and functional properties of human nociceptors,including tetrodotoxin (TTX)-resistant,SCN10A-dependent sodium currents and response to nociceptive stimuli such as ATP and capsaicin. Neuronal fate acquisition occurs about threefold faster than during in vivo development,suggesting that use of small-molecule pathway inhibitors could become a general strategy for accelerating developmental timing in vitro. The quick and high-efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain. View Publication

Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value,thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity,only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2,PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb,heart and embryonic development related transcription factors and biological processes. Moreover,this study uncovered novel possible mechanisms,such as the inhibition of RANBP1,that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1,GSTA2),that protect the cell from secondary oxidative stress. As a proof of principle,we demonstrated that a combination of transcriptomics and proteomics,along with consistent differentiation of hESCs,enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide. View Publication

Human embryonic stem cells (hESCs) provide a valuable window into the dissection of the molecular circuitry underlying the early formation of the human forebrain. However,dissection of signaling events in forebrain development using current protocols is complicated by non-neural contamination and fluctuation of extrinsic influences. Here,we show that SMAD7,a cell-intrinsic inhibitor of transforming growth factor-β (TGFβ) signaling,is sufficient to directly convert pluripotent hESCs to an anterior neural fate. Time course gene expression revealed downregulation of MAPK components,and combining MEK1/2 inhibition with SMAD7-mediated TGFβ inhibition promoted telencephalic conversion. Fibroblast growth factor-MEK and TGFβ-SMAD signaling maintain hESCs by promoting pluripotency genes and repressing neural genes. Our findings suggest that in the absence of these cues,pluripotent cells simply revert to a program of neural conversion. Hence,the primed" state of hESCs requires inhibition of the "default" state of neural fate acquisition. This has parallels in amphibians View Publication

Spinocerebellar ataxia type 2 (SCA2) is caused by triple nucleotidebackslashnrepeat (CAG) expansion in the coding region of the ATAXN2 gene onbackslashnchromosome 12,which produces an elongated,toxic polyglutamine tract,backslashnleading to Purkinje cell loss. There is currently no effective therapy.backslashnOne of the main obstacles that hampers therapeutic development is lackbackslashnof an ideal disease model. In this study,we have generated andbackslashncharacterized SCA2-induced pluripotent stem (iPS) cell lines as an inbackslashnvitro cell model. Dermal fibroblasts (FBs) were harvested from primarybackslashncultures of skin explants obtained from a SCA2 subject and a healthybackslashnsubject. For reprogramming,hOct4,hSox2,hKlf4,and hc-Myc werebackslashntransduced to passage-3 FBs by retroviral infection. Both SCA2 iPS andbackslashncontrol iPS cells were successfully generated and showed typical stembackslashncell growth patterns with normal karyotype. All iPS cell lines expressedbackslashnstem cell markers and differentiated in vitro into cells from threebackslashnembryonic germ layers. Upon in vitro neural differentiation,SCA2 iPSbackslashncells showed abnormality in neural rosette formation but successfullybackslashndifferentiated into neural stem cells (NSCs) and subsequent neuralbackslashncells. SCA2 and normal FBs showed a comparable level of ataxin-2backslashnexpression; whereas SCA2 NSCs showed less ataxin-2 expression thanbackslashnnormal NSCs and SCA2 FBs. Within the neural lineage,neurons had thebackslashnmost abundant expression of ataxin-2. Time-lapsed neural growth assaybackslashnindicated terminally differentiated SCA2 neural cells were short-livedbackslashncompared with control neural cells. The expanded CAG repeats of SCA2backslashnwere stable throughout reprogramming and neural differentiation. Inbackslashnconclusion,we have established the first disease-specific human SCA2backslashniPS cell line. These mutant iPS cells have the potential for neuralbackslashndifferentiation. These differentiated neural cells harboring mutationsbackslashnare invaluable for the study of SCA2 pathogenesis and therapeutic drugbackslashndevelopment. View Publication

Mutations in the RP2 gene lead to a severe form of X-linked retinitis pigmentosa. RP2 patients frequently present with nonsense mutations and no treatments are currently available to restore RP2 function. In this study,we reprogrammed fibroblasts from an RP2 patient carrying the nonsense mutation c.519CtextgreaterT (p.R120X) into induced pluripotent stem cells (iPSC),and differentiated these cells into retinal pigment epithelial cells (RPE) to study the mechanisms of disease and test potential therapies. RP2 protein was undetectable in the RP2 R120X patient cells,suggesting a disease mechanism caused by complete lack of RP2 protein. The RP2 patient fibroblasts and iPSC-derived RPE cells showed phenotypic defects in IFT20 localization,Golgi cohesion and G$\$1 trafficking. These phenotypes were corrected by over-expressing GFP-tagged RP2. Using the translational read-through inducing drugs (TRIDs) G418 and PTC124 (Ataluren),we were able to restore up to 20% of endogenous,full-length RP2 protein in R120X cells. This level of restored RP2 was sufficient to reverse the cellular phenotypic defects observed in both the R120X patient fibroblasts and iPSC-RPE cells. This is the first proof-of-concept study to demonstrate successful read-through and restoration of RP2 function for the R120X nonsense mutation. The ability of the restored RP2 protein level to reverse the observed cellular phenotypes in cells lacking RP2 indicates that translational read-through could be clinically beneficial for patients. View Publication

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant protein-folding disorder caused by over 100 distinct mutations in the transthyretin (TTR) gene. In ATTR,protein secreted from the liver aggregates and forms fibrils in target organs,chiefly the heart and peripheral nervous system,highlighting the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Here,we describe detailed methodologies for the directed differentiation of protein folding disease-specific iPSCs into hepatocytes that produce mutant protein,and neural-lineage cells often targeted in disease. Methodologies are also described for the construction of multisystem models and drug screening using iPSCs. View Publication

Characterizing clinically relevant brain metastasis models and assessing the therapeutic efficacy in such models are fundamental for the development of novel therapies for metastatic brain cancers. In this study,we have developed an in vivo imageable breast-to-brain metastasis mouse model. Using real time in vivo imaging and subsequent composite fluorescence imaging,we show a widespread distribution of micro- and macro-metastasis in different stages of metastatic progression. We also show extravasation of tumour cells and the close association of tumour cells with blood vessels in the brain thus mimicking the multi-foci metastases observed in the clinics. Next,we explored the ability of engineered adult stem cells to track metastatic deposits in this model and show that engineered stem cells either implanted or injected via circulation efficiently home to metastatic tumour deposits in the brain. Based on the recent findings that metastatic tumour cells adopt unique mechanisms of evading apoptosis to successfully colonize in the brain,we reasoned that TNF receptor superfamily member 10A/10B apoptosis-inducing ligand (TRAIL) based pro-apoptotic therapies that induce death receptor signalling within the metastatic tumour cells might be a favourable therapeutic approach. We engineered stem cells to express a tumour selective,potent and secretable variant of a TRAIL,S-TRAIL,and show that these cells significantly suppressed metastatic tumour growth and prolonged the survival of mice bearing metastatic breast tumours. Furthermore,the incorporation of pro-drug converting enzyme,herpes simplex virus thymidine kinase,into therapeutic S-TRAIL secreting stem cells allowed their eradication post-tumour treatment. These studies are the first of their kind that provide insight into targeting brain metastasis with stem-cell mediated delivery of pro-apoptotic ligands and have important clinical implications. View Publication

Hematopoietic progenitor cells arising from bone marrow (BM) are known to contribute to the formation and expansion of tumor vasculature. However,whether different subsets of these cells have different roles in this process is unclear. To investigate the roles of BM-derived progenitor cell subpopulations in the formation of tumor vasculature in a Ewing's sarcoma model,we used a functional assay based on endothelial cell and pericyte differentiation in vivo. Fluorescence-activated cell sorting of human cord blood/BM or mouse BM from green fluorescent protein transgenic mice was used to isolate human CD34+/CD38(-),CD34+/CD45+,and CD34(-)/CD45+ cells and mouse Sca1+/Gr1+,Sca1(-)/Gr1+,VEGFR1+,and VEGFR2+ cells. Each of these progenitor subpopulations was separately injected intravenously into nude mice bearing Ewing's sarcoma tumors. Tumors were resected 1 week later and analyzed using immunohistochemistry and confocal microscopy for the presence of migrated progenitor cells expressing endothelial,pericyte,or inflammatory cell surface markers. We showed two distinct patterns of stem cell infiltration. Human CD34+/CD45+ and CD34+/CD38(-) and murine VEGFR2+ and Sca1+/Gr1+ cells migrated to Ewing's tumors,colocalized with the tumor vascular network,and differentiated into cells expressing either endothelial markers (mouse CD31 or human vascular endothelial cadherin) or the pericyte markers desmin and alpha-smooth muscle actin. By contrast,human CD34(-)/CD45+ and mouse Sca1(-)/Gr1+ cells migrated predominantly to sites outside of the tumor vasculature and differentiated into monocytes/macrophages expressing F4/80 or CD14. Our data indicate that only specific BM stem/progenitor subpopulations participate in Ewing's sarcoma tumor vasculogenesis. View Publication