Product Information

Although recent advances have enabled hematopoietic stem cells (HSCs) to be enriched to near purity,more information about their characteristics will improve our understanding of their development and stage-related functions. Here,using microarray technology,we identified endothelial cell-selective adhesion molecule (ESAM) as a novel marker for murine HSCs in fetal liver. Esam was expressed at high levels within a Rag1(-) c-kit(Hi) Sca1(+) HSC-enriched fraction,but sharply down-regulated with activation of the Rag1 locus,a valid marker for the most primitive lymphoid progenitors in E14.5 liver. The HSC-enriched fraction could be subdivided into 2 on the basis of ESAM levels. Among endothelial antigens on hematopoietic progenitors,ESAM expression showed intimate correlation with HSC activity. The ESAM(Hi) population was highly enriched for multipotent myeloid-erythroid progenitors and primitive progenitors with lymphopoietic activity,and exclusively reconstituted long-term lymphohematopoiesis in lethally irradiated recipients. Tie2(+) c-kit(+) lymphohematopoietic cells in the E9.5-10.5 aorta-gonad-mesonephros region also expressed high levels of ESAM. Furthermore,ESAM was detected on primitive hematopoietic progenitors in adult bone marrow. Interestingly,ESAM expression in the HSC-enriched fraction was up-regulated in aged mice. We conclude that ESAM marks HSC in murine fetal liver and will facilitate studies of hematopoiesis throughout life. View Publication

Current sources of mesenchymal cells,including bone marrow,fat and muscle,all require invasive procurement procedures,and provide relatively low frequencies of progenitors. Here,we describe the non-invasive isolation,and characterization,of a rich source of mesenchymal progenitor cells,which we call human umbilical cord perivascular cells (HUCPVCs). HUCPVCs show a similar immunological phenotype to bone marrow-derived mesenchymal stromal cells (BM-MSCs),since they are non-alloreactive,exhibit immunosuppression,and significantly reduce lymphocyte activation,in vitro. They present a non-hematopoietic myofibroblastic mesenchymal phenotype (CD45-,CD34-,CD105+,CD73+,CD90+,CD44+,CD106+,3G5+,CD146+); with a 1:300 frequency at harvest,a short-doubling time,and a clonogenic frequency of textgreater1:3 in culture. Furthermore,in addition to robust quinti-potential differentiation capacity in vitro,HUCPVCs have been shown to contribute to both musculo-skeletal and dermal wound healing in vivo. View Publication

Megakaryocytopoiesis gives rise to platelets by proliferation and differentiation of lineage-specific progenitors,identified in vitro as Colony Forming Unit-Megakaryocytes (CFU-Mk). The aim of this study was to refine and optimize the in vitro Standard Operating Procedure (SOP) of the CFU-Mk assay for detecting drug-induced thrombocytopenia and to prevalidate a model for predicting the acute exposure levels that cause maximum tolerated decreases in the platelets count,based on the correlation with the maximal plasma concentrations (C max) in vivo. The assay was linear under the SOP conditions,and the in vitro endpoints (percentage of colonies growing) were reproducible within and across laboratories. The protocol performance phase was carried out testing 10 drugs (selected on the base of their recognised or potential in vivo haematotoxicity,according to the literature). Results showed that a relationship can be established between the maximal concentration in plasma (C max) and the in vitro concentrations that inhibited the 10-50-90 percent of colonies growth (ICs). When C max is lower than IC10,it is possible to predict that the chemicals have no direct toxicity effect on CFU-Mk and could not induce thrombocytopenia due to bone marrow damage. When the C max is higher than IC90 and/or IC50,thrombocytopenia can occur due to direct toxicity of chemicals on CFU-Mk progenitors. View Publication

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However,direct injection of hESCs,and cells differentiated from hESCs,into living organisms has thus far been hampered by significant cell death,teratoma formation,and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization,proliferation,and viability. Molecular imaging has given investigators a high-throughput,inexpensive,and sensitive means for tracking in vivo cell proliferation over days,weeks,and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment,proliferation,and teratoma-formation in living subjects. A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes,under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery,are introduced into cells using a variety of vector and non-vector methods. Once in the cell,reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions,depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive,noninvasive instrumentation (e.g.,CCD cameras) using signal-generating probes such as D-luciferin. To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging,bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase,derived from the firefly Photinus pyralis,encodes an enzyme that catalyzes D-luciferin to the optically active metabolite,oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells,allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore,because expression of the reporter gene product is required for signal generation,only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. In this video,the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described. View Publication

Large-scale manufacture of human embryonic stem cells (hESCs) is prerequisite to their widespread use in biomedical applications. However,current hESC culture strategies are labor-intensive and employ highly variable processes,presenting challenges for scaled production and commercial development. Here we demonstrate that passaging of the hESC lines,HUES7,and NOTT1,with trypsin in feeder-free conditions,is compatible with complete automation on the CompacT SelecT,a commercially available and industrially relevant robotic platform. Pluripotency was successfully retained,as evidenced by consistent proliferation during serial passage,expression of stem cell markers (OCT4,NANOG,TRA1-81,and SSEA-4),stable karyotype,and multi-germlayer differentiation in vitro,including to pharmacologically responsive cardiomyocytes. Automation of hESC culture will expedite cell-use in clinical,scientific,and industrial applications. View Publication

Neurotrophins (NTs) and their receptors play a key role in neurogenesis and survival. The TRK (tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA,TRKB,TRKC) are high-affinity NT receptors that are expressed in a variety of human tissues. Their role in normal and malignant hematopoiesis is poorly understood. In a prospective study involving 94 adult patients we demonstrate for the first time cell-surface expression of the 3 TRKs and constitutive activation in blasts from patients with de novo or secondary acute leukemia. At least one TRK was expressed in 55% of the analyzed cases. We establish a clear correlation between the TRK expression pattern and FAB classification. Although only few point mutations were found in TRK sequences by reverse-transcriptase-polymerase chain reaction (RT-PCR),we observed coexpression of BDNF (ligand for TRKB) in more than 50% of TRKB(+) cases (16/30). Activation of TRKA or TRKB by NGF and BDNF,respectively,efficiently rescued murine myeloid cells from irradiation-induced apoptosis. Coexpression of TRKB/BDNF or TRKA/NGF in murine hematopoietic cells induced leukemia. Moreover,activation of TRKs was important for survival of both human and murine leukemic cells. Our findings suggest that TRKs play an important role in leukemogenesis and may serve as a new drug target. View Publication

BACKGROUND Efficient slow freezing protocols within serum-free and feeder-free culture systems are crucial for the clinical application of human embryonic stem (hES) cells. Frequently,however,hES cells must be cryopreserved as clumps when using conventional slow freezing protocols,leading to lower survival rates during freeze-thaw and limiting their recovery and growth efficiency after thawing,as well as limiting downstream applications that require single cell suspensions. We describe a novel method to increase freeze-thaw survival and proliferation rate of single hES cells in serum-free and feeder-free culture conditions. METHODS hES cells maintained on Matrigel-coated dishes were dissociated into single cells with Accutase and slow freezing. After thawing at 37 degrees C,cells were cultured in mTeSR medium supplemented with 10 microM of Rho-associated kinase inhibitor Y-27632 for 1 day. RESULTS The use of Y-27632 and Accutase significantly increases the survival of single hES cells after thawing compared with a control group (P textless 0.01). Furthermore,by treatment of hES cell aggregates with EGTA to disrupt cell-cell interaction,we show that Y-27632 treatment does not directly affect hES cell apoptosis. Even in the presence of Y-27632,hES cells deficient in cell-cell interaction undergo apoptosis. Y-27632-treated freeze-thawed hES cells retain typical morphology,stable karyotype,expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture. CONCLUSIONS The method described here allows for cryopreservation of single hES cells in serum-free and feeder-free conditions and therefore we believe this method will be ideal for current and future hES cell applications that are targeted towards a therapeutic end-point. View Publication

We developed a real-time copy number polymerase chain reaction assay for deletions on chromosome 20q (del20q),screened peripheral blood granulocytes from 664 patients with myeloproliferative disorders,and identified 19 patients with del20q (2.9%),of which 14 (74%) were also positive for JAK2-V617F. To examine the temporal relationship between the occurrence of del20q and JAK2-V617F,we performed colony assays in methylcellulose,picked individual burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte (CFU-G) colonies,and genotyped each colony individually for del20q and JAK2-V617F. In 2 of 9 patients,we found that some colonies with del20q carried only wild-type JAK2,whereas other del20q colonies were JAK2-V617F positive,indicating that del20q occurred before the acquisition of JAK2-V617F. However,in colonies from 3 of 9 patients,we observed the opposite order of events. The lack of a strict temporal order of occurrence makes it doubtful that del20q represents a predisposing event for JAK2-V617F. In 2 patients with JAK2-V617F and 1 patient with MPL-W515L,microsatellite analysis revealed that del20q affected chromosomes of different parental origin and/or 9pLOH occurred at least twice. The fact that rare somatic events,such as del20q or 9pLOH,occurred more than once in subclones from the same patients suggests that the myeloproliferative disorder clone carries a predisposition to acquiring such genetic alterations. View Publication

Defects in apoptosis contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL),calling for novel strategies that counter apoptosis resistance. Here,we demonstrate for the first time that small molecule inhibitors of the antiapoptotic protein XIAP cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells. XIAP inhibitors at subtoxic concentrations,but not a structurally related control compound,synergize with TRAIL to trigger apoptosis and to inhibit clonogenic survival of acute leukemia cells,whereas they do not affect viability of normal peripheral blood lymphocytes,suggesting some tumor selectivity. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases,loss of mitochondrial membrane potential,and cytochrome c release in a caspase-dependent manner,indicating that they promote a caspase-dependent feedback mitochondrial amplification loop. Of note,XIAP inhibitors even overcome Bcl-2-mediated resistance to TRAIL by enhancing Bcl-2 cleavage and Bak conformational change. Importantly,XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and cooperate with TRAIL to induce apoptosis. In vivo,they significantly reduce leukemic burden in a mouse model of pediatric ALL engrafted in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Thus,XIAP inhibitors present a promising novel approach for apoptosis-based therapy of childhood ALL. View Publication

Previous studies have demonstrated that normal mouse mammary tissue contains a rare subset of mammary stem cells. We now describe a method for detecting an analogous subpopulation in normal human mammary tissue. Dissociated cells are suspended with fibroblasts in collagen gels,which are then implanted under the kidney capsule of hormone-treated immunodeficient mice. After 2-8 weeks,the gels contain bilayered mammary epithelial structures,including luminal and myoepithelial cells,their in vitro clonogenic progenitors and cells that produce similar structures in secondary transplants. The regenerated clonogenic progenitors provide an objective indicator of input mammary stem cell activity and allow the frequency and phenotype of these human mammary stem cells to be determined by limiting-dilution analysis. This new assay procedure sets the stage for investigations of mechanisms regulating normal human mammary stem cells (and possibly stem cells in other tissues) and their relationship to human cancer stem cell populations. View Publication

Human embryonic stem (hES) cells represent a potential source for cell replacement therapy of many degenerative diseases. Most frequently,hES cell lines are derived from surplus embryos from assisted reproduction cycles,independent of their quality or morphology. Here,we show that hES cell lines can be obtained from poor-quality blastocysts with the same efficiency as that obtained from good- or intermediate-quality blastocysts. Furthermore,we show that the self-renewal,pluripotency,and differentiation ability of hES cell lines derived from either source are comparable. Finally,we present a simple and reproducible embryoid body-based protocol for the differentiation of hES cells into functional cardiomyocytes. The five new hES cell lines derived here should widen the spectrum of available resources for investigating the biology of hES cells and advancing toward efficient strategies of regenerative medicine. View Publication

Earlier reports have suggested that the BCR/ABL oncogene,associated with chronic myeloid leukemia,induces a mutator phenotype; however,it is unclear whether this leads to long-term changes in chromosomes and whether the phenotype is found in primary chronic myelogeneous leukemia (CML) cells. We have addressed both these issues. BCR/ABL-expressing cell lines show an increase in DNA breaks after treatment with etoposide as compared to control cells. However,although BCR/ABL-expressing cell lines have an equivalent cell survival,they have an increase in chromosomal translocations after DNA repair as compared to control cells. This demonstrates that BCR/ABL expression decreases the fidelity of DNA repair. To see whether this is true in primary CML samples,normal CD34+ progenitor cells and CML progenitor cells were treated with etoposide. CML progenitor cells have equivalent survival but have an increase in DNA double-strand breaks (DSBs). Spectral karyotyping demonstrates new chromosomal translocations in CML cells,but not normal progenitor cells,consistent with error-prone DNA repair. Taken together,these data demonstrate that BCR/ABL enhances the accumulation of DSBs and alters the apoptotic threshold in CML leading to error-prone DNA repair. View Publication