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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

Recently it has been demonstrated that CD30 expression was rather specific for transformed than for normal human ES cells and therefore CD30 maybe suggested as a potential marker for human ES cells bearing chromosomal abnormalities. Using immunohistochemistry and RT-PCR analysis we examined �?¡D30 expression in 10 hESCs lines with normal and abberant karyotypes. All hESC lines expressed CD30 antigen and RNA in undifferentiated state whether cell line beared chromosomal abnormalities or not. In contrast to previous notions our data demonstrate that CD30 could be considered as marker of undifferentiated hESCs without respect to karyotype changes. View Publication

We have demonstrated previously that cord blood CD133(+) cells isolated in the G(0) phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity,in contrast to CD133(+)G(1) cells. Here,we have analysed the phenotype and functional properties of this population in more detail. Our data demonstrate that a large proportion of the CD133(+)G(0) cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133(+)G(1) counterparts (13.5 and 4.1%,respectively). This suggests that stem cell activity resides in the CD133(+)G(0) population. In long-term BM cultures,the CD133(+)G(0) cells generate significantly more progenitors than the CD34(+)G(0) population (Ptextless0.001) throughout the culture period. Furthermore,a comparison of CD133(+)G(0) versus CD133(+)G(1) cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G(0) cells. We conclude that CD133(+) cells in the quiescent phase of the cell cycle have a phenotype consistent with HSCs and are highly enriched for repopulating activity when compared with their G(1) counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols. View Publication

Cyclophosphamide (CYC) can control diffuse proliferative lupus nephritis (DPLN) by potent immunosuppression but remains associated with serious and life-threatening complications. Drugs that specifically target mediators of DPLN may help to reduce CYC dose and side effects. Monocyte chemoattractant protein (MCP-1)/CCL2 mediates monocyte and T cell recruitment in DPLN and Ccl2-specific l-enantiomeric RNA Spiegelmer mNOX-E36 neutralizes the biological effects of murine Ccl2 in vitro and in vivo. We injected MRL(lpr/lpr) mice with DPLN from 14 weeks of age with vehicle,weekly 30 mg/kg CYC (full dose),monthly 30 mg/kg CYC (one-fourth full dose),pegylated control Spiegelmer,pegylated anti-Ccl2 Spiegelmer (3/week),pegylated anti-Ccl2 Spiegelmer plus CYC one-fourth full dose and mycophenolate mofetil. At week 24,DPLN and autoimmune lung injury were virtually abolished with CYC full dose but not with CYC one-fourth full dose. The CYC one-fourth full dose/Spiegelmer combination was equipotent to CYC full dose on kidney and lung injury. CD3(+)CD4(-)CD8(-) and CD3(+)CD4(+)CD25(+) T cells and serum interleukin-12p40 and tumor necrosis factor-alpha levels were all markedly affected by CYC full dose but not by CYC one-fourth full dose. No additive effects of anti-Ccl2 Spiegelmer were noted on bone marrow colony-forming unit-granulocyte macrophage counts and 7/4(high) monocyte counts,lymphoproliferation,and spleen T cell depletion. In summary,anti-Ccl2 Spiegelmer permits 75% dose reduction of CYC for controlling DPLN and pneumonitis in MRL-Fas(lpr) mice,sparing suppressive effects of full-dose CYC on myelosuppression and T cell depletion. We propose anti-Ccl2 Spiegelmer therapy as a novel strategy to reduce CYC toxicity in the treatment of severe lupus. View Publication

High levels of aldehyde dehydrogenase (ALDH) activity have been proposed to be a common feature of stem cells. Adult hematopoietic,neural,and cancer stem cells have all been reported to have high ALDH activity,detected using Aldefluor,a fluorogenic substrate for ALDH. This activity has been attributed to Aldh1a1,an enzyme that is expressed at high levels in stem cells and that has been suggested to regulate stem cell function. Nonetheless,Aldh1a1 function in stem cells has never been tested genetically. We observed that Aldh1a1 was preferentially expressed in mouse hematopoietic stem cells (HSCs) and expression increased with age. Hematopoietic cells from Aldh1a1-deficient mice exhibited increased sensitivity to cyclophosphamide in a non-cell-autonomous manner,consistent with its role in cyclophosphamide metabolism in the liver. However,Aldh1a1 deficiency did not affect hematopoiesis,HSC function,or the capacity to reconstitute irradiated recipients in young or old adult mice. Aldh1a1 deficiency also did not affect Aldefluor staining of hematopoietic cells. Finally,Aldh1a1 deficiency did not affect the function of stem cells from the adult central or peripheral nervous systems. Aldh1a1 is not a critical regulator of adult stem cell function or Aldefluor staining in mice. View Publication

BACKGROUND: Excessive maturation of hematopoietic cells leads to a reduction of long-term proliferative capability during cord blood (CB) expansion. In this study,we report the effects of anit-CD52 (Alemtuzumab,Campath) on both short- and long-term ex vivo expansion of CB hematopoietic stem cells (HSC) by evaluating the potential role of Alemtuzumab in preserving the repopulating capability in CB HSC and nonlymphoid progenitors. METHODS: Ex vivo expansion experiments were carried out using freshly purified CB CD34(+)cells in StemSpantrade mark SFEM medium in the presence of stem cell factor,Flt3-Ligand and thrombopoietin at 50 ng/ml. Alemtuzumab (10 microg/ml) was used to deplete CD52(+) cells during the cultures. Flow cytometry was used to monitor CB HSC and their differentiation. Colony forming unit (CFU) assays and long term culture-initiating cell (LTC-IC) assays were performed on cells obtained from day 0 (before culture) and day 14 after cultures. Secondary cultures was performed using CD34(+) cells isolated at 35 days from primary cultures and further cultured in StemSpantrade mark SFEM medium for another 14 days to confirm the long term effect of alemtuzumab in liquid cultures. RESULTS: Compared to cytokines alone,addition of alemtuzumab resulted in a significant increase in total nucleated cells,absolute CD34(+) cells,myeloid and megakaryocytic progenitors,multi-lineage and myeloid CFU and LTC-IC. CONCLUSION: The results from current study suggested that the use of alemtuzumab for ex vivo expansion of CBHSC maybe advantageous. Our findings may improve current technologies for CBHSC expansion and increase the availability of CB units for transplantation. However,in vivo studies using animal models are likely needed in further studies to test the hematopoietic effects using such expanded CB products. View Publication