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Somatic cells can be induced into pluripotent stem cells (iPSCs) with a combination of four transcription factors,Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28. This provides an enabling platform to obtain patient-specific cells for various therapeutic and research applications. However,several problems remain for this approach to be therapeutically relevant due to drawbacks associated with efficiency and viral genome integration. Recently,it was shown that neural progenitor cells (NPCs) transduced with Oct4/Klf4 can be reprogrammed into iPSCs. However,NPCs express Sox2 endogenously,possibly facilitating reprogramming in the absence of exogenous Sox2. In this study,we identified a small-molecule combination,BIX-01294 and BayK8644,that enables reprogramming of Oct4/Klf4-transduced mouse embryonic fibroblasts,which do not endogenously express the factors essential for reprogramming. This study demonstrates that small molecules identified through a phenotypic screen can compensate for viral transduction of critical factors,such as Sox2,and improve reprogramming efficiency. 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

The mechanisms underlying the decision of a stem or progenitor cell to either self-renew or differentiate are incompletely understood. To address the role of Myc in this process,we expressed different forms of the proto-oncogene Myc in multipotent neural progenitor cells (NPCs) using retroviral transduction. Expression of Myc in neurospheres increased the proportion of self-renewing cells fivefold,and 1% of the Myc-overexpressing cells,but none of the control cells,retained self-renewal capacity even under differentiation-inducing conditions. A Myc mutant (MycV394D) deficient in binding to Miz-1,did not increase the percentage of self-renewing cells but was able to stimulate proliferation of NPCs as efficiently as wild-type Myc,indicating that these two cellular phenomena are regulated by at least partially different pathways. Our results suggest that Myc,through Miz-1,enhances self-renewal of NPCs and influences the way progenitor cells react to the environmental cues that normally dictate the cellular identity of tissues containing self-renewing cells. 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

Using endothelial cells for therapeutic angiogenesis/vasculogenesis of ischemia diseases has led to exploring human embryonic stem cells (hESCs) as a potentially unlimited source for endothelial progenitor cells. With their capacity for self-renewal and pluripotency,hESCs and their derived endothelial cells (hESC-ECs) may be more advantageous than other endothelial cells obtained from diseased populations. However,hESC-ECs' poor differentiation efficiency and poorly characterized in vivo function after transplantation present significant challenges for their future clinical application. This review will focus on the differentiation pathways of hESCs and their therapeutic potential for vascular diseases,as well as the monitoring of transplanted cells' fate via molecular imaging. Finally,cell enhancement strategies to improve the engraftment efficiency of hESC-ECs will be discussed. View Publication

Histone deacetylase inhibitors (HdI) could potentially improve the differentiation of leukemic dendritic cells (DC). Therefore,bone marrow samples from 100 children with acute lymphoblastic leukemia (ALL) were cultured in the cytokines TNF-alpha,GM-CSF,c-kit ligand,and fetal liver tyrosine kinase 3 ligand,with or without IL-3 and -4 and after administration of HdI valproic acid (VAL),suberoylanilide hydroxamic acid (SAHA),isobutyramid,or trichostatin A. Among the tested samples,25 were positive for the chromosomal translocation t(12;21),encoding the fusion gene translocation ETS-like leukemia/acute myeloid leukemia 1 (TEL/AML1). SAHA increased CD83 expression of TEL/AML1-positive blasts in conditions without ILs,and SAHA and VAL increased the number of CD86(+)80(-) cells in the presence of ILs. VAL and isobutyramid supported the allostimulatory capacities of TEL/AML1-positive,leukemic DC; VAL and SAHA reduced those of TEL/AML1-negative DC. Cytotoxic T cells sensitized with leukemic DC produced more IFN-gamma and TNF-alpha upon presentation of the TEL/AML1 peptide. They also induced the cytotoxic lysis of nondifferentiated blasts,which was enhanced when TEL/AML1-positive DC had developed after addition of VAL or SAHA. Therefore,the use of HdI in the differentiation of leukemic DC from patients with TEL/AML1-positive ALL is recommended. View Publication

Protein tyrosine kinases of the Janus kinase (JAK) family are associated with many cytokine receptors,which,on ligand binding,regulate important cellular functions such as proliferation,survival,and differentiation. In multiple myeloma,JAKs may be persistently activated due to a constant stimulation by interleukin (IL)-6,which is produced in the bone marrow environment. INCB20 is a synthetic molecule that potently inhibits all members of the JAK family with a 100- to 1,000-fold selectivity for JAKs over textgreater70 other kinases. Treatment of multiple myeloma cell lines and patient tumor cells with INCB20 resulted in a significant and dose-dependent inhibition of spontaneous as well as IL-6-induced cell growth. Importantly,multiple myeloma cell growth was inhibited in the presence of bone marrow stromal cells. The IL-6 dependent cell line INA-6 was particularly sensitive to the drug (IC50textless1 micromol/L). Growth suppression of INA-6 correlated with an increase in the percentage of apoptotic cells and inhibition of signal transducer and activator of transcription 3 phosphorylation. INCB20 also abrogated the protective effect of IL-6 against dexamethasone by blocking phosphorylation of SHP-2 and AKT. In contrast,AKT phosphorylation induced by insulin-like growth factor-I remained unchanged,showing selectivity of the compound. In a s.c. severe combined immunodeficient mouse model with INA-6,INCB20 significantly delayed INA-6 tumor growth. Our studies show that disruption of JAKs and downstream signaling pathways may both inhibit multiple myeloma cell growth and survival and overcome cytokine-mediated drug resistance,thereby providing the preclinical rationale for the use of JAK inhibitors as a novel therapeutic approach in multiple myeloma. View Publication

PTPN11,which encodes the tyrosine phosphatase SHP2,is mutated in approximately 35% of patients with juvenile myelomonocytic leukemia (JMML) and at a lower incidence in other neoplasms. To model JMML pathogenesis,we generated knockin mice that conditionally express the leukemia-associated mutant Ptpn11(D61Y). Expression of Ptpn11(D61Y) in all hematopoietic cells evokes a fatal myeloproliferative disorder (MPD),featuring leukocytosis,anemia,hepatosplenomegaly,and factor-independent colony formation by bone marrow (BM) and spleen cells. The Lin(-)Sca1(+)cKit(+) (LSK) compartment is expanded and right-shifted� View Publication

The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation,we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex,pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion,respectively,thereby demonstrating the potential of this forward screening approach in primary human stem cell populations. View Publication

BACKGROUND: Hematopoietic progenitors are generated in the yolk sac and aorta-gonad-mesonephros region during early mouse development. At embryonic day 10.5 the first hematopoietic stem cells emerge in the aorta-gonad-mesonephros. Subsequently,hematopoietic stem cells and progenitors are found in the fetal liver. The fetal liver is a potent hematopoietic site,playing an important role in the expansion and differentiation of hematopoietic progenitors and hematopoietic stem cells. However,little is known concerning the regulation of fetal liver hematopoietic stem cells. In particular,the role of cytokines such as interleukin-1 in the regulation of hematopoietic stem cells in the embryo has been largely unexplored. Recently,we observed that the adult pro-inflammatory cytokine interleukin-1 is involved in regulating aorta-gonad-mesonephros hematopoietic progenitor and hematopoietic stem cell activity. Therefore,we set out to investigate whether interleukin-1 also plays a role in regulating fetal liver progenitor cells and hematopoietic stem cells. DESIGN AND METHODS: We examined the interleukin-1 ligand and receptor expression pattern in the fetal liver. The effects of interleukin-1 on hematopoietic progenitor cells and hematopoietic stem cells were studied by FACS and transplantation analyses of fetal liver explants,and in vivo effects on hematopoietic stem cell and progenitors were studied in Il1r1(-/-) embryos. RESULTS: We show that fetal liver hematopoietic progenitor cells express the IL-1RI and that interleukin-1 increases fetal liver hematopoiesis,progenitor cell activity and promotes hematopoietic cell survival. Moreover,we show that in Il1r1(-/-) embryos,hematopoietic stem cell activity is impaired and myeloid progenitor activity is increased. CONCLUSIONS: The IL-1 ligand and receptor are expressed in the midgestation liver and act in the physiological regulation of fetal liver hematopoietic progenitor cells and hematopoietic stem cells. View Publication

Interactions between developmental signaling pathways govern the formation and function of stem cells. Prostaglandin (PG) E2 regulates vertebrate hematopoietic stem cells (HSC). Similarly,the Wnt signaling pathway controls HSC self-renewal and bone marrow repopulation. Here,we show that wnt reporter activity in zebrafish HSCs is responsive to PGE2 modulation,demonstrating a direct interaction in vivo. Inhibition of PGE2 synthesis blocked wnt-induced alterations in HSC formation. PGE2 modified the wnt signaling cascade at the level of beta-catenin degradation through cAMP/PKA-mediated stabilizing phosphorylation events. The PGE2/Wnt interaction regulated murine stem and progenitor populations in vitro in hematopoietic ES cell assays and in vivo following transplantation. The relationship between PGE2 and Wnt was also conserved during regeneration of other organ systems. Our work provides in vivo evidence that Wnt activation in stem cells requires PGE2,and suggests the PGE2/Wnt interaction is a master regulator of vertebrate regeneration and recovery. View Publication

The conventional method of culturing human embryonic stem cells (hESC) is on two-dimensional (2D) surfaces,which is not amenable for scale up to therapeutic quantities in bioreactors. We have developed a facile and robust method for maintaining undifferentiated hESC in three-dimensional (3D) suspension cultures on matrigel-coated microcarriers achieving 2- to 4-fold higher cell densities than those in 2D colony cultures. Stable,continuous propagation of two hESC lines on microcarriers has been demonstrated in conditioned media for 6 months. Microcarrier cultures (MC) were also demonstrated in two serum-free defined media (StemPro and mTeSR1). MC achieved even higher cell concentrations in suspension spinner flasks,thus opening the prospect of propagation in controlled bioreactors. ?? 2009 Elsevier B.V. All rights reserved. View Publication