技术资料

Hematopoietic stem cells (HSC) can be harmed by disease,chemotherapy,radiation,and normal aging. We show in this study that damage also occurs in mice repeatedly treated with very low doses of LPS. Overall health of the animals was good,and there were relatively minor changes in marrow hematopoietic progenitors. However,HSC were unable to maintain quiescence,and transplantation revealed them to be myeloid skewed. Moreover,HSC from treated mice were not sustained in serial transplants and produced lymphoid progenitors with low levels of the E47 transcription factor. This phenomenon was previously seen in normal aging. Screening identified mAbs that resolve HSC subsets,and relative proportions of these HSC changed with age and/or chronic LPS treatment. For example,minor CD150(Hi)CD48(-) populations lacking CD86 or CD18 expanded. Simultaneous loss of CD150(Lo/-)CD48(-) HSC and gain of the normally rare subsets,in parallel with diminished transplantation potential,would be consistent with age- or TLR-related injury. In contrast,HSC in old mice differed from those in LPS-treated animals with respect to VCAM-1 or CD41 expression and lacked proliferation abnormalities. HSC can be exposed to endogenous and pathogen-derived TLR ligands during persistent low-grade infections. This stimulation might contribute in part to HSC senescence and ultimately compromise immunity. View Publication

In the present study,the trilineage differentiation capacity of human foreskin-derived precursor cells (hSKP) was evaluated upon exposure to various (non)commercial (i and ii) ectodermal,(iii) mesodermal and (iv) endodermal differentiation media. (i) Upon sequential exposure of the cells to keratinocyte growth (CnT-07® or CnT-057®) and differentiation (CnT-02® or Epilife®) media,keratinocyte-like cells (filaggrin(+)/involucrin(+)) were obtained. The preferred keratinocyte differentiation strategy was exposure to CnT-07®. (ii) When hSKP were subsequently exposed to NeuroCult® media,cells underwent a weak neuro-ectodermal differentiation expressing nestin,myelin binding protein (MBP),vimentin and alpha-foetoprotein (AFP). Sequential exposure to NPMM® and NPDM® generated cells with an inferior neuro-ectodermal phenotype (nestin(+)/vimentin(+)/MBP(-)/AFP(-)). (iii) Upon exposure of hSKP to insulin-transferrin-selenite (ITS) and dexamethasone,small lipid droplets were observed,suggesting their differentiation potential towards adipocyte-like cells. (iv) Finally,after sequential exposure to hepatogenic growth factors and cytokines,an immature hepatic cell population was generated. The presence of pre-albumin suggests that a sequential exposure strategy is here superior to a cocktail approach. In summary,a considerable impact of different (non)commercial media on the lineage-specific differentiation efficiency of hSKP is shown. In addition,we demonstrate here for the first time that,in a suitable keratinocyte stimulating micro-environment,hSKP can generate keratinocyte-like progeny in vitro. View Publication

Neural stem cells comprise a small population of subependymal cells in the adult brain that divide asymmetrically under baseline conditions to maintain the stem cell pool and divide symmetrically in response to injury to increase their numbers. Using in vivo and in vitro models,we demonstrate that Wnt signaling plays a role in regulating the symmetric divisions of adult neural stem cells with no change in the proliferation kinetics of the progenitor population. Using BAT-gal transgenic reporter mice to identify cells with active Wnt signaling,we demonstrate that Wnt signaling is absent in stem cells in conditions where they are dividing asymmetrically and that it is upregulated when stem cells are dividing symmetrically,such as (a) during subependymal regeneration in vivo,(b) in response to stroke,and (c) during colony formation in vitro. Moreover,we demonstrate that blocking Wnt signaling in conditions where neural stem cells are dividing symmetrically inhibits neural stem cell expansion both in vivo and in vitro. Together,these findings reveal that the mechanism by which Wnt signaling modulates the size of the stem cell pool is by regulating the symmetry of stem cell division. View Publication

The neurosphere assay (NSA) is one of the most frequently used methods to isolate,expand and also calculate the frequency of neural stem cells (NSCs). Furthermore,this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied,overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay,the neural-colony forming cell assay (N-CFCA),which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential,and thus provides a method to enumerate NSC frequency. In the N-CFCA,colonies ≥2 mm in diameter are derived from cells that meet all the functional criteria of a NSC,while colonies textless 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are textless 2mm and the ones that are ≥2mm in reference to the number of cells that were initially plated. View Publication

Disruptor of telomeric silencing 1-like (Dot1l) is a histone 3 lysine 79 methyltransferase. Studies of constitutive Dot1l knockout mice show that Dot1l is essential for embryonic development and prenatal hematopoiesis. DOT1L also interacts with translocation partners of Mixed Lineage Leukemia (MLL) gene,which is commonly translocated in human leukemia. However,the requirement of Dot1l in postnatal hematopoiesis and leukemogenesis of MLL translocation proteins has not been conclusively shown. With a conditional Dot1l knockout mouse model,we examined the consequences of Dot1l loss in postnatal hematopoiesis and MLL translocation leukemia. Deletion of Dot1l led to pancytopenia and failure of hematopoietic homeostasis,and Dot1l-deficient cells minimally reconstituted recipient bone marrow in competitive transplantation experiments. In addition,MLL-AF9 cells required Dot1l for oncogenic transformation,whereas cells with other leukemic oncogenes,such as Hoxa9/Meis1 and E2A-HLF,did not. These findings illustrate a crucial role of Dot1l in normal hematopoiesis and leukemogenesis of specific oncogenes. View Publication

Src homology 2 domain-containing phosphatase 2 (Shp2),encoded by Ptpn11,is a member of the nonreceptor protein-tyrosine phosphatase family,and functions in cell survival,proliferation,migration,and differentiation in many tissues. Here we report that loss of Ptpn11 in murine hematopoietic cells leads to bone marrow aplasia and lethality. Mutant mice show rapid loss of hematopoietic stem cells (HSCs) and immature progenitors of all hematopoietic lineages in a gene dosage-dependent and cell-autonomous manner. Ptpn11-deficient HSCs and progenitors undergo apoptosis concomitant with increased Noxa expression. Mutant HSCs/progenitors also show defective Erk and Akt activation in response to stem cell factor and diminished thrombopoietin-evoked Erk activation. Activated Kras alleviates the Ptpn11 requirement for colony formation by progenitors and cytokine/growth factor responsiveness of HSCs,indicating that Ras is functionally downstream of Shp2 in these cells. Thus,Shp2 plays a critical role in controlling the survival and maintenance of HSCs and immature progenitors in vivo. View Publication

Both the nonreceptor tyrosine kinase Src and the receptors for transforming growth factor (TGF)-β (TβRI,TβRII) play major roles during tumorigenesis by regulating cell growth,migration/invasion and metastasis. The common Src family kinase inhibitors PP2 and PP1 effectively block Src activity in vitro and in vivo,however,they may exert non-specific effects on other kinases. In this study,we have evaluated PP2 and PP1 for their ability to inhibit TGFβ1-mediated responses in the TGF-β-responsive pancreatic adenocarcinoma cell line Panc1. We show that PP2 and PP1 but not the more specific Src inhibitor SU6656 effectively relieved TGF-b1-induced growth arrest and p21(WAF1) induction,while basal growth was enhanced by PP2 and PP1,and suppressed by SU6656. PP2 and PP1 but not SU6656 also suppressed TGF-β1-induced epithelial-to-mesenchymal transition (EMT) as evidenced by their ability to inhibit downregulation of the epithelial marker E-cadherin,and upregulation of the EMT-associated transcription factor Slug. Likewise,PP2 and PP1 but not SU6656 effectively blocked TGF-β1-induced activation of Smad2 and p38 MAPK and partially suppressed Smad activation and transcriptional activity on TGF-β/Smad-responsive reporters of a kinase-active TβRI mutant ectopically expressed in Panc1 cells. Interestingly,PP2 and PP1 strongly inhibited recombinant TβRI in an in vitro kinase assay,with PP1 being more potent and PP2 being nearly as potent as the established TβRI inhibitor SB431542. PP2 but not PP1 also weakly inhibited the TβRII kinase. Together,these data provide evidence that PP2 and PP1 are powerful inhibitors of TβR function that can block TGF-β/Smad signaling in a Src-unrelated fashion. Both agents may be useful as dual TGF-β/Src inhibitors in experimental therapeutics of late stage metastatic disease. View Publication

Genetic reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) could offer replenishable cell sources for transplantation therapies. To fulfill their promises,human iPSCs will ideally be free of exogenous DNA (footprint-free),and be derived and cultured in chemically defined media free of feeder cells. Currently,methods are available to enable efficient derivation of footprint-free human iPSCs. However,each of these methods has its limitations. We have previously derived footprint-free human iPSCs by employing episomal vectors for transgene delivery,but the process was inefficient and required feeder cells. Here,we have greatly improved the episomal reprogramming efficiency using a cocktail containing MEK inhibitor PD0325901,GSK3β inhibitor CHIR99021,TGF-β/Activin/Nodal receptor inhibitor A-83-01,ROCK inhibitor HA-100 and human leukemia inhibitory factor. Moreover,we have successfully established a feeder-free reprogramming condition using chemically defined medium with bFGF and N2B27 supplements and chemically defined human ESC medium mTeSR1 for the derivation of footprint-free human iPSCs. These improvements enabled the routine derivation of footprint-free human iPSCs from skin fibroblasts,adipose tissue-derived cells and cord blood cells. This technology will likely be valuable for the production of clinical-grade human iPSCs. View Publication

PURPOSE: The combination of erlotinib and gemcitabine has shown a small but statistically significant survival advantage when compared with gemcitabine alone in patients with advanced pancreatic cancer. However,the overall survival rate with the erlotinib and gemcitabine combination is still low. In this study,we sought to identify gene targets that,when inhibited,would enhance the activity of epidermal growth factor receptor (EGFR)-targeted therapies in pancreatic cancer cells. EXPERIMENTAL DESIGN: A high-throughput RNA interference (RNAi) screen was carried out to identify candidate genes. Selected gene hits were further confirmed and mechanisms of action were further investigated using various assays. RESULTS: Six gene hits from siRNA screening were confirmed to significantly sensitize BxPC-3 pancreatic cancer cells to erlotinib. One of the hits,mitogen-activated protein kinase (MAPK) 1,was selected for further mechanistic studies. Combination treatments of erlotinib and two MAP kinase kinase (MEK) inhibitors,RDEA119 and AZD6244,showed significant synergistic effect for both combinations (RDEA119-erlotinib and AZD6244-erlotinib) compared with the corresponding single drug treatments in pancreatic cancer cell lines with wild-type KRAS (BxPC-3 and Hs 700T) but not in cell lines with mutant KRAS (MIA PaCa-2 and PANC-1). The enhanced antitumor activity of the combination treatment was further verified in the BxPC-3 and MIA PaCa-2 mouse xenograft model. Examination of the MAPK signaling pathway by Western blotting indicated effective inhibition of the EGFR signaling by the drug combination in KRAS wild-type cells but not in KRAS mutant cells. CONCLUSIONS: Overall,our results suggest that combination therapy of an EGFR and MEK inhibitors may have enhanced efficacy in patients with pancreatic cancer. View Publication

One of the key consequences of exposure of human cells to genotoxic agents is the activation of DNA damage responses (DDR). While the mechanisms underpinning DDR in fully differentiated somatic human cells have been studied extensively,molecular signaling events and pathways involved in DDR in pluripotent human embryonic stem cells (hESC) remain largely unexplored. We studied changes in the human genome-wide transcriptome of H9 hESC line following exposures to 1Gy of gamma-radiation at 2h and 16h post-irradiation. Quantitative real-time PCR was performed to verify the expression data for a subset of genes. In parallel,the cell growth,DDR kinetics,and expression of pluripotency markers in irradiated hESC were monitored. The changes in gene expression in hESC after exposure to ionizing radiation (IR) are substantially different from those observed in somatic human cell lines. Gene expression patterns at 2h post-IR showed almost an exclusively p53-dependent,predominantly pro-apoptotic,signature with a total of only 30 up-regulated genes. In contrast,the gene expression patterns at 16h post-IR showed 354 differentially expressed genes,mostly involved in pro-survival pathways,such as increased expression of metallothioneins,ubiquitin cycle,and general metabolism signaling. Cell growth data paralleled trends in gene expression changes. DDR in hESC followed the kinetics reported for human somatic differentiated cells. The expression of pluripotency markers characteristic of undifferentiated hESC was not affected by exposure to IR during the time course of our analysis. Our data on dynamics of transcriptome response of irradiated hESCs may provide a valuable tool to screen for markers of IR exposure of human cells in their most naive state; thus unmasking the key elements of DDR; at the same time,avoiding the complexity of interpreting distinct cell type-dependent genotoxic stress responses of terminally differentiated cells. View Publication

A major road-block in stem cell therapy is the poor homing and integration of transplanted stem cells with the targeted host tissue. Human induced pluripotent stem (hiPS) cells are considered an excellent alternative to embryonic stem (ES) cells and we tested the feasibility of using small,physiological electric fields (EFs) to guide hiPS cells to their target. Applied EFs stimulated and guided migration of cultured hiPS cells toward the anode,with a stimulation threshold of textless30 mV/mm; in three-dimensional (3D) culture hiPS cells remained stationary,whereas in an applied EF they migrated directionally. This is of significance as the therapeutic use of hiPS cells occurs in a 3D environment. EF exposure did not alter expression of the pluripotency markers SSEA-4 and Oct-4 in hiPS cells. We compared EF-directed migration (galvanotaxis) of hiPS cells and hES cells and found that hiPS cells showed greater sensitivity and directedness than those of hES cells in an EF,while hES cells migrated toward cathode. Rho-kinase (ROCK) inhibition,a method to aid expansion and survival of stem cells,significantly increased the motility,but reduced directionality of iPS cells in an EF by 70-80%. Thus,our study has revealed that physiological EF is an effective guidance cue for the migration of hiPS cells in either 2D or 3D environments and that will occur in a ROCK-dependent manner. Our current finding may lead to techniques for applying EFs in vivo to guide migration of transplanted stem cells. View Publication

Adipose tissue is an abundantly available source of proliferative and multipotent mesenchymal stem cells with promising potential for regenerative therapeutics. We previously demonstrated that both human and mouse adipose-derived stem cells (ASCs) can be reprogrammed into induced pluripotent stem cells (iPSCs) with efficiencies higher than those that have been reported for other cell types. The ASC-derived iPSCs can be generated in a feeder-independent manner,representing a unique model to study reprogramming and an important step toward establishing a safe,clinical grade of cells for therapeutic use. In this study,we provide a detailed protocol for isolation,preparation and transformation of ASCs from fat tissue into mouse iPSCs in feeder-free conditions and human iPSCs using feeder-dependent or feeder/xenobiotic-free processes. This protocol also describes how ASCs can be used as feeder cells for maintenance of other pluripotent stem cells. ASC derivation is rapid and can be completed in textless1 week,with mouse and human iPS reprogramming times averaging 1.5 and 2.5 weeks,respectively. View Publication