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In the three decades since pluripotent mouse embryonic stem (ES) cells were first described they have been derived and maintained by using various empirical combinations of feeder cells,conditioned media,cytokines,growth factors,hormones,fetal calf serum,and serum extracts. Consequently ES-cell self-renewal is generally considered to be dependent on multifactorial stimulation of dedicated transcriptional circuitries,pre-eminent among which is the activation of STAT3 by cytokines (ref. 8). Here we show,however,that extrinsic stimuli are dispensable for the derivation,propagation and pluripotency of ES cells. Self-renewal is enabled by the elimination of differentiation-inducing signalling from mitogen-activated protein kinase. Additional inhibition of glycogen synthase kinase 3 consolidates biosynthetic capacity and suppresses residual differentiation. Complete bypass of cytokine signalling is confirmed by isolating ES cells genetically devoid of STAT3. These findings reveal that ES cells have an innate programme for self-replication that does not require extrinsic instruction. This property may account for their latent tumorigenicity. The delineation of minimal requirements for self-renewal now provides a defined platform for the precise description and dissection of the pluripotent state. View Publication

OBJECTIVE: We used genetically engineered mouse hematopoietic progenitor cells (HPCs) to investigate the therapeutic effects of human apoAI on atherosclerosis in apoE(-/-) mice. METHODS AND RESULTS: Lentiviral constructs expressing either human apoAI (LV-apoAI) or green fluorescent protein (LV-GFP) cDNA under a macrophage specific promoter (CD68) were generated and used for ex vivo transduction of mouse HPCs and macrophages. The transduction efficiency was textgreater25% for HPCs and textgreater70% for macrophages. ApoAI was found in the macrophage culture media,mostly associated with the HDL fraction. Interestingly,a significant increase in mRNA and protein levels for ATP binding cassette A1 (ABCA1) and ABCG1 were found in apoAI-expressing macrophages after acLDL loading. Expression of apoAI significantly increased cholesterol efflux in wild-type and apoE(-/-) macrophages. HPCs transduced with LV-apoAI ex vivo and then transplanted into apoE(-/-) mice caused a 50% reduction in atherosclerotic lesion area compared to GFP controls,without influencing plasma HDL-C levels. CONCLUSIONS: Lentiviral transduction of apoAI into HPCs reduces atherosclerosis in apoE(-/-) mice. Expression of apoAI in macrophages improves cholesterol trafficking in wild-type apoE-producing macrophages and causes upregulation of ABCA1 and ABCG1. These novel observations set the stage for a cell therapy approach to atherosclerosis regression,exploiting the cooperation between apoE and apoAI to maximize cholesterol exit from the plaque. View Publication

B lymphocyte stimulator (BLyS) is a well-known direct costimulator of adaptive immune cells,particularly B lineage cells. However,we have reported recently that BLyS is also able to activate monocytes. Other innate immune cells,such as dendritic cells (DCs),play a key role in the initiation of adaptive immune responses and the purpose of the current study was to assess whether there is a direct role for BLyS in modulating human DC functions. In this study,we show that BLyS induces DC activation and maturation. Thus,BLyS strongly induced up-regulation of surface costimulatory molecule expression and secretion of specific cytokines and chemokines in DCs. BLyS-stimulated DCs (BLyS-DCs) were also able to augment allogeneic CD4 T cell proliferation to a greater extent than control DCs. BLyS-DCs secreted elevated levels of the major Th1-polarizing cytokine,IL-12p70,and they promoted naive CD4 T cell differentiation into Th1 T cells. Regarding BLyS receptor expression,DCs primarily express cytoplasmic transmembrane activator and CAML interactor; however,low levels of cell surface transmembrane activator and CAML interactor are expressed as well. Collectively,our data suggest that BLyS may modulate adaptive immune cells indirectly by inducing DC maturation. View Publication

BACKGROUND: Patients with type 2 diabetes mellitus are at increased risk for the development of atherosclerosis. A pivotal event in the development of atherosclerosis is macrophage foam cell formation. The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 regulate macrophage cholesterol efflux and hence play a vital role in macrophage foam cell formation. We have previously found that chronic elevated glucose reduces ABCG1 expression. In the present study,we examined whether patients with type 2 diabetes mellitus had decreased ABCG1 and/or ABCA1,impaired cholesterol efflux,and increased macrophage foam cell formation. METHODS AND RESULTS: Blood was collected from patients with and without type 2 diabetes mellitus. Peripheral blood monocytes were differentiated into macrophages,and cholesterol efflux assays,immunoblots,histological analysis,and intracellular cholesteryl ester measurements were performed. Macrophages from patients with type 2 diabetes mellitus had a 30% reduction in cholesterol efflux with a corresponding 60% increase in cholesterol accumulation relative to control subjects. ABCG1 was present in macrophages from control subjects but was undetectable in macrophages from patients with type 2 diabetes mellitus. In contrast,ABCA1 expression in macrophages was similar in both control subjects and patients with type 2 diabetes mellitus. Macrophage expression of ABCG1 in both patients and control subjects was induced by treatment with the liver X receptor agonist TO-901317. Upregulation of liver X receptor dramatically reduced foam cell formation in macrophages from patients with type 2 diabetes mellitus. CONCLUSIONS: ABCG1 expression and cholesterol efflux are reduced in patients with type 2 diabetes mellitus. This impaired ABCG1-mediated cholesterol efflux significantly correlates with increased intracellular cholesterol accumulation. Strategies to upregulate ABCG1 expression and function in type 2 diabetes mellitus could have therapeutic potential for limiting the accelerated vascular disease observed in patients with type 2 diabetes mellitus. View Publication

Recent studies have shown that mesenchymal stem cells (MSC) with the potential for cell-mediated therapies and tissue engineering applications can be isolated from extracted dental tissues. Here,we investigated the collection,processing,and cryobiological characteristics of MSC from human teeth processed under current good tissue practices (cGTP). Viable dental pulp-derived MSC (DPSC) cultures were isolated from 31 of 40 teeth examined. Of eight DPSC cultures examined more thoroughly,all expressed appropriate cell surface markers and underwent osteogenic,adipogenic,and chondrogenic differentiation in appropriate differentiation medium,thus meeting criteria to be called MSC. Viable DPSC were obtained up to 120 h postextraction. Efficient recovery of DPSC from cryopreserved intact teeth and second-passage DPSC cultures was achieved. These studies indicate that DPSC isolation is feasible for at least 5 days after tooth extraction,and imply that processing immediately after extraction may not be required for successful banking of DPSC. Further,the recovery of viable DPSC after cryopreservation of intact teeth suggests that minimal processing may be needed for the banking of samples with no immediate plans for expansion and use. These initial studies will facilitate the development of future cGTP protocols for the clinical banking of MSC. View Publication

The demonstration that the small synthetic molecule reversine [2-(4-morpholinoanilino)-N6-cyclohexyladenine] promotes the dedifferentiation of committed cells into multipotent progenitor-type cells has raised hopes on the exploitation of this small chemical tool for the generation of stem cells. Here,we show that reversine causes a failure in cytokinesis and induces polyploidization. These effects of reversine are due to the inhibition of Aurora A and B,two related kinases that are implicated in several aspects of mitosis and that are frequently amplified and overexpressed in human tumors. Reversine inhibits the phosphorylation of histone H3,a direct downstream target of Aurora kinases. Similarly to the Aurora kinase inhibitor VX-680,which has recently entered phase II clinical trials for cancer treatment,reversine inhibited colony formation of leukemic cells from patients with acute myeloid leukemia but was significantly less toxic than VX-680 on cells from healthy donors. The crystal structure of the reversine-Aurora B kinase complex shows that reversine is a novel class of ATP-competitive Aurora kinase inhibitors. Thus,although our studies raise serious doubts on the application of reversine in regenerative medicine,they support the paradigm that reversine might be a useful agent in cancer chemotherapy. View Publication

The regulated production of neurons in the hippocampus throughout life underpins important brain functions such as learning and memory. Surprisingly,however,studies have so far failed to identify a resident hippocampal stem cell capable of providing the renewable source of these neurons. Here,we report that depolarizing levels of KCl produce a threefold increase in the number of neurospheres generated from the adult mouse hippocampus. Most interestingly,however,depolarizing levels of KCl led to the emergence of a small subpopulation of precursors (approximately eight per hippocampus) with the capacity to generate very large neurospheres (textgreater 250 microm in diameter). Many of these contained cells that displayed the cardinal properties of stem cells: multipotentiality and self-renewal. In contrast,the same conditions led to the opposite effect in the other main neurogenic region of the brain,the subventricular zone,in which neurosphere numbers decreased by approximately 40% in response to depolarizing levels of KCl. Most importantly,we also show that the latent hippocampal progenitor population can be activated in vivo in response to prolonged neural activity found in status epilepticus. This work provides the first direct evidence of a latent precursor and stem cell population in the adult hippocampus,which is able to be activated by neural activity. Because the latent population is also demonstrated to reside in the aged animal,defining the precise mechanisms that underlie its activation may provide a means to combat the cognitive deficits associated with a decline in neurogenesis. View Publication

MDS is characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Development of effective treatments has been impeded by limited insight into pathogenic pathways governing dysplastic growth of hematopoietic progenitors. We demonstrate that smad2,a downstream mediator of transforming growth factor-beta (TGF-beta) receptor I kinase (TBRI) activation,is constitutively activated in MDS bone marrow (BM) precursors and is overexpressed in gene expression profiles of MDS CD34(+) cells,providing direct evidence of overactivation of TGF-beta pathway in this disease. Suppression of the TGF-beta signaling by lentiviral shRNA-mediated down-regulation of TBRI leads to in vitro enhancement of hematopoiesis in MDS progenitors. Pharmacologic inhibition of TBRI (alk5) kinase by a small molecule inhibitor,SD-208,inhibits smad2 activation in hematopoietic progenitors,suppresses TGF-beta-mediated gene activation in BM stromal cells,and reverses TGF-beta-mediated cell-cycle arrest in BM CD34(+) cells. Furthermore,SD-208 treatment alleviates anemia and stimulates hematopoiesis in vivo in a novel murine model of bone marrow failure generated by constitutive hepatic expression of TGF-beta1. Moreover,in vitro pharmacologic inhibition of TBRI kinase leads to enhancement of hematopoiesis in varied morphologic MDS subtypes. These data directly implicate TGF-beta signaling in the pathobiology of ineffective hematopoiesis and identify TBRI as a potential therapeutic target in low-risk MDS. View Publication

It has recently been shown that interleukin (IL)-21 is produced by Th17 cells,functions as an autocrine growth factor for Th17 cells,and plays critical roles in autoimmune diseases. In this study,we investigated the differentiation and characteristics of IL-21-producing CD4(+) T cells by intracellular staining. Unexpectedly,we found that under Th17-polarizing conditions,the majority of IL-21-producing CD4(+) T cells did not produce IL-17A and -17F. We also found that IL-6 and -21 potently induced the development of IL-21-producing CD4(+) T cells without the induction of IL-4,IFN-gamma,IL-17A,or IL-17F production. On the other hand,TGF-beta inhibited IL-6- and IL-21-induced development of IL-21-producing CD4(+) T cells. IL-2 enhanced the development of IL-21-producing CD4(+) T cells under Th17-polarizing conditions. Finally,IL-21-producing CD4(+) T cells exhibited a stable phenotype of IL-21 production in the presence of IL-6,but retained the potential to produce IL-4 under Th2-polarizing conditions and IL-17A under Th17-polarizing conditions. These results suggest that IL-21-producing CD4(+) T cells exhibit distinct characteristics from Th17 cells and develop preferentially in an IL-6-rich environment devoid of TGF-beta,and that IL-21 functions as an autocrine growth factor for IL-21-producing CD4(+) T cells. View Publication

BACKGROUND: Human embryonic stem cells (hESC) provide a unique model to study early events in human development. The hESC-derived cells can potentially be used to replace or restore different tissues including neuronal that have been damaged by disease or injury.backslashnbackslashnMETHODOLOGY AND PRINCIPAL FINDINGS: The cells of two different hESC lines were converted to neural rosettes using adherent and chemically defined conditions. The progenitor cells were exposed to retinoic acid (RA) or to human recombinant basic fibroblast growth factor (bFGF) in the late phase of the rosette formation. Exposing the progenitor cells to RA suppressed differentiation to rostral forebrain dopamine neural lineage and promoted that of spinal neural tissue including motor neurons. The functional characteristics of these differentiated neuronal precursors under both,rostral (bFGF) and caudalizing (RA) signals were confirmed by patch clamp analysis.backslashnbackslashnCONCLUSIONS/SIGNIFICANCE: These findings suggest that our differentiation protocol has the capacity to generate region-specific and electrophysiologically active neurons under in vitro conditions without embryoid body formation,co-culture with stromal cells and without presence of cells of mesodermal or endodermal lineages. View Publication

Embryonic stem cells (ESCs),which have characteristics such as self-renewal,indefinite proliferation,and pluripotency,are thought to hold great promise for regenerative medicine. ESCs are generally cultured on mouse embryonic fibroblast (MEF) or MEF conditioned medium (MEF-CM). However,for therapeutic applications,it is preferable for ESCs to be cultured under chemically defined conditions. Here,we report synthetic compounds that allow expansion of undifferentiated mouse ESCs in the absence of MEF,Leukemia Inhibitory Factor (LIF),and Fetal Bovine Serum (FBS). ESCs cultured for more than 30 d in a serum-free medium supplemented with indole derivertives retained their characteristic morphology and expressed markers such as SSEA-1,OCT3/4,Rex-1,Sox2,and Nanog. They consistently differentiated into many types of cells,including neurons,muscle cells,and hepatocytes. These results indicate that our compounds provide a more efficient and safer large-scale culture system for pluripotent ESCs,and hence might contribute to the use of ESCs in therapeutic applications. View Publication

Vitamin D has been known to be important for skeletal development and growth but the mechanism whereby it affects these processes is not well understood. We report now that the hormonal metabolite of vitamin D3,namely 1,25-dihydroxyvitamin D3,stimulates chondrogenesis in cultures of stage 24 chick embryo limb bud mesenchymal cells,as evidenced by morphologic changes as well as by increased transcription of collagen type II and core protein genes. This effect appears to be specific to 1,25(OH)2D3 since 24,25(OH)2D3 or D3 does not influence chondrogenesis in this system,and is probably mediated via the specific 1,25(OH)2D3 receptor protein which is undetectable in untreated cells but appears following exposure to the hormone. View Publication