技术资料

Mesenchymal stem cells (MSCs) are being tested in a wide range of human diseases; however,loss of potency and inconsistent quality severely limit their use. To overcome these issues,we have utilized a developmental precursor called the hemangioblast as an intermediate cell type in the derivation of a highly potent and replenishable population of MSCs from human embryonic stem cells (hESCs). This method circumvents the need for labor-intensive hand-picking,scraping,and sorting that other hESC-MSC derivation methods require. Moreover,unlike previous reports on hESC-MSCs,we have systematically evaluated their immunomodulatory properties and in vivo potency. As expected,they dynamically secrete a range of bioactive factors,display enzymatic activity,and suppress T-cell proliferation that is induced by either allogeneic cells or mitogenic stimuli. However,they also display unique immunophenotypic properties,as well as a smaller size and textgreater30,000-fold proliferative capacity than bone marrow-derived MSCs. In addition,this is the first report which demonstrates that hESC-MSCs can inhibit CD83 up-regulation and IL-12p70 secretion from dendritic cells and enhance regulatory T-cell populations induced by interleukin 2 (IL-2). This is also the first report which shows that hESC-MSCs have therapeutic efficacy in two different autoimmune disorder models,including a marked increase in survival of lupus-prone mice and a reduction of symptoms in an autoimmune model of uveitis. Our data suggest that this novel and therapeutically active population of MSCs could overcome many of the obstacles that plague the use of MSCs in regenerative medicine and serve as a scalable alternative to current MSC sources. View Publication

GABAergic interneurons regulate cortical neural networks by providing inhibitory inputs,and their malfunction,resulting in failure to intricately regulate neural circuit balance,is implicated in brain diseases such as Schizophrenia,Autism,and Epilepsy. During early development,GABAergic interneuron progenitors arise from the ventral telencephalic area such as medial ganglionic eminence (MGE) and caudal ganglionic eminence (CGE) by the actions of secreted signaling molecules from nearby organizers,and migrate to their target sites where they form local synaptic connections. In this study,using combinatorial and temporal modulation of developmentally relevant dorsoventral and rostrocaudal signaling pathways (SHH,Wnt,and FGF8),we efficiently generated MGE cells from multiple human pluripotent stem cells. Most importantly,modulation of FGF8/FGF19 signaling efficiently directed MGE versus CGE differentiation. Human MGE cells spontaneously differentiated into Lhx6-expressing GABAergic interneurons and showed migratory properties. These human MGE-derived neurons generated GABA,fired action potentials,and displayed robust GABAergic postsynaptic activity. Transplantation into rodent brains results in well-contained neural grafts enriched with GABAergic interneurons that migrate in the host and mature to express somatostatin or parvalbumin. Thus,we propose that signaling modulation recapitulating normal developmental patterns efficiently generate human GABAergic interneurons. This strategy represents a novel tool in regenerative medicine,developmental studies,disease modeling,bioassay,and drug screening. View Publication

A major concern in Pluripotent Stem Cell (PSC)-derived cell replacement therapy is the risk of teratoma formation from contaminating undifferentiated cells. Removal of undifferentiated cells from differentiated cultures is an essential step before PSC-based cell therapies can be safely deployed in a clinical setting. We report a group of novel small molecules that are cytotoxic to PSCs. Our data indicates that these molecules are specific and potent in their activity allowing rapid eradication of undifferentiated cells. Experiments utilizing mixed PSC and primary human neuronal and cardiomyocyte cultures demonstrate that up to a 6-fold enrichment for specialized cells can be obtained without adversely affecting cell viability and function. Several structural variants were synthesized to identify key functional groups and to improve specificity and efficacy. Comparative microarray analysis and ensuing RNA knockdown studies revealed involvement of the PERK/ATF4/DDIT3 ER stress pathway. Surprisingly,cell death following ER stress induction was associated with a concomitant decrease in endogenous ROS levels in PSCs. Undifferentiated cells treated with these molecules preceding transplantation fail to form teratomas in SCID mice. Furthermore,these molecules remain non-toxic and non-teratogenic to zebrafish embryos suggesting that they may be safely used in vivo. View Publication

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients can be a good model for studying human diseases and for future therapeutic regenerative medicine. Current initiatives to establish human iPSC (hiPSC) banking face challenges in recruiting large numbers of donors with diverse diseased,genetic,and phenotypic representations. In this study,we describe the efficient derivation of transgene-free hiPSCs from human finger-prick blood. Finger-prick sample collection can be performed on a do-it-yourself" basis by donors and sent to the hiPSC facility for reprogramming. We show that single-drop volumes of finger-prick samples are sufficient for performing cellular reprogramming� View Publication

The expansion of human pluripotent stem cells (hPSC) for biomedical applications generally compels a defined,reliable,and scalable platform. Bioreactors offer a three-dimensional culture environment that relies on the implementation of microcarriers (MC),as supports for cell anchorage and their subsequent growth. Polystyrene microspheres/MC coated with adhesion-promoting extracellular matrix (ECM) protein,vitronectin (VN),or laminin (LN) have been shown to support hPSC expansion in a static environment. However,they are insufficient to promote human embryonic stem cells (hESC) seeding and their expansion in an agitated environment. The present study describes an innovative technology,consisting of a cationic charge that underlies the ECM coatings. By combining poly-L-lysine (PLL) with a coating of ECM protein,cell attachment efficiency and cell spreading are improved,thus enabling seeding under agitation in a serum-free medium. This coating combination also critically enables the subsequent formation and evolution of hPSC/MC aggregates,which ensure cell viability and generate high yields. Aggregate dimensions of at least 300 $\$ during early cell growth give rise to ≈15-fold expansion at 7 days' culture. Increasing aggregate numbers at a quasi-constant size of ≈300 $\$ indicates hESC growth within a self-regulating microenvironment. PLL+LN enables cell seeding and aggregate evolution under constant agitation,whereas PLL+VN requires an intermediate 2-day static pause to attain comparable aggregate sizes and correspondingly high expansion yields. The cells' highly reproducible bioresponse to these defined and characterized MC surface properties is universal across multiple cell lines,thus confirming the robustness of this scalable expansion process in a defined environment. View Publication

NK cells are key regulators of innate defense against mouse CMV (MCMV). Like NK cells,NKT cells also produce high levels of IFN-γ rapidly after MCMV infection. However,whether similar mechanisms govern activation of these two cell types,as well as the significance of NKT cells for host resistance,remain unknown. In this article,we show that,although both NKT and NK cells are activated via cytokines,their particular cytokine requirements differ significantly in vitro and in vivo. IL-12 is required for NKT cell activation in vitro but is not sufficient,whereas NK cells have the capacity to be activated more promiscuously in response to individual cytokines from innate cells. In line with these results,GM-CSF-derived dendritic cells activated only NK cells upon MCMV infection,consistent with their virtual lack of IL-12 production,whereas Flt3 ligand-derived dendritic cells produced IL-12 and activated both NK and NKT cells. In vivo,NKT cell activation was abolished in IL-12(-/-) mice infected with MCMV,whereas NK cells were still activated. In turn,splenic NK cell activation was more IL-18 dependent. The differential requirements for IL-12 and IL-18 correlated with the levels of cytokine receptor expression by NK and NKT cells. Finally,mice lacking NKT cells showed reduced control of MCMV,and depleting NK cells further enhanced viral replication. Taken together,our results show that NKT and NK cells have differing requirements for cytokine-mediated activation,and both can contribute nonredundantly to MCMV defense,revealing that these two innate lymphocyte subsets function together to fine-tune antiviral responses. View Publication

One of the differences between murine and human embryonic stem cells (ESCs) is the epigenetic state of the X chromosomes in female lines. Murine ESCs (mESCs) present two transcriptionally active Xs that will undergo the dosage compensation process of XCI upon differentiation,whereas most human ESCs (hESCs) spontaneously inactivate one X while keeping their pluripotency. Whether this reflects differences in embryonic development of mice and humans,or distinct culture requirements for the two kinds of pluripotent cells is not known. Recently it has been shown that hESCs established in physiological oxygen levels are in a stable pre-XCI state equivalent to that of mESCs,suggesting that culture in low oxygen concentration is enough to preserve that epigenetic state of the X chromosomes. Here we describe the establishment of two new lines of hESCs under physiological oxygen level and the characterization of the XCI state in the 46,XX line BR-5. We show that a fraction of undifferentiated cells present XIST RNA accumulation and single H3K27me foci,characteristic of the inactive X. Moreover,analysis of allele specific gene expression suggests that pluripotent BR-5 cells present completely skewed XCI. Our data indicate that physiological levels of oxygen are not sufficient for the stabilization of the pre-XCI state in hESCs. View Publication

Plasmacytoid dendritic cells (pDCs) play a vital role in activation of anti-HIV-1 immunity,and suppression of pDCs might mitigate immune responses against HIV-1. HIV-1 gp120 high-mannose has been attributed immunosuppressive roles in human myeloid DCs,but no receptors for high-mannose have so far been reported on human pDCs. Here we show that upon activation with HIV-1 or by a synthetic compound triggering the same receptor in human pDCs as single-stranded RNA,human pDCs upregulate the mannose receptor (MR,CD206). To examine the functional outcome of this upregulation,inactivated intact or viable HIV-1 particles with various degrees of mannosylation were cultured with pDCs. Activation of pDCs was determined by assaying secretion of IFN-alpha,viability,and upregulation of several pDC-activation markers: CD40,CD86,HLA-DR,CCR7,and PD-L1. The level of activation negatively correlated with degree of mannosylation,however,subsequent reduction in the original mannosylation level had no effect on the pDC phenotype. Furthermore,two of the infectious HIV-1 strains induced profound necrosis in pDCs,also in a mannose-independent manner. We therefore conclude that natural mannosylation of HIV-1 is not involved in HIV-1-mediated immune suppression of pDCs. View Publication

Cancer stem cells (CSCs) have been identified in a variety of cancers and emerged as a new target for cancer therapy. CSCs are resistant to many current cancer treatments,including chemotherapy and radiation therapy. Therefore,eradication of this cell population is a primary objective in cancer therapy. Here,we report gold nanorods (AuNRs) mediated photothermal treatment can selectively eliminate CSCs in MCF-7 breast cancer cells. It significantly reduced the aldehyde dehydrogenase positive (ALDH(+)) cells subpopulation and the mammosphere formation ability of treated cells. Also,the gene expression of stem cell markers was decreased. Cellular uptake assay revealed that polyelectrolyte conjugated AuNRs could be internalized by CSCs much more and faster than non cancer stem cells (NCSCs),which might be the main reason for the selective elimination of CSCs. We further loaded salinomycin (SA),a CSCs inhibitor with polyelectrolyte conjugated AuNRs to get a synergistic CSCs inhibition. Enhanced inhibition of CSCs was obtained by NIR light triggered drug release and hyperthermia. This CSCs-targeted thermo-chemotherapy platform provides a new combinatorial strategy for efficient inhibition of CSCs,which is promising to improve cancer treatment and may overcome the chemoresistance and recurrence of cancer. View Publication

Mesenchymal stem cells (MSCs) have a high potential for therapeutic efficacy in treating diverse musculoskeletal injuries and cardiovascular diseases,and for ameliorating the severity of graft-versus-host and autoimmune diseases. While most of these clinical applications require substantial cell quantities,the number of MSCs that can be obtained initially from a single donor is limited. Reports on the derivation of MSC-like cells from pluripotent stem cells (PSCs) are,thus,of interest,as the infinite proliferative capacity of PSCs opens the possibility to generate large amounts of uniform batches of MSCs. However,characterization of such MSC-like cells is currently inadequate,especially with regard to the question of whether these cells are equivalent or identical to MSCs. In this study,we have derived MSC-like cells [induced PSC-derived MSC-like progenitor cells (iMPCs)] using four different methodologies from a newly established induced PSC line reprogrammed from human bone marrow stromal cells (BMSCs),and compared the iMPCs directly with the originating parental BMSCs. The iMPCs exhibited typical MSC/fibroblastic morphology and MSC-typical surface marker profile,and they were capable of differentiation in vitro along the osteogenic,chondrogenic,and adipogenic lineages. However,compared with the parental BMSCs,iMPCs displayed a unique expression pattern of mesenchymal and pluripotency genes and were less responsive to traditional BMSC differentiation protocols. We,therefore,conclude that iMPCs generated from PSCs via spontaneous differentiation represent a distinct population of cells which exhibit MSC-like characteristics. View Publication

Approximately 70% of KRAS-positive colorectal cancers (CRCs) have a CpG island methylator phenotype (CIMP) characterized by aberrant DNA hypermethylation and transcriptional silencing of many genes. The factors involved in,and the mechanistic basis of,CIMP is not understood. Among the CIMP genes are the tumor suppressors p14(ARF),p15(INK4B),and p16(INK4A),encoded by the INK4-ARF locus. In this study,we perform an RNA interference screen and identify ZNF304,a zinc-finger DNA-binding protein,as the pivotal factor required for INK4-ARF silencing and CIMP in CRCs containing activated KRAS. In KRAS-positive human CRC cell lines and tumors,ZNF304 is bound at the promoters of INK4-ARF and other CIMP genes. Promoter-bound ZNF304 recruits a corepressor complex that includes the DNA methyltransferase DNMT1,resulting in DNA hypermethylation and transcriptional silencing. KRAS promotes silencing through upregulation of ZNF304,which drives DNA binding. Finally,we show that ZNF304 also directs transcriptional silencing of INK4-ARF in human embryonic stem cells. DOI: http://dx.doi.org/10.7554/eLife.02313.001. View Publication

Efficient derivation of large-scale motor neurons (MNs) from human pluripotent stem cells is central to the understanding of MN development,modelling of MN disorders in vitro and development of cell-replacement therapies. Here we develop a method for rapid (20 days) and highly efficient (˜70%) differentiation of mature and functional MNs from human pluripotent stem cells by tightly modulating neural patterning temporally at a previously undefined primitive neural progenitor stage. This method also allows high-yield (textgreater250%) MN production in chemically defined adherent cultures. Furthermore,we show that Islet-1 is essential for formation of mature and functional human MNs,but,unlike its mouse counterpart,does not regulate cell survival or suppress the V2a interneuron fate. Together,our discoveries improve the strategy for MN derivation,advance our understanding of human neural specification and MN development,and provide invaluable tools for human developmental studies,drug discovery and regenerative medicine. View Publication