技术资料

Genome editing with engineered site-specific endonucleases involves nonhomologous end-joining,leading to reading frame disruption. The approach is applicable to dominant negative disorders,which can be treated simply by knocking out the mutant allele,while leaving the normal allele intact. We applied this strategy to dominant dystrophic epidermolysis bullosa (DDEB),which is caused by a dominant negative mutation in the COL7A1 gene encoding type VII collagen (COL7). We performed genome editing with TALENs and CRISPR/Cas9 targeting the mutation,c.80688084delinsGA. We then cotransfected Cas9 and guide RNA expression vectors expressed with GFP and DsRed,respectively,into induced pluripotent stem cells (iPSCs) generated from DDEB fibroblasts. After sorting,90% of the iPSCs were edited,and we selected four gene-edited iPSC lines for further study. These iPSCs were differentiated into keratinocytes and fibroblasts secreting COL7. RT-PCR and Western blot analyses revealed gene-edited COL7 with frameshift mutations degraded at the protein level. In addition,we confirmed that the gene-edited truncated COL7 could neither associate with normal COL7 nor undergo triple helix formation. Our data establish the feasibility of mutation site-specific genome editing in dominant negative disorders. View Publication

Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including,synthetic and structural biology,cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity,thus impeding unrestricted multigene expression. We developed MultiPrime,a modular,non-cytotoxic,non-integrating,baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types,including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming,and for genome editing and engineering by CRISPR/Cas9. Moreover,we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool,to deliver multiple genes for a wide range of applications in primary and established mammalian cells. View Publication

We have previously demonstrated that mouse embryonic stem (ES) cells differentiated on three-dimensional (3D),highly porous,tantalum-based scaffolds (Cytomatrixtrade mark) have significantly higher hematopoietic differentiation efficiency than those cultured under conventional two-dimensional (2D) tissue culture conditions. In addition,ES cell-seeded scaffolds cultured inside spinner bioreactors showed further enhancement in hematopoiesis compared to static conditions. In the present study,we evaluated how these various biomaterial-based culture conditions,e.g. 2D vs. 3D scaffolds and static vs. dynamic,influence the global gene expression profile of differentiated ES cells. We report that compared to 2D tissue culture plates,cells differentiated on porous,Cytomatrixtrade mark scaffolds possess significantly higher expression levels of extracellular matrix (ECM)-related genes,as well as genes that regulate cell growth,proliferation and differentiation. In addition,these differences in gene expression were more pronounced in 3D dynamic culture compared to 3D static culture. We report specific genes that are either uniquely expressed under each condition or are quantitatively regulated,i.e. over expressed or inhibited by a specific culture environment. We conclude that that biomaterial-based 3D cultures,especially under dynamic conditions,might favor efficient hematopoietic differentiation of ES cells by stimulating increased expression of specific ECM proteins,growth factors and cell adhesion related genes while significantly down-regulating genes that act to inhibit expression of these molecules. View Publication

Neural progenitor cells (NPCs) can be induced from somatic cells by defined factors. Here we report that NPCs can be generated from mouse embryonic fibroblasts by a chemical cocktail,namely VCR (V,VPA,an inhibitor of HDACs; C,CHIR99021,an inhibitor of GSK-3 kinases and R,Repsox,an inhibitor of TGF-β pathways),under a physiological hypoxic condition. These chemical-induced NPCs (ciNPCs) resemble mouse brain-derived NPCs re- garding their proliferative and self-renewing abilities,gene expression profiles,and multipotency for different neu- roectodermal lineages in vitro and in vivo. Further experiments reveal that alternative cocktails with inhibitors of histone deacetylation,glycogen synthase kinase,and TGF-β pathways show similar efficacies for ciNPC induction. Moreover,ciNPCs can also be induced from mouse tail-tip fibroblasts and human urinary cells with the same chemi- cal cocktail VCR. Thus our study demonstrates that lineage-specific conversion of somatic cells to NPCs could be achieved by chemical cocktails without introducing exogenous factors. View Publication

The physiologic function of BUBR1,a key component of the spindle checkpoint,was examined by generating BUBR1-mutant mice. BUBR1(-/-) embryos failed to survive beyond day 8.5 in utero as a result of extensive apoptosis. Whereas BUBR1(+/-) blastocysts grew relatively normally in vitro,BUBR1(-/-) blastocysts exhibited impaired proliferation and atrophied. Adult BUBR1(+/-) mice manifested splenomegaly and abnormal megakaryopoiesis. BUBR1 haploinsufficiency resulted in an increase in the number of splenic megakaryocytes,which was correlated with an increase in megakaryocytic,but a decrease in erythroid,progenitors in bone marrow cells. RNA interference-mediated down-regulation of BUBR1 also caused an increase in polyploidy formation in murine embryonic fibroblast cells and enhanced megakaryopoiesis in bone marrow progenitor cells. However,enhanced megakaryopoiesis in BUBR1(+/-) mice was not correlated with a significant increase in platelets in peripheral blood,which was at least partly due to a defect in the formation of proplatelet-producing megakaryocytes. Together,these results indicate that BUBR1 is essential for early embryonic development and normal hematopoiesis. View Publication

Embryonic stem cells can be induced in vitro,by coculture with the stromal line RP.0.10 and a mixture of interleukins 3,6,and 7,to differentiate into T (Joro75+) and B (B-220+) lymphocyte progenitors and other (Thy-1+,PgP-1+,c-kit+,Joro75-,B-220-,F4/80-,Mac-1-) hemopoietic precursors. The progeny of in vitro-induced embryonic stem cells can reconstitute the lymphoid compartments of T- and B-lymphocyte-deficient scid mice and generate mature T and B lymphocytes in sublethally irradiated normal mice. Exogenous cytokines can dramatically alter the developmental fate of embryonic stem cells in culture. The in vitro system described here should facilitate the study of molecular events leading to cell-lineage commitment and to the formation of hemopoietic stem cells and their immediate lymphoid progeny. View Publication

Undifferentiated pluripotent stem cells with flexible developmental potentials are not normally found in peripheral blood. However,such cells have recently been reported to reside in the bone marrow. Herein are reported methods of inducing pluripotency in cells derived from unmobilised adult human peripheral blood. In response to the inclusion of purified CR3/43 monoclonal antibody (mAb) to well-established culture conditions,mononuclear cells (MNC) obtained from a single blood donor are converted into pluripotent haematopoietic,neuronal and cardiomyogenic progenitor stem cells or undifferentiated stem cells. The haematopoietic stem cells are CD34+,clonogenic and have been shown to repopulate non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The neuronal precursors transcribe the primitive stem cell markers OCT-4 and nestin,and on maturation,differentially stain positive for neuronal,glial or oligodendrocyte-specific antigens. The cardiomyogenic progenitor stem cells form large bodies of asynchronously beating cells and differentiate into mature cardiomyocytes which transcribe GATA-4. The undifferentiated stem cells do not express haematopoietic-associated markers,are negative for major histocompatibility complex (MHC) class I and II antigens,transcribe high levels of OCT-4 and form embryoid body (EB)-like structures. This induction of stem cell-like plasticity in MNC may have proceeded by a process of retrodifferentiation but,in any case,could have profound clinical and pharmacological implications. Finally,the flexibility and the speed by which a variety of stem cell classes can be generated ex vivo from donor blood could potentially transfer this novel process into a less invasive automated clinical procedure. View Publication

Embryonic stem (ES) cells have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that influence cell fate decisions and to develop scalable methods of cell production. We compared four standard ES cell differentiation culture systems by measuring aspects of embryoid body (EB) formation efficiency and cell proliferation,and by tracking development of a specific differentiated tissue type-blood-using functional (colony-forming cell) and phenotypic (Flk-1 and CD34 expression) assays. We report that individual murine ES cells form EBs with an efficiency of 42 +/- 9%,but this value is rarely obtained because of EB aggregation-a process whereby two or more individual ES cells or EBs fuse to form a single,larger cell aggregate. Regardless of whether EBs were generated from a single ES cell in methylcellulose or liquid suspension culture,or aggregates of ES cells in hanging drop culture,they grew to a similar maximum cell number of 28,000 +/- 9,000 cells per EB. Among the three methods for EB generation in suspension culture there were no differences in the kinetics or frequency of hematopoietic development. Thus,initiating EBs with a single ES cell and preventing EB aggregation should allow for maximum yield of differentiated cells in the EB system. EB differentiation cultures were also compared to attached differentiation culture using the same outputs. Attached colonies were not similarly limited in cell number; however,hematopoietic development in attached culture was impaired. The percentage of early Flk-1 and CD34 expressing cells was dramatically lower than in EBs cultured in suspension,whereas hematopoietic colony formation was almost completely inhibited. These results provide a foundation for development of efficient,scalable bioprocesses for ES cell differentiation,and inform novel methods for the production of hematopoietic tissues. View Publication

BACKGROUND Fabry disease (FD) is a lysosomal storage disease in which glycosphingolipids (GB3) accumulate in organs of the human body,leading to idiopathic hypertrophic cardiomyopathy and target organ damage. Its pathophysiology is still poorly understood. OBJECTIVES We aimed to generate patient-specific induced pluripotent stem cells (iPSC) from FD patients presenting cardiomyopathy to determine whether the model could recapitulate key features of the disease phenotype and to investigate the energy metabolism in Fabry disease. METHODS Peripheral blood mononuclear cells from a 30-year-old Chinese man with a diagnosis of Fabry disease,GLA gene (IVS4+919G>A) mutation were reprogrammed into iPSCs and differentiated into iPSC-CMs and energy metabolism was analyzed in iPSC-CMs. RESULTS The FD-iPSC-CMs recapitulated numerous aspects of the FD phenotype including reduced GLA activity,cellular hypertrophy,GB3 accumulation and impaired contractility. Decreased energy metabolism with energy utilization shift to glycolysis was observed,but the decreased energy metabolism was not modified by enzyme rescue replacement (ERT) in FD-iPSCs-CMs. CONCLUSION This model provided a promising in vitro model for the investigation of the underlying disease mechanism and development of novel therapeutic strategies for FD. This potential remedy for enhancing the energetic network and utility efficiency warrants further study to identify novel therapies for the disease. View Publication

Tissue-resident macrophages,such as microglia,Kupffer cells,and Langerhans cells,derive from Myb-independent yolk sac (YS) progenitors generated before the emergence of hematopoietic stem cells (HSCs). Myb-independent YS-derived resident macrophages self-renew locally,independently of circulating monocytes and HSCs. In contrast,adult blood monocytes,as well as infiltrating,gut,and dermal macrophages,derive from Myb-dependent HSCs. These findings are derived from the mouse,using gene knockouts and lineage tracing,but their applicability to human development has not been formally demonstrated. Here,we use human induced pluripotent stem cells (iPSCs) as a tool to model human hematopoietic development. By using a CRISPR-Cas9 knockout strategy,we show that human iPSC-derived monocytes/macrophages develop in an MYB-independent,RUNX1-,and SPI1 (PU.1)-dependent fashion. This result makes human iPSC-derived macrophages developmentally related to and a good model for MYB-independent tissue-resident macrophages,such as alveolar and kidney macrophages,microglia,Kupffer cells,and Langerhans cells. View Publication

Classical lissencephaly is a genetic neurological disorder associated with mental retardation and intractable epilepsy,and Miller-Dieker syndrome (MDS) is the most severe form of the disease. In this study,to investigate the effects of MDS on human progenitor subtypes that control neuronal output and influence brain topology,we analyzed cerebral organoids derived from control and MDS-induced pluripotent stem cells (iPSCs) using time-lapse imaging,immunostaining,and single-cell RNA sequencing. We saw a cell migration defect that was rescued when we corrected the MDS causative chromosomal deletion and severe apoptosis of the founder neuroepithelial stem cells,accompanied by increased horizontal cell divisions. We also identified a mitotic defect in outer radial glia,a progenitor subtype that is largely absent from lissencephalic rodents but critical for human neocortical expansion. Our study,therefore,deepens our understanding of MDS cellular pathogenesis and highlights the broad utility of cerebral organoids for modeling human neurodevelopmental disorders. View Publication

Many neuropsychiatric disorders are thought to result from subtle changes in neural circuit formation. We used human embryonic stem cells and induced pluripotent stem cells (hiPSCs) to model mature,post-mitotic excitatory neurons and examine effects of fibroblast growth factor 2 (FGF2). FGF2 gene expression is known to be altered in brain regions of major depressive disorder (MDD) patients and FGF2 has anti-depressive effects in animal models of depression. We generated stable inducible neurons (siNeurons) conditionally expressing human neurogenin-2 (NEUROG2) to generate a homogenous population of post-mitotic excitatory neurons and study the functional as well as the transcriptional effects of FGF2. Upon induction of NEUROG2 with doxycycline,the vast majority of cells are post-mitotic,and the gene expression profile recapitulates that of excitatory neurons within 6 days. Using hES cell lines that inducibly express NEUROG2 as well as GCaMP6f,we were able to characterize spontaneous calcium activity in these neurons and show that calcium transients increase in the presence of FGF2. The FGF2-responsive genes were determined by RNA-Seq. FGF2-regulated genes previously identified in non-neuronal cell types were up-regulated (EGR1,ETV4,SPRY4,and DUSP6) as a result of chronic FGF2 treatment of siNeurons. Novel neuron-specific genes were also identified that may mediate FGF2-dependent increases in synaptic efficacy including NRXN3,SYT2,and GALR1. Since several of these genes have been implicated in MDD previously,these results will provide the basis for more mechanistic studies of the role of FGF2 in MDD. View Publication