技术资料

Generation of induced pluripotent stem (iPS) cells from somatic cells has been successfully achieved by ectopic expression of four transcription factors,Oct4,Sox2,Klf4 and c-Myc,also known as the Yamanaka factors. In practice,initial iPS colonies are picked based on their embryonic stem (ES) cell-like morphology,but often may go on to fail subsequent assays,such as the alkaline phosphate (AP) assay. In this study,we co-expressed through lenti-viral delivery the Yamanaka factors in amniotic fluid-derived (AF) cells. ES-like colonies were picked onto a traditional feeder layer and a high percentage AF-iPS with partial to no AP activity was found. Interestingly,we obtained an overwhelming majority of fully stained AP positive (AP+) AF-iPS colonies when colonies were first seeded on a feeder-free culture system,and then transferred to a feeder layer for expansion. Furthermore,colonies with no AP activity were not detected. This screening step decreased the variation seen between morphology and AP assay. We observed the AF-iPS colonies grown on the feeder layer with 28% AP+ colonies,45% AP partially positive (AP+/-) colonies and 27% AP negative (AP-) colonies,while colonies screened by the feeder-free system were 84% AP+ colonies,16% AP+/- colonies and no AP- colonies. The feeder-free screened AP+ AF-iPS colonies were also positive for pluripotent markers,OCT4,SOX2,NANOG,TRA-1-60,TRA-1-81,SSEA-3 and SSEA-4 as well as having differentiation abilities into three germ layers in vitro and in vivo. In this study,we report a simplistic,one-step method for selection of AP+ AF-iPS cells via feeder-free screening. View Publication

Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs),and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the $\beta$-globin gene (HBB). Sickle hemoglobin damages erythrocytes,causing vasoocclusion,severe pain,progressive organ damage,and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA,together with a single-stranded DNA oligonucleotide donor (ssODN),to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice,ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing,enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells,and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases. View Publication

The use of human pluripotent stem cells (hPSCs) in cell therapy is hindered by the tumorigenic risk from residual undifferentiated cells. Here we performed a high-throughput screen of over 52,000 small molecules and identified 15 pluripotent cell-specific inhibitors (PluriSIns),nine of which share a common structural moiety. The PluriSIns selectively eliminated hPSCs while sparing a large array of progenitor and differentiated cells. Cellular and molecular analyses demonstrated that the most selective compound,PluriSIn 1,induces ER stress,protein synthesis attenuation,and apoptosis in hPSCs. Close examination identified this molecule as an inhibitor of stearoyl-coA desaturase (SCD1),the key enzyme in oleic acid biosynthesis,revealing a unique role for lipid metabolism in hPSCs. PluriSIn 1 was also cytotoxic to mouse blastocysts,indicating that the dependence on oleate is inherent to the pluripotent state. Finally,application of PluriSIn 1 prevented teratoma formation from tumorigenic undifferentiated cells. These findings should increase the safety of hPSC-based treatments. ?? 2013 Elsevier Inc. View Publication

Hematologic malignancies such as acute and chronic myeloid leukemia are characterized by the malignant transformation of immature CD34(+) progenitor cells. Transformation is associated with elevated expression of the Wilm's tumor gene encoded transcription factor (WT1). Here we demonstrate that WT1 can serve as a target for cytotoxic T lymphocytes (CTL) with exquisite specificity for leukemic progenitor cells. HLA-A0201- restricted CTL specific for WT1 kill leukemia cell lines and inhibit colony formation by transformed CD34(+) progenitor cells isolated from patients with chronic myeloid leukemia (CML),whereas colony formation by normal CD34(+) progenitor cells is unaffected. Thus,the tissue-specific transcription factor WT1 is an ideal target for CTL-mediated purging of leukemic progenitor cells in vitro and for antigen-specific therapy of leukemia and other WT1-expressing malignancies in vivo. View Publication

The translocation t(11;18)(q21;q21) that generates an API2-MALT1 fusion protein is the most common structural abnormality among the genetic defects reported in mucosa-associated lymphoid tissue (MALT)-type lymphomas,and its presence correlates with the apparent lack of further genetic instability or chromosomal imbalances. Hence,constitutive nuclear factor-kappaB (NF-kappaB) activation induced by the API2-MALT1 fusion protein is considered essential for B-cell transformation. To examine its role in B-cell development and lymphomagenesis,Emu-API2-MALT1 transgenic mice were produced. Our data show that expression of the API2-MALT1 fusion protein alone is not sufficient for the development of lymphoma masses within 50 weeks. Nevertheless,API2-MALT1 expression affected B-cell maturation in the bone marrow and triggered the specific expansion of splenic marginal zone B cells. Polyubiquitination of IkappaB kinase gamma (IKKgamma),indicative for enhanced NF-kappaB activation,was increased in splenic lymphocytes and promoted the survival of B cells ex vivo. In addition,we show that the API2-MALT1 fusion resided in the cholesterol- and sphingolipid-enriched membrane microdomains,termed lipid rafts. We provide evidence that association of the MALT1 COOH terminal with the lipid rafts,which is mediated by the API2 portion,is sufficient to trigger NF-kappaB activation via enhanced polyubiquitination of IKKgamma. Taken together,these data support the hypothesis that the API2-MALT1 fusion protein can contribute to MALT lymphoma formation via increased NF-kappaB activation. View Publication

Accidental insertional activation of proto-oncogenes and potential vector mobilization pose serious challenges for human gene therapy using retroviral vectors. Comparative analyses of integration sites of different retroviral vectors have elucidated distinct target site preferences,highlighting vectors based on the alpharetrovirus Rous sarcoma virus (RSV) as those with the most neutral integration spectrum. To date,alpharetroviral vector systems are based mainly on single constructs containing viral coding sequences and intact long terminal repeats (LTR). Even though they are considered to be replication incompetent in mammalian cells,the transfer of intact viral genomes is unacceptable for clinical applications,due to the risk of vector mobilization and the potentially immunogenic expression of viral proteins,which we minimized by setting up a split-packaging system expressing the necessary viral proteins in trans. Moreover,intact LTRs containing transcriptional elements are capable of activating cellular genes. By removing most of these transcriptional elements,we were able to generate a self-inactivating (SIN) alpharetroviral vector,whose LTR transcriptional activity is strongly reduced and whose transgene expression can be driven by an internal promoter of choice. Codon optimization of the alpharetroviral Gag/Pol expression construct and further optimization steps allowed the production of high-titer self-inactivating vector particles in human cells. We demonstrate proof of principle for the versatility of alpharetroviral SIN vectors for the genetic modification of murine and human hematopoietic cells at a low multiplicity of infection. View Publication

Fate allocation in the gastrulating embryo is spatially organized as cells differentiate into specialized cell types depending on their positions with respect to the body axes. There is a need for in vitro protocols that allow the study of spatial organization associated with this developmental transition. Although embryoid bodies and organoids can exhibit some spatial organization of differentiated cells,methods that generate embryoid bodies or organoids do not yield consistent and fully reproducible results. Here,we describe a micropatterning approach in which human embryonic stem cells are confined to disk-shaped,submillimeter colonies. After 42 h of BMP4 stimulation,cells form self-organized differentiation patterns in concentric radial domains,which express specific markers associated with the embryonic germ layers,reminiscent of gastrulating embryos. Our protocol takes 3 d; it uses commercial microfabricated slides (from CYTOO),human laminin-521 (LN-521) as extracellular matrix coating,and either conditioned or chemically defined medium (mTeSR). Differentiation patterns within individual colonies can be determined by immunofluorescence and analyzed with cellular resolution. Both the size of the micropattern and the type of medium affect the patterning outcome. The protocol is appropriate for personnel with basic stem cell culture training. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation. View Publication

Remodelling of the human embryo at implantation is indispensable for successful pregnancy. Yet it has remained mysterious because of the experimental hurdles that beset the study of this developmental phase. Here,we establish an in vitro system to culture human embryos through implantation stages in the absence of maternal tissues and reveal the key events of early human morphogenesis. These include segregation of the pluripotent embryonic and extra-embryonic lineages,and morphogenetic rearrangements leading to generation of a bilaminar disc,formation of a pro-amniotic cavity within the embryonic lineage,appearance of the prospective yolk sac,and trophoblast differentiation. Using human embryos and human pluripotent stem cells,we show that the reorganization of the embryonic lineage is mediated by cellular polarization leading to cavity formation. Together,our results indicate that the critical remodelling events at this stage of human development are embryo-autonomous,highlighting the remarkable and unanticipated self-organizing properties of human embryos. View Publication

Amniogenesis-the development of amnion-is a critical developmental milestone for early human embryogenesis and successful pregnancy. However,human amniogenesis is poorly understood due to limited accessibility to peri-implantation embryos and a lack of in vitro models. Here we report an efficient biomaterial system to generate human amnion-like tissue in vitro through self-organized development of human pluripotent stem cells (hPSCs) in a bioengineered niche mimicking the in vivo implantation environment. We show that biophysical niche factors act as a switch to toggle hPSC self-renewal versus amniogenesis under self-renewal-permissive biochemical conditions. We identify a unique molecular signature of hPSC-derived amnion-like cells and show that endogenously activated BMP-SMAD signalling is required for the amnion-like tissue development by hPSCs. This study unveils the self-organizing and mechanosensitive nature of human amniogenesis and establishes the first hPSC-based model for investigating peri-implantation human amnion development,thereby helping advance human embryology and reproductive medicine. View Publication

Cerebral organoids recapitulate human brain development at a considerable level of detail,even in the absence of externally added signaling factors. The patterning events driving this self-organization are currently unknown. Here,we examine the developmental and differentiative capacity of cerebral organoids. Focusing on forebrain regions,we demonstrate the presence of a variety of discrete ventral and dorsal regions. Clearing and subsequent 3D reconstruction of entire organoids reveal that many of these regions are interconnected,suggesting that the entire range of dorso-ventral identities can be generated within continuous neuroepithelia. Consistent with this,we demonstrate the presence of forebrain organizing centers that express secreted growth factors,which may be involved in dorso-ventral patterning within organoids. Furthermore,we demonstrate the timed generation of neurons with mature morphologies,as well as the subsequent generation of astrocytes and oligodendrocytes. Our work provides the methodology and quality criteria for phenotypic analysis of brain organoids and shows that the spatial and temporal patterning events governing human brain development can be recapitulated in vitro. View Publication

Tissue morphogenesis and organ formation are the consequences of biochemical and biophysical cues that lead to cellular spatial patterning in development. To model such events in vitro,we use PEG-patterned substrates to geometrically confine human pluripotent stem cell colonies and spatially present mechanical stress. Modulation of the WNT/β-catenin pathway promotes spatial patterning via geometric confinement of the cell condensation process during epithelial-mesenchymal transition,forcing cells at the perimeter to express an OCT4+ annulus,which is coincident with a region of higher cell density and E-cadherin expression. The biochemical and biophysical cues synergistically induce self-organizing lineage specification and creation of a beating human cardiac microchamber confined by the pattern geometry. These highly defined human cardiac microchambers can be used to study aspects of embryonic spatial patterning,early cardiac development and drug-induced developmental toxicity. View Publication

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