技术资料

It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells to Purkinje cells. In this study,we derived iPSCs from human fibroblasts and directed the specification of iPSCs first to Purkinje progenitors,by adding Fgf2 and insulin to the embryoid bodies (EBs) in a time-sensitive manner,which activates the endogenous production of Wnt1 and Fgf8 from EBs that further patterned the cells towards a midbrain-hindbrain-boundary tissue identity. Neph3-positive human Purkinje progenitors were sorted out by using flow cytometry and cultured either alone or with granule cell precursors,in a 2-dimensional or 3-dimensional environment. However,Purkinje progenitors failed to mature further under above conditions. By co-culturing human Purkinje progenitors with rat cerebellar slices,we observed mature Purkinje-like cells with right morphology and marker expression patterns,which yet showed no appropriate membrane properties. Co-culture with human fetal cerebellar slices drove the progenitors to not only morphologically correct but also electrophysiologically functional Purkinje neurons. Neph3-posotive human cells could also survive transplantation into the cerebellum of newborn immunodeficient mice and differentiate to L7- and Calbindin-positive neurons. Obtaining mature human Purkinje cells in vitro has significant implications in studying the mechanisms of spinocerebellar ataxias and other cerebellar diseases. View Publication

OBJECTIVES Kidney disease is emerging as a critical medical problem worldwide. Because of limited treatment options for the damaged kidney,stem cell treatment is becoming an alternative therapeutic approach. Of many possible human stem cell sources,pluripotent stem cells are most attractive due to their self-renewal and pluripotent capacity. However,little is known about the derivation of renal lineage cells from human pluripotent stem cells (hPSCs). In this study,we developed a novel protocol for differentiation of nephron progenitor cells (NPCs) from hPSCs in a serum- and feeder-free system. MATERIALS AND METHODS We designed step-wise protocols for differentiation of human pluripotent stem cells toward primitive streak,intermediate mesoderm and NPCs by recapitulating normal nephrogenesis. Expression of key marker genes was examined by RT-PCR,real time RT-PCR and immunocytochemistry. Each experiment was independently performed three times to confirm its reproducibility. RESULTS After modification of culture period and concentration of exogenous factors,hPSCs can differentiate into NPCs that markedly express specific marker genes such as SIX2,GDNF,HOXD11,WT1 and CITED1 in addition to OSR1,PAX2,SALL1 and EYA1. Moreover,NPCs possess the potential of bidirectional differentiation into both renal tubular epithelial cells and glomerular podocytes in defined culture conditions. In particular,approximately 70% of SYN-positive cells were obtained from hPSC-derived NPCs after podocytes induction. NPCs can also form in vitro tubule-like structures in three dimensional culture systems. CONCLUSIONS Our novel protocol for hPSCs differentiation into NPCs can be useful for producing alternative sources of cell replacement therapy and disease modeling for human kidney diseases. View Publication

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can differentiate to cardiomyocytes in vitro,offering unique opportunities to investigate cardiac development and disease as well as providing a platform to perform drug and toxicity tests. Initial cardiac differentiation methods were based on either inductive co-culture or aggregation as embryoid bodies,often in the presence of fetal calf serum. More recently,monolayer differentiation protocols have evolved as feasible alternatives and are often performed in completely defined culture medium and substrates. Thus,our ability to efficiently and reproducibly generate cardiomyocytes from multiple different hESC and hiPSC lines has improved significantly.We have developed a directed differentiation monolayer protocol that can be used to generate cultures comprising ˜50% cardiomyocytes,in which both the culture of the undifferentiated human pluripotent stem cells (hPSCs) and the differentiation procedure itself are defined and serum-free. The differentiation method is also effective for hPSCs maintained in other culture systems. In this chapter,we outline the differentiation protocol and describe methods to assess cardiac differentiation efficiency as well as to identify and quantify the yield of cardiomyocytes. View Publication

The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony-forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel-forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of textgreater10(8) ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb,and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells. View Publication

In this unit,we describe a simple coculture-free method for obtaining mast cells from mouse and human embryonic stem (ES) cells. Much of our knowledge regarding the mechanisms by which mast cells are activated comes from studies of mouse bone marrow-derived mast cells. Studies of human mast cells have been hampered by the limited sources from which they can be cultured,the difficulty in introducing specific genetic changes into these cells,and differences between established cultures that reflect the unique genetic makeup of the tissue donor. Derivation of mast cells from embryonic stem cells addresses these limitations. ES-derived mast cells can be generated in numbers sufficient for studies of the pathways involved in mast cell effector functions. These ES cell-derived mast cells respond to antigens and other stimuli by releasing histamine,cytokines,lipids,and other bioactive mediators. The derivation of human mast cells from ES cells carrying mutations introduced by homologous recombination should provide a novel means of testing the function of genes in both the development and the effector functions of mast cells. View Publication

Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid,beta-glycerophosphate,and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after,but not before,14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair. View Publication

Embryonic stem (ES) cells have been established as permanent lines of undifferentiated pluripotent cells from early mouse embryos. ES cells provide a unique system for the genetic manipulation and the creation of knockout strains of mice through gene targeting. By cultivation in vitro as 3D aggregates called embryoid bodies,ES cells can differentiate into derivatives of all 3 primary germ layers,including cardiomyocytes. Protocols for the in vitro differentiation of ES cells into cardiomyocytes representing all specialized cell types of the heart,such as atrial-like,ventricular-like,sinus nodal-like,and Purkinje-like cells,have been established. During differentiation,cardiac-specific genes as well as proteins,receptors,and ion channels are expressed in a developmental continuum,which closely recapitulates the developmental pattern of early cardiogenesis. Exploitation of ES cell-derived cardiomyocytes has facilitated the analysis of early cardiac development and has permitted in vitro gain-of-function" or "loss-of-function" genetic studies. Recently� View Publication

Congenital heart defects are the most common birth defect. The limiting factor in tissue engineering repair strategies is an autologous source of functional cardiomyocytes. Amniotic fluid contains an ideal cell source for prenatal harvest and use in correction of congenital heart defects. This study aims to investigate the potential of amniotic fluid-derived stem cells (AFSC) to undergo non-viral reprogramming into induced pluripotent stem cells (iPSC) followed by growth-factor-free differentiation into functional cardiomyocytes. AFSC from human second trimester amniotic fluid were transfected by non-viral vesicle fusion with modified mRNA of OCT4,KLF4,SOX2,LIN28,cMYC and nuclear GFP over 18 days,then differentiated using inhibitors of GSK3 followed 48 hours later by inhibition of WNT. AFSC-derived iPSC had high expression of OCT4,NANOG,TRA-1-60,and TRA-1-81 after 18 days of mRNA transfection and formed teratomas containing mesodermal,ectodermal,and endodermal germ layers in immunodeficient mice. By Day 30 of cardiomyocyte differentiation,cells contracted spontaneously,expressed connexin 43 and β-myosin heavy chain organized in sarcomeric banding patterns,expressed cardiac troponin T and β-myosin heavy chain,showed upregulation of NKX2.5,ISL-1 and cardiac troponin T with downregulation of POU5F1,and displayed calcium and voltage transients similar to those in developing cardiomyocytes. These results demonstrate that cells from human amniotic fluid can be differentiated through a pluripotent state into functional cardiomyocytes. View Publication

The spinal cord consists of multiple neuronal cell types that are critical to motor control and arise from distinct progenitor domains in the developing neural tube. Excitatory V2a interneurons in particular are an integral component of central pattern generators that control respiration and locomotion; however,the lack of a robust source of human V2a interneurons limits the ability to molecularly profile these cells and examine their therapeutic potential to treat spinal cord injury (SCI). Here,we report the directed differentiation of CHX10(+) V2a interneurons from human pluripotent stem cells (hPSCs). Signaling pathways (retinoic acid,sonic hedgehog,and Notch) that pattern the neural tube were sequentially perturbed to identify an optimized combination of small molecules that yielded ∼25% CHX10(+) cells in four hPSC lines. Differentiated cultures expressed much higher levels of V2a phenotypic markers (CHX10 and SOX14) than other neural lineage markers. Over time,CHX10(+) cells expressed neuronal markers [neurofilament,NeuN,and vesicular glutamate transporter 2 (VGlut2)],and cultures exhibited increased action potential frequency. Single-cell RNAseq analysis confirmed CHX10(+) cells within the differentiated population,which consisted primarily of neurons with some glial and neural progenitor cells. At 2 wk after transplantation into the spinal cord of mice,hPSC-derived V2a cultures survived at the site of injection,coexpressed NeuN and VGlut2,extended neurites textgreater5 mm,and formed putative synapses with host neurons. These results provide a description of V2a interneurons differentiated from hPSCs that may be used to model central nervous system development and serve as a potential cell therapy for SCI. View Publication

The reprogramming of somatic cells into induced pluripotent stem (iPS) cells upon overexpression of the transcription factors Oct4,Sox2,Klf4 and cMyc is inefficient. It has been assumed that the somatic differentiation state provides a barrier for efficient reprogramming; however,direct evidence for this notion is lacking. Here,we tested the potential of mouse hematopoietic cells at different stages of differentiation to be reprogrammed into iPS cells. We show that hematopoietic stem and progenitor cells give rise to iPS cells up to 300 times more efficiently than terminally differentiated B and T cells do,yielding reprogramming efficiencies of up to 28%. Our data provide evidence that the differentiation stage of the starting cell has a critical influence on the efficiency of reprogramming into iPS cells. Moreover,we identify hematopoietic progenitors as an attractive cell type for applications of iPS cell technology in research and therapy. View Publication

Human embryonic stem cells (hESCs) and their differentiated progeny allow for investigation of important changes/events during normal embryonic development. Currently most of the research is focused on proteinacous changes occurring as a result of differentiation of stem cells and little is known about changes in cell surface glycosylation patterns. Identification of cell lineage specific glycans can help in understanding their role in maintenance,proliferation and differentiation. Furthermore,these glycans can serve as markers for isolation of homogenous populations of cells. Using a panel of eight biotinylated lectins,the glycan expression of hESCs,hESCs-derived human neural progenitors (hNP) cells,and hESCs-derived mesenchymal progenitor (hMP) cells was investigated. Our goal was to identify glycans that are unique for hNP cells and use the corresponding lectins for cell isolation. Flow cytometry and immunocytochemistry were used to determine expression and localization of glycans,respectively,in each cell type. These results show that the glycan expression changes upon differentiation of hESCs and is different for neural and mesenchymal lineage. For example,binding of PHA-L lectin is low in hESCs (14±4.4%) but significantly higher in differentiated hNP cells (99±0.4%) and hMP cells (90±3%). Three lectins: VVA,DBA and LTL have low binding in hESCs and hMP cells,but significantly higher binding in hNP cells. Finally,VVA lectin binding was used to isolate hNP cells from a mixed population of hESCs,hNP cells and hMP cells. This is the first report that compares glycan expression across these human stem cell lineages and identifies significant differences. Also,this is the first study that uses VVA lectin for isolation for human neural progenitor cells. View Publication

Long noncoding RNAs (lncRNAs) exhibit diverse functions,including regulation of development. Here,we combine genome-wide mapping of SMAD3 occupancy with expression analysis to identify lncRNAs induced by activin signaling during endoderm differentiation of human embryonic stem cells (hESCs). We find that DIGIT is divergent to Goosecoid (GSC) and expressed during endoderm differentiation. Deletion of the SMAD3-occupied enhancer proximal to DIGIT inhibits DIGIT and GSC expression and definitive endoderm differentiation. Disruption of the gene encoding DIGIT and depletion of the DIGIT transcript reveal that DIGIT is required for definitive endoderm differentiation. In addition,we identify the mouse ortholog of DIGIT and show that it is expressed during development and promotes definitive endoderm differentiation of mouse ESCs. DIGIT regulates GSC in trans,and activation of endogenous GSC expression is sufficient to rescue definitive endoderm differentiation in DIGIT-deficient hESCs. Our study defines DIGIT as a conserved noncoding developmental regulator of definitive endoderm. View Publication