技术资料

Culturing human embryonic stem and induced pluripotent stem cells (hESCs/iPSCs) is one of the most costly and labor-intensive tissue cultures,as media containing expensive factors/cytokines should be changed every day to maintain and propagate undifferentiated hESCs/iPSCs in vitro. We recently reported that doxycycline,an anti-bacterial agent,had dramatic effects on hESC/iPSC survival and promoted self-renewal. In this study,we extended the effects of doxycycline to a more practical issue to save cost and labor in hESC/iPSC cultures. Regardless of cultured cell conditions,hESCs/iPSCs in doxycycline-supplemented media were viable and proliferating for at least 3 days without media change,while none or few viable cells were detected in the absence of doxycycline in the same conditions. Thus,hESCs/iPSCs supplemented with doxycycline can be cultured for a long period of time with media changes at 3-day intervals without altering their self-renewal and pluripotent properties,indicating that doxycycline supplementation can reduce the frequency of media changes and the amount of media required by 1/3. These findings strongly encourage the use of doxycycline to save cost and labor in culturing hESCs/iPSCs. View Publication

Although endothelial cells (ECs) have been derived from human pluripotent stem cells (hPSCs),large-scale generation of hPSC-ECs remains challenging and their functions are not well characterized. Here we report a simple and efficient three-stage method that allows generation of approximately 98 and 9500 ECs on day 16 and day 34,respectively,from each human embryonic stem cell (hESC) input. The functional properties of hESC-ECs derived in the presence and absence of a TGF$$-inhibitory molecule SB431542 were characterized and compared with those of human umbilical vein endothelial cells (HUVECs). Confluent monolayers formed by SB431542(+) hESC-ECs,SB431542(-) hESC-ECs,and HUVECs showed similar permeability to 10,000 Da dextran,but these cells exhibited striking differences in forming tube-like structures in 3D fibrin gels. The SB431542(+) hESC-ECs were most potent in forming tube-like structures regardless of whether VEGF and bFGF were present in the medium; less potent SB431542(-) hESC-ECs and HUVECs responded differently to VEGF and bFGF,which significantly enhanced the ability of HUVECs to form tube-like structures but had little impact on SB431542(-) hESC-ECs. This study offers an efficient approach to large-scale hPSC-EC production and suggests that the phenotypes and functions of hPSC-ECs derived under different conditions need to be thoroughly examined before their use in technology development. This article is protected by copyright. All rights reserved. View Publication

The diverse progenitors that give rise to the human neocortex have been difficult to characterize because progenitors,particularly radial glia (RG),are rare and are defined by a combination of intracellular markers,position and morphology. To circumvent these problems,we developed Fixed and Recovered Intact Single-cell RNA (FRISCR),a method for profiling the transcriptomes of individual fixed,stained and sorted cells. Using FRISCR,we profiled primary human RG that constitute only 1% of the midgestation cortex and classified them as ventricular zone-enriched RG (vRG) that express ANXA1 and CRYAB,and outer subventricular zone-localized RG (oRG) that express HOPX. Our study identified vRG and oRG markers and molecular profiles,an essential step for understanding human neocortical progenitor development. FRISCR allows targeted single-cell profiling of any tissues that lack live-cell markers. View Publication

Bipolar disorder is a complex neuropsychiatric disorder that is characterized by intermittent episodes of mania and depression; without treatment,15% of patients commit suicide. Hence,it has been ranked by the World Health Organization as a top disorder of morbidity and lost productivity. Previous neuropathological studies have revealed a series of alterations in the brains of patients with bipolar disorder or animal models,such as reduced glial cell number in the prefrontal cortex of patients,upregulated activities of the protein kinase A and C pathways and changes in neurotransmission. However,the roles and causation of these changes in bipolar disorder have been too complex to exactly determine the pathology of the disease. Furthermore,although some patients show remarkable improvement with lithium treatment for yet unknown reasons,others are refractory to lithium treatment. Therefore,developing an accurate and powerful biological model for bipolar disorder has been a challenge. The introduction of induced pluripotent stem-cell (iPSC) technology has provided a new approach. Here we have developed an iPSC model for human bipolar disorder and investigated the cellular phenotypes of hippocampal dentate gyrus-like neurons derived from iPSCs of patients with bipolar disorder. Guided by RNA sequencing expression profiling,we have detected mitochondrial abnormalities in young neurons from patients with bipolar disorder by using mitochondrial assays; in addition,using both patch-clamp recording and somatic Ca(2+) imaging,we have observed hyperactive action-potential firing. This hyperexcitability phenotype of young neurons in bipolar disorder was selectively reversed by lithium treatment only in neurons derived from patients who also responded to lithium treatment. Therefore,hyperexcitability is one early endophenotype of bipolar disorder,and our model of iPSCs in this disease might be useful in developing new therapies and drugs aimed at its clinical treatment. View Publication

Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report,we employ electron,immunofluorescence microscopy,and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 $$g/$$g proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 $$g/$$g proteins) reported in human cancer cell lines. Moreover,we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states,similar to those naïve-like hPSCs,with increased glycogen synthesis. Furthermore,we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus,our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions. View Publication

Hyaluronic acid (HA) has been cross-linked to form hydrogel for potential applications in the self-renewal and differentiation of human pluripotent stem cells (hPSCs) for years. However,HA hydrogel with improved residence time and mechanical integrity that allows the survival of hPSCs under defined conditions is still much needed for clinical applications. In this study,HA was modified with methacrylate functional groups (MeHA) and cross-linked by photo-crosslinking method. After subsequent conjugation with adhesive peptide,these MeHA surfaces demonstrated performance in facilitating human induced pluripotent stem cells (hiPSCs) proliferation,and good pluripotency maintenance of hiPSCs under defined conditions. Moreover,MeHA films on glass-slides exhibited long residence time and mechanical stability throughout hiPSC culture. Our photo-crosslinkable MeHA possesses great value in accelerating the application of HA hydrogel in hiPSCs proliferation and differentiation with the conjugation of adhesive peptides. View Publication

Patient safety is a major concern in the application of induced pluripotent stem cells (iPSCs) in cell-based therapy. Efforts are being made to reprogram,maintain,and differentiate iPSCs in defined conditions to provide a safe source of stem cells for regenerative medicine. Recently,human fibroblasts were successfully reprogrammed into pluripotent stem cells using four recombinant proteins (OCT4,c-Myc,KLF4,and SOX2) fused with a cell-penetrating peptide (9R). These protein-induced pluripotent stem cells (piPSCs) are maintained and propagated on a feeder layer of mouse embryonic fibroblasts. Use of animal-derived products in maintenance and differentiation of iPSCs poses risks of zoonotic disease transmission and immune rejection when transplanted into humans. To avoid potential incorporation of xenogenic products,we cultured piPSCs on recombinant human matrix proteins. We then tested whether recombinant human matrix proteins can support self-renewal and pluripotency of piPSCs. After long-term culture on recombinant human vitronectin in xeno-free conditions,piPSCs retained the expression of pluripotent markers. The pluripotency of these cells was further evaluated by differentiating toward ectoderm,mesoderm,and endoderm lineages in vitro. In conclusion,recombinant human vitronectin can support the long-term culture and maintain the stemness of piPSCs in defined nonxenogenic conditions. View Publication

Stem cell-based neuronal differentiation has provided a unique opportunity for disease modeling and regenerative medicine. Neurospheres are the most commonly used neuroprogenitors for neuronal differentiation,but they often clump in culture,which has always represented a challenge for neurodifferentiation. In this study,we report a novel method and defined culture conditions for generating sub-type or region-specific neurons from human embryonic and induced pluripotent stem cells derived neurosphere without any genetic manipulation. Round and bright-edged neurospheres were generated in a supplemented knockout serum replacement medium (SKSRM) with 10% CO2,which doubled the expression of the NESTIN,PAX6 and FOXG1 genes compared with those cultured with 5% CO2. Furthermore,an additional step (AdSTEP) was introduced to fragment the neurospheres and facilitate the formation of a neuroepithelial-type monolayer that we termed the neurosphederm". The large neural tube-type rosette (NTTR) structure formed from the neurosphederm� View Publication

Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders,highlighted by their successful therapeutic use in inherent immunodeficiencies. However,biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here,we report an RNA-based episomal vector system,amenable for long-term transgene expression in stem cells. Specifically,we used a unique intranuclear RNA virus,Borna disease virus (BDV),as the gene transfer vehicle,capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology,cell surface CD105 expression,or the adipogenicity of MSCs. Similarly,replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells (iPSCs),while maintaining the ability to differentiate into three embryonic germ layers. Thus,the BDV-based vectors offer a genomic modification-free,episomal RNA delivery system for sustained stem cell transduction.Gene Therapy accepted article preview online,03 December 2015. doi:10.1038/gt.2015.108. View Publication

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However,upgrading them to pluripotency confers refractoriness toward senescence,higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling,such as Down syndrome or $\$-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing,feeder-dependent culture. Here,we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium,a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4,Nanog,Sox2,SSEA-1,SSEA-4,TRA-1-60,TRA-1-81 in a pattern typical for human primed PSC. Additionally,the cells formed teratomas,and were deemed pluripotent by PluriTest,a global expression microarray-based in-silico pluripotency assay. However,we found that the PluriTest scores were borderline,indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology,non-integrating reprogramming and chemically defined culture are more acceptable. View Publication

Elevated levels of the proinflammatory cytokine IL6 are associated with poor survival outcomes in many cancers. Antibodies targeting IL6 and its receptor have been developed for chronic inflammatory disease,but they have not yet been shown to clearly benefit cancer patients,possibly due to antibody potency or the settings in which they have been tested. In this study,we describe the development of a novel high-affinity anti-IL6 antibody,MEDI5117,which features an extended half-life and potent inhibitory effects on IL6 biologic activity. MEDI5117 inhibited IL6-mediated activation of STAT3,suppressing the growth of several tumor types driven by IL6 autocrine signaling. In the same models,MEDI5117 displayed superior preclinical activity relative to a previously developed anti-IL6 antibody. Consistent with roles for IL6 in promoting tumor angiogenesis,we found that MEDI5117 inhibited the growth of endothelial cells,which can produce IL6 and support tumorigenesis. Notably,in tumor xenograft assays in mice,we documented the ability of MEDI5117 to enhance the antitumor activities of chemotherapy or gefitinib in combination treatment regimens. MEDI5117 also displayed robust activity on its own against trastuzumab-resistant HER2(+) tumor cells by targeting the CD44(+)CD24(-) cancer stem cell population. Collectively,our findings extend the evidence of important pleiotropic roles of IL6 in tumorigenesis and drug resistance,and offer a preclinical proof of concept for the use of IL6 antibodies in combination regimens to heighten therapeutic responses and overcome drug resistance. View Publication

Embryonic stem cells (ESCs) exhibit unrestricted and indefinite,but stringently controlled,proliferation,and can differentiate into any lineage in the body. In the current study,we test the hypothesis that expression of ribosomal RNA (rRNA) and ribosomal protein genes (RPGs) contribute to the ability of hESCs to proliferate indefinitely. Consistent with the accelerated growth rate of hESCs,we find that hESC lines H1 and H9 both exhibit significantly higher levels of rRNA when compared to a panel of normal and cancer human cell lines. Although many RPGs are expressed at levels that comparable to other human cell lines,a few RPGs also exhibit higher expression levels. In situ nuclear run-on assays reveal that both nucleoli in hESCs actively transcribe nascent rRNA. Employing genome-wide chromatin immunoprecipitation-deep sequencing and bioinformatics approaches,we discovered that,RPGs are dominantly marked by the activating H3K4me3 histone mark in the G1,M,and G2 phases of the cell cycle. Interestingly,the rDNA repeats are marked by the activating H3K4me3 only in the M phase,and repressive H3K27me3 histone mark in all three cell cycle phases. Bioinformatics analyses also reveal that Myc,a known regulator of cell growth and proliferation,occupies both the rRNA genes and RPGs. Functionally,down-regulation of Myc expression by siRNA results in a concomitant decrease in rRNA levels. Together,our results show that expression of rRNA,which is regulated by the Myc pluripotency transcription factor,and of RPGs in hESCs is associated with the activating H3K4me3 modification. J. Cell. Physiol. 231: 2007-2013,2016. textcopyright 2016 Wiley Periodicals,Inc. View Publication