技术资料

The breakthrough development of induced pluripotent stem cells (iPSCs) raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells. However,whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear. In this study,we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with autogenous peripheral blood mononuclear cells (PBMCs),we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation. However,a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs. Furthermore,no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells (CD3+CD8− T cells,CD3+CD8+ T cells or CD3−CD56+ NK cells) by NPCs in both PBMC and T cell co-culture systems. These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants,and thus set a base for further preclinical evaluation of human iPSCs. View Publication

Tumorigenicity of human pluripotent stem cells is a major threat limiting their application in cell therapy protocols. It remains unclear,however,whether suppression of tumorigenic potential can be achieved without critically affecting pluripotency. A previous study has identified hyperexpressed genes in cancer stem cells,among which is E2F2,a gene involved in malignant transformation and stem cell self-renewal. Here we tested whether E2F2 knockdown would affect the proliferative capacity and tumorigenicity of human embryonic stem cells (hESC). Transient E2F2 silencing in hESC significantly inhibited expression of the proto-oncogenes BMI1 and HMGA1,in addition to proliferation of hESC,indicated by a higher proportion of cells in G1,fewer cells in G2/M phase,and a reduced capacity to generate hESC colonies in vitro. Nonetheless,E2F2-silenced cells kept expression of typical pluripotency markers and displayed differentiation capacity in vitro. More importantly,E2F2 knockdown in hESC significantly inhibited tumor growth in vivo,which was considerably smaller than tumors generated from control hESC,although displaying typical teratoma traits,a major indicator of pluripotency retention in E2F2-silenced cells. These results suggest that E2F2 knockdown can inhibit hESC proliferation and tumorigenicity without significantly harming stemness,providing a rationale to future protocols aiming at minimizing risks related to therapeutic application of cells and/or products derived from human pluripotent cells. View Publication

The reliable derivation of induced pluripotent stem cells (iPSCs) from a noninvasive autologous source at birth would facilitate the study of patient-specific in vitro modeling of congenital diseases and would enhance ongoing efforts aimed at developing novel cell-based treatments for a wide array of fetal and pediatric disorders. Accordingly,we have successfully generated iPSCs from human fetal chorionic somatic cells extracted from term pregnancies by ectopic expression of OCT4,SOX2,KLF4,and cMYC. The isolated parental somatic cells exhibited an immunophenotypic profile consistent with that of chorionic mesenchymal stromal cells (CMSCs). CMSC-iPSCs maintained pluripotency in feeder-free systems for more than 15 passages based on morphology,immunocytochemistry,and gene expression studies and were capable of embryoid body formation with spontaneous trilineage differentiation. CMSC-iPSCs could be selectively differentiated in vitro into various germ layer derivatives,including neural stem cells,beating cardiomyocytes,and definitive endoderm. This study demonstrates the feasibility of term placental chorion as a novel noninvasive alternative to dermal fibroblasts and cord blood for human perinatal iPSC derivation and may provide additional insights regarding the reprogramming capabilities of extra-embryonic tissues as they relate to developmental ontogeny and perinatal tissue engineering applications. View Publication

SIRT1,an NAD-dependent deacetylase,plays a role in regulation of autophagy. SIRT1 increases mitochondrial function and reduces oxidative stress,and has been linked to age-related reactive oxygen species (ROS) generation,which is highly dependent on mitochondrial metabolism. H2O2 induces oxidative stress and autophagic cell death through interference with Beclin 1 and the mTOR signaling pathways. We evaluated connections between SIRT1 activity and induction of autophagy in murine (m) and human (h) embryonic stem cells (ESCs) upon ROS challenge. Exogenous H2O2 (1 mM) induced apoptosis and autophagy in wild-type (WT) and Sirt1-/- mESCs. High concentrations of H2O2 (1 mM) induced more apoptosis in Sirt1-/-,than in WT mESCs. However,addition of 3-methyladenine,a widely used autophagy inhibitor,in combination with H2O2 induced more cell death in WT than in Sirt1-/- mESCs. Decreased induction of autophagy in Sirt1-/- mESCs was demonstrated by decreased conversion of LC3-I to LC3-II,lowered expression of Beclin-1,and decreased LC3 punctae and LysoTracker staining. H2O2 induced autophagy with loss of mitochondrial membrane potential and disruption of mitochondrial dynamics in Sirt1-/- mESCs. Increased phosphorylation of P70/85-S6 kinase and ribosomal S6 was noted in Sirt1-/- mESCs,suggesting that SIRT1 regulates the mTOR pathway. Consistent with effects in mESCs,inhibition of SIRT1 using Lentivirus-mediated SIRT1 shRNA in hESCs demonstrated that knockdown of SIRT1 decreased H2O2-induced autophagy. This suggests a role for SIRT1 in regulating autophagy and mitochondria function in ESCs upon oxidative stress,effects mediated at least in part by the class III PI3K/Beclin 1 and mTOR pathways. Stem Cells 2014;32:1183-1194 View Publication

UNLABELLED Congenital human cytomegalovirus (HCMV) infection is a major cause of central nervous system structural anomalies and sensory impairments. It is likely that the stage of fetal development,as well as the state of differentiation of susceptible cells at the time of infection,affects the severity of the disease. We used human embryonic stem (ES) cell-derived primitive prerosette neural stem cells (pNSCs) and neural progenitor cells (NPCs) maintained in chemically defined conditions to study HCMV replication in cells at the early stages of neural development. In contrast to what was observed previously using fetus-derived NPCs,infection of ES cell-derived pNSCs with HCMV was nonprogressive. At a low multiplicity of infection,we observed only a small percentage of cells expressing immediate-early genes (IE) and early genes. IE expression was found to be restricted to cells negative for the anterior marker FORSE-1,and treatment of pNSCs with retinoic acid restored IE expression. Differentiation of pNSCs into NPCs restored IE expression but not the transactivation of early genes. Virions produced in NPCs and pNSCs were exclusively cell associated and were mostly non-neural tropic. Finally,we found that viral genomes could persist in pNSC cultures for up to a month after infection despite the absence of detectable IE expression by immunofluorescence,and infectious virus could be produced upon differentiation of pNSCs to neurons. In conclusion,our results highlight the complex array of hurdles that HCMV must overcome in order to infect primitive neural stem cells and suggest that these cells might act as a reservoir for the virus. IMPORTANCE Human cytomegalovirus (HCMV) is a betaherpesvirus that is highly prevalent in the population. HCMV infection is usually asymptomatic but can lead to severe consequences in immunosuppressed individuals. HCMV is also the most important infectious cause of congenital developmental birth defects. Manifestations of fetal HCMV disease range from deafness and learning disabilities to more severe symptoms such as microcephaly. In this study,we have used embryonic stem cells to generate primitive neural stem cells and have used these to model HCMV infection of the fetal central nervous system (CNS) in vitro. Our results reveal that these cells,which are similar to those present in the developing neural tube,do not support viral replication but instead likely constitute a viral reservoir. Future work will define the effect of viral persistence on cellular functions as well as the exogenous signals leading to the reactivation of viral replication in the CNS. View Publication

The first step in developing regenerative medicine approaches to treat renal diseases using pluripotent stem cells must be the generation of intermediate mesoderm (IM),an embryonic germ layer that gives rise to kidneys. In order to achieve this goal,establishing an efficient,stable and low-cost method for differentiating IM cells using small molecules is required. In this study,we identified two retinoids,AM580 and TTNPB,as potent IM inducers by high-throughput chemical screening,and established rapid (five days) and efficient (80% induction rate) IM differentiation from human iPSCs using only two small molecules: a Wnt pathway activator,CHIR99021,combined with either AM580 or TTNPB. The resulting human IM cells showed the ability to differentiate into multiple cell types that constitute adult kidneys,and to form renal tubule-like structures. These small molecule differentiation methods can bypass the mesendoderm step,directly inducing IM cells by activating Wnt,retinoic acid (RA),and bone morphogenetic protein (BMP) pathways. Such methods are powerful tools for studying kidney development and may potentially provide cell sources to generate renal lineage cells for regenerative therapy. View Publication

Human cell transformation is a key step for oncogenic development,which involves multiple pathways; however,the mechanism remains unclear. To test our hypothesis whether cell oncogenic transformation shares some mechanisms with the process of reprogramming non-stem cells to induced pluripotent stem cells (iPSC),we studied the relationship among the key factors for promoting or inhibiting iPSC in radiation-transformed human epithelial cell lines derived from different tissues (lung,breast and colon). We unexpectedly found that p63 and OCT4 were highly expressed (accompanied by low expressed p53 and miR-34a) in all transformed cell lines examined when compared with their non-transformed counterparts. We further elucidated the relationship of these factors: the 3p strand of miR-34a directly targeted OCT4 by binding to the 3′ untranslated region (3′-UTR) of OCT4 and,OCT4,in turn,stimulated p63 but inhibited p53 expression by binding to a specific region of the p63 or p53 promoter. Moreover,we revealed that the effects of OCT4 on promoting cell oncogenic transformation were by affecting p63 and p53. These results support that a positive loop exists in human cells: OCT4 upregulation as a consequence of inhibition of miR-34a,promotes p63 but suppresses p53 expression,which further stimulates OCT4 upregulation by downregulating miR-34a. This functional loop contributes significantly to cell transformation and,most likely,also to the iPSC process. View Publication

Following haematopoietic stem cell transplantation,monitoring the proportion of donor and recipient haematopoiesis in the patient (chimerism) is an influential tool in directing further treatment choices. Short tandem repeat (STR) analysis is a method of chimerism monitoring using DNA isolated from peripheral blood,bone marrow or specific isolated cell lineages such as CD3+ T cells. For lineage-specific STR analysis on cell populations isolated from peripheral blood,a qualitative estimation of the purity of each isolated population is essential for the correct interpretation of the test data. We describe a rapid,inexpensive method for the determination of purity using a simple flow cytometry method. The method described for assessing the purity of sorted CD3+ cells can be applied to any cell population isolated using the same technology. Data obtained were comparable to results from a commercial polymerase chain reaction (PCR)-based method for the assessment of purity (Non-T Genomic Detection Kit,Accumol,Calgary,AB,Canada) (P = 0.59). Of the 303 samples tested by flow cytometry,290 (95.7%) exceeded 90% purity,and 215 (70.95%) were over 99% pure. There were some outlying samples,showing diversity between samples and the unpredictability of purity of isolated cell populations. This flow cytometry method can be easily assimilated into routine testing protocols,allowing purity assessment in multiple-sorted cell populations for lineage-specific chimerism monitoring using a single secondary antibody and giving results comparable to a PCR-based method. As purity of isolated cell lineages is affected by time after venepuncture and storage temperature,assessment of each sample is recommended to give a reliable indication of sample quality and confidence in the interpretation of the results. View Publication

The purpose of this study was to investigate the chondrogenic features of human induced pluripotent stem cells (hiPSCs) and examine the differences in the chondrogenesis between hiPSCs and human bone marrow-derived MSCs (hBMMSCs). Embryoid bodies (EBs) were formed from undifferentiated hiPSCs. After EBs were dissociated into single cells,chondrogenic culture was performed in pellets and alginate hydrogel. Chondro-induced hiPSCs were implanted in osteochondral defects created on the patellar groove of immunosuppressed rats and evaluated after 12 weeks. The ESC markers NANOG,SSEA4 and OCT3/4 disappeared while the mesodermal marker BMP-4 appeared in chondro-induced hiPSCs. After 21 days of culture,greater glycosaminoglycan contents and better chondrocytic features including lacuna and abundant matrix formation were observed from chondro-induced hiPSCs compared to chondro-induced hBMMSCs. The expression of chondrogenic markers including SOX-9,type II collagen,and aggrecan in chondro-induced hiPSCs was comparable to or greater than chondro-induced hBMMSCs. A remarkably low level of hypertrophic and osteogenic markers including type X collagen,type I collagen and Runx-2 was noted in chondro-induced hiPSCs compared to chondro-induced hBMMSCs. hiPSCs had significantly greater methylation of several CpG sites in COL10A1 promoter than hBMMSCs in either undifferentiated or chondro-induced state,suggesting an epigenetic cause of the difference in hypertrophy. The defects implanted with chondro-induced hiPSCs showed a significantly better quality of cartilage repair than the control defects,and the majority of cells in the regenerated cartilage consisted of implanted hiPSCs. ?? 2014 Elsevier Ltd. View Publication

Recent genomic approaches have revealed that the repertoire of RNA Pol III-transcribed genes varies in different human cell types,and that this variation is likely determined by a combination of the chromatin landscape,cell-specific DNA-binding transcription factors,and collaboration with RNA Pol II. Although much is known about this regulation in differentiated human cells,there is presently little understanding of this aspect of the Pol III system in human ES cells. Here,we determine the occupancy profiles of Pol III components in human H1 ES cells,and also induced pluripotent cells,and compare to known profiles of chromatin,transcription factors,and RNA expression. We find a relatively large fraction of the Pol III repertoire occupied in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In ES cells we find clear correlations between Pol III occupancy and active chromatin. Interestingly,we find a highly significant fraction of Pol III-occupied genes with adjacent binding events by pluripotency factors in ES cells,especially NANOG. Notably,in human ES cells we find H3K27me3 adjacent to but not overlapping many active Pol III loci. We observe in all such cases,a peak of H3K4me3 and/or RNA Pol II,between the H3K27me3 and Pol III binding peaks,suggesting that H3K4me3 and Pol II activity may “insulate�? Pol III from neighboring repressive H3K27me3. Further,we find iPSCs have a larger Pol III repertoire than their precursors. Finally,the active Pol III genome in iPSCs is not completely reprogrammed to a hESC like state and partially retains the transcriptional repertoire of the precursor. Together,our correlative results are consistent with Pol III binding and activity in human ES cells being enabled by active/permissive chromatin that is shaped in part by the pluripotency network of transcription factors and RNA Pol II activity. View Publication

A priori,a common receptor induced in tumor microvessels,cancer cells and cancer stem-like cells (CSCs) that is involved in tumor angiogenesis,invasiveness,and CSC anoikis resistance and survival,could underlie contemporaneous coordination of these events rather than assume stochasticity. Here we show that functional analysis of the dual endothelin1/VEGFsignal peptide receptor,DEspR,(formerly named Dear,Chr.4q31.2) supports the putative common receptor paradigm in pancreatic ductal adenocarcinoma (PDAC) and glioblastoma (GBM) selected for their invasiveness,CD133+CSCs,and polar angiogenic features. Unlike normal tissue,DEspR is detected in PDAC and GBM microvessels,tumor cells,and CSCs isolated from PDAC-Panc1 and GBM-U87 cells. DEspR-inhibition decreased angiogenesis,invasiveness,CSC-survival and anoikis resistance in vitro,and decreased Panc1-CSC and U87-CSC xenograft tumor growth,vasculo-angiogenesis and invasiveness in nude(nu/nu) rats,suggesting that DEspR activation would coordinate these tumor progression events. As an accessible,cell-surface 'common receptor coordinator',DEspR-inhibition defines a novel targeted-therapy paradigm for pancreatic cancer and glioblastoma. View Publication

New mouse models with specific drivers of genetic alterations are needed for preclinical studies. Herein,we created and characterized at the genetic level a new syngeneic model for lung cancer and metastasis in Balb-c mice. Tumor cell lines were obtained from a silica-mediated airway chronic inflammation that promotes tumorigenesis when combined with low doses of N-nitrosodimethylamine,a tobacco smoke carcinogen. Orthotopic transplantation of these cells induced lung adenocarcinomas,and their intracardiac injection led to prominent colonization of various organs (bone,lung,liver and brain). Driver gene alterations included a mutation in the codon 12 of KRAS (G-A transition),accompanied by a homozygous deletion of the WW domain-containing oxidoreductase (WWOX) gene. The mutant form of WWOX lacked exons 5-8 and displayed reduced protein expression level and activity. WWOX gene restoration decreased the in vitro and in vivo tumorigenicity,confirming the tumor suppressor function of this gene in this particular model. Interestingly,we found that cells displayed remarkable sphere formation ability with expression of specific lung cancer stem cell markers. Study of non-small-cell lung cancer patient cohorts demonstrated a deletion of WWOX in 30% of cases,with significant reduction in protein levels as compared to normal tissues. Overall,our new syngeneic mouse model provides a most valuable tool to study lung cancer metastasis in balb-c mice background and highlights the importance of WWOX deletion in lung carcinogenesis. View Publication