技术资料

Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) gene,which codes for a chloride/bicarbonate channel in the apical epithelial membranes. CFTR dysfunction results in a multisystem disease including the development of life limiting lung disease. The possibility of a cure for CF by replacing defective CFTR has led to different approaches for CF gene therapy; all of which ultimately have to be tested in preclinical model systems. Primary human nasal epithelial cultures (HNECs) derived from nasal turbinate brushing were used to test the efficiency of a helper-dependent adenoviral (HD-Ad) vector expressing CFTR. HD-Ad-CFTR transduction resulted in functional expression of CFTR at the apical membrane in nasal epithelial cells obtained from CF patients. These results suggest that HNECs can be used for preclinical testing of gene therapy vectors in CF. View Publication

Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR,cyclic enrichment strategy gave ˜100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that,when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways. View Publication

Mutations in SCN1A,the gene encoding the α subunit of Nav1.1 channel,can cause epilepsies with wide ranges of clinical phenotypes,which are associated with the contrasting effects of channel loss-of-function or gain-of-function. In this project,CRISPR/Cas9- and TALEN-mediated genome-editing techniques were applied to induced pluripotent stem cell (iPSC)-based-disease model to explore the mechanism of epilepsy caused by SCN1A loss-of-function mutation. By fluorescently labeling GABAergic subtype in iPSC-derived neurons using CRISPR/Cas9,we for the first time performed electrophysiological studies on SCN1A-expressing neural subtype and monitored the postsynaptic activity of both inhibitory and excitatory types. We found that the mutation c.A5768G,which led to no current of Nav1.1 in exogenously transfected system,influenced the properties of not only Nav current amount,but also Nav activation in Nav1.1-expressing GABAergic neurons. The two alterations in Nav further reduced the amplitudes and enhanced the thresholds of action potential in patient-derived GABAergic neurons,and led to weakened spontaneous inhibitory postsynaptic currents (sIPSCs) in the patient-derived neuronal network. Although the spontaneous excitatory postsynaptic currents (sEPSCs) did not change significantly,when the frequencies of both sIPSCs and sEPSCs were further analyzed,we found the whole postsynaptic activity transferred from the inhibition-dominated state to excitation in patient-derived neuronal networks,suggesting that changes in sIPSCs alone were sufficient to significantly reverse the excitatory level of spontaneous postsynaptic activity. In summary,our findings fill the gap of our knowledge regarding the relationship between SCN1A mutation effect recorded on exogenously transfected cells and on Nav1.1-expressing neurons,and reveal the physiological basis underlying epileptogenesis caused by SCN1A loss-of-function mutation. View Publication

Human induced pluripotent stem cells (hiPSCs) are capable of unlimited self-renewal and can generate nearly all cells in the body. Changes induced by different LSD1 activities on the regulation of hiPSC self-renewal and differentiation and the mechanism underlying such changes were determined. We used two different LSD1 inhibitors (phenelzine sulfate and tranylcypromine) and RNAi technique to inhibit LSD1 activity,and we obtained hiPSCs showing 71.3%,53.28%,and 31.33% of the LSD1 activity in normal hiPSCs. The cells still maintained satisfactory self-renewal capacity when LSD1 activity was at 71.3%. The growth rate of hiPSCs decreased and cells differentiated when LSD1 activity was at approximately 53.28%. The hiPSCs were mainly arrested in the G0/G1 phase and simultaneously differentiated into endodermal tissue when LSD1 activity was at 31.33%. Teratoma experiments showed that the downregulation of LSD1 resulted in low teratoma volume. When LSD1 activity was below 50%,pluripotency of hiPSCs was impaired,and the teratomas mainly comprised endodermal and mesodermal tissues. This phenomenon was achieved by regulating the critical balance between histone methylation and demethylation at regulatory regions of several key pluripotent and developmental genes. View Publication

Oct4 is considered a key transcription factor for pluripotent stem cell self-renewal. It binds to specific regions within target genes to regulate their expression and is downregulated upon induction of differentiation of pluripotent stem cells; however,the mechanisms that regulate the levels of human Oct4 expression remain poorly understood. Here we show that expression of human Oct4 is directly repressed by germ cell nuclear factor (GCNF),an orphan nuclear receptor,in hES cells. Knockdown of GCNF by siRNA resulted in maintenance of Oct4 expression during RA-induced hES cell differentiation. While overexpression of GCNF promoted repression of Oct4 expression in both undifferentiated and differentiated hES cells. The level of Oct4 repression was dependent on the level of GCNF expression in a dose-dependent manner. mRNA microarray analysis demonstrated that overexpression of GCNF globally regulates gene expression in undifferentiated and differentiated hES cells. Within the group of altered genes,GCNF down-regulated 36% of the genes,and up-regulated 64% in undifferentiated hES cells. In addition,GCNF also showed a regulatory gene pattern that is different from RA treatment during hES cell differentiation. These findings increase our understanding of the mechanisms that maintain hES cell pluripotency and regulate gene expression during the differentiation process. View Publication

Strategies to prevent organ transplant rejection whilst minimizing long-term immunosuppression are currently under intense investigation with regulatory T cells (Tregs) nearing clinical application. The clinical trial,ThRIL,recently commenced at King's College London,proposes to use Treg cell therapy to induce tolerance in liver transplant recipients,the success of which has the potential to revolutionize the management of these patients and enable a future of drug-free transplants. This is the first report of the manufacture of clinical grade Tregs from prospective liver transplant recipients via a CliniMACS-based GMP isolation technique and expanded using anti-CD3/CD28 beads,IL-2 and rapamycin. We report the enrichment of a pure,stable population of Tregs (textgreater95% CD4(+)CD25(+)FOXP3(+)),reaching adequate numbers for their clinical application. Our protocol proved successful in,influencing the expansion of superior functional Tregs,as compared to freshly isolated cells,whilst also preventing their conversion to Th17 cells under pro-inflammatory conditions. We conclude with the manufacture of the final Treg product in the clinical research facility (CRF),a prerequisite for the clinical application of these cells. The data presented in this manuscript together with the much-anticipated clinical results from ThRIL,will undoubtedly inform the improved management of the liver transplant recipient. View Publication

Spinal muscular atrophy (SMA) is an inherited neuromuscular disease primarily characterized by degeneration of spinal motor neurons,and caused by reduced levels of the SMN protein. Previous studies to understand the proteomic consequences of reduced SMN have mostly utilized patient fibroblasts and animal models. We have derived human motor neurons from type I SMA and healthy controls by creating their induced pluripotent stem cells (iPSCs). Quantitative mass spectrometry of these cells revealed increased expression of 63 proteins in control motor neurons compared to respective fibroblasts,whereas 30 proteins were increased in SMA motor neurons vs. their fibroblasts. Notably,UBA1 was significantly decreased in SMA motor neurons,supporting evidence for ubiquitin pathway defects. Subcellular distribution of UBA1 was predominantly cytoplasmic in SMA motor neurons in contrast to nuclear in control motor neurons; suggestive of neurodevelopmental abnormalities. Many of the proteins that were decreased in SMA motor neurons,including beta III-tubulin and UCHL1,were associated with neurodevelopment and differentiation. These neuron-specific consequences of SMN depletion were not evident in fibroblasts,highlighting the importance of iPSC technology. The proteomic profiles identified here provide a useful resource to explore the molecular consequences of reduced SMN in motor neurons,and for the identification of novel biomarker and therapeutic targets for SMA. View Publication

Pluripotency makes human pluripotent stem cells (hPSCs) promising for regenerative medicine,but the teratoma formation has been considered to be a major obstacle for their clinical applications. Here,we determined that the downregulation of miR-302 suppresses the teratoma formation,hampers the self-renewal and pluripotency,and promotes hPSC differentiation. The underlying mechanism is that the high endogenous expression of miR-302 suppresses the AKT1 expression by directly targeting its 3'UTR and subsequently maintains the pluripotent factor OCT4 at high level. Our findings reveal that miR-302 regulates OCT4 by suppressing AKT1,which provides hPSCs two characteristics related to their potential for clinical applications: the benefit of pluripotency and the hindrance of teratoma formation. More importantly,we demonstrate that miR-302 upregulation cannot lead OCT4 negative human adult mesenchymal stem cells (hMSCs) to acquire the teratoma formation in vivo. Whether miR-302 upregulation can drive hMSCs to acquire a higher differentiation potential is worthy of deep investigation. View Publication

Myasthenia gravis (MG) is a prototypical autoimmune disease that is among the few for which the target Ag and the pathogenic autoantibodies are clearly defined. The pathology of the disease is affected by autoantibodies directed toward the acetylcholine receptor (AChR). Mature,Ag-experienced B cells rely on the action of Th cells to produce these pathogenic Abs. The phenotype of the MG Ag-reactive T cell compartment is not well defined; thus,we sought to determine whether such cells exhibit both a proinflammatory and a pathogenic phenotype. A novel T cell library assay that affords multiparameter interrogation of rare Ag-reactive CD4(+) T cells was applied. Proliferation and cytokine production in response to both AChR and control Ags were measured from 3120 T cell libraries derived from 11 MG patients and paired healthy control subjects. The frequency of CCR6(+) memory T cells from MG patients proliferating in response to AChR-derived peptides was significantly higher than that of healthy control subjects. Production of both IFN-γ and IL-17,in response to AChR,was also restricted to the CCR6(+) memory T cell compartment in the MG cohort,indicating a proinflammatory phenotype. These T cells also included an elevated expression of GM-CSF and absence of IL-10 expression,indicating a proinflammatory and pathogenic phenotype. This component of the autoimmune response in MG is of particular importance when considering the durability of MG treatment strategies that eliminate B cells,because the autoreactive T cells could renew autoimmunity in the reconstituted B cell compartment with ensuing clinical manifestations. View Publication

There is a foundational need for quality control tools in stem cell laboratories engaged in basic research,regenerative therapies,and toxicological studies. These tools require automated methods for evaluating cell processes and quality during in vitro passaging,expansion,maintenance,and differentiation. In this paper,an unbiased,automated high-content profiling toolkit,StemCellQC,is presented that non-invasively extracts information on cell quality and cellular processes from time-lapse phase-contrast videos. Twenty four (24) morphological and dynamic features were analyzed in healthy,unhealthy,and dying human embryonic stem cell (hESC) colonies to identify those features that were affected in each group. Multiple features differed in the healthy versus unhealthy/dying groups,and these features were linked to growth,motility,and death. Biomarkers were discovered that predicted cell processes before they were detectable by manual observation. StemCellQC distinguished healthy and unhealthy/dying hESC colonies with 96% accuracy by non-invasively measuring and tracking dynamic and morphological features over 48 hours. Changes in cellular processes can be monitored by StemCellQC and predictions can be made about the quality of pluripotent stem cell colonies. This toolkit reduced the time and resources required to track multiple pluripotent stem cell colonies and eliminated handling errors and false classifications due to human bias. StemCellQC provided both user-specified and classifier-determined analysis in cases where the affected features are not intuitive or anticipated. Video analysis algorithms allowed assessment of biological phenomena using automatic detection analysis,which can aid facilities where maintaining stem cell quality and/or monitoring changes in cellular processes are essential. In the future StemCellQC can be expanded to include other features,cell types,treatments,and differentiating cells. View Publication

CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which,however,has focused on HDR-based strategies and was proven inefficient. Here,we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs,and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy,integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells,and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells. View Publication

Heterologous immunity is recognized as a significant barrier to transplant tolerance. Whereas it has been established that pathogen-elicited memory T cells can have high or low affinity for cross-reactive allogeneic peptide-MHC,the role of TCR affinity during heterologous immunity has not been explored. We established a model with which to investigate the impact of TCR-priming affinity on memory T cell populations following a graft rechallenge. In contrast to high-affinity priming,low-affinity priming elicited fully differentiated memory T cells with a CD45RB(hi) status. High CD45RB status enabled robust secondary responses in vivo,as demonstrated by faster graft rejection kinetics and greater proliferative responses. CD45RB blockade prolonged graft survival in low affinity-primed mice,but not in high affinity-primed mice. Mechanistically,low affinity-primed memory CD8(+) T cells produced more IL-2 and significantly upregulated IL-2Rα expression during rechallenge. We found that CD45RB(hi) status was also a stable marker of priming affinity within polyclonal CD8(+) T cell populations. Following high-affinity rechallenge,low affinity-primed CD45RB(hi) cells became CD45RB(lo),demonstrating that CD45RB status acts as an affinity-based differentiation switch on CD8(+) T cells. Thus,these data establish a novel mechanism by which CD45 isoforms tune low affinity-primed memory CD8(+) T cells to become potent secondary effectors following heterologous rechallenge. These findings have direct implications for allogeneic heterologous immunity by demonstrating that despite a lower precursor frequency,low-affinity priming is sufficient to generate memory cells that mediate potent secondary responses against a cross-reactive graft challenge. View Publication