技术资料

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature,differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF,NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases. View Publication

Mouse embryonic fibroblasts (MEFs) are commonly used as feeder cells for the generation of human induced pluripotent stem cells (hiPSCs). However,medical applications of cell derivatives of hiPSCs generated with a MEF feeder system run the risk of having xeno-factor contamination due to long-term cell culturing under an animal factor-containing environment. We developed a new method for the derivation of human fibroblast-like cells (FLCs) from a previously established hiPSC line in an FLC differentiation medium. The method was based on direct differentiation of hiPSCs seeded on Matrigel followed by expansion of differentiating cells on gelatin. Using inactivated FLCs as feeder layers,primary human foreskin fibroblasts were successfully reprogrammed into a state of pluripotency by Oct4,Sox2 Klf4,and c-Myc (OSKM) transcription factor genes,with a reprogramming efficiency under an optimized condition superior to that obtained on MEF feeder layers. Furthermore,the FLCs were more effective in supporting the growth of human pluripotent stem cells. The pluripotency and differentiation capability of the cells cultured on FLC feeder layers were well retained. Our results suggest that FLCs are a safe alternative to MEFs for hiPSC generation and expansion,especially in the clinical settings wherein hiPSC derivatives will be used for medical treatment. View Publication

Human NK cells are characterized by their ability to initiate an immediate and direct cytolytic response to virally infected or malignantly transformed cells. Within human peripheral blood,the more mature CD56(dim) NK cell efficiently kills malignant targets at rest,whereas the less mature CD56(bright) NK cells cannot. In this study,we show that resting CD56(bright) NK cells express significantly more phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein when compared with CD56(dim) NK cells. Consistent with this,forced overexpression of PTEN in NK cells resulted in decreased cytolytic activity,and loss of PTEN in CD56(bright) NK cells resulted in elevated cytolytic activity. Comparable studies in mice showed PTEN overexpression did not alter NK cell development or NK cell-activating and inhibitory receptor expression yet,as in humans,did decrease expression of downstream NK activation targets MAPK and AKT during early cytolysis of tumor target cells. Confocal microscopy revealed that PTEN overexpression disrupts the NK cell's ability to organize immunological synapse components including decreases in actin accumulation,polarization of the microtubule organizing center,and the convergence of cytolytic granules. In summary,our data suggest that PTEN normally works to limit the NK cell's PI3K/AKT and MAPK pathway activation and the consequent mobilization of cytolytic mediators toward the target cell and suggest that PTEN is among the active regulatory components prior to human NK cells transitioning from the noncytolytic CD56(bright) NK cell to the cytolytic CD56(dim) NK cells. View Publication

OBJECTIVE: The molecular events that lead to human thyroid cell speciation remain incompletely characterized. It has been shown that overexpression of the regulatory transcription factors Pax8 and Nkx2-1 (ttf-1) directs murine embryonic stem (mES) cells to differentiate into thyroid follicular cells by initiating a transcriptional regulatory network. Such cells subsequently organized into three-dimensional follicular structures in the presence of extracellular matrix. In the current study,human embryonic stem (hES) cells were studied with the aim of recapitulating this scenario and producing functional human thyroid cell lines. METHODS: Reporter gene tagged pEZ-lentiviral vectors were used to express human PAX8-eGFP and NKX2-1-mCherry in the H9 hES cell line followed by differentiation into thyroid cells directed by Activin A and thyrotropin (TSH). RESULTS: Both transcription factors were expressed efficiently in hES cells expressing either PAX8,NKX2-1,or in combination in the hES cells,which had low endogenous expression of these transcription factors. Further differentiation of the double transfected cells showed the expression of thyroid-specific genes,including thyroglobulin (TG),thyroid peroxidase (TPO),the sodium/iodide symporter (NIS),and the TSH receptor (TSHR) as assessed by reverse transcription polymerase chain reaction and immunostaining. Most notably,the Activin/TSH-induced differentiation approach resulted in thyroid follicle formation and abundant TG protein expression within the follicular lumens. On stimulation with TSH,these hES-derived follicles were also capable of dose-dependent cAMP generation and radioiodine uptake,indicating functional thyroid epithelial cells. CONCLUSION: The induced expression of PAX8 and NKX2-1 in hES cells was followed by differentiation into thyroid epithelial cells and their commitment to form functional three-dimensional neo-follicular structures. The data provide proof of principal that hES cells can be committed to thyroid cell speciation under appropriate conditions. View Publication

Terahertz (THz) radiation was proposed recently for use in various applications,including medical imaging and security scanners. However,there are concerns regarding the possible biological effects of non-ionising electromagnetic radiation in the THz range on cells. Human embryonic stem cells (hESCs) are extremely sensitive to environmental stimuli,and we therefore utilised this cell model to investigate the non-thermal effects of THz irradiation. We studied DNA damage and transcriptome responses in hESCs exposed to narrow-band THz radiation (2.3 THz) under strict temperature control. The transcription of approximately 1% of genes was subtly increased following THz irradiation. Functional annotation enrichment analysis of differentially expressed genes revealed 15 functional classes,which were mostly related to mitochondria. Terahertz irradiation did not induce the formation of γH2AX foci or structural chromosomal aberrations in hESCs. We did not observe any effect on the mitotic index or morphology of the hESCs following THz exposure. View Publication

It is likely that arrhythmias should be avoided for therapies based on human pluripotent stem cell (hPSC)-derived cardiomyocytes (CM) to be effective. Towards achieving this goal,we introduced light-activated channelrhodopsin-2 (ChR2),a cation channel activated with 480 nm light,into human embryonic stem cells (hESC). By using in vitro approaches,hESC-CM are able to be activated with light. ChR2 is stably transduced into undifferentiated hESC via a lentiviral vector. Via directed differentiation,hESCChR2-CM are produced and subjected to optical stimulation. hESCChR2-CM respond to traditional electrical stimulation and produce similar contractility features as their wild-type counterparts but only hESCChR2-CM can be activated by optical stimulation. Here it is shown that a light sensitive protein can enable in vitro optical control of hESC-CM and that this activation occurs optimally above specific light stimulation intensity and pulse width thresholds. For future therapy,in vivo optical stimulation along with optical inhibition could allow for acute synchronization of implanted hPSC-CM with patient cardiac rhythms. View Publication

To address existing limitations in live neuron imaging,we have developed NeuO,a novel cell-permeable fluorescent probe with an unprecedented ability to label and image live neurons selectively over other cells in the brain. NeuO enables robust live neuron imaging and isolation in vivo and in vitro across species; its versatility and ease of use sets the basis for its development in a myriad of neuronal targeting applications. View Publication

Type 1 diabetes mellitus (T1DM) is the most common type of diabetes in children and adolescents. Diabetic subjects are more likely to experience a myocardial infarction compared to nondiabetic subjects. In recent years,induced pluripotent stem cells (iPSCs) have received increasing attention from basic scientists and clinicians and hold promise for myocardial regeneration due to their unlimited proliferation potential and differentiation capacity. However,cardiomyogenesis of type 1 diabetic donor-derived iPSCs (T1DM-iPSCs) has not been investigated yet. The aim of the study was to comparatively analyze cardiomyocyte (CM) differentiation capacity of nondiabetic donor-derived iPSCs (N-iPSCs) and T1DM-iPSCs. The differentiated CMs were confirmed by both expression of cardiac-specific markers and presence of cardiac action potential. Since mitochondrial bioenergetics is vital to every aspect of CM function,extracellular acidification rates and oxygen consumption rates were measured using Seahorse extracellular flux analyzer. The results showed that N-iPSCs and T1DMiPSCs demonstrated similar capacity of differentiation into spontaneously contracting CMs exhibiting nodal-,atrial-,or ventricular-like action potentials. Differentiation efficiency was up to 90%. In addition,the CMs differentiated from N-iPSCs and T1DM-iPSCs (N-iPSC-CMs and T1DM-iPSC-CMs,respectively) showed 1) well-regulated glucose utilization at the level of glycolysis and mitochondrial oxidative phosphorylation and 2) the ability to switch metabolic pathways independent of extracellular glucose concentration. Collectively,we demonstrate for the first time that T1DM-iPSCs can differentiate into functional CMs with well-regulated glucose utilization as shown in N-iPSCs,suggesting that T1DM-iPSC-CMs might be a promising autologous cell source for myocardial regeneration in type 1 diabetes patients. View Publication

Monge's disease,also known as chronic mountain sickness (CMS),is a disease that potentially threatens more than 140 million highlanders during extended time living at high altitudes (over 2500m). The prevalence of CMS in Andeans is about 15-20%,suggesting that the majority of highlanders (non-CMS) are rather healthy at high altitudes; however,CMS subjects experience severe hypoxemia,erythrocytosis and many neurologic manifestations including migraine,headache,mental fatigue,confusion,and memory loss. The underlying mechanisms of CMS neuropathology are not well understood and no ideal treatment is available to prevent or cure CMS,except for phlebotomy. In the current study,we reprogrammed fibroblast cells from both CMS and non-CMS subjects' skin biopsies into the induced pluripotent stem cells (iPSCs),then differentiated into neurons and compared their neuronal properties. We discovered that CMS neurons were much less excitable (higher rheobase) than non-CMS neurons. This decreased excitability was not caused by differences in passive neuronal properties,but instead by a significantly lowered Na+ channel current density and by a shift of the voltage-conductance curve in the depolarization direction. Our findings provide,for the first time,evidence of a neuronal abnormality in CMS subjects as compared to non-CMS subjects,hoping that such studies can pave the way to a better understanding of the neuropathology in CMS. View Publication

Gene- and cell-based therapies are promising strategies for the treatment of degenerative retinal diseases such as age-related macular degeneration,Stargardt disease,and retinitis pigmentosa. Cellular engineering before transplantation may allow the delivery of cellular factors that can promote functional improvements,such as increased engraftment or survival of transplanted cells. A current challenge in traditional DNA-based vector transfection is to find a delivery system that is both safe and efficient,but using mRNA as an alternative to DNA can circumvent these major roadblocks. In this study,we show that both unmodified and modified mRNA can be delivered to retinal pigmented epithelial (RPE) cells with a high efficiency compared with conventional plasmid delivery systems. On the other hand,administration of unmodified mRNA induced a strong innate immune response that was almost absent when using modified mRNA. Importantly,transfection of mRNA encoding a key regulator of RPE gene expression,microphthalmia-associated transcription factor (MITF),confirmed the functionality of the delivered mRNA. Immunostaining showed that transfection with either type of mRNA led to the expression of roughly equal levels of MITF,primarily localized in the nucleus. Despite these findings,quantitative RT-PCR analyses showed that the activation of the expression of MITF target genes was higher following transfection with modified mRNA compared with unmodified mRNA. Our findings,therefore,show that modified mRNA transfection can be applied to human embryonic stem cell-derived RPE cells and that the method is safe,efficient,and functional. View Publication

TOX is a nuclear factor essential for the development of CD4(+) T cells in the thymus. It is normally expressed in low amounts in mature CD4(+) T cells of the skin and the peripheral blood. We have recently discovered that the transcript levels of TOX were significantly increased in mycosis fungoides,the most common type of cutaneous T-cell lymphoma (CTCL),as compared to normal skin or benign inflammatory dermatoses. However,its involvement in advanced CTCL and its biological effects on CTCL pathogenesis have not been explored. In this study,we demonstrate that TOX expression is also enhanced significantly in primary CD4(+)CD7(-) cells from patients with Sézary syndrome,a leukemic variant of CTCL,and that high TOX transcript levels correlate with increased disease-specific mortality. Stable knockdown of TOX in CTCL cells promoted apoptosis and reduced cell cycle progression,leading to less cell viability and colony-forming ability in vitro and to reduced tumor growth in vivo. Furthermore,TOX knockdown significantly increased 2 cyclin-dependent kinase (CDK) inhibitors,CDKN1B and CDKN1C. Lastly,blocking CDKN1B and CDKN1C reversed growth inhibition of TOX knockdown. Collectively,these findings provide strong evidence that aberrant TOX activation is a critical oncogenic event for CTCL. View Publication

U0126 is a potent and selective inhibitor of MEK1 and MEK2 kinases. It has been widely used as an inhibitor for the Ras/Raf/MEK/ERK signaling pathway with over 5000 references on the NCBI PubMed database. In particular,U0126 has been used in a number of studies to show that inhibition of the Raf/MEK/ERK pathway protects neuronal cells against oxidative stress. Here,we report that U0126 can function as an antioxidant that protects PC12 cells against a number of different oxidative-stress inducers. This protective effect of U0126 is independent of its function as a MEK inhibitor,as several other MEK inhibitors failed to show similar protective effects. U0126 reduces reactive oxygen species (ROS) in cells. We further demonstrate that U0126 is a direct ROS scavenger in vitro,and the oxidation products of U0126 exhibit fluorescence. Our finding that U0126 is a strong antioxidant signals caution for its future usage as a MEK inhibitor and for interpreting some previous results. View Publication