技术资料

The future of safe cell-based therapy rests on overcoming teratoma/tumor formation,in particular when using human pluripotent stem cells (hPSCs),such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Because the presence of a few remaining undifferentiated hPSCs can cause undesirable teratomas after transplantation,complete removal of these cells with no/minimal damage to differentiated cells is a prerequisite for clinical application of hPSC-based therapy. Having identified a unique hESC signature of pro- and antiapoptotic gene expression profile,we hypothesized that targeting hPSC-specific antiapoptotic factor(s) (i.e.,survivin or Bcl10) represents an efficient strategy to selectively eliminate pluripotent cells with teratoma potential. Here we report the successful identification of small molecules that can effectively inhibit these antiapoptotic factors,leading to selective and efficient removal of pluripotent stem cells through apoptotic cell death. In particular,a single treatment of hESC-derived mixed population with chemical inhibitors of survivin (e.g.,quercetin or YM155) induced selective and complete cell death of undifferentiated hPSCs. In contrast,differentiated cell types (e.g.,dopamine neurons and smooth-muscle cells) derived from hPSCs survived well and maintained their functionality. We found that quercetin-induced selective cell death is caused by mitochondrial accumulation of p53 and is sufficient to prevent teratoma formation after transplantation of hESC- or hiPSC-derived cells. Taken together,these results provide the proof of concept" that small-molecule targeting of hPSC-specific antiapoptotic pathway(s) is a viable strategy to prevent tumor formation by selectively eliminating remaining undifferentiated pluripotent cells for safe hPSC-based therapy." View Publication

It has been known for over 20 years that foetal calf serum can induce hypertrophy in cultured cardiomyocytes but this is rarely considered when examining cardiomyocytes derived from pluripotent stem cells (PSC). Here,we determined how serum affected cardiomyocytes from human embryonic- (hESC) and induced pluripotent stem cells (hiPSC) and hiPSC from patients with hypertrophic cardiomyopathy linked to a mutation in the MYBPC3 gene. We first confirmed previously published hypertrophic effects of serum on cultured neonatal rat cardiomyocytes demonstrated as increased cell surface area and beating frequency. We then found that serum increased the cell surface area of hESC- and hiPSC-derived cardiomyocytes and their spontaneous contraction rate. Phenylephrine,which normally induces cardiac hypertrophy,had no additional effects under serum conditions. Likewise,hiPSC-derived cardiomyocytes from three MYBPC3 patients which had a greater surface area than controls in the absence of serum as predicted by their genotype,did not show this difference in the presence of serum. Serum can thus alter the phenotype of human PSC derived cardiomyocytes under otherwise defined conditions such that the effects of hypertrophic drugs and gene mutations are underestimated. It is therefore pertinent to examine cardiac phenotypes in culture media without or in low concentrations of serum. View Publication

Clinical trials are currently used to test therapeutic efficacies for lung cancer,infections and diseases. Animal models are also used as surrogates for human disease. Both approaches are expensive and time-consuming. The utility of human biospecimens as models is limited by specialized tissue processing methods that preserve subclasses of analytes (e.g. RNA,protein,morphology) at the expense of others. We present a rapid and reproducible method for the cryopreservation of viable lung tissue from patients undergoing lobectomy or transplant. This method involves the pseudo-diaphragmatic expansion of pieces of fresh lung tissue with cryoprotectant formulation (pseudo-diaphragmatic expansion-cryoprotectant perfusion or PDX-CP) followed by controlled-rate freezing in cryovials. Expansion-perfusion rates,volumes and cryoprotectant formulation were optimized to maintain tissue architecture,decrease crystal formation and increase long-term cell viability. Rates of expansion of 4 cc/min or less and volumes ranging from 0.8-1.2 × tissue volume were well-tolerated by lung tissue obtained from patients with chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis,showing minimal differences compared to standard histopathology. Morphology was greatly improved by the PDX-CP procedure compared to simple fixation. Fresh versus post-thawed lung tissue showed minimal differences in histology,RNA integrity numbers and post-translational modified protein integrity (2-dimensional differential gel electrophoresis). It was possible to derive numerous cell types,including alveolar epithelial cells,fibroblasts and stem cells,from the tissue for at least three months after cryopreservation. This new method should provide a uniform,cost-effective approach to the banking of biospecimens,with versatility to be amenable to any post-acquisition process applicable to fresh tissue samples. View Publication

Protein arginine methyltransferases (PRMTs) are responsible for symmetric and asymmetric methylation of arginine residues of nuclear and cytoplasmic proteins. In the nucleus,PRMTs belong to important chromatin modifying enzymes of immense functional significance that affect gene expression,splicing and DNA repair. By time-lapse microscopy we have studied the sub-cellular localization and kinetics of PRMT1 after inhibition of PRMT1 and after irradiation. Both transiently expressed and endogenous PRMT1 accumulated in cytoplasmic bodies that were located in the proximity of the cell nucleus. The shape and number of these bodies were stable in untreated cells. However,when cell nuclei were microirradiated by UV-A,the mobility of PRMT1 cytoplasmic bodies increased,size was reduced,and disappeared within approximately 20 min. The same response occurred after $$-irradiation of the whole cell population,but with delayed kinetics. Treatment with PRMT1 inhibitors induced disintegration of these PRMT1 cytoplasmic bodies and prevented formation of 53BP1 nuclear bodies (NBs) that play a role during DNA damage repair. The formation of 53BP1 NBs was not influenced by PRMT1 overexpression. Taken together,we show that PRMT1 concentrates in cytoplasmic bodies,which respond to DNA injury in the cell nucleus,and to treatment with various PRMT1 inhibitors. View Publication

INTRODUCTION: Ischemic stroke is a leading cause of death and disability,but treatment options are severely limited. Cell therapy offers an attractive strategy for regenerating lost tissues and enhancing the endogenous healing process. In this study,we investigated the use of human embryonic stem cell-derived neural precursors as a cell therapy in a murine stroke model.backslashnbackslashnMETHODS: Neural precursors were derived from human embryonic stem cells by using a fully adherent SMAD inhibition protocol employing small molecules. The efficiency of neural induction and the ability of these cells to further differentiate into neurons were assessed by using immunocytochemistry. Whole-cell patch-clamp recording was used to demonstrate the electrophysiological activity of human embryonic stem cell-derived neurons. Neural precursors were transplanted into the core and penumbra regions of a focal ischemic stroke in the barrel cortex of mice. Animals received injections of bromodeoxyuridine to track regeneration. Neural differentiation of the transplanted cells and regenerative markers were measured by using immunohistochemistry. The adhesive removal test was used to determine functional improvement after stroke and intervention.backslashnbackslashnRESULTS: After 11 days of neural induction by using the small-molecule protocol,over 95% of human embryonic stem-derived cells expressed at least one neural marker. Further in vitro differentiation yielded cells that stained for mature neuronal markers and exhibited high-amplitude,repetitive action potentials in response to depolarization. Neuronal differentiation also occurred after transplantation into the ischemic cortex. A greater level of bromodeoxyuridine co-localization with neurons was observed in the penumbra region of animals receiving cell transplantation. Transplantation also improved sensory recovery in transplant animals over that in control animals.backslashnbackslashnCONCLUSIONS: Human embryonic stem cell-derived neural precursors derived by using a highly efficient small-molecule SMAD inhibition protocol can differentiate into electrophysiologically functional neurons in vitro. These cells also differentiate into neurons in vivo,enhance regenerative activities,and improve sensory recovery after ischemic stroke. View Publication

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells,retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE),and fetal RPE. The protocols include collection of RNA for analysis by microarray,and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally,cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types. View Publication

Anterior foregut endoderm (AFE) gives rise to therapeutically relevant cell types in tissues such as the esophagus,salivary glands,lung,thymus,parathyroid and thyroid. Despite its importance,reports describing the generation of AFE from pluripotent stem cells (PSCs) by directed differentiation have mainly focused on the Nkx2.1(+) lung and thyroid lineages. Here,we describe a novel protocol to derive a subdomain of AFE,identified by expression of Pax9,from PSCs using small molecules and defined media conditions. We generated a reporter PSC line for isolation and characterization of Pax9(+) AFE cells,which when transplanted in vivo,can form several distinct complex AFE-derived epithelia,including mucosal glands and stratified squamous epithelium. Finally,we show that the directed differentiation protocol can be used to generate AFE from human PSCs. Thus,this work both broadens the range of PSC-derived AFE tissues and creates a platform enabling the study of AFE disorders. View Publication

Induced pluripotent stem cells (IPS) are an artificially derived type of pluripotent stem cell,showing many of the same characteristics as natural pluripotent stem cells. IPS are a hopeful therapeutic model; however there is a critical need to determine their response to environmental toxins. Effects of arsenic on cells have been studied extensively; however,its effect on IPS is yet to be elucidated. Arsenic trioxide (ATO) has been shown to inhibit cell proliferation,induce apoptosis and genotoxicity in many cells. Based on ATOs action in other cells,we hypothesize that it will induce alterations in morphology,inhibit cell viability and induce a genotoxic effect on IPS. Cells were treated for 24 hours with ATO (0-9 µg/mL). Cell morphology,viability and DNA damage were documented. Results indicated sufficient changes in morphology of cell colonies mainly in cell ability to maintain grouping and ability to remain adherent. Cell viability decreased in a dose dependent manner. There were significant increases in tail length and moment as well as destruction of intact DNA as concentration increased. Exposure to ATO resulted in a reproducible dose dependent sequence of events marked by changes in morphology,decrease of cell viability,and induction of genotoxicity in IPS. View Publication

Primitive erythroid cells,the first red blood cells produced in the mammalian embryo,are necessary for embryonic survival. Erythropoietin and its receptor EpoR,are absolutely required for survival of late-stage definitive erythroid progenitors in the fetal liver and adult bone marrow. Epo- and Epor-null mice die at E13.5 with a lack of definitive erythrocytes. However,the persistence of circulating primitive erythroblasts raises questions about the role of erythropoietin/EpoR in primitive erythropoiesis. Using Epor-null mice and a novel primitive erythroid 2-step culture we found that erythropoietin is not necessary for specification of primitive erythroid progenitors. However,Epor-null embryos develop a progressive,profound anemia by E12.5 as primitive erythroblasts mature as a synchronous cohort. This anemia results from reduced primitive erythroblast proliferation associated with increased p27 expression,from advanced cellular maturation,and from markedly elevated rates of apoptosis associated with an imbalance in pro- and anti-apoptotic gene expression. Both mouse and human primitive erythroblasts cultured without erythropoietin also undergo accelerated maturation and apoptosis at later stages of maturation. We conclude that erythropoietin plays an evolutionarily conserved role in promoting the proliferation,survival,and appropriate timing of terminal maturation of primitive erythroid precursors. View Publication

We here report that doxycycline,an antibacterial agent,exerts dramatic effects on human embryonic stem and induced pluripotent stem cells (hESC/iPSCs) survival and self-renewal. The survival-promoting effect was also manifest in cultures of neural stem cells (NSCs) derived from hESC/iPSCs. These doxycycline effects are not associated with its antibacterial action,but mediated by direct activation of a PI3K-AKT intracellular signal. These findings indicate doxycycline as a useful supplement for stem cell cultures,facilitating their growth and maintenance. View Publication

The development of novel platforms for large scale production of human embryonic stem cells (hESC) derived cardiomyocytes (CM) becomes more crucial as the demand for CMs in preclinical trials,high throughput cardio toxicity assays and future regenerative therapeutics rises. To this end,we have designed a microcarrier (MC) suspension agitated platform that integrates pluripotent hESC expansion followed by CM differentiation in a continuous,homogenous process.Hydrodynamic shear stresses applied during the hESC expansion and CM differentiation steps drastically reduced the capability of the cells to differentiate into CMs. Applying vigorous stirring during pluripotent hESC expansion on Cytodex 1 MC in spinner cultures resulted in low CM yields in the following differentiation step (cardiac troponin-T (cTnT): 22.83. ??. 2.56%; myosin heavy chain (MHC): 19.30. ??. 5.31%). Whereas the lower shear experienced in side to side rocker (wave type) platform resulted in higher CM yields (cTNT: 47.50. ??. 7.35%; MHC: 42.85. ??. 2.64%). The efficiency of CM differentiation is also affected by the hydrodynamic shear stress applied during the first 3. days of the differentiation stage. Even low shear applied continuously by side to side rocker agitation resulted in very low CM differentiation efficiency (cTnT. textless. 5%; MHC. textless. 2%). Simply by applying intermittent agitation during these 3. days followed by continuous agitation for the subsequent 9. days,CM differentiation efficiency can be substantially increased (cTNT: 65.73. ??. 10.73%; MHC: 59.73. ??. 9.17%). These yields are 38.3% and 39.3% higher (for cTnT and MHC respectively) than static culture control.During the hESC expansion phase,cells grew on continuously agitated rocker platform as pluripotent cell/MC aggregates (166??88??105??m2) achieving a cell concentration of 3.74??0.55??106cells/mL (18.89??2.82 fold expansion) in 7days. These aggregates were further differentiated into CMs using a WNT modulation differentiation protocol for the subsequent 12days on a rocking platform with an intermittent agitation regime during the first 3days. Collectively,the integrated MC rocker platform produced 190.5??58.8??106 CMs per run (31.75??9.74 CM/hESC seeded). The robustness of the system was demonstrated by using 2 cells lines,hESC (HES-3) and human induced pluripotent stem cell (hiPSC) IMR-90. The CM/MC aggregates formed extensive sarcomeres that exhibited cross-striations confirming cardiac ontogeny. Functionality of the CMs was demonstrated by monitoring the effect of inotropic drug,Isoproterenol on beating frequency.In conclusion,we have developed a simple robust and scalable platform that integrates both hESC expansion and CM differentiation in one unit process which is capable of meeting the need for large amounts of CMs. ?? 2014. View Publication

Stirred-suspension bioreactors are a promising modality for large-scale culture of 3D aggregates of pluripotent stem cells and their progeny. Yet,cells within these clusters experience limitations in the transfer of factors and particularly O2 which is characterized by low solubility in aqueous media. Cultured stem cells under different O2 levels may exhibit significantly different proliferation,viability and differentiation potential. Here,a transient diffusion-reaction model was built encompassing the size distribution and ultrastructural characteristics of embryonic stem cell (ESC) aggregates. The model was coupled to experimental data from bioreactor and static cultures for extracting the effective diffusivity and kinetics of consumption of O2 within mouse (mESC) and human ESC (hESC) clusters. Under agitation,mESC aggregates exhibited a higher maximum consumption rate than hESC aggregates. Moreover,the reaction-diffusion model was integrated with a population balance equation (PBE) for the temporal distribution of ESC clusters changing due to aggregation and cell proliferation. Hypoxia was found to be negligible for ESCs with a smaller radius than 100 µm but became appreciable for aggregates larger than 300 µm. The integrated model not only captured the O2 profile both in the bioreactor bulk and inside ESC aggregates but also led to the calculation of the duration that fractions of cells experience a certain range of O2 concentrations. The approach described in this study can be employed for gaining a deeper understanding of the effects of O2 on the physiology of stem cells organized in 3D structures. Such frameworks can be extended to encompass the spatial and temporal availability of nutrients and differentiation factors and facilitate the design and control of relevant bioprocesses for the production of stem cell therapeutics. View Publication