技术资料

Several transcription factors (TFs) have been implicated in neuroectoderm (NE) development,and recently,the TF PAX6 was shown to be critical for human NE specification. However,microRNA networks regulating human NE development have been poorly documented. We hypothesized that microRNAs activated by PAX6 should promote NE development. Using a genomics approach,we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells (hESC). PAX6 binding to active enhancers was found in the proximity of several microRNAs,including hsa-miR-135b. MiR-135b was activated during NE development,and ectopic expression of miR-135b in hESC promoted differentiation toward NE. MiR-135b promotes neural conversion by targeting components of the TGF-β and BMP signaling pathways,thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-miRNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage. View Publication

Induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) has widely been appreciated as a promising tool to model human ocular disease emanating from primary RPE pathology. Here,we describe the successful reprogramming of adult human dermal fibroblasts to iPSCs and their differentiation to pure expandable RPE cells with structural and functional features characteristic for native RPE. Fibroblast cultures were established from skin biopsy material and subsequently reprogrammed following polycistronic lentiviral transduction with OCT4,SOX2,KLF4 and L-Myc. Fibroblast-derived iPSCs showed typical morphology,chromosomal integrity and a distinctive stem cell marker profile. Subsequent differentiation resulted in expandable pigmented hexagonal RPE cells. The cells revealed stable RNA expression of mature RPE markers RPE65,RLBP and BEST1. Immunolabelling verified localisation of BEST1 at the basolateral plasma membrane,and scanning electron microscopy showed typical microvilli at the apical side of iPSC-derived RPE cells. Transepithelial resistance was maintained at high levels during cell culture indicating functional formation of tight junctions. Secretion capacity was demonstrated for VEGF-A. Feeding of porcine photoreceptor outer segments revealed the proper ability of these cells for phagocytosis. IPSC-derived RPE cells largely maintained these properties after cryopreservation. Together,our study underlines that adult dermal fibroblasts can serve as a valuable resource for iPSC-derived RPE with characteristics highly reminiscent of true RPE cells. This will allow its broad application to establish cellular models for RPE-related human diseases. View Publication

Teratoma formation,the standard in vivo pluripotency assay,is also frequently used as a tumorigenicity assay. A common concern in therapeutic stem cell applications is the tumorigenicity potential of a small number of cell impurities in the final product. Estimation of this small number is hampered by the inaccurate methodology of the tumorigenicity assay. Hence,a protocol for tumorigenicity assay that can deliver a defined number of cells,without error introduced by leakage or migration of cells is needed. In this study,we tested our modified transplantation method that allows for transplant of small numbers of pluripotent stem cells (PSCs) under the kidney capsule with minimal cell leakage. A glass capillary with a finely shaped tip and an attached mouth pipette was used to inject PSCs into the rodent kidney capsule. H9 embryonic and induced PSCs were tagged with Fluc and green fluorescence protein reporter genes and divided in different cell doses for transplantation. Bioluminescence imaging (BLI) on the day of surgery showed that the cell signal was confined to the kidney and signal intensity correlated with increasing transplant cell numbers. The overall cell leakage rate was 17% and the rodent survival rate was 96%. Teratoma formation was observed in rodents transplanted with cell numbers between 1 × 10(5)-2 × 10(6). We conclude that this modified procedure for transplanting PSCs under the kidney capsule allows for transplantation of a defined number of PSCs with significant reduction of error associated with cell leakage from the transplant site. View Publication

The SU5416 combined with hypoxia (SuHx) rat model features angio-obliterative pulmonary hypertension resembling human pulmonary arterial hypertension. Despite increasing use of this model,a comprehensive haemodynamic characterisation in conscious rats has not been reported. We used telemetry to characterise haemodynamic responses in SuHx rats and associated these with serial histology. Right ventricular systolic pressure (RVSP) increased to a mean±sd of 106±7 mmHg in response to SuHx and decreased but remained elevated at 72±8 mmHg upon return to normoxia. Hypoxia-only exposed rats showed a similar initial increase in RVSP,a lower maximum RVSP and near-normalisation of RVSP during subsequent normoxia. Progressive vascular remodelling consisted of a four-fold increase in intima thickness,while only minimal changes in media thickness were found. The circadian range in RVSP provided an accurate longitudinal estimate of vascular remodelling. In conclusion,in SuHx rats,re-exposure to normoxia leads to a partial decrease in pulmonary artery pressure,with persisting hypertension and pulmonary vascular remodelling characterised by progressive intima obstruction. View Publication

Spleen tyrosine kinase (Syk) is an attractive drug target in autoimmune,inflammatory,and oncology disease indications. The most advanced Syk inhibitor,R406,1 (or its prodrug form fostamatinib,2),has shown efficacy in multiple therapeutic indications,but its clinical progress has been hampered by dose-limiting adverse effects that have been attributed,at least in part,to the off-target activities of 1. It is expected that a more selective Syk inhibitor would provide a greater therapeutic window. Herein we report the discovery and optimization of a novel series of imidazo[1,2-a]pyrazine Syk inhibitors. This work culminated in the identification of GS-9973,68,a highly selective and orally efficacious Syk inhibitor which is currently undergoing clinical evaluation for autoimmune and oncology indications. View Publication

The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells,holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research,numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported,potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects,we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol,including the use of both kinase and translation inhibitors,and cell culture in the presence of histone deacetylase inhibitors,promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors,and was inversely related to the number of days of hormonal stimulation required for oocyte maturation,whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration,causing premature oocyte activation,we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol,we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and,for the first time,an adult,a female with type 1 diabetes. View Publication

Human stem cells are promising sources for bladder regeneration. Among several possible sources,pluripotent stem cells are the most fascinating because they can differentiate into any cell type,and proliferate limitlessly in vitro. Here,we developed a protocol for differentiation of human pluripotent stem cells (hPSCs) into bladder urothelial cells (BUCs) under a chemically defined culture system. We first differentiated hPSCs into definitive endoderm (DE),and further specified DE cells into BUCs by treating retinoic acid under a keratinocyte-specific serum free medium. hPSC-derived DE cells showed significantly expressed DE-specific genes,but did not express mesodermal or ectodermal genes. After DE cells were specified into BUCs,they notably expressed urothelium-specific genes such as UPIb,UPII,UPIIIa,P63 and CK7. Immunocytochemistry showed that BUCs expressed UPII,CK8/18 and P63 as well as tight junction molecules,E-CADHERIN and ZO-1. Additionally,hPSCs-derived BUCs exhibited low permeability in a FITC-dextran permeability assay,indicating BUCs possessed the functional units of barrier on their surfaces. However,BUCs did not express the marker genes of other endodermal lineage cells (intestine and liver) as well as mesodermal or ectodermal lineage cells. In summary,we sequentially differentiated hPSCs into DE and BUCs in a serum- and feeder-free condition. Our differentiation protocol will be useful for producing cells for bladder regeneration and studying normal and pathological development of the human bladder urothelium in vitro. View Publication

The genetic reprogramming technology allows one to generate pluripotent stem cells for individual patients. These cells,called induced pluripotent stem cells (iPSCs),can be an unlimited source of specialized cell types for the body. Thus,autologous somatic cell replacement therapy becomes possible,as well as the generation of in vitro cell models for studying the mechanisms of disease pathogenesis and drug discovery. Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder that leads to a loss of upper and lower motor neurons. About 10% of cases are genetically inherited,and the most common familial form of ALS is associated with mutations in the SOD1 gene. We used the reprogramming technology to generate induced pluripotent stem cells with patients with familial ALS. Patient-specific iPS cells were obtained by both integration and transgene-free delivery methods of reprogramming transcription factors. These iPS cells have the properties of pluripotent cells and are capable of direct differentiation into motor neurons. View Publication

We measured the dynamics of an essential epigenetic modifier,HP1β,in human cells at different stages of differentiation using Fluorescence Recovery After Photobleaching (FRAP). We found that HP1β mobility is similar in human embryonic stem cells (hES) and iPS cells where it is more mobile compared to fibroblasts; HP1β is less mobile in senescent fibroblasts than in young (dividing) fibroblasts. Introduction of reprogramming factors"� View Publication

Cord blood (CB) cells that express CD34 have extensive hematopoietic capacity and rapidly divide ex vivo in the presence of cytokine combinations; however,many of these CB CD34+ cells lose their marrow-repopulating potential. To overcome this decline in function,we treated dividing CB CD34+ cells ex vivo with several histone deacetylase inhibitors (HDACIs). Treatment of CB CD34+ cells with the most active HDACI,valproic acid (VPA),following an initial 16-hour cytokine priming,increased the number of multipotent cells (CD34+CD90+) generated; however,the degree of expansion was substantially greater in the presence of both VPA and cytokines for a full 7 days. Treated CD34+ cells were characterized based on the upregulation of pluripotency genes,increased aldehyde dehydrogenase activity,and enhanced expression of CD90,c-Kit (CD117),integrin α6 (CD49f),and CXCR4 (CD184). Furthermore,siRNA-mediated inhibition of pluripotency gene expression reduced the generation of CD34+CD90+ cells by 89%. Compared with CB CD34+ cells,VPA-treated CD34+ cells produced a greater number of SCID-repopulating cells and established multilineage hematopoiesis in primary and secondary immune-deficient recipient mice. These data indicate that dividing CB CD34+ cells can be epigenetically reprogrammed by treatment with VPA so as to generate greater numbers of functional CB stem cells for use as transplantation grafts. View Publication

We have previously reported the genetic correction of Huntington's disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work,we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR. View Publication

During mammalian embryonic development,the primitive streak initiates the differentiation of pluripotent epiblast cells into germ layers. Pluripotency can be reacquired in committed somatic cells using a combination of a handful of transcription factors,such as OCT3/4,SOX2,KLF4 and c-MYC (hereafter referred to as OSKM),albeit with low efficiency. Here we show that during OSKM-induced reprogramming towards pluripotency in human cells,intermediate cells transiently show gene expression profiles resembling mesendoderm,which is a major component of the primitive streak. Based on these findings,we discover that forkhead box H1 (FOXH1),a transcription factor required for anterior primitive streak specification during early development,significantly enhances the reprogramming efficiency of human fibroblasts by promoting their maturation,including mesenchymal to epithelial transition and the activation of late pluripotency markers. These results demonstrate that during the reprogramming process,human somatic cells go through a transient state that resembles mesendoderm. View Publication