技术资料

PURPOSE Nonexudative (dry) age-related macular degeneration (AMD),a leading cause of blindness in the elderly,is associated with the loss of retinal pigmented epithelium (RPE) cells and the development of geographic atrophy,which are areas devoid of RPE cells and photoreceptors. One possible treatment option would be to stimulate RPE attachment and proliferation to replace dying/dysfunctional RPE and bring about wound repair. Clinical trials are underway testing injections of RPE cells derived from pluripotent stem cells to determine their safety and efficacy in treating AMD. However,the factors regulating RPE responses to AMD-associated lesions are not well understood. Here,we use cell culture to investigate the role of RhoA coiled coil kinases (ROCKs) in human embryonic stem cell-derived RPE (hESC-RPE) attachment,proliferation,and wound closure. METHODS H9 hESC were spontaneously differentiated into RPE cells. hESC-RPE cells were treated with a pan ROCK1/2 or a ROCK2 only inhibitor; attachment,and proliferation and cell size within an in vitro scratch assay were examined. RESULTS Pharmacological inhibition of ROCKs promoted hESC-RPE attachment and proliferation,and increased the rate of closure of in vitro wounds. ROCK inhibition decreased phosphorylation of cofilin and myosin light chain,suggesting that regulation of the cytoskeleton underlies the mechanism of action of ROCK inhibition. CONCLUSIONS ROCK inhibition promotes attachment,proliferation,and wound closure in H9 hESC-RPE cells. ROCK isoforms may have different roles in wound healing. TRANSLATIONAL RELEVANCE Modulation of the ROCK-cytoskeletal axis has potential in stimulating wound repair in transplanted RPE cells and attachment in cellular therapies. View Publication

Gap junction-mediated cell-cell interactions are highly conserved and play essential roles in cell survival,proliferation,differentiation and patterning. We report that Connexin 32 (Cx32)-mediated gap junctional intercellular communication (GJIC) is necessary for human embryonic stem cell-derived hepatocytes (hESC-Heps) during step-wise hepatic lineage restriction and maturation. Vitamin K2,previously shown to promote Cx32 expression in mature hepatocytes,up-regulated Cx32 expression and GJIC activation during hepatic differentiation and maturation,resulting in significant increases of hepatic markers expression and hepatocyte functions. In contrast,negative Cx32 regulator 2-aminoethoxydiphenyl borate blocked hESC-to-hepatocyte maturation and muted hepatocyte functions through disruption of GJIC activities. Dynamic gap junction organization and internalization are phosphorylation-dependent and the p38 mitogen-activated protein kinases pathway (MAPK) can negatively regulate Cxs through phosphorylation-dependent degradation of Cxs. We found that p38 MAPK inhibitor SB203580 improved maturation of hESC-Heps correlating with up-regulation of Cx32; by contrast,the p38 MAPK activator,anisomycin,blocked hESC-Heps maturation correlating with down-regulation of Cx32. These results suggested that Cx32 is essential for cell-cell interactions that facilitate driving hESCs through hepatic-lineage maturation. Regulators of both Cx32 and other members of its pathways maybe used as a promising approach on regulating hepatic lineage restriction of pluripotent stem cells and optimizing their functional maturation. View Publication

Biophysical properties of the scaffolds such as the elastic modulus,have been recently shown to impact stem cell lineage commitment. On the other hand,the contribution of the Poisson's ratio,another important biophysical property,to the stem cell fate decision,has not been studied. Scaffolds with tunable Poisson's ratio (ν) (termed as auxetic scaffolds when Poisson's ratio is zero or negative) are anticipated to provide a spectrum of unique biophysical 3-D microenvironments to influence stem cell fate. To test this hypothesis,in the present work we fabricated auxetic polyurethane scaffolds (ν=0 to -0.45) and evaluated their effects on neural differentiation of mouse embryonic stem cells (ESCs) and human induced pluripotent stem cells (hiPSCs). Compared to the regular scaffolds (ν=+0.30) before auxetic conversion,the auxetic scaffolds supported smaller aggregate formation and higher expression of β-tubulin III upon neural differentiation. The influences of pore structure,Poisson's ratio,and elastic modulus on neural lineage commitment were further evaluated using a series of auxetic scaffolds. The results indicate that Poisson's ratio may confound the effects of elastic modulus,and auxetic scaffolds with proper pore structure and Poisson's ratio enhance neural differentiation. This study demonstrates that tuning the Poisson's ratio of the scaffolds together with elastic modulus and microstructure would enhance the capability to generate broader,more diversified ranges of biophysical 3-D microenvironments for the modulation of cellular differentiation. STATEMENT OF SIGNIFICANCE Biophysical signaling from the substrates and scaffolds plays a critical role in neural lineage commitment of pluripotent stem cells. While the contribution of elastic modulus has been well studied,the influence of Poisson's ratio along with microstructure of the scaffolds remains unknown largely due to the lack of technology to produce materials with tailorable Poisson's ratio. This study fabricated auxetic polyurethane scaffolds with different elastic modulus,Poisson's ratio and microstructure and evaluated neural differentiation of pluripotent stem cells. The findings add a novel angle to understand the impact of biophysical microenvironment on stem cell fate decisions. View Publication

OBJECTIVE The differentiation program for human thyroid follicular cells (TFCs) relies on the interplay between sequence-specific transcription factors and transcriptional co-regulators. Transcriptional co-activator with PDZ-binding motif (TAZ) is a co-activator that regulates several transcription factors,including PAX8 and NKX2-1,which play a central role in thyroid-specific gene transcription. TAZ and PAX8/NKX2-1 are co-expressed in the nuclei of thyroid cells,and TAZ interacts directly with both PAX8 and NKX2-1,leading to their enhanced transcriptional activity on the thyroglobulin (TG) promoter and additional genes. METHODS The use of a small molecule,ethacridine,recently identified as a TAZ activator,in the differentiation of thyroid cells from human embryonic stem (hES) cells was studied. First,endodermal cells were derived from hES cells using Activin A,followed by induction of differentiation into thyroid cells directed by ethacridine and thyrotropin (TSH). RESULTS The expression of TAZ was increased in the Activin A-derived endodermal cells by ethacridine in a dose-dependent manner and followed by increases in PAX8 and NKX2-1 when assessed by both quantitative polymerase chain reaction and immunostaining. Following further differentiation with the combination of ethacridine and TSH,the thyroid-specific genes TG,TPO,TSHR,and NIS were all induced in the differentiated hES cells. When these cells were cultured with extracellular matrix-coated dishes,thyroid follicle formation and abundant TG protein expression were observed. Furthermore,such hES cell-derived thyroid follicles showed a marked TSH-induced and dose-dependent increase in radioiodine uptake and protein-bound iodine accumulation. CONCLUSION These data show that fully functional human thyroid cells can be derived from hES cells using ethacridine,a TAZ activator,which induces thyroid-specific gene expression and promotes thyroid cell differentiation from the hES cells. These studies again demonstrate the importance of transcriptional regulation in thyroid cell development. This approach also yields functional human thyrocytes,without any gene transfection or complex culture conditions,by directly manipulating the transcriptional machinery without interfering with intermediate signaling events. View Publication

No treatments exist to effectively treat many retinal diseases. Retinal pigmented epithelium (RPE) and neural retina can be generated from human embryonic stem cells/induced pluripotent stem cells (hESCs/hiPSCs). The efficacy of current protocols is,however,limited. It was hypothesised that generation of laminated neural retina and/or RPE from hiPSCs/hESCs could be enhanced by three dimensional (3D) culture in hydrogels. hiPSC- and hESC-derived embryoid bodies (EBs) were encapsulated in 0.5% RGD-alginate; 1% RGD-alginate; hyaluronic acid (HA) or HA/gelatin hydrogels and maintained until day 45. Compared with controls (no gel),0.5% RGD-alginate increased: the percentage of EBs with pigmented RPE foci; the percentage EBs with optic vesicles (OVs) and pigmented RPE simultaneously; the area covered by RPE; frequency of RPE cells (CRALBP+); expression of RPE markers (TYR and RPE65) and the retinal ganglion cell marker,MATH5. Furthermore,0.5% RGD-alginate hydrogel encapsulation did not adversely affect the expression of other neural retina markers (PROX1,CRX,RCVRN,AP2α or VSX2) as determined by qRT-PCR,or the percentage of VSX2 positive cells as determined by flow cytometry. 1% RGD-alginate increased the percentage of EBs with OVs and/or RPE,but did not significantly influence any other measures of retinal differentiation. HA-based hydrogels had no significant effect on retinal tissue development. The results indicated that derivation of retinal tissue from hESCs/hiPSCs can be enhanced by culture in 0.5% RGD-alginate hydrogel. This RGD-alginate scaffold may be useful for derivation,transport and transplantation of neural retina and RPE,and may also enhance formation of other pigmented,neural or epithelial tissue. STATEMENT OF SIGNIFICANCE The burden of retinal disease is ever growing with the increasing age of the world-wide population. Transplantation of retinal tissue derived from human pluripotent stem cells (PSCs) is considered a promising treatment. However,derivation of retinal tissue from PSCs using defined media is a lengthy process and often variable between different cell lines. This study indicated that alginate hydrogels enhanced retinal tissue development from PSCs,whereas hyaluronic acid-based hydrogels did not. This is the first study to show that 3D culture with a biomaterial scaffold can improve retinal tissue derivation from PSCs. These findings indicate potential for the clinical application of alginate hydrogels for the derivation and subsequent transplantation retinal tissue. This work may also have implications for the derivation of other pigmented,neural or epithelial tissue. View Publication

In this study,because excessive polycythemia is a predominant trait in some high-altitude dwellers (chronic mountain sickness [CMS] or Monge's disease) but not others living at the same altitude in the Andes,we took advantage of this human experiment of nature and used a combination of induced pluripotent stem cell technology,genomics,and molecular biology in this unique population to understand the molecular basis for hypoxia-induced excessive polycythemia. As compared with sea-level controls and non-CMS subjects who responded to hypoxia by increasing their RBCs modestly or not at all,respectively,CMS cells increased theirs remarkably (up to 60-fold). Although there was a switch from fetal to adult HgbA0 in all populations and a concomitant shift in oxygen binding,we found that CMS cells matured faster and had a higher efficiency and proliferative potential than non-CMS cells. We also established that SENP1 plays a critical role in the differential erythropoietic response of CMS and non-CMS subjects: we can convert the CMS phenotype into that of non-CMS and vice versa by altering SENP1 levels. We also demonstrated that GATA1 is an essential downstream target of SENP1 and that the differential expression and response of GATA1 and Bcl-xL are a key mechanism underlying CMS pathology. View Publication

The differentiation of human pluripotent stem cells toward the hepatocyte lineage can potentially provide an unlimited source of functional hepatocytes for transplantation and extracorporeal bioartificial liver applications. It is anticipated that the quantities of cells needed for these applications will be in the order of 10(9)-10(10) cells,because of the size of the liver. An ideal differentiation protocol would be to enable directed differentiation to the hepatocyte lineage with simultaneous cell expansion. We introduced a cell expansion stage after the commitment of human embryonic stem cells to the endodermal lineage,to allow for at least an eightfold increase in cell number,with continuation of cell maturation toward the hepatocyte lineage. The progressive changes in the transcriptome were measured by expression array,and the expression dynamics of certain lineage markers was measured by mass cytometry during the differentiation and expansion process. The findings revealed that while cells were expanding they were also capable of progressing in their differentiation toward the hepatocyte lineage. In addition,our transcriptome,protein and functional studies,including albumin secretion,drug-induced CYP450 expression and urea production,all indicated that the hepatocyte-like cells obtained with or without cell expansion are very similar. This method of simultaneous cell expansion and hepatocyte differentiation should facilitate obtaining large quantities of cells for liver cell applications. View Publication

Obtaining highly purified differentiated cells via directed differentiation from human pluripotent stem cells (hPSCs) is an essential step for their clinical application. Among the various conditions that should be optimized,the precise role and contribution of the extracellular matrix (ECM) during differentiation are relatively unclear. Here,using a short fragment of laminin 411 (LM411-E8),an ECM predominantly expressed in the vascular endothelial basement membrane,we demonstrate that the directed switching of defined ECMs robustly yields highly-purified (textgreater95%) endothelial progenitor cells (PSC-EPCs) without cell sorting from hPSCs in an integrin-laminin axis-dependent manner. Single-cell RNA-seq analysis revealed that LM411-E8 resolved intercellular transcriptional heterogeneity and escorted the progenitor cells to the appropriate differentiation pathway. The PSC-EPCs gave rise to functional endothelial cells both in vivo and in vitro. We therefore propose that sequential switching of defined matrices is an important concept for guiding cells towards desired fate. View Publication

Purpose The purpose of this study was to characterize the secretion profile of induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. iPS-RPE was used to develop an in vitro wound healing model. We hypothesized that iPS-RPE secretes cytokines and growth factors which act in an autocrine manner to promote migration and proliferation of cells during wound healing. Methods iPS-RPE was grown in transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Levels of Ki-67,$$-catenin,e-cadherin,n-cadherin,and S100A4 expression were analyzed by immunofluorescent labeling. Cell culture medium samples were collected from both the apical and basolateral sides of the transwells every 72 hours for 21 days. The medium samples were analyzed using multiplex ELISA to detect secreted growth factors and cytokines. The effects of conditioned medium on collagen gel contraction,cell proliferation,and migration were measured. Results iPS-RPE underwent epithelial-mesenchymal transition (EMT) during wound healing as indicated by the translocation of $$-catenin to the nucleus,cadherin switch,and expression of S100A4. GRO,GM-CSF,MCP-1,IL-6,and IL-8 were secreted by both the control and the wounded cell cultures. VEGF,FGF-2,and TGF$$ expression were detected at higher levels after wounding than those in control. The proteins were found to be secreted in a polarized manner. The conditioned medium from wounded monolayers promoted collagen gel contraction,as well as proliferation and migration of ARPE 19 cells. Conclusions These results indicate that after the monolayer is wounded,iPS-RPE secretes proteins into the culture medium that promote increased proliferation,contraction,and migration. View Publication

OBJECTIVE The aim of this study was to evaluate the distribution,concentration and toxicity of cinnamaldehyde in electronic cigarette (e-cigarette) refill fluids and aerosols. METHODS The distribution and concentration of cinnamaldehyde were determined in 39 e-cigarette refill fluids plus 6 duplicates using gas chromatography and mass spectrometry (GC/MS). A cinnamaldehyde toxicity profile was established for embryonic and adult cells using a live cell imaging assay,immunocytochemistry,the comet assay and a recovery assay. RESULTS Twenty of the 39 refill fluids contained cinnamaldehyde at concentrations that are cytotoxic to human embryonic and lung cells in the MTT assay. Cinnamon Ceylon aerosol produced in a cartomizer-style e-cigarette was cytotoxic. Cinnamon Ceylon aerosols and refill fluid aerosols (80% propylene glycol or cinnamaldehyde/propylene glycol) made using a tank/boxmod e-cigarette were more cytotoxic at 5 V than 3 V. Using GC/MS,aerosols produced at 5 V contained 10 additional peaks not present in aerosol generated at 3 V. One of these,2,3-butandione (diacetyl),was confirmed with an authentic standard. Cinnamaldehyde depolymerised microtubules in human pulmonary fibroblasts. At concentrations that produced no effect in the MTT assay,cinnamaldehyde decreased growth,attachment and spreading; altered cell morphology and motility; increased DNA strand breaks; and increased cell death. At the MTT IC50 concentration,lung cells were unable to recover from cinnamaldehyde after 2 hours of treatment,whereas embryonic cells recovered after 8 hours. CONCLUSIONS Cinnamaldehyde-containing refill fluids and aerosols are cytotoxic,genotoxic and low concentrations adversely affect cell processes and survival. These data indicate that cinnamaldehyde in e-cigarette refill fluids/aerosols may impair homeostasis in the respiratory system. View Publication

Engineered heart tissues made from human pluripotent stem cell-derived cardiomyocytes have been used for modeling cardiac pathologies,screening new therapeutics,and providing replacement cardiac tissue. Current methods measure the functional performance of engineered heart tissue by their twitch force and beating frequency,typically obtained by optical measurements. In this article,we describe a novel method for assessing twitch force and beating frequency of engineered heart tissue using magnetic field sensing,which enables multiple tissues to be measured simultaneously. The tissues are formed as thin structures suspended between two silicone posts,where one post is rigid and another is flexible and contains an embedded magnet. When the tissue contracts it causes the flexible post to bend in proportion to its twitch force. We measured the bending of the post using giant magnetoresistive (GMR) sensors located underneath a 24-well plate containing the tissues. We validated the accuracy of the readings from the GMR sensors against optical measurements. We demonstrated the utility and sensitivity of our approach by testing the effects of three concentrations of isoproterenol and verapamil on twitch force and beating frequency in real-time,parallel experiments. This system should be scalable beyond the 24-well format,enabling greater automation in assessing the contractile function of cardiomyocytes in a tissue-engineered environment. View Publication

Haploid human pluripotent stem cells (PSCs) integrate haploidy and pluripotency,providing a novel system for functional genomics and developmental research in humans. We have recently derived haploid human embryonic stem cells (ESCs) by parthenogenesis and demonstrated their wide differentiation potential and applicability for genetic screening. Because haploid cells can spontaneously become diploid,their enrichment at an early passage is key for successful derivation. In this protocol,we describe two methodologies,namely metaphase spread analysis and cell sorting,for the identification of haploid human cells within parthenogenetic ESC lines. The cell sorting approach also enables the isolation of haploid cells at low percentages,as well as the maintenance of highly enriched haploid ESC lines throughout passaging. The isolation of essentially pure populations of haploid human ESCs by this protocol requires basic PSC culture expertise and can be achieved within 4-6 weeks. View Publication