Scientific Resources

We investigate the effects of myocardial transplantation of human induced pluripotent stem cell (iPSC)-derived progenitors and cardiomyocytes into acutely infarcted myocardium in severe combined immune deficiency mice. A total of 2 × 10(5) progenitors,cardiomyocytes or cell-free saline were injected into peri-infarcted anterior free wall. Sham-operated animals received no injection. Myocardial function was assessed at 2-week and 4-week post-infarction by using echocardiography and pressure-volume catheterization. Early myocardial remodelling was observed at 2-week with echocardiography derived stroke volume (SV) in saline (20.45 ± 7.36 $\$,P textless 0.05) and cardiomyocyte (19.52 ± 3.97 $\$,P textless 0.05) groups,but not in progenitor group (25.65 ± 3.61 $\$),significantly deteriorated as compared to sham control group (28.41 ± 4.41 $\$). Consistently,pressure-volume haemodynamic measurements showed worsening chamber dilation in saline (EDV: 23.24 ± 5.01 $\$,P textless 0.05; ESV: 17.08 ± 5.82 $\$,P textless 0.05) and cardiomyocyte (EDV: 26.45 ± 5.69 $\$,P textless 0.05; ESV: 18.03 ± 6.58 $\$,P textless 0.05) groups by 4-week post-infarction as compared to control (EDV: 15.26 ± 2.96 $\$; ESV: 8.41 ± 2.94 $\$). In contrast,cardiac progenitors (EDV: 20.09 ± 7.76 $\$; ESV: 13.98 ± 6.74 $\$) persistently protected chamber geometry against negative cardiac remodelling. Similarly,as compared to sham control (54.64 ± 11.37%),LV ejection fraction was preserved in progenitor group from 2-(38.68 ± 7.34%) to 4-week (39.56 ± 13.26%) while cardiomyocyte (36.52 ± 11.39%,P textless 0.05) and saline (35.34 ± 11.86%,P textless 0.05) groups deteriorated early at 2-week. Improvements of myocardial function in the progenitor group corresponded to increased vascularization (16.12 ± 1.49/mm(2) to 25.48 ± 2.08/mm(2) myocardial tissue,P textless 0.05) and coincided with augmented networking of cardiac telocytes in the interstitial space of infarcted zone. View Publication

Hyaluronic acid (HA) has been cross-linked to form hydrogel for potential applications in the self-renewal and differentiation of human pluripotent stem cells (hPSCs) for years. However,HA hydrogel with improved residence time and mechanical integrity that allows the survival of hPSCs under defined conditions is still much needed for clinical applications. In this study,HA was modified with methacrylate functional groups (MeHA) and cross-linked by photo-crosslinking method. After subsequent conjugation with adhesive peptide,these MeHA surfaces demonstrated performance in facilitating human induced pluripotent stem cells (hiPSCs) proliferation,and good pluripotency maintenance of hiPSCs under defined conditions. Moreover,MeHA films on glass-slides exhibited long residence time and mechanical stability throughout hiPSC culture. Our photo-crosslinkable MeHA possesses great value in accelerating the application of HA hydrogel in hiPSCs proliferation and differentiation with the conjugation of adhesive peptides. View Publication

We have previously demonstrated the existence of two phenotypically distinct cell subsets in estrogen receptor (ER)-positive breast cancer (BC) based on their differential response to a Sox2 reporter (SRR2),with reporter responsive (RR) cells being more tumorigenic and stem-like than reporter unresponsive (RU) cells. To delineate the molecular mechanisms underlying this phenotypic dichotomy,we tested our hypothesis that Sox2,which is a key regulator of the RR phenotype,is under the control of its binding partners. In this study,we focused on DDX17,known to be a transcription co-activator and found to be a Sox2 binding partner by liquid chromatography-mass spectrometry. Using immunoprecipitation,we confirmed the binding between DDX17 and Sox2,although this interaction was largely restricted to RR cells. While DDX17 was found in both the cytoplasm and nuclei in RU cells,it is confined to the nuclei in RR cells. siRNA knockdown of DDX17 in RR cells substantially decreased the Sox2-SRR2 binding and significantly decreased the SRR2 reporter activity without affecting the protein level of Sox2. Using ChIP-PCR,DDX17 knockdown also significantly decreased the binding of Sox2 to genomic SRR2,as well as 3 of its specific gene targets including MUC15,CCND1 and CD133. Correlating with these findings,siRNA knockdown of DDX17 significantly reduced soft agar colony formation and mammosphere formation in RR cells but not RU cells. To conclude,DDX17 is a Sox2-binding protein in ER-positive BC. In RR but not RU cells,DDX17 enhances the tumorigenic and stem-like features of Sox2 by promoting its binding to its target genes. View Publication

Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report,we employ electron,immunofluorescence microscopy,and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 $$g/$$g proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 $$g/$$g proteins) reported in human cancer cell lines. Moreover,we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states,similar to those naïve-like hPSCs,with increased glycogen synthesis. Furthermore,we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus,our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions. View Publication

The translation of laboratory processes into scaled production systems suitable for manufacture is a significant challenge for cell based therapies; in particular there is a lack of analytical methods that are informative and efficient for process control. Here the potential of image analysis as one part of the solution to this issue is explored,using pluripotent stem cell colonies as a valuable and challenging exemplar. The Cell-IQ live cell imaging platform was used to build image libraries of morphological culture attributes such as colony edge� View Publication

Fibroblasts from patients with Type I bipolar disorder (BPD) and their unaffected siblings were obtained from an Old Order Amish pedigree with a high incidence of BPD and reprogrammed to induced pluripotent stem cells (iPSCs). Established iPSCs were subsequently differentiated into neuroprogenitors (NPs) and then to neurons. Transcriptomic microarray analysis was conducted on RNA samples from iPSCs,NPs and neurons matured in culture for either 2 weeks (termed early neurons,E) or 4 weeks (termed late neurons,L). Global RNA profiling indicated that BPD and control iPSCs differentiated into NPs and neurons at a similar rate,enabling studies of differentially expressed genes in neurons from controls and BPD cases. Significant disease-associated differences in gene expression were observed only in L neurons. Specifically,328 genes were differentially expressed between BPD and control L neurons including GAD1,glutamate decarboxylase 1 (2.5 fold) and SCN4B,the voltage gated type IV sodium channel beta subunit (-14.6 fold). Quantitative RT-PCR confirmed the up-regulation of GAD1 in BPD compared to control L neurons. Gene Ontology,GeneGo and Ingenuity Pathway Analysis of differentially regulated genes in L neurons suggest that alterations in RNA biosynthesis and metabolism,protein trafficking as well as receptor signaling pathways may play an important role in the pathophysiology of BPD. View Publication

The epigenetics of early commitment to embryonal cardiomyocyte is poorly understood. In this work,we compared the effect of thyroid hormone and that of anacardic acid,a naturally occurring histone acetylase inhibitor,or both in combination,on mouse embryonic stem cells (mES) differentiating into embryonal cardiomyocyte by embryoid bodies (EBs) formation. Although the results indicated that anacardic acid (AA) and thyroid hormone were both efficient in promoting cardiomyocyte differentiation,we noticed that a transient exposure of mES to AA alone was sufficient to enlarge the beating areas of EBs compared to those of untreated controls. This effect was associated with changes in the chromatin structure at the promoters of specific cardiomyogenic genes. Among them,a rapid induction of the transcription factor Castor 1 (CASZ1),important for cardiomyocytes differentiation and maturation during embryonic development,was observed in the presence of AA. In contrast,thyroid hormone (T 3) was more effective in stimulating spontaneous firing,thus suggesting a role in the production of a population of cardiomyocyte with pacemaker properties. In conclusion,AA and thyroid hormone both enhanced cardiomyocyte formation along in apparently distinct pathways. View Publication

The diverse progenitors that give rise to the human neocortex have been difficult to characterize because progenitors,particularly radial glia (RG),are rare and are defined by a combination of intracellular markers,position and morphology. To circumvent these problems,we developed Fixed and Recovered Intact Single-cell RNA (FRISCR),a method for profiling the transcriptomes of individual fixed,stained and sorted cells. Using FRISCR,we profiled primary human RG that constitute only 1% of the midgestation cortex and classified them as ventricular zone-enriched RG (vRG) that express ANXA1 and CRYAB,and outer subventricular zone-localized RG (oRG) that express HOPX. Our study identified vRG and oRG markers and molecular profiles,an essential step for understanding human neocortical progenitor development. FRISCR allows targeted single-cell profiling of any tissues that lack live-cell markers. View Publication

Focal malformations of cortical development (FMCDs) account for the majority of drug-resistant pediatric epilepsy. Postzygotic somatic mutations activating the phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway are found in a wide range of brain diseases,including FMCDs. It remains unclear how a mutation in a small fraction of cells disrupts the architecture of the entire hemisphere. Within human FMCD-affected brain,we found that cells showing activation of the PI3K-AKT-mTOR pathway were enriched for the AKT3(E17K) mutation. Introducing the FMCD-causing mutation into mouse brain resulted in electrographic seizures and impaired hemispheric architecture. Mutation-expressing neural progenitors showed misexpression of reelin,which led to a non-cell autonomous migration defect in neighboring cells,due at least in part to derepression of reelin transcription in a manner dependent on the forkhead box (FOX) transcription factor FOXG1. Treatments aimed at either blocking downstream AKT signaling or inactivating reelin restored migration. These findings suggest a central AKT-FOXG1-reelin signaling pathway in FMCD and support pathway inhibitors as potential treatments or therapies for some forms of focal epilepsy. View Publication

The blood-brain barrier (BBB) is a critical component of the central nervous system (CNS) that regulates the flux of material between the blood and the brain. Because of its barrier properties,the BBB creates a bottleneck to CNS drug delivery. Human in vitro BBB models offer a potential tool to screen pharmaceutical libraries for CNS penetration as well as for BBB modulators in development and disease,yet primary and immortalized models respectively lack scalability and robust phenotypes. Recently,in vitro BBB models derived from human pluripotent stem cells (hPSCs) have helped overcome these challenges by providing a scalable and renewable source of human brain microvascular endothelial cells (BMECs). We have demonstrated that hPSC-derived BMECs exhibit robust structural and functional characteristics reminiscent of the in vivo BBB. Here,we provide a detailed description of the methods required to differentiate and functionally characterize hPSC-derived BMECs to facilitate their widespread use in downstream applications. View Publication

Replication stress causes DNA damage at fragile sites in the genome. DNA damage at telomeres can initiate breakage-fusion-bridge cycles and chromosome instability,which can result in replicative senescence or tumor formation. Little is known about the extent of replication stress or telomere dysfunction in human embryonic stem cells (hESCs). hESCs are grown in culture with the expectation of being used therapeutically in humans,making it important to minimize the levels of replication stress and telomere dysfunction. Here,the hESC line UCSF4 was cultured in a defined medium with growth factor Activin A,exogenous nucleosides,or DNA polymerase inhibitor aphidicolin. We used quantitative fluorescence in situ hybridization to analyze individual telomeres for dysfunction and observed that it can be increased by aphidicolin or Activin A. In contrast,adding exogenous nucleosides relieved dysfunction,suggesting that telomere dysfunction results from replication stress. Whether these findings can be applied to other hESC lines remains to be determined. However,because the loss of telomeres can lead to chromosome instability and cancer,we conclude that hESCs grown in culture for future therapeutic purposes should be routinely checked for replication stress and telomere dysfunction. View Publication

Although embryonal proteins have been used as tumor marker,most are not useful for detection of early malignancy. In the present study,we developed mouse monoclonal antibodies against fetal lung of miniature swine,and screened them to find an embryonal protein that is produced at the early stage of malignancy,focusing on lung adenocarcinoma. We found an antibody clone that specifically stained stroma of lung adenocarcinoma. LC-MS/MS identified the protein recognized by this clone as dimethylarginine dimethylaminohydrolase 2 (DDAH2),an enzyme known for antiatherosclerotic activity. DDAH2 was found to be expressed in fibroblasts of stroma of malignancies,with higher expression in minimally invasive adenocarcinoma (MIA) and invasive adenocarcinoma than in adenocarcinoma in situ (AIS). Moreover,tumors with high stromal expression of DDAH2 had a poorer prognosis than those without. In vitro analysis showed that DDAH2 increases expression of endothelial nitric oxide synthase (eNOS),inducing proliferation and capillary-like tube formation of vascular endothelial cells. In resected human tissues,eNOS also showed higher expression in invasive adenocarcinoma than in AIS and normal lung,similarly to DDAH2. Our data indicate that expression of DDAH2 is associated with invasiveness of lung adenocarcinoma via tumor angiogenesis. DDAH2 expression might be a prognostic factor in lung adenocarcinoma. View Publication