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BACKGROUND Whole-lung scaffolds can be created by perfusion decellularization of cadaveric donor lungs. The resulting matrices can then be recellularized to regenerate functional organs. This study evaluated the capacity of acellular lung scaffolds to support recellularization with lung progenitors derived from human induced pluripotent stem cells (iPSCs). METHODS Whole rat and human lungs were decellularized by constant-pressure perfusion with 0.1% sodium dodecyl sulfate solution. Resulting lung scaffolds were cryosectioned into slices or left intact. Human iPSCs were differentiated to definitive endoderm,anteriorized to a foregut fate,and then ventralized to a population expressing NK2 homeobox 1 (Nkx2.1). Cells were seeded onto slices and whole lungs,which were maintained under constant perfusion biomimetic culture. Lineage specification was assessed by quantitative polymerase chain reaction and immunofluorescent staining. Regenerated left lungs were transplanted in an orthotopic position. RESULTS Activin-A treatment,followed by transforming growth factor-$\$,induced differentiation of human iPSCs to anterior foregut endoderm as confirmed by forkhead box protein A2 (FOXA2),SRY (Sex Determining Region Y)-Box 17 (SOX17),and SOX2 expression. Cells cultured on decellularized lung slices demonstrated proliferation and lineage commitment after 5 days. Cells expressing Nkx2.1 were identified at 40% to 60% efficiency. Within whole-lung scaffolds and under perfusion culture,cells further upregulated Nkx2.1 expression. After orthotopic transplantation,grafts were perfused and ventilated by host vasculature and airways. CONCLUSIONS Decellularized lung matrix supports the culture and lineage commitment of human iPSC-derived lung progenitor cells. Whole-organ scaffolds and biomimetic culture enable coseeding of iPSC-derived endothelial and epithelial progenitors and enhance early lung fate. Orthotopic transplantation may enable further in vivo graft maturation. View Publication

Although hepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) are considered a promising model for predicting hepatotoxicity,their application has been restricted because of the low activity of drug metabolizing enzymes (DMEs). Here we found that the low expression of xenobiotic receptors (constitutive androstane receptor,CAR; and pregnane X receptor,PXR) contributes to the low activity of DMEs in hPSC-HLCs. Most CAR- and PXR-regulated DMEs and transporters were transcriptionally down-regulated in hPSC-HLC. Transcriptional expression of CAR and PXR was highly repressed in hPSC-HLCs,whereas mRNA levels of aryl hydrocarbon receptor (AHR) were comparable to those of adult liver. Furthermore,ligand-induced transcriptional activation was observed only at AHR in hPSC-HLCs. Bisulfite sequencing analysis demonstrated that promoter hypermethylation of CAR and PXR was associated with diminished transcriptional activity in hPSC-HLCs. Treatment with AHR-selective ligands increased the transcription of AHR-dependent target genes by direct AHR-DNA binding at the xenobiotic response element. In addition,an antagonist of AHR significantly inhibited AHR-dependent target gene expression. Thus,AHR may function intrinsically as a xenosensor as well as a ligand-dependent transcription factor in hPSC-HLCs. Our results indicate that hPSC-HLCs can be used to screen toxic substances related to AHR signaling and to identify potential AHR-targeted therapeutics. View Publication

Synthetic biology has advanced the design of standardized transcription control devices that programme cellular behaviour. By coupling synthetic signalling cascade- and transcription factor-based gene switches with reverse and differential sensitivity to the licensed food additive vanillic acid,we designed a synthetic lineage-control network combining vanillic acid-triggered mutually exclusive expression switches for the transcription factors Ngn3 (neurogenin 3; OFF-ON-OFF) and Pdx1 (pancreatic and duodenal homeobox 1; ON-OFF-ON) with the concomitant induction of MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A; OFF-ON). This designer network consisting of different network topologies orchestrating the timely control of transgenic and genomic Ngn3,Pdx1 and MafA variants is able to programme human induced pluripotent stem cells (hIPSCs)-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells,whose glucose-stimulated insulin-release dynamics are comparable to human pancreatic islets. Synthetic lineage-control networks may provide the missing link to genetically programme somatic cells into autologous cell phenotypes for regenerative medicine. View Publication

Predictive toxicology using stem cells or their derived tissues has gained increasing importance in biomedical and pharmaceutical research. Here,we show that toxicity category prediction by support vector machines (SVMs),which uses qRT-PCR data from 20 categorized chemicals based on a human embryonic stem cell (hESC) system,is improved by the adoption of gene networks,in which network edge weights are added as feature vectors when noisy qRT-PCR data fail to make accurate predictions. The accuracies of our system were 97.5-100% for three toxicity categories: neurotoxins (NTs),genotoxic carcinogens (GCs) and non-genotoxic carcinogens (NGCs). For two uncategorized chemicals,bisphenol-A and permethrin,our system yielded reasonable results: bisphenol-A was categorized as an NGC,and permethrin was categorized as an NT; both predictions were supported by recently published papers. Our study has two important features: (i) as the first study to employ gene networks without using conventional quantitative structure-activity relationships (QSARs) as input data for SVMs to analyze toxicogenomics data in an hESC validation system,it uses additional information of gene-to-gene interactions to significantly increase prediction accuracies for noisy gene expression data; and (ii) using only undifferentiated hESCs,our study has considerable potential to predict late-onset chemical toxicities,including abnormalities that occur during embryonic development. View Publication

Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4,SOX2,C-MYC,and KLF4). Patient-specific iPSC derivatives (e.g.,neuronal,cardiac,hepatic,muscular,and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study,we aimed to evaluate whether the cellular origin can affect the differentiation,in vivo behavior,and single-cell gene expression signatures of human iPSC-derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs),ECs (EC-iPSCs),and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin,BMP4,bFGF,and VEGF. EC-iPSCs at early passage (10 textless P textless 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC-ECs were recovered with a higher percentage of CD31(+) population and expressed higher EC-specific gene expression markers (PECAM1,KDR,and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC-ECs maintained a higher CD31(+) population than FB-iPSC-ECs and CPC-iPSC-ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence,the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation. View Publication

Human pluripotent stem cells (hPSCs) provide a valuable model for the study of human development and a means to generate a scalable source of cells for therapeutic applications. This protocol specifies cell fate efficiently into cardiac and endothelial lineages from hPSCs. The protocol takes 2 weeks to complete and requires experience in hPSC culture and differentiation techniques. Building on lessons taken from early development,this monolayer-directed differentiation protocol uses different concentrations of activin A and bone morphogenetic protein 4 (BMP4) to polarize cells into mesodermal subtypes that reflect mid-primitive-streak cardiogenic mesoderm and posterior-primitive-streak hemogenic mesoderm. This differentiation platform provides a basis for generating distinct cardiovascular progenitor populations that enable the derivation of cardiomyocytes and functionally distinct endothelial cell (EC) subtypes from cardiogenic versus hemogenic mesoderm with high efficiency without cell sorting. ECs derived from cardiogenic and hemogenic mesoderm can be matured into textgreater90% CD31(+)/VE-cadherin(+) definitive ECs. To test the functionality of ECs at different stages of differentiation,we provide methods for assaying the blood-forming potential and de novo lumen-forming activity of ECs. To our knowledge,this is the first protocol that provides a common platform for directed differentiation of cardiomyocytes and endothelial subtypes from hPSCs. This protocol yields endothelial differentiation efficiencies exceeding those of previously published protocols. Derivation of these cell types is a critical step toward understanding the basis of disease and generating cells with therapeutic potential. View Publication

The effects of nicotinamide (NIC) on human fetal and adult endocrine pancreatic cells were studied in tissue culture. Treatment of the fetal cells with 10 mM NIC resulted in a twofold increase in DNA content and a threefold increase in insulin content. This was associated with the development of beta cell outgrowths from undifferentiated epithelial cell clusters and an increase in the expression of the insulin,glucagon,and somatostatin genes. DNA synthesis was stimulated only in the undifferentiated cells. Half-maximal doses for the insulinotropic and mitogenic effects of NIC were 5-10 and 1-2 mM,respectively. Islet-like cell clusters cultured with NIC responded to glucose stimulation with a biphasic increase in insulin release (fourfold peak),whereas control cells were unresponsive to glucose. Both control and NIC-treated cells developed into functional islet tissue after transplantation into athymic nude mice. As compared with adult islets,the insulinotropic action of NIC could only be demonstrated in the fetal cells. Our results indicate that NIC induces differentiation and maturation of human fetal pancreatic islet cells. This model should be useful for the study of molecular mechanisms involved in beta cell development. View Publication

One objective of clinical gene marking trials in multiple myeloma (MM) is to determine the extent to which relapse after stem cell transplant is attributable to contamination of the autograft with myeloma cells. A requirement in these studies is ex vivo genetic marking of malignant cells present in autografts which are derived from patients exposed to significant prior chemotherapy. We evaluated gene marking of cloonogenic myeloma cells in marrow aspirates from 14 patients with MM. To effect gene transfer we utilized a long-term marrow culture (LTMC) system previously shown to facilitate gene transfer into a spectrum of hematopoietic progenitor and stem cells. Transduction of cells in LTMC was performed by multiple supernatant exposure. At LTMC initiation and after 21 days of culture malignant cells were assessed by morphology,flow cytometry,and polymerase chain reaction (PCR). The mean number of day 21 LTMC adherent layer-derived granulocyte/macrophage progenitors as a percentage of the original inoculum was within the normal range for this technique. The efficiency of transduction of normal hematopoietic progenitors as determined by the number of colonies positive for proviral DNA by PCR,G418 resistance,and X-gal staining was also within the expected range; 65%,44% and 23%,respectively. Thus,there was no evidence that prior chemotherapy exposure or malignant cell contamination compromised cell survival or gene transfer efficiency in LTMC. All patients retained plasma cells in LTMCs for the duration of the 21-day culture period. Molecular analysis confirmed the persistence of clonal IgVH gene rearrangements in day 21 LTMC-derived DNA from 6 of 12 informative patients (50%). PCR using allele-specific primers when available confirmed the specificity of IgVH rearrangements for the myeloma clone. In 2 of the 14 patients,expansion of clonogenic cells was demonstrated in LTMC. In both cases there was strong evidence for transfer of reporter genes (neo and LacZ) into the myeloma clone: morphologically abnormal G418-resistant colonies demonstrated intense staining for beta-galactosidase,and cytospin preparations showed 100% plasma cells with monoclonal heavy and light chain restriction. In one patient,individual colonies positive for beta-galactosidase bore a cytogenetic abnormality characteristic of the patient's myeloma clone. PCR of DNA from pooled plasma cell colonies using tumor-specific CDR3 primers was positive. Our results demonstrate the maintenance of myeloma cells in vitro for up to 21 days in LTMC. They further illustrate that these cells can be genetically marked using transduction protocols currently being tested in clinical trials of hematopoietic cell gene transfer. View Publication

Realization of clinical potential of human pluripotent stem cells (hPSCs) in bone regenerative medicine requires development of simple and safe biomaterials for expansion of hPSCs followed by directing their lineage commitment to osteoblasts. In the present study,a chemically defined peptide-decorated polycaprolactone (PCL) nanofibrous microenvironment was prepared through electrospinning technology and subsequent conjugation with vitronectin peptide to promote the culture and osteogenic potential of hPSCs in vitro. The results indicated that hPSCs successfully proliferated and maintained their pluripotency on the biointerface of peptide-conjugated nanofibers without Matrigel under defined conditions. Moreover,the prepared niche exhibited an appealing ability in promoting directed differentiation of hPSCs to osteoblastic phenotype without embryoid body formation step,determined from the cell morphological alteration,alkaline phosphate activity,and osteogenesis-related gene expression,as well as protein production. Such well-defined,xeno-free,and safe nanofiber scaffolds that allow the survival and facilitate osteo-differentiation of hPSCs provide a novel platform for hPSCs differentiation via cell-nanofiber interplay,and possess great value in accelerating the translational perspectives of hPSCs in bone tissue engineering. View Publication

Retinal degenerative diseases are among the leading causes of blindness worldwide,and cell replacement is considered as a promising therapeutic. However,the resources of seed cells are scarce. To further explore this type of therapy,we adopted a culture system that could harvest a substantial quantity of retinal progenitor cells (RPCs) from human embryonic stem cells (hESCs) within a relatively short period of time. Furthermore,we transplanted these RPCs into the subretinal spaces of Royal College of Surgeons (RCS) rats. We quantified the thickness of the treated rats' outer nuclear layers (ONLs) and explored the visual function via electroretinography (ERG). It was found that the differentiated cells expressed RPC markers and photoreceptor progenitor markers. The transplanted RPCs survived for at least 12 weeks,resulting in beneficial effects on the morphology of the host retina,and led to a significant improvement in the visual function of the treated animals. These therapeutic effects suggest that the hESCs-derived RPCs could delay degeneration of the retina and partially restore visual function. View Publication

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However,direct injection of hESCs,and cells differentiated from hESCs,into living organisms has thus far been hampered by significant cell death,teratoma formation,and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization,proliferation,and viability. Molecular imaging has given investigators a high-throughput,inexpensive,and sensitive means for tracking in vivo cell proliferation over days,weeks,and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment,proliferation,and teratoma-formation in living subjects. A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes,under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery,are introduced into cells using a variety of vector and non-vector methods. Once in the cell,reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions,depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive,noninvasive instrumentation (e.g.,CCD cameras) using signal-generating probes such as D-luciferin. To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging,bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase,derived from the firefly Photinus pyralis,encodes an enzyme that catalyzes D-luciferin to the optically active metabolite,oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells,allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore,because expression of the reporter gene product is required for signal generation,only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. In this video,the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described. View Publication