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Production of human embryonic stem cell (hESC)-derived lung progenitors has broad applicability for drug screening and cell therapy; however,this is complicated by limitations in demarcating phenotypic changes with functional validation of airway cell types. In this paper,we reveal the potential of hESCs to produce multipotent lung progenitors using a combined growth factor and physical culture approach,guided by the use of novel markers LIFRα and NRP1. Lung specification of hESCs was achieved by priming differentiation via matrix-specific support,followed by air-liquid interface to allow generation of lung progenitors capable of in vitro maturation into airway epithelial cell types,resulting in functional characteristics such as secretion of pulmonary surfactant,ciliation,polarization,and acquisition of innate immune activity. This approach provided a robust expansion of lung progenitors,allowing in vivo assessment,which demonstrated that only fully differentiated hESC-derived airway cells were retained in the distal airway,where they aided in physiological recovery in immunocompromised mice receiving airway injury. Our study provides a basis for translational applications of hESCs for lung diseases. View Publication

Transplantation of photoreceptor precursor cells (PPCs) differentiated from human embryonic stem cells (hESCs) is a promising approach to treat common blinding diseases such as age-related macular degeneration and retinitis pigmentosa. However,existing PPC generation methods are inefficient. To enhance differentiation protocols for rapid and high-yield production of PPCs,we focused on optimizing the handling of the cells by including feeder-independent growth of hESCs,using size-controlled embryoid bodies (EBs),and addition of triiodothyronine (T3) and taurine to the differentiation medium,with subsequent removal of undifferentiated cells via negative cell-selection. Our novel protocol produces higher yields of PPCs than previously reported while reducing the time required for differentiation,which will help understand retinal diseases and facilitate large-scale preclinical trials. View Publication

In recent years,studies of cancer development and recurrence have been influenced by the cancer stem cells (CSCs)/cancer-initiating cells (CICs) hypothesis. According to this,cancer is sustained by highly positioned,chemoresistant cells with extensive capacity of self renewal,which are responsible for disease relapse after chemotherapy. Growth of cancer cells as three-dimensional non-adherent spheroids is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from malignant pleural effusions (MPEs) of patients affected by adenocarcinoma of the lung are able to efficiently form spheroids in non-adherent conditions supplemented with growth factors. By expression profiling,we were able to identify a set of genes whose expression is significantly upregulated in lung tumor spheroids versus adherent cultures. One of the most strongly upregulated gene was stearoyl-CoA desaturase (SCD1),the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study,we show both by RNA interference and through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in MPE-derived primary tumor cultures. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker,most of them positive also for the stemness marker ALDH1A1,thus suggesting that the SCD1 inhibitor is selectively killing cells with stem-like properties. Furthermore,SCD1-inhibited lung cancer cells were strongly impaired in their in vivo tumorigenicity and ALDH1A1 expression. These results suggest that SCD1 is a critical target in lung cancer tumor-initiating cells. View Publication

Smooth muscle cells (SMCs) and endothelial cells (ECs) are typically derived separately,with low efficiencies,from human pluripotent stem cells (hPSCs). The concurrent generation of these cell types might lead to potential applications in regenerative medicine to model,elucidate,and eventually treat vascular diseases. Here we report a robust two-step protocol that can be used to simultaneously generate large numbers of functional SMCs and ECs from a common proliferative vascular progenitor population via a two-dimensional culture system. We show here that coculturing hPSCs with OP9 cells in media supplemented with vascular endothelial growth factor,basic fibroblast growth factor,and bone morphogenetic protein 4 yields a higher percentage of CD31(+)CD34(+) cells on day 8 of differentiation. Upon exposure to endothelial differentiation media and SM differentiation media,these vascular progenitors were able to differentiate and mature into functional endothelial cells and smooth muscle cells,respectively. Furthermore,we were able to expand the intermediate population more than a billion fold to generate sufficient numbers of ECs and SMCs in parallel for potential therapeutic transplantations. View Publication

Mesenchymal stem cells (MSCs) modulate cardiac healing after myocardial injury through the release of paracrine factors,but the exact mechanisms are still unknown. One possible mechanism is through mobilization of endogenous cardiac stem cells (CSCs). This study aimed to test the pro-migratory effect of MSC conditioned medium (MSC-CM) on endogenous CSCs from human cardiac tissue. By using a three-dimensional collagen assay,we found that MSC-CM improved migration of cells from human cardiac tissue. Cell counts,perimeter and area measurements were utilized to quantify migration effects. To examine whether resident stem cells were among the migrating cells,specific stem cell properties were investigated. The migrating cells displayed strong similarities with resident Cardiac Atrial appendage Stem Cells (CASCs),including a clonogenic potential of ˜21.5% and expression of pluripotency associated genes like Oct-4,Nanog,c-Myc and Klf-4. Similar to CASCs,migrating cells demonstrated high aldehyde dehydrogenase activity and were able to differentiate towards cardiomyocytes. Receptor tyrosine kinase analysis and collagen assays performed with recombinant platelet derived growth factor (PDGF)-AA and Imatinib Mesylate,a PDGF receptor inhibitor,suggested a role for the PDGF-AA/PDGF receptor $$ axis in enhancing the migration process of CASCs. In conclusion,our findings demonstrate that factors present in MSC-CM improve migration of resident stem cells from human cardiac tissue. These data open doors towards future therapies in which MSC secreted factors,like PDGF-AA,can be utilized to enhance the recruitment of CASCs towards the site of myocardial injury. View Publication

High telomerase activity is a characteristic of human embryonic stem cells (hESCs),however,the regulation and maintenance of correct telomere length in hESCs is unclear. In this study we investigated telomere elongation in hESCs in vitro and found that telomeres lengthened from their derivation in blastocysts through early expansion,but stabilized at later passages. We report that the core unit of telomerase,hTERT,was highly expressed in hESCs in blastocysts and throughout long-term culture; furthermore,this was regulated in a Wnt-β-catenin-signaling-dependent manner. Our observations that the alternative lengthening of telomeres (ALT) pathway was suppressed in hESCs and that hTERT knockdown partially inhibited telomere elongation,demonstrated that high telomerase activity was required for telomere elongation. We observed that chromatin modification through trimethylation of H3K9 and H4K20 at telomeric regions decreased during early culture. This was concurrent with telomere elongation,suggesting that epigenetic regulation of telomeric chromatin may influence telomerase function. By measuring telomere length in 96 hESC lines,we were able to establish that telomere length remained relatively stable at 12.02±1.01 kb during later passages (15-95). In contrast,telomere length varied in hESCs with genomic instability and hESC-derived teratomas. In summary,we propose that correct,stable telomere length may serve as a potential biomarker for genetically stable hESCs. View Publication

BACKGROUND: Gene delivery can potentially be used as a therapeutic for treating genetic diseases,including neurodegenerative diseases,as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts.backslashnbackslashnMETHODS: A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling.backslashnbackslashnRESULTS: 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents,including Lipofectamine® 2000,FuGENE® HD,and 25 kDa branched polyethylenimine,for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa,and enabled coexpression of exogenously delivered genes,as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation,but not by poly(beta-amino ester) reprogramming,could be differentiated toward the neuronal lineage,specifically pseudostratified optic cups.backslashnbackslashnCONCLUSION: This study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming. View Publication

Unresectable metastatic bone sarcoma and soft-tissue sarcomas (STS) are incurable due to the inability to eradicate chemoresistant cancer stem-like cells (sCSC) that are likely responsible for relapses and drug resistance. In this study,we investigated the preclinical activity of patient-derived cytokine-induced killer (CIK) cells against autologous bone sarcoma and STS,including against putative sCSCs. Tumor killing was evaluated both in vitro and within an immunodeficient mouse model of autologous sarcoma. To identify putative sCSCs,autologous bone sarcoma and STS cells were engineered with a CSC detector vector encoding eGFP under the control of the human promoter for OCT4,a stem cell gene activated in putative sCSCs. Using CIK cells expanded from 21 patients,we found that CIK cells efficiently killed allogeneic and autologous sarcoma cells in vitro. Intravenous infusion of CIK cells delayed autologous tumor growth in immunodeficient mice. Further in vivo analyses established that CIK cells could infiltrate tumors and that tumor growth inhibition occurred without an enrichment of sCSCs relative to control-treated animals. These results provide preclinical proof-of-concept for an effective strategy to attack autologous sarcomas,including putative sCSCs,supporting the clinical development of CIK cells as a novel class of immunotherapy for use in settings of untreatable metastatic disease. View Publication

Human pluripotent stem cells (hPSCs) constitute a promising resource for use in cell-based therapies and a valuable in vitro model for studying early human development and disease. Despite significant advancements in the derivation of specific fates from hPSCs,the generation of skeletal muscle remains challenging and is mostly dependent on transgene expression. Here,we describe a method based on the use of a small-molecule GSK3?? inhibitor to derive skeletal muscle from several hPSC lines. We show that early GSK3?? inhibition is sufficient to create the conditions necessary for highly effective derivation of muscle cells. Moreover,we developed a strategy for stringent fluorescence-activated cell sorting-based purification of emerging PAX3+/PAX7+ muscle precursors that are able to differentiate in postsort cultures into mature myocytes. This transgene-free,efficient protocol provides an essential tool for producing myogenic cells for in vivo preclinical studies,in vitro screenings,and disease modeling. ?? 2013 The Authors. View Publication

Induced pluripotent stem cells (iPS) can differentiate into cardiomyocytes (CM) and represent a promising form of cellular therapy for heart regeneration. However,residual undifferentiated iPS derivates (iPSD),which are not fully eliminated by cell differentiation or purification protocols,may form tumors after transplantation,thus compromising therapeutic application. Inhibition of stearoyl-coA desaturase (SCD) has recently been reported to eliminate undifferentiated human embryonic stem cells,which share many features with iPSD. Here,we tested the effects of PluriSin1,a small-molecule inhibitor of SCD,on iPS-derived CM. We found that plurisin1 treatment significantly decreased the mRNA and protein level of Nanog,a marker for both cell pluripotency and tumor progression; importantly,we provide evidence that PluriSin1 treatment at 20 µM for 1 day significantly induces the apoptosis of Nanog-positive iPSD. In addition,PluriSin1 treatment at 20 µM for 4 days diminished Nanog-positive stem cells in cultured iPSD while not increasing apoptosis of iPS-derived CM. To investigate whether PluriSin1 treatment prevents tumorigenicity of iPSD after cell transplantation,we intramyocardially injected PluriSin1- or DMSO-treated iPSD in a mouse model of myocardial infarction (MI). DMSO-treated iPSD readily formed Nanog-expressing tumors 2 weeks after injection,which was prevented by treatment with PluriSin1. Moreover,treatment with PluriSin1 did not change the expression of cTnI,α-MHC,or MLC-2v,markers of cardiac differentiation (Ptextgreater0.05,n = 4). Importantly,pluriSin1-treated iPS-derived CM exhibited the ability to engraft and survive in the infarcted myocardium. We conclude that inhibition of SCD holds the potential to enhance the safety of therapeutic application of iPS cells for heart regeneration. View Publication

The reliable derivation of induced pluripotent stem cells (iPSCs) from a noninvasive autologous source at birth would facilitate the study of patient-specific in vitro modeling of congenital diseases and would enhance ongoing efforts aimed at developing novel cell-based treatments for a wide array of fetal and pediatric disorders. Accordingly,we have successfully generated iPSCs from human fetal chorionic somatic cells extracted from term pregnancies by ectopic expression of OCT4,SOX2,KLF4,and cMYC. The isolated parental somatic cells exhibited an immunophenotypic profile consistent with that of chorionic mesenchymal stromal cells (CMSCs). CMSC-iPSCs maintained pluripotency in feeder-free systems for more than 15 passages based on morphology,immunocytochemistry,and gene expression studies and were capable of embryoid body formation with spontaneous trilineage differentiation. CMSC-iPSCs could be selectively differentiated in vitro into various germ layer derivatives,including neural stem cells,beating cardiomyocytes,and definitive endoderm. This study demonstrates the feasibility of term placental chorion as a novel noninvasive alternative to dermal fibroblasts and cord blood for human perinatal iPSC derivation and may provide additional insights regarding the reprogramming capabilities of extra-embryonic tissues as they relate to developmental ontogeny and perinatal tissue engineering applications. View Publication