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Magnesium (Mg) is a promising biodegradable metallic material for applications in cellular/tissue engineering and biomedical implants/devices. To advance clinical translation of Mg-based biomaterials,we investigated the effects and mechanisms of Mg degradation on the proliferation and pluripotency of human embryonic stem cells (hESCs). We used hESCs as the in vitro model system to study cellular responses to Mg degradation because they are sensitive to toxicants and capable of differentiating into any cell types of interest for regenerative medicine. In a previous study when hESCs were cultured in vitro with either polished metallic Mg (99.9% purity) or pre-degraded Mg,cell death was observed within the first 30 hours of culture. Excess Mg ions and hydroxide ions induced by Mg degradation may have been the causes for the observed cell death; hence,their respective effects on hESCs were investigated for the first time to reveal the potential mechanisms. For this purpose,the mTeSR®1 hESC culture media was either modified to an alkaline pH of 8.1 or supplemented with 0.4-40 mM of Mg ions. We showed that the initial increase of media pH to 8.1 had no adverse effect on hESC proliferation. At all tested Mg ion dosages,the hESCs grew to confluency and retained pluripotency as indicated by the expression of OCT4,SSEA3,and SOX2. When the supplemental Mg ion dosages increased to greater than 10 mM,however,hESC colony morphology changed and cell counts decreased. These results suggest that Mg-based implants or scaffolds are promising in combination with hESCs for regenerative medicine applications,providing their degradation rate is moderate. Additionally,the hESC culture system could serve as a standard model for cytocompatibility studies of Mg in vitro,and an identified 10 mM critical dosage of Mg ions could serve as a design guideline for safe degradation of Mg-based implants/scaffolds. View Publication

Human embryonic stem cells and induced pluripotent stem cells have great potential in research and thera-pies. The current in vitro culture systems for human pluripotent stem cells (hPSCs) do not mimic the three-dimensional (3D) in vivo stem cell niche that transiently supports stem cell proliferation and is subject to changes which facilitate subsequent differentiation during development. Here,we demonstrate,for the first time,that a novel plant-derived nanofibrillar cellulose (NFC) hydrogel creates a flexible 3D environment for hPSC culture. The pluripotency of hPSCs cultured in the NFC hydrogel was maintained for 26 days as evidenced by the expression of OCT4,NANOG,and SSEA-4,in vitro embryoid body formation and in vivo teratoma formation. The use of a cellulose enzyme,cellulase,enables easy cell propagation in 3D culture as well as a shift between 3D and two-dimensional cultures. More importantly,the removal of the NFC hydrogel facilitates differentiation while retaining 3D cell organization. Thus,the NFC hydrogel represents a flexible,xeno-free 3D culture system that supports pluripotency and will be useful in hPSC-based drug research and regenerative medicine. View Publication

Low oxygen tension (hypoxia) contributes critically to pluripotency of human embryonic stem cells (hESCs) by preventing spontaneous differentiation and supporting self-renewal. However,it is not well understood how hESCs respond to reduced oxygen availability and what are the molecular mechanisms maintaining pluripotency in these conditions. In this study we characterized the transcriptional and molecular responses of three hESC lines (H9,HS401 and HS360) on short (2 hours),intermediate (24 hours) and prolonged (7 days) exposure to low oxygen conditions (4% O2). In response to prolonged hypoxia the expression of pluripotency surface marker SSEA-3 was increased. Furthermore,the genome wide gene-expression analysis revealed that a substantial proportion (12%) of all hypoxia-regulated genes in hESCs,were directly linked to the mechanisms controlling pluripotency or differentiation. Moreover,transcription of MYC oncogene was induced in response to continuous hypoxia. At the protein level MYC was stabilized through phosphorylation already in response to a short hypoxic exposure. Total MYC protein levels remained elevated throughout all the time points studied. Further,MYC protein expression in hypoxia was affected by silencing HIF2α,but not HIF1α. Since MYC has a crucial role in regulating pluripotency we propose that induction of sustained MYC expression in hypoxia contributes to activation of transcriptional programs critical for hESC self-renewal and maintenance of enhanced pluripotent state. View Publication

We previously demonstrated that leukemia cell lines expressing CD44 and hematopoietic progenitor cells (HPC) from umbilical cord blood (CB) showed rolling on hyaluronic acid (HA)-coated surfaces under physiological shear stress. In the present study,we quantitatively assessed the interaction of HPC derived from CB,mobilized peripheral blood (mPB) and bone marrow (BM) from healthy donors,as well as primary leukemia blasts from PB and BM of patients with acute myeloid leukemia (AML) with HA. We have demonstrated that HPC derived from healthy donors showed relative homogeneous rolling and adhesion to HA. In contrast,highly diverse behavioral patterns were found for leukemia blasts under identical conditions. The monoclonal CD44 antibody (clone BU52) abrogated the shear stress-induced rolling of HPC and leukemia blasts,confirming the significance of CD44 in this context. On the other hand,the immobile adhesion of leukemia blasts to the HA-coated surface was,in some cases,not or incompletely inhibited by BU52. The latter property was associated with non-responsiveness to induction chemotherapy and subsequently poor clinical outcome. View Publication

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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

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