Ethical issues in nanomedicine: Tempest in a teapot?

Nanomedicine offers remarkable options for new therapeutic avenues. As methods in nanomedicine advance, ethical questions conjunctly arise. Nanomedicine is an exceptional niche in several aspects as it reflects risks and uncertainties not encountered in other areas of medical research or practice. Nanomedicine partially overlaps, partially interlocks and partially exceeds other medical disciplines. Some interpreters agree that advances in nanotechnology may pose varied ethical challenges, whilst others argue that these challenges are not new and that nanotechnology basically echoes recurrent bioethical dilemmas. The purpose of this article is to discuss some of the ethical issues related to nanomedicine and to reflect on the question whether nanomedicine generates ethical challenges of new and unique nature. Such a determination should have implications on regulatory processes and professional conducts and protocols in the future.

Let-7d microRNA affects mesenchymal phenotypic properties of lung fibroblasts.

MicroRNAs are small noncoding RNAs that inhibit protein expression. We have previously shown that the inhibition of the microRNA let-7d in epithelial cells caused changes consistent with epithelial-to-mesenchymal transition (EMT) both in vitro and in vivo. The aim of this study was to determine whether the introduction of let-7d into fibroblasts alters their mesenchymal properties. Transfection of primary fibroblasts with let-7d caused a decrease in expression of the mesenchymal markers α-smooth muscle actin, N-cadherin, fibroblast-specific protein-1, and fibronectin, as well as an increase in the epithelial markers tight junction protein-1 and keratin 19. Phenotypic changes were also present, including a delay in wound healing, reduced motility, and proliferation of fibroblasts following transfection. In addition, we examined the effects of transfection on fibroblast responsiveness to TGF-ß, an important factor in many fibrotic processes such as lung fibrosis and found that let-7d transfection significantly attenuated high-mobility group-A2 protein induction by TGF-ß. Our results indicate that administration of the epithelial microRNA let-7d can significantly alter the phenotype of primary fibroblasts.

Nicotine exposure during differentiation causes inhibition of N-myc expression.

BACKGROUND: The ability of chemicals to disrupt neonatal development can be studied using embryonic stem cells (ESC). One such chemical is nicotine. Prenatal nicotine exposure is known to affect postnatal lung function, although the mechanisms by which it has this effect are not clear. Since fibroblasts are a critical component of the developing lung, providing structure and secreting paracrine factors that are essential to epithelialization, this study focuses on the differentiation of ESC into fibroblasts using a directed differentiation protocol. METHODS: Fibroblasts obtained from non-human primate ESC (nhpESC) differentiation were analyzed by immunohistochemistry, immunostaining, Affymetrix gene expression array, qPCR, and immunoblotting. RESULTS: Results of these analyses demonstrated that although nhpESCs differentiate into fibroblasts in the presence of nicotine and appear normal by some measures, including H&E and SMA staining, they have an altered gene expression profile. Network analysis of expression changes demonstrated an over-representation of cell-cycle related genes with downregulation of N-myc as a central regulator in the pathway. Further investigation demonstrated that cells differentiated in the presence of nicotine had decreased N-myc mRNA and protein expression and longer doubling times, a biological effect consistent with downregulation of N-myc. CONCLUSIONS: This study is the first to use primate ESC to demonstrate that nicotine can affect cellular differentiation from pluripotency into fibroblasts, and in particular, mediate N-myc expression in differentiating ESCs. Given the crucial role of fibroblasts throughout the body, this has important implications for the effect of cigarette smoke exposure on human development not only in the lung, but in organogenesis in general.

Sexually dimorphic gene expression in non-human primate ESCs analyzed stringently.

Human exhibit sexual dimorphism early in development and throughout life. Here we stringently analyzed gene expression in inbred non-human primate embryonic stem cells (nhpESCs) searching for sexually dimorphisms. We utilized location-specific probes solely, thus avoiding probe cross-reactivity between members of gene families and genomic gene duplications. Seventeen sexually dimorphic transcripts (15 genes, out of which 9 autosomals) were identified, of which five were verified using real-time q-PCR. We compared these results from pedigreed nhpESCs with available human ESCs datasets. Three human X-linked genes show sexual dimorphism. Thus, these results enhance our knowledge and deepen our understanding on early development processes for sexual dimorphism.

Inhibition and role of let-7d in idiopathic pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation. OBJECTIVES: To determine changes in expression and role of microRNAs in IPF. METHODS: RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-beta. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry. MEASUREMENTS AND MAIN RESULTS: Eighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-beta down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and alpha-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells. CONCLUSIONS: Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT 00258544).

WNT5A is a regulator of fibroblast proliferation and resistance to apoptosis.

Usual interstitial pneumonia (UIP) is a specific histopathologic pattern of interstitial lung fibrosis that may be idiopathic or secondary to autoimmune diseases and environmental exposures. In this study, we compared gene expression patterns in primary fibroblasts isolated from lung tissues with UIP histology and fibroblasts isolated from lung tissues with normal histology using expression microarrays. We found that WNT5A was significantly increased in fibroblasts obtained from UIP lung tissues compared with normal lung fibroblasts, an observation verified by quantitative real-time RT-PCR and Western blot. Because the role of WNT5A in UIP is unknown, we treated normal lung fibroblasts or UIP lung fibroblasts with WNT5A, and found that WNT5A increased proliferation as well as relative resistance to H2O2-induced apoptosis. This effect was not mediated through the canonical WNT/beta-catenin pathway, as WNT5A induced a decrease in beta-catenin levels in the same cells. In addition, WNT5A induced increases in fibronectin and alpha(5)-integrin in normal lung fibroblasts. Collectively, our data suggest that WNT5A may play a role in fibroblast expansion and survival characteristics of idiopathic pulmonary fibrosis and other fibrotic interstitial lung diseases that exhibit UIP histological patterns.

Pedigreed primate embryonic stem cells express homogeneous familial gene profiles.

Human embryonic stem cells (hESCs) hold great biomedical promise, but experiments comparing them produce heterogeneous results, raising concerns regarding their reliability and utility, although these variations may result from their disparate and anonymous origins. To determine whether primate ESCs have intrinsic biological limitations compared with mouse ESCs, we examined expression profiles and pluripotency of newly established nonhuman primate ESC (nhpESCs). Ten pedigreed nhpESC lines, seven full siblings (fraternal quadruplets and fraternal triplets), and nine half siblings were derived from 41 rhesus embryos; derivation success correlated with embryo quality. Each line has been growing continuously for approximately 1 year with stable diploid karyotype (except for one stable trisomy) and expresses in vitro pluripotency markers, and eight have already formed teratomas. Unlike the heterogeneous gene expression profiles found among hESCs, these nhpESCs display remarkably homogeneous profiles (>97%), with full-sibling lines nearly identical (>98.2%). Female nhpESCs express genes distinct from their brother lines; these sensitive analyses are enabled because of the very low background differences. Experimental comparisons among these primate ESCs may prove more reliable than currently available hESCs, since they are akin to inbred mouse strains in which genetic variables are also nearly eliminated. Finally, contrasting the biological similarities among these lines with the heterogeneous hESCs might suggest that additional, more uniform hESC lines are justified. Taken together, pedigreed primate ESCs display homogeneous and reliable expression profiles. These similarities to mouse ESCs suggest that heterogeneities found among hESCs likely result from their disparate origins rather than intrinsic biological limitations with primate embryonic stem cells.

Utilizing a GnRH-based chimeric protein for the detection of adenocarcinoma.

Since early diagnosis of many types of cancer greatly improves the chances for successful treatment, high-quality methods for cancer detection are necessary. Our laboratory develops chimeric proteins for targeted therapy, such as gonadotropin releasing hormone (GnRH)-based chimeric proteins for the targeted therapy of adenocarcinomas in humans. For chimeric proteins to cause specific cell death, they must first recognize specific receptors/binding sites expressed on the surface of target cells. Thus, we examined whether we could exploit these binding sites not only as targets for the killing of specific cells but also as a diagnostic marker for identifying adenocarcinomas, using the same chimeric proteins. In this report, we show that one such GnRH-based chimeric protein, GnRH-Caspase3, can indeed serve as a diagnostic tool. GnRH-Caspase3 was able to specifically bind adenocarcinoma cells, as measured by FACS analysis and demonstrated with the aid of confocal microscopy and specific antibodies. Moreover, we found a correlation between cell sensitivity to treatment and the binding level of the chimeric protein to the cells. Hence, we suggest that in addition to their therapeutic potential, GnRH-based chimeric proteins can be used as a diagnostic tool for the detection of adenocarcinomas.

Using apoptosis for targeted cancer therapy by a new gonadotropin releasing hormone-DNA fragmentation factor 40 chimeric protein.

PURPOSE: GnRH-based chimeric proteins have been shown to specifically target and kill adenocarcinomas both in vitro and in vivo. The purpose of this study is to construct a new GnRH-based chimeric protein for the treatment of adenocarcinomas in humans. RESULTS: In this study, we constructed and characterized a new chimeric protein, GnRH-DFF40, composed of a new human killing moiety: the apoptotic DNase-DFF40 (DNA fragmentation factor), known also as caspase-activated DNase (CAD). GnRH-DFF40 exhibits DNase activity in vitro. We found that this chimeric protein can target and kill adenocarcinoma cells. Such death occurs via apoptotic pathways, resulting in an increase in the sub-G(1) population, DNA fragmentation, terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-positive cells, and morphology typical of apoptotic cells. These apoptotic events involve the mitochondria because we found cytochrome c depletion and caspase-9 and caspase-3 activation. Preliminary in vivo results showed that treatment of colon adenocarcinoma xenografts in nude mice with the new chimeric protein caused a reduction in tumor weight. CONCLUSIONS: Because GnRH-DFF40 is a whole human-based chimeric protein when applied to humans, the nonspecific toxicity and immunogenicity seen with bacterial/plant-based chimeric proteins should be avoided. Thus, GnRH-DFF40 is a promising candidate for the treatment of adenocarcinomas in humans.

Tryptase activates peripheral blood mononuclear cells causing the synthesis and release of TNF-alpha, IL-6 and IL-1 beta: possible relevance to multiple sclerosis.

Presence of mast cells and an increase in the concentration of their products has been reported in multiple sclerosis (MS) plaques. The most abundant secretory mediator of the human mast cell is the tetrameric protease tryptase. We demonstrate that tryptase can activate peripheral mononuclear cells (PBMCs), isolated from healthy donors as well as MS patients for the release of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-1beta. Cytokine secretion was significantly higher in secondary progressive (SP) MS patients and healthy control (HC) individuals than in relapsing-remitting (RR) patients. Our findings suggest that tryptase is, most probably, an important mediator of inflammation in MS.

Targeted cancer therapy with gonadotropin-releasing hormone chimeric proteins.

Tumor-associated antigens (TAAs) have been identified mainly to determine cancer prognosis. In the past few years, TAAs have been used in the development of treatment modalities such as tumor vaccination. This review describes an additional application of TAAs: as a target for specific antitumor treatment. Since TAAs are overexpressed on the tumor cell surface, they can be targeted to deliver drugs directly to cancer cells. One such delivery system exploits chimeric proteins. Chimeric proteins are a class of targeted molecules designed to recognize and specifically destroy cells that overexpress specific receptors. These molecules, designed and constructed by gene fusion techniques, comprise both cell-targeting and cell-killing moieties. The authors' laboratory has developed a number of chimeric proteins using gonadotropin-releasing hormone (GnRH) as the targeting moiety. These chimeras recognize a GnRH binding site that is expressed on adenocarcinoma cells. GnRH was fused to a large number of killing moieties, including bacterial and human proapoptotic proteins. All GnRH-based chimeric proteins selectively killed adenocarcinoma cells both in vitro and in vivo. Utilizing chimeric proteins for targeted therapy represents a new and exciting therapeutic modality for the treatment of cancer in humans.

Cartilage oligomeric matrix protein in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and life threatening disease with median survival of 2.5-3 years. The IPF lung is characterized by abnormal lung remodeling, epithelial cell hyperplasia, myofibroblast foci formation, and extracellular matrix deposition. Analysis of gene expression microarray data revealed that cartilage oligomeric matrix protein (COMP), a non-collagenous extracellular matrix protein is among the most significantly up-regulated genes (Fold change 13, p-value <0.05) in IPF lungs. This finding was confirmed at the mRNA level by nCounter® expression analysis in additional 115 IPF lungs and 154 control lungs as well as at the protein level by western blot analysis. Immunohistochemical analysis revealed that COMP was expressed in dense fibrotic regions of IPF lungs and co-localized with vimentin and around pSMAD3 expressing cells. Stimulation of normal human lung fibroblasts with TGF-ß1 induced an increase in COMP mRNA and protein expression. Silencing COMP in normal human lung fibroblasts significantly inhibited cell proliferation and negatively impacted the effects of TGF-ß1 on COL1A1 and PAI1. COMP protein concentration measured by ELISA assay was significantly increased in serum of IPF patients compared to controls. Analysis of serum COMP concentrations in 23 patients who had prospective blood draws revealed that COMP levels increased in a time dependent fashion and correlated with declines in force vital capacity (FVC). Taken together, our results should encourage more research into the potential use of COMP as a biomarker for disease activity and TGF-ß1 activity in patients with IPF. Hence, studies that explore modalities that affect COMP expression, alleviate extracellular matrix rigidity and lung restriction in IPF and interfere with the amplification of TGF-ß1 signaling should be persuaded.

LifeMap Discovery™: the embryonic development, stem cells, and regenerative medicine research portal.

LifeMap Discovery™ provides investigators with an integrated database of embryonic development, stem cell biology and regenerative medicine. The hand-curated reconstruction of cell ontology with stem cell biology; including molecular, cellular, anatomical and disease-related information, provides efficient and easy-to-use, searchable research tools. The database collates in vivo and in vitro gene expression and guides translation from in vitro data to the clinical utility, and thus can be utilized as a powerful tool for research and discovery in stem cell biology, developmental biology, disease mechanisms and therapeutic discovery. LifeMap Discovery is freely available to academic nonprofit institutions at http://discovery.lifemapsc.com.

Mutated human embryonic stem cells for the study of human genetic disorders.

Human embryonic stem cells (HESCs) are of great interest in biology and medicine due to their ability to grow indefinitely in culture while maintaining their ability to differentiate into all different cell types in the human body. In addition, HESCs can be used for better understanding the key developmental processes and can, therefore, serve for studying genetic disorders for which no good research model exists. Preimplantation genetic diagnosis of in vitro derived embryos results in affected-spare blastocysts with specific known inherited mutations.These affected blastocysts can be used for the derivation of disease-bearing HESCs, which would serve for studying the molecular and pathophysiological mechanisms underlying the genetic disease for which they were diagnosed. This chapter describes the methods to derive HESCs carrying mutations for inherited disorders.

Human amniotic epithelial cells are reprogrammed more efficiently by induced pluripotency than adult fibroblasts.

Cellular reprogramming from adult somatic cells into an embryonic cell-like state, termed induced pluripotency, has been achieved in several cell types. However, the ability to reprogram human amniotic epithelial cells (hAECs), an abundant cell source derived from discarded placental tissue, has only recently been investigated. Here we show that not only are hAECs easily reprogrammed into induced pluripotent stem cells (AE-iPSCs), but hAECs reprogram faster and more efficiently than adult and neonatal somatic dermal fibroblasts. Furthermore, AE-iPSCs express higher levels of NANOG and OCT4 compared to human foreskin fibroblast iPSCs (HFF1-iPSCs) and express decreased levels of genes associated with differentiation, including NEUROD1 and SOX17, markers of neuronal differentiation. To elucidate the mechanism behind the higher reprogramming efficiency of hAECs, we analyzed global DNA methylation, global histone acetylation, and the mitochondrial DNA A3243G point mutation. Whereas hAECs show no differences in global histone acetylation or mitochondrial point mutation accumulation compared to adult and neonatal dermal fibroblasts, hAECs demonstrate a decreased global DNA methylation compared to dermal fibroblasts. Likewise, quantitative gene expression analyses show that hAECs endogenously express OCT4, SOX2, KLF4, and c-MYC, all four factors used in cellular reprogramming. Thus, hAECs represent an ideal cell type for testing novel approaches for generating clinically viable iPSCs and offer significant advantages over postnatal cells that more likely may be contaminated by environmental exposures and infectious agents.

Female sex bias in human embryonic stem cell lines.

The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism.

Systems biology discoveries using non-human primate pluripotent stem and germ cells: novel gene and genomic imprinting interactions as well as unique expression patterns.

The study of pluripotent stem cells has generated much interest in both biology and medicine. Understanding the fundamentals of biological decisions, including what permits a cell to maintain pluripotency, that is, its ability to self-renew and thereby remain immortal, or to differentiate into multiple types of cells, is of profound importance. For clinical applications, pluripotent cells, including both embryonic stem cells and adult stem cells, have been proposed for cell replacement therapy for a number of human diseases and disorders, including Alzheimer's, Parkinson's, spinal cord injury and diabetes. One challenge in their usage for such therapies is understanding the mechanisms that allow the maintenance of pluripotency and controlling the specific differentiation into required functional target cells. Because of regulatory restrictions and biological feasibilities, there are many crucial investigations that are just impossible to perform using pluripotent stem cells (PSCs) from humans (for example, direct comparisons among panels of inbred embryonic stem cells from prime embryos obtained from pedigreed and fertile donors; genomic analysis of parent versus progeny PSCs and their identical differentiated tissues; intraspecific chimera analyses for pluripotency testing; and so on). However, PSCs from nonhuman primates are being investigated to bridge these knowledge gaps between discoveries in mice and vital information necessary for appropriate clinical evaluations. In this review, we consider the mRNAs and novel genes with unique expression and imprinting patterns that were discovered using systems biology approaches with primate pluripotent stem and germ cells.

mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

Deciding to exit pluripotency and undergo differentiation is of singular importance for pluripotent cells, including embryonic stem cells (ESCs). The molecular mechanisms for these decisions to differentiate, as well as reversing those decisions during induced pluripotency (iPS), have focused largely on transcriptomic controls. Here, we explore the role of translational control for the maintenance of pluripotency and the decisions to differentiate. Global protein translation is significantly reduced in hESCs compared to their differentiated progeny. Furthermore, p70 S6K activation is restricted in hESCs compared to differentiated fibroblast-like cells. Disruption of p70 S6K-mediated translation by rapamycin or siRNA knockdown in undifferentiated hESCs does not alter cell viability or expression of the pluripotency markers Oct4 and Nanog. However, expression of constitutively active p70 S6K, but not wild-type p70 S6K, induces differentiation. Additionally, hESCs exhibit high levels of the mTORC1/p70 S6K inhibitory complex TSC1/TSC2 and preferentially express more rapamycin insensitive mTORC2 compared to differentiated cells. siRNA-mediated knockdown of both TSC2 and Rictor elevates p70 S6K activation and induces differentiation of hESCs. These results suggest that hESCs tightly regulate mTORC1/p70 S6K-mediated protein translation to maintain a pluripotent state as well as implicate a novel role for protein synthesis as a driving force behind hESC differentiation.

Pluripotency genes overexpressed in primate embryonic stem cells are localized on homologues of human chromosomes 16, 17, 19, and X.

While human embryonic stem cells (hESCs) are predisposed toward chromosomal aneploidities on 12, 17, 20, and X, rendering them susceptible to transformation, the specific genes expressed are not yet known. Here, by identifying the genes overexpressed in pluripotent rhesus ESCs (nhpESCs) and comparing them both to their genetically identical differentiated progeny (teratoma fibroblasts) and to genetically related differentiated parental cells (parental skin fibroblasts from whom gametes were used for ESC derivation), we find that some of those overexpressed genes in nhpESCs cluster preferentially on rhesus chromosomes 16, 19, 20, and X, homologues of human chromosomes 17, 19, 16, and X, respectively. Differentiated parental skin fibroblasts display gene expression profiles closer to nhpESC profiles than to teratoma cells, which are genetically identical to the pluripotent nhpESCs. Twenty over- and underexpressed pluripotency modulators, some implicated in neurogenesis, have been identified. The overexpression of some of these genes discovered using pedigreed nhpESCs derived from prime embryos generated by fertile primates, which is impossible to perform with the anonymously donated clinically discarded embryos from which hESCs are derived, independently confirms the importance of chromosome 17 and X regions in pluripotency and suggests specific candidates for targeting differentiation and transformation decisions.

Semiquantitative histopathology and 3D magnetic resonance microscopy as collaborative platforms for tissue identification and comparison within teratomas derived from pedigreed primate embryonic stem cells.

Teratoma formation in xenografts is a sufficiently stringent pluripotency assay for stem cells. However, little is known about the composition and spatial relationships of tissues within teratomas that may provide clues about development and platforms for studying organ development. Additionally, teratoma formation and analysis lack standards for reporting as assays of pluripotency. Three of 27 total teratomas derived from pedigreed primate embryonic stem cells underwent quantitative three-dimensional high-resolution magnetic resonance microscopy (MRM). Teratomas were subsequently serially sectioned and tissue types identified, semiquantitated, and correlated with MRM images. All teratomas demonstrated tissue derivatives from the three germ layers and approximately 23 different tissue types were identified. Certain tissue groups attempted to form organs more frequently (e.g., trachea/bronchi, small intestine). MRM discriminated some tissues readily (e.g., bone, adipose, cartilage) while other tissue types with like MR intensities could not be distinguished. Semiquantitative histopathological analysis of teratomas demonstrates the ability to delineate multiple tissues as derived from ectoderm, mesoderm, or endoderm and to use this information for comparison to other teratomas. MRM provides rapid quantitative imaging of intact teratomas that complements histology and identifies sites of interest for additional biological studies.