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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

Evaluating protocols for embryonic stem cell differentiation into insulin-secreting beta-cells using insulin II-GFP as a specific and noninvasive reporter.

Stable and full differentiation of pluripotent stem cells into functional beta-cells offers the potential to treat type I diabetes with a theoretically inexhaustible source of replacement cells. In addition to the difficulties in directed differentiation, progress toward an optimized and reliable protocol has been hampered by the complication that cultured cells will concentrate insulin from the media, thus making it difficult to tell which, if any, cells are producing insulin. To address this, we utilized a novel murine embryonic stem cell (mESC) research model, in which the green fluorescent protein (GFP) has been inserted within the C-peptide of the mouse insulinII gene (InsulinII-GFP). Using this method, cells producing insulin are easily identified. We then compared four published protocols for differentiating mESCs into beta-cells to evaluate their relative efficiency by assaying intrinsic insulin production. Cells differentiated using each protocol were easily distinguished based on culture conditions and morphology. This comparison is strengthened because all testing is performed within the same laboratory by the same researchers, thereby removing interlaboratory variability in culture, cells, or analysis. Differentiated cells were analyzed and sorted based on GFP fluorescence as compared to wild type cells. Each differentiation protocol increased GFP fluorescence but only modestly. None of these protocols yielded more than 3% of cells capable of insulin biosynthesis indicating the relative inefficiency of all analyzed protocols. Therefore, improved beta-cells differentiation protocols are needed, and these insulin II GFP cells may prove to be an important tool to accelerate this process.

Establishment and characterization of baboon embryonic stem cell lines: an Old World Primate model for regeneration and transplantation research.

Here we have developed protocols using the baboon as a complementary alternative Old World Primate to rhesus and other macaques which have severe limitations in their availability. Baboons are not limited as research resources, they are evolutionarily closer to humans, and the multiple generations of pedigreed colonies which display complex human disease phenotypes all support their further optimization as an invaluable primate model. Since neither baboon-assisted reproductive technologies nor baboon embryonic stem cells (ESCs) have been reported, here we describe the first derivations and characterization of baboon ESC lines from IVF-generated blastocysts. Two ESCs lines (BabESC-4 and BabESC-15) display ESC morphology, express pluripotency markers (Oct-4, hTert, Nanog, Sox-2, Rex-1, TRA1-60, TRA1-81), and maintain stable euploid female karyotypes with parentage confirmed independently. They have been grown continuously for >430 and 290 days, respectively. Teratomas from both lines have all three germ layers. Availabilities of these BabESCs represent another important resource for stem cell biologists.

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.

Derivation and characterization of nonhuman primate embryonic stem cells.

Embryonic stem (ES) cells are a powerful research tool enabling the generation of mice with custom genetics, the study of the earliest stages of mammalian differentiation in vitro and, with the isolation of human ES cells, the potential of cell-based therapies for a number of diseases including Parkinson's and Type 1 diabetes. ES cells isolated from nonhuman primates (nhpES cells) offer the opportunity to ethically test the developmental potential of primate ES cells in chimeric offspring. If these cells have similar potency to mouse ES cells, this may open a new era of primate models of human disease. Nonhuman primates are the perfect model system for the preclinical testing of ES cell-derived therapies. In this unit, we describe methods for the derivation and characterization of nonhuman primate ES cells. With these protocols, the investigator will be able to isolate nhpES cells and perform the necessary tests to confirm the pluripotent phenotype.

Specific dynamic and noninvasive labeling of pancreatic beta cells in reporter mice.

Noninvasive detection of differentiated cells is increasingly demanded for accurate and reliable assessments of both in vitro and in vivo experimental systems. Here we present an efficient, innovative approach for imaging the beta cells of the pancreatic islets of Langerhans. The main physiologic function of beta cells is glucose-stimulated insulin secretion. This function is facilitated through the synthesis and storage of insulin in secretory vesicles of beta cells, which then release their contents when beta cells are exposed to hyperglycemic conditions. To visualize beta cells in vivo in the mouse, we used targeted mutagenesis techniques to construct a modified insulin II (InsII) gene allele, InsII(EGFP), that expresses a proinsulin-EGFP (enhanced green fluorescent protein) fusion peptide. The EGFP portion of this fusion is entirely within the C-peptide portion of the proinsulin peptide. This fusion protein is processed in beta cells to insulin and EGFP-tagged C peptide, which are stored together in cytoplasmic secretory vesicles. The large amount of vesicular EGFP-tagged C peptide is evident as a characteristic robust and specific fluorescence pattern in the beta cells of InsII(EGFP) mice. This innovative method of visualizing beta cells will be a useful tool in the study of both beta cell physiology and the development of the endocrine cells of the pancreas.