Mechanisms regulating the initiation of porcine conceptus elongation.

Significant increases in litter size within commercial swine production over the past decades have led to increases in preweaning piglet mortality due to increase within-litter birthweight variation, typically due to mortality of the smallest littermate piglets. Therefore, identifying mechanisms to reduce variation in placental development and subsequent fetal growth are critical to normalizing birthweight variation and improving piglet survivability in high-producing commercial pigs. A major contributing factor to induction of within-litter variation occurs during the peri-implantation period as the pig blastocyst elongates from spherical to filamentous morphology in a short period of time and rapidly begins superficial implantation. During this period, there is significant within-litter variation in the timing and extent of elongation among littermates. As a result, delays and deficiencies in conceptus elongation not only contribute directly to early embryonic mortality, but also influence subsequent within-litter birthweight variation. This study will highlight key aspects of conceptus elongation and provide some recent evidence pertaining to specific mechanisms from -omics studies (i.e., metabolomics of the uterine environment and transcriptomics of the conceptus) that may specifically regulate the initiation of conceptus elongation to identify potential factors to reduce within-litter variation and improve piglet survivability.

Secreted metabolome of porcine blastocysts encapsulated within in vitro 3D alginate hydrogel culture systems undergoing morphological changes provides insights into specific mechanisms involved in the initiation of porcine conceptus elongation.

CONTEXT: The exact mechanisms regulating the initiation of porcine conceptus elongation are not known due to the complexity of the uterine environment. AIMS: To identify contributing factors for initiation of conceptus elongation in vitro , this study evaluated differential metabolite abundance within media following culture of blastocysts within unmodified alginate (ALG) or Arg-Gly-Asp (RGD)-modified alginate hydrogel culture systems. METHODS: Blastocysts were harvested from pregnant gilts, encapsulated within ALG or RGD or as non-encapsulated control blastocysts (CONT), and cultured. At the termination of 96h culture, media were separated into blastocyst media groups: non-encapsulated control blastocysts (CONT); ALG and RGD blastocysts with no morphological change (ALG- and RGD-); ALG and RGD blastocysts with morphological changes (ALG+ and RGD+) and evaluated for non-targeted metabolomic profiling by liquid chromatography (LC)-mass spectrometry (MS) techniques and gas chromatography-(GC-MS). KEY RESULTS: Analysis of variance identified 280 (LC-MS) and 1 (GC-MS) compounds that differed (P <0.05), of which 134 (LC-MS) and 1 (GC-MS) were annotated. Metabolites abundance between ALG+ vs ALG-, RGD+ vs RGD-, and RGD+ vs ALG+ were further investigated to identify potential differences in metabolic processes during the initiation of elongation. CONCLUSIONS: This study identified changes in phospholipid, glycosphingolipid, lipid signalling, and amino acid metabolic processes as potential RGD-independent mechanisms of elongation and identified changes in lysophosphatidylcholine and sphingolipid secretions during RGD-mediated elongation. IMPLICATIONS: These results illustrate changes in phospholipid and sphingolipid metabolic processes and secretions may act as mediators of the RGD-integrin adhesion that promotes porcine conceptus elongation.

Global analysis of differential gene expression within the porcine conceptus transcriptome as it transitions through spherical, ovoid, and tubular morphologies during the initiation of elongation.

This study aimed to identify transcriptome differences between distinct or transitional stage spherical, ovoid, and tubular porcine blastocysts throughout the initiation of elongation. We performed a global transcriptome analysis of differential gene expression using RNA-Seq with high temporal resolution between spherical, ovoid, and tubular stage blastocysts at specific sequential stages of development from litters containing conceptus populations of distinct or transitional blastocysts. After RNA-Seq analysis, significant differentially expressed genes (DEGs) and pathways were identified between distinct morphologies or sequential development stages. Overall, 1898 significant DEGs were identified between distinct spherical and ovoid morphologies, with 311 total DEGs between developmental stages throughout this first morphological transition, while 15 were identified between distinct ovoid and tubular, with eight total throughout these second morphological transition developmental stages. The high quantity of DEGs and pathways between conceptus stages throughout the spherical to ovoid transition suggests the importance of gene regulation during this first morphological transition for initiating elongation. Further, extensive DEG coverage of known elongation signaling pathways was illustrated from spherical to ovoid, and regulation of lipid signaling and membrane/ECM remodeling across these early conceptus stages were implicated as essential to this process, providing novel insights into potential mechanisms governing this rapid morphological change.

High- and low-molecular-weight chitosan act as adjuvants during single-dose influenza A virus protein vaccination through distinct mechanisms.

The investigation of new adjuvants is essential for the development of efficacious vaccines. Chitosan (CS), a derivative of chitin, has been shown to act as an adjuvant, improving vaccine-induced immune responses. However, the effect of CS molecular weight (MW) on this adjuvanticity has not been investigated, despite MW having been shown to impact CS biological properties. Here, two MW variants of CS were investigated for their ability to enhance vaccine-elicited immune responses in vitro and in vivo, using a single-dose influenza A virus (IAV) protein vaccine model. Both low-molecular-weight (LMW) and high-molecular-weight (HMW) CS-induced interferon regulatory factor pathway signaling, antigen-presenting cell activation, and cytokine messenger RNA (mRNA) production, with LMW inducing higher mRNA levels at 24 h and HMW elevating mRNA responses at 48 h. LMW and HMW CS also induced adaptive immune responses after vaccination, indicated by enhanced immunoglobulin G production in mice receiving LMW CS and increased CD4 interleukin 4 (IL-4) and IL-2 production in mice receiving HMW CS. Importantly, both LMW and HMW CS adjuvantation reduced morbidity following homologous IAV challenge. Taken together, these results support that LMW and HMW CS can act as adjuvants, although this protection may be mediated through distinct mechanisms based on CS MW.

Combined TLR4 and TLR9 agonists induce distinct phenotypic changes in innate immunity in vitro and in vivo.

Toll-like receptor (TLR)4 and TLR9 agonists, MPL and CpG, are used as adjuvants in vaccines and have been investigated for their combined potential. However, how these two combined agonists regulate transcriptional changes in innate immune cells and cells at the site of vaccination has not been thoroughly investigated. Here, we utilized transcriptomics to investigate how CpG, MPL, and CpG + MPL impact gene expression in dendritic cells (DC) in vitro. Principal component analysis of transcriptional changes after single and combined treatment indicated that CpG, MPL, and CpG + MPL caused distinct gene signatures. CpG + MPL induced antiviral gene expression and activated the interferon regulatory factor pathway. In vitro changes were associated with lower in vivo morbidity upon viral challenge, elevated systemic cytokine protein production, local cytokine mRNA expression, and increased migratory monocyte derived DC populations in the draining lymph node following vaccination with CpG + MPL. This report suggests that CpG + MPL enhances transcription of antiviral and inflammatory genes and increases DC migration.

Metabolic compounds within the porcine uterine environment are unique to the type of conceptus present during the early stages of blastocyst elongation.

The objective of this study was to identify metabolites within the porcine uterine milieu during the early stages of blastocyst elongation. At Days 9, 10, or 11 of gestation, reproductive tracts of White cross-bred gilts (n = 38) were collected immediately following harvest and flushed with Roswell Park Memorial Institute-1640 medium. Conceptus morphologies were assessed from each pregnancy and corresponding uterine flushings were assigned to one of five treatment groups based on these morphologies: (a) uniform spherical (n = 8); (b) heterogeneous spherical and ovoid (n = 8); (c) uniform ovoid (n = 8); (d) heterogeneous ovoid and tubular (n = 8); and (e) uniform tubular (n = 6). Uterine flushings from these pregnancies were submitted for nontargeted profiling by gas chromatography-mass spectrometry (GC-MS) and ultra performance liquid chromatography (UPLC)-MS techniques. Unsupervised multivariate principal component analysis (PCA) was performed using pcaMethods and univariate analysis of variance was performed in R with false discovery rate (FDR) adjustment. PCA analysis of the GC-MS and UPLC-MS data identified 153 and 104 metabolites, respectively. After FDR adjustment of the GC-MS and UPLC-MS data, 38 and 59 metabolites, respectively, differed (p < .05) in uterine flushings from pregnancies across the five conceptus stages. Some metabolites were greater (p < .05) in abundance for uterine flushings containing earlier stage conceptuses (i.e., spherical), such as uric acid, tryptophan, and tyrosine. In contrast, some metabolites were greater (p < .05) in abundance for uterine flushings containing later stage conceptuses (i.e., tubular), such as creatinine, serine, and urea. These data illustrate several putative metabolites that change within the uterine milieu during early porcine blastocyst elongation.

Mechanisms of unprimed and dexamethasone-primed nonviral gene delivery to human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are under intense study for applications of cell and gene therapeutics because of their unique immunomodulatory and regenerative properties. Safe and efficient genetic modification of hMSCs could increase their clinical potential by allowing functional expression of therapeutic transgenes or control over behavior and differentiation. Viral gene delivery is efficient, but suffers from safety issues, while nonviral methods are safe, but highly inefficient, especially in hMSCs. Our lab previously demonstrated that priming cells before delivery of DNA complexes with dexamethasone (DEX), an anti-inflammatory glucocorticoid drug, significantly increases hMSC transfection success. This work systematically investigates the mechanisms of hMSC transfection and DEX-mediated enhancement of transfection. Our results show that hMSC transfection and its enhancement by DEX are decreased by inhibiting classical intracellular transport and nuclear import pathways, but DEX transfection priming does not increase cellular or nuclear internalization of plasmid DNA (pDNA). We also show that hMSC transgene expression is largely affected by pDNA promoter and enhancer sequence changes, but DEX-mediated enhancement of transfection is unaffected by any pDNA sequence changes. Furthermore, DEX-mediated transfection enhancement is not the result of increased transgene messenger RNA transcription or stability. However, DEX-priming increases total protein synthesis by preventing hMSC apoptosis induced by transfection, resulting in increased translation of transgenic protein. DEX may also promote further enhancement of transgenic reporter enzyme activity by other downstream mechanisms. Mechanistic studies of nonviral gene delivery will inform future rationally designed technologies for safe and efficient genetic modification of clinically relevant cell types.

In vitro porcine blastocyst development in three-dimensional alginate hydrogels.

Appropriate embryonic and fetal development significantly impact pregnancy success and, therefore, the efficiency of swine production. The pre-implantation period of porcine pregnancy is characterized by several developmental hallmarks, which are initiated by the dramatic morphological change that occurs as pig blastocysts elongate from spherical to filamentous blastocysts. Deficiencies in blastocyst elongation contribute to approximately 20% of embryonic loss, and have a direct influence on within-litter birth weight variation. Although factors identified within the uterine environment may play a role in blastocyst elongation, little is known about the exact mechanisms by which porcine (or other species') blastocysts initiate and progress through the elongation process. This is partly due to the difficulty of replicating elongation in vitro, which would allow for its study in a controlled environment and in real-time. We developed a three dimensional (3-D) culture system using alginate hydrogel matrices that can encapsulate pig blastocysts, maintain viability and blastocyst architecture, and facilitate reproducible morphological changes with corresponding expression of steroidogenic enzyme transcripts and estrogen production, consistent with the initiation of elongation in vivo. This review highlights key aspects of the pre-implantation period of porcine pregnancy and the difficulty of studying blastocyst elongation in vivo or by using in vitro systems. This review also provides insights on the utility of 3-D hydrogels to study blastocyst elongation continuously and in real-time as a complementary and confirmatory approach to in vivo analysis.

Glucocorticoid Cell Priming Enhances Transfection Outcomes in Adult Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) are one of the most widely researched stem cell types with broad applications from basic research to therapeutics, the majority of which require introduction of exogenous DNA. However, safety and scalability issues hinder viral delivery, while poor efficiency hinders nonviral gene delivery, particularly to hMSCs. Here, we present the use of a pharmacologic agent (glucocorticoid) to overcome barriers to hMSC DNA transfer to enhance transfection using three common nonviral vectors. Glucocorticoid priming significantly enhances transfection in hMSCs, demonstrated by a 3-fold increase in efficiency, 4-15-fold increase in transgene expression, and prolonged transgene expression when compared to transfection without glucocorticoids. These effects are dependent on glucocorticoid receptor binding and caused in part by maintenance of normal metabolic function and increased cellular (5-fold) and nuclear (6-10-fold) DNA uptake over hMSCs transfected without glucocorticoids. Results were consistent across five human donors and in cells up to passage five. Glucocorticoid cell priming is a simple and effective technique to significantly enhance nonviral transfection of hMSCs that should enhance their clinical use, accelerate new research, and decrease reliance on early passage cells.

Development of pre-implantation porcine blastocysts cultured within alginate hydrogel systems either supplemented with secreted phosphoprotein 1 or conjugated with Arg-Gly-Asp Peptide.

Although deficiencies in porcine blastocyst elongation play a significant role in early embryonic mortality and establishment of within-litter developmental variation, the exact mechanisms of elongation are poorly understood. Secreted phosphoprotein 1 (SPP1) is increased within the uterine milieu during early porcine pregnancy and contains an Arg-Gly-Asp (RGD) peptide sequence that binds to cell surface integrins on the uterine endometrium and trophectoderm, promoting cell adhesion and migration. The aim of the present study was to evaluate the development of preimplantation porcine blastocysts encapsulated and cultured within alginate hydrogels either supplemented with SPP1 or conjugated with RGD. Blastocysts encapsulated within alginate hydrogels supplemented with SPP1 or conjugated with RGD had increased survival compared with non-encapsulated control blastocysts. In addition, the percentage of blastocysts encapsulated within RGD hydrogels that underwent morphological changes was greater than that of blastocysts encapsulated within standard alginate hydrogels or SPP1-supplemented hydrogels. Finally, only blastocysts encapsulated within RGD hydrogels had both increased expression of steroidogenic and immune responsiveness transcripts and increased 17ß-oestradiol production, consistent with blastocysts undergoing elongation in vivo. These results illustrate the importance of the integrin-binding RGD peptide sequence for stimulating the initiation of blastocyst elongation.

Temporal endogenous gene expression profiles in response to lipid-mediated transfection.

BACKGROUND: Design of efficient nonviral gene delivery systems is limited as a result of the rudimentary understanding of the specific molecules and processes that facilitate DNA transfer. METHODS: Lipoplexes formed with Lipofectamine 2000 (LF2000) and plasmid-encoding green fluorescent protein (GFP) were delivered to the HEK 293T cell line. After treating cells with lipoplexes, HG-U133 Affymetrix microarrays were used to identify endogenous genes differentially expressed between treated and untreated cells (2 h exposure) or between flow-separated transfected cells (GFP+) and treated, untransfected cells (GFP-) at 8, 16 and 24 h after lipoplex treatment. Cell priming studies were conducted using pharmacologic agents to alter endogenous levels of the identified differentially expressed genes to determine effect on transfection levels. RESULTS: Relative to untreated cells 2 h after lipoplex treatment, only downregulated genes were identified ≥ 30-fold: ALMS1, ITGB1, FCGR3A, DOCK10 and ZDDHC13. Subsequently, relative to GFP- cells, the GFP+ cell population showed at least a five-fold upregulation of RAP1A and PACSIN3 (8 h) or HSPA6 and RAP1A (16 and 24 h). Pharmacologic studies altering endogenous levels for ALMS1, FCGR3A, and DOCK10 (involved in filopodia protrusions), ITGB1 (integrin signaling), ZDDHC13 (membrane trafficking) and PACSIN3 (proteolytic shedding of membrane receptors) were able to increase or decrease transgene production. CONCLUSIONS: RAP1A, PACSIN3 and HSPA6 may help lipoplex-treated cells overcome a transcriptional shutdown due to treatment with lipoplexes and provide new targets for investigating molecular mechanisms of transfection or for enhancing transfection through cell priming or engineering of the nonviral gene delivery system.

Temporal endogenous gene expression profiles in response to polymer-mediated transfection and profile comparison to lipid-mediated transfection.

BACKGROUND: Design of efficient nonviral gene delivery systems is limited by the rudimentary understanding of specific molecules that facilitate transfection. METHODS: Polyplexes using 25-kDa polyethylenimine (PEI) and plasmid-encoding green fluorescent protein (GFP) were delivered to HEK 293T cells. After treating cells with polyplexes, microarrays were used to identify endogenous genes differentially expressed between treated and untreated cells (2 h of exposure) or between flow-separated transfected cells (GFP+) and treated, untransfected cells (GFP-) at 8, 16 and 24 h after lipoplex treatment. Cell priming studies were conducted using pharmacologic agents to alter endogenous levels of the identified differentially expressed genes to determine effect on transfection levels. Differentially expressed genes in polyplex-mediated transfection were compared with those differentially expressed in lipoplex transfection to identify DNA carrier-dependent molecular factors. RESULTS: Differentially expressed genes were RGS1, ARHGAP24, PDZD2, SNX24, GSN and IGF2BP1 after 2 h; RAP1A and ACTA1 after 8 h; RAP1A, WDR78 and ACTA1 after 16 h; and RAP1A, SCG5, ATF3, IREB2 and ACTA1 after 24 h. Pharmacologic studies altering endogenous levels for ARHGAP24, GSN, IGF2BP1, PDZD2 and RGS1 were able to increase or decrease transgene production. Comparing differentially expressed genes for polyplexes and lipoplexes, no common genes were identified at the 2-h time point, whereas, after the 8-h time point, RAP1A, ATF3 and HSPA6 were similarly expressed. SCG5 and PGAP1 were only upregulated in polyplex-transfected cells. CONCLUSIONS: The identified genes and pharmacologic agents provide targets for improving transfection systems, although polyplex or lipoplex dependencies must be considered.

Integrating mitosis, toxicity, and transgene expression in a telecommunications packet-switched network model of lipoplex-mediated gene delivery.

Gene delivery systems transport exogenous genetic information to cells or biological systems with the potential to directly alter endogenous gene expression and behavior with applications in functional genomics, tissue engineering, medical devices, and gene therapy. Nonviral systems offer advantages over viral systems because of their low immunogenicity, inexpensive synthesis, and easy modification but suffer from lower transfection levels. The representation of gene transfer using models offers perspective and interpretation of complex cellular mechanisms,including nonviral gene delivery where exact mechanisms are unknown. Here, we introduce a novel telecommunications model of the nonviral gene delivery process in which the delivery of the gene to a cell is synonymous with delivery of a packet of information to a destination computer within a packet-switched computer network. Such a model uses nodes and layers to simplify the complexity of modeling the transfection process and to overcome several challenges of existing models. These challenges include a limited scope and limited time frame, which often does not incorporate biological effects known to affect transfection. The telecommunication model was constructed in MATLAB to model lipoplex delivery of the gene encoding the green fluorescent protein to HeLa cells. Mitosis and toxicity events were included in the model resulting in simulation outputs of nuclear internalization and transfection efficiency that correlated with experimental data. A priori predictions based on model sensitivity analysis suggest that increasing endosomal escape and decreasing lysosomal degradation, protein degradation, and GFP-induced toxicity can improve transfection efficiency by three-fold. Application of the telecommunications model to nonviral gene delivery offers insight into the development of new gene delivery systems with therapeutically relevant transfection levels.

Combined QCM-D/GE as a tool to characterize stimuli-responsive swelling of and protein adsorption on polymer brushes grafted onto 3D-nanostructures.

A combined setup of quartz crystal microbalance and generalized ellipsometry can be used to comprehensively investigate complex functional coatings comprising stimuli-responsive polymer brushes and 3D nanostructures in a dynamic, noninvasive in situ measurement. While the quartz crystal microbalance detects the overall change in areal mass, for instance, during a swelling or adsorption process, the generalized ellipsometry data can be evaluated in terms of a layered model to distinguish between processes occurring within the intercolumnar space or on top of the anisotropic nanocolumns. Silicon films with anisotropic nanocolumnar morphology were prepared by the glancing angle deposition technique and further functionalized by grafting of poly-(acrylic acid) or poly-(N- isopropylacrylamide) chains. Investigations of the thermoresponsive swelling of the poly-(N-isopropylacrylamide) brush on the Si nanocolumns proved the successful preparation of a stimuli-responsive coating. Furthermore, the potential of these novel coatings in the field of biotechnology was explored by investigation of the adsorption of the model protein bovine serum albumin. Adsorption, retention, and desorption triggered by a change in the pH value is observed using poly-(acrylic acid) functionalized nanostructures, although generalized ellipsometry data revealed that this process occurs only on top of the nanostructures. Poly-(N-isopropylacrylamide) is found to render the nanostructures non-fouling properties.

In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix.

Between Days 10 and 12 of gestation, porcine embryos undergo a dramatic morphological change, known as elongation, with a corresponding increase in oestrogen production that triggers maternal recognition of pregnancy. Elongation deficiencies contribute to embryonic loss, but exact mechanisms of elongation are poorly understood due to the lack of an effective in vitro culture system. Our objective was to use alginate hydrogels as three-dimensional scaffolds that can mechanically support the in vitro development of preimplantation porcine embryos. White cross-bred gilts were bred at oestrus (Day 0) to Duroc boars and embryos were recovered on Days 9, 10 or 11 of gestation. Spherical embryos were randomly assigned to be encapsulated within double-layered 0.7% alginate beads or remain as non-encapsulated controls (ENC and CONT treatment groups, respectively) and were cultured for 96h. Every 24h, half the medium was replaced with fresh medium and an image of each embryo was recorded. At the termination of culture, embryo images were used to assess morphological changes and cell survival. 17ß-Oestradiol levels were measured in the removed media by radioimmunoassay. Real-time polymerase chain reaction was used to analyse steroidogenic transcript expression at 96h in ENC and CONT embryos, as well as in vivo-developed control embryos (i.e. spherical, ovoid and tubular). Although no differences in cell survival were observed, 32% (P<0.001) of the surviving ENC embryos underwent morphological changes characterised by tubal formation with subsequent flattening, whereas none of the CONT embryos exhibited morphological changes. Expression of steroidogenic transcripts STAR, CYP11A1 and CYP19A1 was greater (P<0.07) in ENC embryos with morphological changes (ENC+) compared with CONT embryos and ENC embryos with no morphological changes (ENC-), and was more similar to expression of later-stage in vivo-developed controls. Furthermore, a time-dependent increase (P<0.001) in 17ß-oestradiol was observed in culture media from ENC+ compared with ENC- and CONT embryos. These results illustrate that preimplantation pig embryos encapsulated in alginate hydrogels can undergo morphological changes with increased expression of steroidogenic transcripts and oestrogen production, consistent with in vivo-developed embryos. This alginate culture system can serve as a tool for evaluating specific mechanisms of embryo elongation that could be targeted to improve pregnancy outcomes.

Network analysis of endogenous gene expression profiles after polyethyleneimine-mediated DNA delivery.

BACKGROUND: DNA delivery systems, which transport exogenous DNA to cells, have applications that include gene therapy, tissue engineering and medical devices. Although the cationic nonviral DNA carrier polyethyleneimine (PEI) has been widely studied, the molecular factors and pathways underlying PEI-mediated DNA transfer remain largely unknown, preventing the design of more efficient delivery systems. METHODS: HEK 293 T cells were treated with polyplexes formed with PEI and pEGFPLuc encoding for green fluorescent protein (GFP). Transfected cells expressing GFP were flow-separated from treated, untransfected cells. Gene expression profiles were obtained using Affymetrix HG-U133 2.0 microarrays and differentially expressed genes were identified using R/Bioconductor. Gene network analysis using EGAN (exploratory gene association network) bioinformatics tools was then used to find interaction among genes and enriched gene ontology (GO) terms related to transfection. Genes identified by this method were perturbed using pharmacologic activators or inhibitors to assess their effect on DNA transfer. RESULTS: Microarray analysis comparing transfected cells to untransfected cells revealed 215 genes to be differentially expressed, with the majority enriched to GO processes including metabolism, response to stimulus, cell cycle, biological regulation and cellular component organization or biogenesis pathways. Gene network analysis revealed a coordinated induction of RAP1A, SCG5, PGAP1, ATF3 and NEB genes implicated in cell stress, cell cycle and cytoskeletal processes. Altering pathways with pharmacologic agents confirmed the potential role of RAP1A, SCG5 and ATF3 in transfection. CONCLUSIONS: Microarray and gene network analyses of the sorted, transfected cell population can identify potential mediators of transfection, providing a basis for the design of improved delivery systems.

Systematic comparison of nonviral gene delivery strategies for efficient co-expression of two transgenes in human mesenchymal stem cells.

BACKGROUND: Human mesenchymal stem cells (hMSCs) are being researched for cell-based therapies due to a host of unique properties, however, genetic modification of hMSCs, accomplished through nonviral gene delivery, could greatly advance their therapeutic potential. Furthermore, expression of multiple transgenes in hMSCs could greatly advance their clinical significance for treatment of multifaceted diseases, as individual transgenes could be expressed that target separate pathogenic drivers of complex diseases. Expressing multiple transgenes can be accomplished by delivering multiple DNA vectors encoding for each transgene, or by delivering a single poly-cistronic vector that encodes for each transgene and accomplishes expression through either use of multiple promoters, an internal ribosome entry site (IRES), or a 2A peptide sequence. These different transgene expression strategies have been used to express multiple transgenes in various mammalian cells, however, they have not been fully evaluated in difficult-to-transfect primary cells, like hMSCs. This study systematically compared four transgene expression and delivery strategies for expression of two reporter transgenes in four donors of hMSCs from two tissue sources using lipid- and polymer-mediate transfection, as follows: (i) delivery of separate DNA vectors in separate nanoparticles; (ii) delivery of separate DNA vectors combined in the same nanoparticle; (iii) delivery of a bi-cistronic DNA vector with an IRES sequence via nanoparticles; and (iv) delivery of a bi-cistronic DNA vector with a dual 2A peptide sequence via nanoparticles. RESULTS: Our results indicate that expression of two transgenes in hMSCs, independent of expression or delivery strategy, is inefficient compared to expressing a single transgene. However, delivery of separate DNA vectors complexed in the same nanoparticle, or delivery of a bi-cistronic DNA vector with a dual 2A peptide sequence, significantly increased the number of hMSCs expressing both transgenes compared to other conditions tested. CONCLUSION: Separate DNA vectors delivered in the same nanoparticle and bi-cistronic DNA vectors with dual 2A peptide sequences are highly efficient at simultaneously expressing two transgenes in multiple donors of hMSCs from different tissue sources. The data presented in this work can guide the development of hMSC transfection systems for delivery of multiple transgenes, with the goal of producing clinically relevant, genetically modified hMSCs.

Generalized ellipsometry in-situ quantification of organic adsorbate attachment within slanted columnar thin films.

We apply generalized ellipsometry, well-known to be sensitive to the optical properties of anisotropic materials, to determine the amount of fibronectin protein that adsorbs onto a Ti slanted columnar thin film from solution. We find that the anisotropic optical properties of the thin film change upon organic adsorption. An optical model for ellipsometry data analysis incorporates an anisotropic Bruggeman effective medium approximation. We find that differences in experimental data from before and after fibronectin adsorption can be solely attributable to the uptake of fibronectin within the slanted columnar thin film. Simultaneous, in-situ generalized ellipsometry and quartz crystal microbalance measurements show excellent agreement on the amount and rate of fibronectin adsorption. Quantitative characterization of organic materials within three-dimensional, optically anisotropic slanted columnar thin films could permit their use in optical sensor applications.

MR elastography monitoring of tissue-engineered constructs.

The objective of tissue engineering (TE) is to create functional replacements for various tissues; the mechanical properties of these engineered constructs are critical to their function. Several techniques have been developed for the measurement of the mechanical properties of tissues and organs; however, current methods are destructive. The field of TE will benefit immensely if biomechanical models developed by these techniques could be combined with existing imaging modalities to enable noninvasive, dynamic assessment of mechanical properties during tissue growth. Specifically, MR elastography (MRE), which is based on the synchronization of a mechanical actuator with a phase contrast imaging pulse sequence, has the capacity to measure tissue strain generated by sonic cyclic displacement. The captured displacement is presented in shear wave images from which the complex shear moduli can be extracted or simplified by a direct measure, termed the shear stiffness. MRE has been extended to the microscopic scale, combining clinical MRE with high-field magnets, stronger magnetic field gradients and smaller, more sensitive, radiofrequency coils, enabling the interrogation of smaller samples, such as tissue-engineered constructs. The following topics are presented in this article: (i) current mechanical measurement techniques and their limitations in TE; (ii) a description of the MRE system, MRE theory and how it can be applied for the measurement of mechanical properties of tissue-engineered constructs; (iii) a summary of in vitro MRE work for the monitoring of osteogenic and adipogenic tissues originating from human adult mesenchymal stem cells (MSCs); (iv) preliminary in vivo studies of MRE of tissues originating from mouse MSCs implanted subcutaneously in immunodeficient mice with an emphasis on in vivo MRE challenges; (v) future directions to resolve current issues with in vivo MRE in the context of how to improve the future role of MRE in TE.

Microarray analysis of gene expression profiles in cells transfected with nonviral vectors.

Inefficient gene delivery is a critical factor limiting the use of nonviral methods in therapeutic applications including gene therapy and tissue engineering. There have been few efforts to understand or engineer the molecular signaling pathways that dictate the efficacy of gene transfer. Microarray analysis was used to determine endogenous gene expression profiles modulated during nonviral gene transfer. Nonviral DNA lipoplexes were delivered to HEK 293T cells. Flow cytometry was used to isolate a population of transfected cells. Expression patterns were compared between transfected and nontransfected samples, which revealed three genes that were significantly upregulated in transfected cells, including RAP1A, a GTPase implicated in integrin-mediated cell adhesion, and HSP70B', a stress-inducible gene that may be important for maintaining cell viability. Furthermore, RAP1A was also significantly upregulated in untransfected cells that were exposed to lipoplexes but that had not expressed the transgene as compared to control, untreated cells. Transfection in the presence of activators of upregulated genes was enhanced, demonstrating the principle of altering endogenous gene expression profiles to enhance transfection. With a greater understanding of signaling pathways involved in gene delivery, more efficient nonviral delivery schemes capitalizing on endogenous factors can be developed to advance therapeutic applications.