The Challenges to Advancing Induced Pluripotent Stem Cell-Dependent Cell Replacement Therapy.

Induced pluripotent stem cells (iPSC) represent a potentially exciting regenerative-medicine cell therapy for several chronic conditions such as macular degeneration, soft tissue and orthopedic conditions, cardiopulmonary disease, cancer, neurodegenerative disorders and metabolic disorders. The field of iPSC therapeutics currently exists at an early stage of development. There are several important stakeholders that include academia, industry, regulatory agencies, financial institutions and patients who are committed to advance the field. Yet, unlike more established therapeutic modalities like small and large molecules, iPSC therapies pose significant unique challenges with respect to safety, potency, genetic stability, immunogenicity, tumorgenicity, cell reproducibility, scalability and engraftment. The aim of this review article is to highlight the unique technical challenges that need to be addressed before iPSC technology can be fully realized as a cell replacement therapy. Additionally, this manuscript offers some potential solutions and identifies areas of focus that should be considered in order for the iPSC field to achieve its promise. The scope of this article covers the following areas: (1) the impact of different iPSC reprogramming methods on immunogenicity and tumorigenicity; (2) the effect of genetic instability on cell reproducibility and differentiation; (3) the role of growth factors and post-translational modification on differentiation and cell scalability; (4) the potential use of gene editing in improving iPSC differentiation; (5) the advantages and disadvantages between autologous and allogeneic cell therapy; (6) the regulatory considerations in developing a viable and reproducible cell product; and (7) the impact of local tissue inflammation on cell engraftment and cell viability.

Virus-free and oncogene-free induced pluripotent stem cell reprogramming in cord blood and peripheral blood in patients with lung disease.

AIM: A virus- and oncogene-free induced pluripotent stem cell (iPSC) reprogramming method was developed with cord blood-derived mononuclear cells (CBDMNC) and peripheral blood mononuclear cells (PBMNC) from patients with genetic lung diseases. METHOD: iPSC reprogramming used small molecules, hematopoietic stem cell (HSC) expansion media and episomal vectors that lacked Myc and Lin28. RESULTS: All iPSC colonies were fully reprogrammed based on SSEA4 expression. A total of 300,000 CBDMNC was the optimal cell number for cell reprogramming, which was associated with a 13-fold increase in CD34+ cells upon exposure to HSC media. Cell reprogramming was not observed in the absence of HSC expansion media. The method also reprogrammed PBMNC in patients with cystic fibrosis or α-1 antitrypsin deficiency. Oncogene-free iPSC cell lines differentiated into all three germ cell lineages. CONCLUSION: This iPSC reprogramming approach satisfies an important regulatory requirement for iPSC-based cell therapies with lower clinical risk from CBDMNC and PBMNC.

Efficient method to create integration-free, virus-free, Myc and Lin28-free human induced pluripotent stem cells from adherent cells.

AIM: Nonviral induced pluripotent stem cell (IPSC) reprogramming is not efficient without the oncogenes, Myc and Lin28. We describe a robust Myc and Lin28-free IPSC reprogramming approach using reprogramming molecules. METHODS: IPSC colony formation was compared in the presence and absence of Myc and Lin28 by the mixture of reprogramming molecules and episomal vectors. RESULTS: While more colonies were observed in cultures transfected with the aforementioned oncogenes, the Myc and Lin28-free method achieved the same reprogramming efficiency as reports that used these oncogenes. Further, all colonies were fully reprogrammed based on expression of SSEA4, even in the absence of Myc and Lin28. CONCLUSION: This approach satisfies an important regulatory pathway for developing IPSC cell therapies with lower clinical risk.

Cellular electrical micro-impedance parameter artifacts produced by passive and active current regulation.

This study analyzes the cellular microelectrode voltage measurement errors produced by active and passive current regulation, and the propagation of these errors into cellular barrier function parameter estimates. The propagation of random and systematic errors into these parameters is accounted for within a Riemannian manifold framework consistent with information geometry. As a result, the full non-linearity of the model parameter state dependence, the instrumental noise distribution, and the systematic errors associated with the voltage to impedance conversion, are accounted for. Specifically, cellular model parameters are treated as the coordinates of a model space manifold that inherits a Riemannian metric from the data space. The model space metric is defined in terms of the pull back of an instrumental noise-dependent Fisher information metric. Additional noise sources produced by the evaluation of the cell-covered electrode model that is a function of a naked electrode random variable are also included in the analysis. Based on a circular cellular micro-impedance model in widespread use, this study shows that cellular barrier function parameter estimates are highly model state dependent. Systematic errors produced by coaxial lead capacitances and circuit loading can also lead to significant and model state-dependent parameter errors and should, therefore, be either reduced or corrected for analytically.

Equilibrium and non-equilibrium charge-dependent quantification of endothelial cell hydrogel scaffolds.

Using equilibrium swelling and non-equilibrium membrane potential measurements, this study assesses the charge density in two representative series of polyelectrolyte hydrogels and examines the morphological and proliferative responses of endothelial cells as a function of the prepared charge offset. The neutral monomers 2-hydroxyethylmethacrylate (HEMA) and poly(ethylene glycol) dimethacrylate (n = 1,000) (PEGDMA) were copolymerized with either the acidic monomer 2-sulfoethyl methacrylate (SEMA) or the basic monomer methacryloxy ethyltrimethylammonium chloride (MAETAC) to make membranes with pregelation charge offset concentrations varying from 0 to +/-200 mM. A thermodynamic analysis of swelling and membrane potential measurements quantified the hydrogel charge density state following equilibration at different ion strengths. Porcine pulmonary artery endothelial cells were seeded on samples of each HEMA and PEGDMA copolymer and the amount of cell coverage was measured over a 4-day period. Cellular attachment and proliferation increased with increasing proportions of charged monomers and showed a threshold pattern of attachment and growth on the positively charged HEMA-MAETAC copolymer hydrogels with increasing proportions of initially prepared charge. The series of PEGDMA copolymer hydrogels remained relatively resistant to cellular attachment and proliferation over the range of prepared charges considered in this study.

A Riemannian manifold analysis of endothelial cell monolayer impedance parameter precision.

Endothelial cell adhesion and barrier function play a critical role in many biological and pathophysiological processes. The decomposition of endothelial cell adhesion and barrier function into cell-cell and cell-matrix components using frequency dependent cellular micro-impedance measurements has, therefore, received widespread application. Few if any studies, however, have examined the precision of these model parameters. This study presents a parameter sensitivity analysis of a representative cellular barrier function model using a concise geometric formulation that includes instrumental data acquisition settings. Both model state dependence and instrumental noise distributions are accounted for within the framework of Riemannian manifold theory. Experimentally acquired microimpedance measurements of attached endothelial cells define the model state domain, while experimentally measured noise statistics define the data space Riemannian metric based on the Fisher information matrix. The results of this analysis show that the sensitivity of cell-cell and cell-matrix impedance components are highly model state dependent and several well defined regions of low precision exist. The results of this study further indicate that membrane resistive components can significantly reduce the precision of the remaining parameters in these models.

Endothelial cell electrical impedance parameter artifacts produced by a gold electrode and phase sensitive detection.

Frequency dependent cellular micro-impedance estimates obtained from a gold two-electrode configuration using phase sensitive detection have become increasingly used to evaluate cellular barrier model parameters. The results of this study show that cellular barrier function parameter estimates optimized using measurements obtained from this biosensor are highly susceptible to both time dependent and systematic instrumental artifacts. Based on a power spectral analysis of experimentally measured microelectrode voltages, synchronous, 60 Hz, and white Gaussian noise were identified as the most significant time dependent instrumental artifacts. The reduction of these artifacts using digital filtering produced a corresponding reduction in the optimized model parameter fluctuations. Using a series of instrumental circuit models, this study also shows that electrode impedance voltage divider effects and circuit capacitances can produce systematic deviations in cellular barrier function parameter estimates. Although the implementation of an active current source reduced the voltage divider effects, artifacts produced by coaxial cable and other circuit capacitive elements at frequencies exceeding 1 kHz still remained. Reducing time dependent instrumental fluctuations and systematic errors produced a significant reduction in cellular model barrier parameter errors and improved the model fit to experimental data.

Growth factor- and heparin-dependent regulation of constitutive and agonist-mediated human endothelial barrier function.

We report functional differences in constitutive and agonist-mediated endothelial barrier function between cultured primary and Clonetics human umbilical vein endothelial cells (pHUVEC and cHUVEC) grown in soluble growth factors and heparin. Basal transendothelial resistance (TER) was much lower in pHUVEC than in cHUVEC grown in medium supplemented with growth factors, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and human epithelial growth factor (EGF), and heparin. On the basis of a numerical model of TER, the increased basal TER in cHUVEC was due to effects on cell-matrix adhesion and membrane capacitance. Heparin and bFGF increased constitutive TER in cultured pHUVEC, and heparin mediated additional increases in constitutive TER in pHUVEC supplemented with bFGF. EGF attenuated bFGF-mediated increases in TER. On the basis of the numerical model, in contrast to cHUVEC, heparin and bFGF augmented TER through effects on cell-cell adhesion and membrane capacitance in pHUVEC. Thrombin mediated quantitatively greater amplitude and a more sustained decline in TER in cultured cHUVEC than pHUVEC. Thrombin-mediated barrier dysfunction was attenuated in pHUVEC conditioned in EGF in the presence or absence of heparin. Thrombin-mediated barrier dysfunction was also attenuated when monolayers were exposed to low concentrations of heparin and further attenuated in the presence of bFGF. cAMP stimulation mediated differential attenuation of thrombin-mediated barrier dysfunction between pHUVEC and cHUVEC. VEGF displayed differential effects in TER in serum-free medium. Taken together, these data demonstrate marked differential regulation of constitutive and agonist-mediated endothelial barrier function in response to mitogens and heparin stimulation.

Involvement of ROCK-mediated endothelial tension development in neutrophil-stimulated microvascular leakage.

Neutrophil-induced coronary microvascular barrier dysfunction is an important pathophysiological event in heart disease. Currently, the precise cellular and molecular mechanisms of neutrophil-induced microvascular leakage are not clear. The aim of this study was to test the hypothesis that rho kinase (ROCK) increases coronary venular permeability in association with elevated endothelial tension. We assessed permeability to albumin (P(a)) in isolated porcine coronary venules and in coronary venular endothelial cell (CVEC) monolayers. Endothelial barrier function was also evaluated by measuring transendothelial electrical resistance (TER) of CVEC monolayers. In parallel, we measured isometric tension of CVECs grown on collagen gels. Transference of constitutively active (ca)-ROCK protein into isolated coronary venules or CVEC monolayers caused a significant increase in P(a) and decreased TER in CVECs. The ROCK inhibitor Y-27632 blocked the ca-ROCK-induced changes. C5a-activated neutrophils (10(6)/ml) also significantly elevated venular P(a), which was dose-dependently inhibited by Y-27632 and a structurally distinct ROCK inhibitor, H-1152. In CVEC monolayers, activated neutrophils increased permeability with a concomitant elevation in isometric tension, both of which were inhibited by Y-27632 or H-1152. Treatment with ca-ROCK also significantly increased CVEC monolayer permeability and isometric tension, coupled with actin polymerization and elevated phosphorylation of myosin regulatory light chain on Thr18/Ser19. The data suggest that during neutrophil activation, ROCK promotes microvascular leakage in association with actin-myosin-mediated tension development in endothelial cells.

Modeling error and stability of endothelial cytoskeletal membrane parameters based on modeling transendothelial impedance as resistor and capacitor in series.

Transendothelial impedance across an endothelial monolayer grown on a microelectrode has previously been modeled as a repeating pattern of disks in which the electrical circuit consists of a resistor and capacitor in series. Although this numerical model breaks down barrier function into measurements of cell-cell adhesion, cell-matrix adhesion, and membrane capacitance, such solution parameters can be inaccurate without understanding model stability and error. In this study, we have evaluated modeling stability and error by using a chi(2) evaluation and Levenberg-Marquardt nonlinear least-squares (LM-NLS) method of the real and/or imaginary data in which the experimental measurement is compared with the calculated measurement derived by the model. Modeling stability and error were dependent on current frequency and the type of experimental data modeled. Solution parameters of cell-matrix adhesion were most susceptible to modeling instability. Furthermore, the LM-NLS method displayed frequency-dependent instability of the solution parameters, regardless of whether the real or imaginary data were analyzed. However, the LM-NLS method identified stable and reproducible solution parameters between all types of experimental data when a defined frequency spectrum of the entire data set was selected on the basis of a criterion of minimizing error. The frequency bandwidth that produced stable solution parameters varied greatly among different data types. Thus a numerical model based on characterizing transendothelial impedance as a resistor and capacitor in series and as a repeating pattern of disks is not sufficient to characterize the entire frequency spectrum of experimental transendothelial impedance.

Gene delivery of l-caldesmon protects cytoskeletal cell membrane integrity against adenovirus infection independently of myosin ATPase and actin assembly.

The cytoskeleton is critical to the viral life cycle. Agents like cytochalasin inhibit viral infections but cannot be used for antiviral therapy because of their toxicity. We report the efficacy, safety, and mechanisms by which gene delivery of human wild-type low-molecular-weight caldesmon (l-CaD) protects cell membrane integrity from adenovirus infection in a DF-1 cell line, an immortalized avian fibroblast that is null for l-CaD. Transfection with an adenovirus (Ad)-controlled construct mediated a dose-dependent decline in transcellular resistance. In accordance with a computational model of cytoskeletal membrane properties, Ad disturbed cell-cell and cell-matrix adhesion and membrane capacitance. Transfection with the Ad-l-CaD construct attenuated adenovirus-mediated loss in transcellular resistance. Quantitation of vinculin-stained plaques revealed an increase in total focal contact mass in monolayers transfected with the Ad-l-CaD construct. Expression of l-CaD protected transcellular resistance through primary effects on membrane capacitance and independently of actin solubility and effects on pre-stress, as measured by the decline in isometric tension in response to cytochalasin D. Expression of l-CaD exhibited less Trypan blue cell toxicity than cytochalasin, and, unlike cytochalasin, it did not interfere with wound closure or adversely effect transcellular resistance. These findings demonstrate the gene delivery of wild-type human l-CaD as a potentially efficacious and safe agent that inhibits some of the cytopathic effects of adenovirus.

Phorbol ester-mediated pulmonary artery endothelial barrier dysfunction through regulation of actin cytoskeletal mechanics.

The mechanisms of phorbol ester- and thrombin-mediated pulmonary artery endothelial barrier dysfunction were compared. Phorbol ester dibutyrate (PDBU) mediated slow force velocity and less force than thrombin. Taxol did not attenuate PDBU-mediated tension, while it reversed nocodazole-mediated tension. PDBU-mediated tension was not affected by acrylamide; PDBU increased cell stiffness and produced greater declines in transendothelial resistance (TER) than acrylamide. Thus PDBU caused a net increase in tension and did not unload microtubule or intermediate filaments. Microfilament remodeling, determined on the basis of immunocytochemistry and actin solubility, lacked the sensitivity and specificity to predict actin-dependent mechanical properties. Thrombin increased myosin light chain (MLC) kinase site-specific MLC phosphorylation, according to peptide map analysis, whereas PDBU did not increase PKC-specific MLC phosphorylation. The initial PDBU-mediated tension development temporally correlated with PDBU-mediated decline in TER and increased low-molecular-weight caldesmon (l-CaD) phosphorylation. PDBU-mediated tension development and decreases in TER were associated with a temporal loss of endothelial cell-matrix adhesion, based on a numerical model of TER. Although, on the basis of immunocytochemistry, thrombin-mediated tension was associated with actin insolubility, actin reorganization, and gap formation, these changes did not predict thrombin-mediated gap formation, based on TER and time-lapse differential interference contrast microscopy. These data suggest that PDBU may disrupt endothelial barrier function through loss of cell-matrix adhesion through l-CaD-dependent actin contraction.

Differential effects of histamine and thrombin on endothelial barrier function through actin-myosin tension.

We compared temporal changes in isometric tension in cultured human umbilical vein endothelial cells inoculated on a polymerized collagen membrane with changes in cell-cell and cell-matrix adhesion derived by a mathematical model of transendothelial cell resistance. Thrombin and histamine disrupt barrier function by targeting a greater loss in cell-cell adhesion, which preceded losses in overall transendothelial resistance. There were minor losses in cell-matrix adhesion, which was temporally slower than the decline in the overall transendothelial resistance. In contrast, thrombin and histamine restored barrier function by initiating a restoration of cell-matrix adhesion, which occurred before an increase in overall transendothelial resistance. Thrombin mediated a second and slower decline in cell-cell adhesion, which was not observed in histamine-treated cells. This decline in cell-cell adhesion temporally correlated with expressed maximal levels of tension development, suggesting that actin-myosin contraction directly strains cell-cell adhesion sites. Pretreatment of cells with ML-7 mediated more rapid recovery of cell-cell adhesion and had no effect on cell-matrix adhesion. Taken together, expression of actin-myosin contraction affects the restoration of barrier function by straining cell-cell adhesion sites.