Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide a promising source for cell therapy and drug screening. Several high-yield protocols exist for hESC-CM production; however, methods to significantly advance hESC-CM maturation are still lacking. Building on our previous experience with mouse ESC-CMs, we investigated the effects of 3-dimensional (3D) tissue-engineered culture environment and cardiomyocyte purity on structural and functional maturation of hESC-CMs. 2D monolayer and 3D fibrin-based cardiac patch cultures were generated using dissociated cells from differentiated Hes2 embryoid bodies containing varying percentage (48-90%) of CD172a (SIRPA)-positive cardiomyocytes. hESC-CMs within the patch were aligned uniformly by locally controlling the direction of passive tension. Compared to hESC-CMs in age (2 weeks) and purity (48-65%) matched 2D monolayers, hESC-CMs in 3D patches exhibited significantly higher conduction velocities (CVs), longer sarcomeres (2.09 ± 0.02 vs. 1.77 ± 0.01 μm), and enhanced expression of genes involved in cardiac contractile function, including cTnT, αMHC, CASQ2 and SERCA2. The CVs in cardiac patches increased with cardiomyocyte purity, reaching 25.1 cm/s in patches constructed with 90% hESC-CMs. Maximum contractile force amplitudes and active stresses of cardiac patches averaged to 3.0 ± 1.1 mN and 11.8 ± 4.5 mN/mm(2), respectively. Moreover, contractile force per input cardiomyocyte averaged to 5.7 ± 1.1 nN/cell and showed a negative correlation with hESC-CM purity. Finally, patches exhibited significant positive inotropy with isoproterenol administration (1.7 ± 0.3-fold force increase, EC50 = 95.1 nm). These results demonstrate highly advanced levels of hESC-CM maturation after 2 weeks of 3D cardiac patch culture and carry important implications for future drug development and cell therapy studies.

Porcine endothelial cells cocultured with smooth muscle cells became procoagulant in vitro.

Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC/smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts.

Chondrogenesis of adult stem cells from adipose tissue and bone marrow: induction by growth factors and cartilage-derived matrix.

OBJECTIVES: Adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) are multipotent adult stem cells with potential for use in cartilage tissue engineering. We hypothesized that these cells show distinct responses to different chondrogenic culture conditions and extracellular matrices, illustrating important differences between cell types. METHODS: Human ASCs and MSCs were chondrogenically differentiated in alginate beads or a novel scaffold of reconstituted native cartilage-derived matrix with a range of growth factors, including dexamethasone, transforming growth factor beta3, and bone morphogenetic protein 6. Constructs were analyzed for gene expression and matrix synthesis. RESULTS: Chondrogenic growth factors induced a chondrocytic phenotype in both ASCs and MSCs in alginate beads or cartilage-derived matrix. MSCs demonstrated enhanced type II collagen gene expression and matrix synthesis as well as a greater propensity for the hypertrophic chondrocyte phenotype. ASCs had higher upregulation of aggrecan gene expression in response to bone morphogenetic protein 6 (857-fold), while MSCs responded more favorably to transforming growth factor beta3 (573-fold increase). CONCLUSIONS: ASCs and MSCs are distinct cell types as illustrated by their unique responses to growth factor-based chondrogenic induction. This chondrogenic induction is affected by the composition of the scaffold and the presence of serum.

Effect of microRNA modulation on bioartificial muscle function.

Cellular therapies have recently employed the use of small RNA molecules, particularly microRNAs (miRNAs), to regulate various cellular processes that may be altered in disease states. In this study, we examined the effect of transient muscle-specific miRNA inhibition on the function of three-dimensional skeletal muscle cultures, or bioartificial muscles (BAMs). Skeletal myoblast differentiation in vitro is enhanced by inhibiting a proliferation-promoting miRNA (miR-133) expressed in muscle tissues. As assessed by functional force measurements in response to electrical stimulation at frequencies ranging from 0 to 20 Hz, peak forces exhibited by BAMs with miR-133 inhibition (anti-miR-133) were on average 20% higher than the corresponding negative control, although dynamic responses to electrical stimulation in miRNA-transfected BAMs and negative controls were similar to nontransfected controls. Immunostaining for alpha-actinin and myosin also showed more distinct striations and myofiber organization in anti-miR-133 BAMs, and fiber diameters were significantly larger in these BAMs over both the nontransfected and negative controls. Compared to the negative control, anti-miR-133 BAMs exhibited more intense nuclear staining for Mef2, a key myogenic differentiation marker. To our knowledge, this study is the first to demonstrate that miRNA mediation has functional effects on tissue-engineered constructs.

Functional properties of cell-seeded three-dimensionally woven poly(epsilon-caprolactone) scaffolds for cartilage tissue engineering.

Articular cartilage possesses complex mechanical properties that provide healthy joints the ability to bear repeated loads and maintain smooth articulating surfaces over an entire lifetime. In this study, we utilized a fiber-reinforced composite scaffold designed to mimic the anisotropic, nonlinear, and viscoelastic biomechanical characteristics of native cartilage as the basis for developing functional tissue-engineered constructs. Three-dimensionally woven poly(epsilon-caprolactone) (PCL) scaffolds were encapsulated with a fibrin hydrogel, seeded with human adipose-derived stem cells, and cultured for 28 days in chondrogenic culture conditions. Biomechanical testing showed that PCL-based constructs exhibited baseline compressive and shear properties similar to those of native cartilage and maintained these properties throughout the culture period, while supporting the synthesis of a collagen-rich extracellular matrix. Further, constructs displayed an equilibrium coefficient of friction similar to that of native articular cartilage (mu(eq) approximately 0.1-0.3) over the prescribed culture period. Our findings show that three-dimensionally woven PCL-fibrin composite scaffolds can be produced with cartilage-like mechanical properties, and that these engineered properties can be maintained in culture while seeded stem cells regenerate a new, functional tissue construct.

Hand-held spectroscopic device for in vivo and intraoperative tumor detection: contrast enhancement, detection sensitivity, and tissue penetration.

Surgery is one of the most effective and widely used procedures in treating human cancers, but a major problem is that the surgeon often fails to remove the entire tumor, leaving behind tumor-positive margins, metastatic lymph nodes, and/or satellite tumor nodules. Here we report the use of a hand-held spectroscopic pen device (termed SpectroPen) and near-infrared contrast agents for intraoperative detection of malignant tumors, based on wavelength-resolved measurements of fluorescence and surface-enhanced Raman scattering (SERS) signals. The SpectroPen utilizes a near-infrared diode laser (emitting at 785 nm) coupled to a compact head unit for light excitation and collection. This pen-shaped device effectively removes silica Raman peaks from the fiber optics and attenuates the reflected excitation light, allowing sensitive analysis of both fluorescence and Raman signals. Its overall performance has been evaluated by using a fluorescent contrast agent (indocyanine green, or ICG) as well as a surface-enhanced Raman scattering (SERS) contrast agent (pegylated colloidal gold). Under in vitro conditions, the detection limits are approximately 2-5 × 10(-11) M for the indocyanine dye and 0.5-1 × 10(-13) M for the SERS contrast agent. Ex vivo tissue penetration data show attenuated but resolvable fluorescence and Raman signals when the contrast agents are buried 5-10 mm deep in fresh animal tissues. In vivo studies using mice bearing bioluminescent 4T1 breast tumors further demonstrate that the tumor borders can be precisely detected preoperatively and intraoperatively, and that the contrast signals are strongly correlated with tumor bioluminescence. After surgery, the SpectroPen device permits further evaluation of both positive and negative tumor margins around the surgical cavity, raising new possibilities for real-time tumor detection and image-guided surgery.

Use of an insulating mask for controlling anisotropy in multilayer electrospun scaffolds for tissue engineering.

Tissue engineering of various musculoskeletal or cardiovascular tissues requires scaffolds with controllable mechanical anisotropy. However, native tissues also exhibit significant inhomogeneity in their mechanical properties, and the principal axes of anisotropy may vary with site or depth from the tissue surface. Thus, techniques to produce multilayered biomaterial scaffolds with controllable anisotropy may provide improved biomimetic properties for functional tissue replacements. In this study, poly(ε-caprolactone) scaffolds were electrospun onto a collecting electrode that was partially covered by rectangular or square shaped insulating masks. The use of a rectangular mask resulted in aligned scaffolds that were significantly stiffer in tension in the axial direction than the transverse direction at 0 strain (22.9 ± 1.3 MPa axial, 16.1 ± 0.9 MPa transverse), and at 0.1 strain (4.8 ± 0.3 MPa axial, 3.5 ± 0.2 MPa transverse). The unaligned scaffolds, produced using a square mask, did not show this anisotropy, with similar stiffness in the axial and transverse directions at 0 strain (19.7 ± 1.4 MPa axial, 20.8 ± 1.3 MPa transverse) and 0.1 strain (4.4 ± 0.2 MPa axial, 4.6 ± 0.3 MPa, transverse). Aligned scaffolds also induced alignment of adipose stem cells near the expected axis on aligned scaffolds (0.015 ± 0.056 rad), while on the unaligned scaffolds, their orientation showed more variation and was not along the expected axis (1.005 ± 0.225 rad). This method provides a novel means of creating multilayered electrospun scaffolds with controlled anisotropy for each layer, potentially providing a means to mimic the complex mechanical properties of various native tissues.

Animal models of soft-tissue sarcoma.

Soft-tissue sarcomas (STSs) are rare mesenchymal tumors that arise from muscle, fat and connective tissue. Currently, over 75 subtypes of STS are recognized. The rarity and heterogeneity of patient samples complicate clinical investigations into sarcoma biology. Model organisms might provide traction to our understanding and treatment of the disease. Over the past 10 years, many successful animal models of STS have been developed, primarily genetically engineered mice and zebrafish. These models are useful for studying the relevant oncogenes, signaling pathways and other cell changes involved in generating STSs. Recently, these model systems have become preclinical platforms in which to evaluate new drugs and treatment regimens. Thus, animal models are useful surrogates for understanding STS disease susceptibility and pathogenesis as well as for testing potential therapeutic strategies.

Noninvasive monitoring of tissue hemoglobin using UV-VIS diffuse reflectance spectroscopy: a pilot study.

We conducted a pilot study on 10 patients undergoing general surgery to test the feasibility of diffuse reflectance spectroscopy in the visible wavelength range as a noninvasive monitoring tool for blood loss during surgery. Ratios of raw diffuse reflectance at wavelength pairs were tested as a first-pass for estimating hemoglobin concentration. Ratios can be calculated easily and rapidly with limited post-processing, and so this can be considered a near real-time monitoring device. We found the best hemoglobin correlations were when ratios at isosbestic points of oxy- and deoxyhemoglobin were used, specifically 529/500 nm. Baseline subtraction improved correlations, specifically at 520/509 nm. These results demonstrate proof-of-concept for the ability of this noninvasive device to monitor hemoglobin concentration changes due to surgical blood loss. The 529/500 nm ratio also appears to account for variations in probe pressure, as determined from measurements on two volunteers.

Magnetic resonance water proton relaxation in protein solutions and tissue: T(1rho) dispersion characterization.

BACKGROUND: Image contrast in clinical MRI is often determined by differences in tissue water proton relaxation behavior. However, many aspects of water proton relaxation in complex biological media, such as protein solutions and tissue are not well understood, perhaps due to the limited empirical data. PRINCIPAL FINDINGS: Water proton T(1), T(2), and T(1rho) of protein solutions and tissue were measured systematically under multiple conditions. Crosslinking or aggregation of protein decreased T(2) and T(1rho), but did not change high-field T(1). T(1rho) dispersion profiles were similar for crosslinked protein solutions, myocardial tissue, and cartilage, and exhibited power law behavior with T(1rho)(0) values that closely approximated T(2). The T(1rho) dispersion of mobile protein solutions was flat above 5 kHz, but showed a steep curve below 5 kHz that was sensitive to changes in pH. The T(1rho) dispersion of crosslinked BSA and cartilage in DMSO solvent closely resembled that of water solvent above 5 kHz but showed decreased dispersion below 5 kHz. CONCLUSIONS: Proton exchange is a minor pathway for tissue T(1) and T(1rho) relaxation above 5 kHz. Potential models for relaxation are discussed, however the same molecular mechanism appears to be responsible across 5 decades of frequencies from T(1rho) to T(1).

Utility of telomerase-pot1 fusion protein in vascular tissue engineering.

While advances in regenerative medicine and vascular tissue engineering have been substantial in recent years, important stumbling blocks remain. In particular, the limited life span of differentiated cells that are harvested from elderly human donors is an important limitation in many areas of regenerative medicine. Recently, a mutant of the human telomerase reverse transcriptase enzyme (TERT) was described, which is highly processive and elongates telomeres more rapidly than conventional telomerase. This mutant, called pot1-TERT, is a chimeric fusion between the DNA binding protein pot1 and TERT. Because pot1-TERT is highly processive, it is possible that transient delivery of this transgene to cells that are utilized in regenerative medicine applications may elongate telomeres and extend cellular life span while avoiding risks that are associated with retroviral or lentiviral vectors. In the present study, adenoviral delivery of pot1-TERT resulted in transient reconstitution of telomerase activity in human smooth muscle cells, as demonstrated by telomeric repeat amplification protocol (TRAP). In addition, human engineered vessels that were cultured using pot1-TERT-expressing cells had greater collagen content and somewhat better performance in vivo than control grafts. Hence, transient delivery of pot1-TERT to elderly human cells may be useful for increasing cellular life span and improving the functional characteristics of resultant tissue-engineered constructs.

Portable, Fiber-Based, Diffuse Reflection Spectroscopy (DRS) Systems for Estimating Tissue Optical Properties.

Steady-state diffuse reflection spectroscopy is a well-studied optical technique that can provide a noninvasive and quantitative method for characterizing the absorption and scattering properties of biological tissues. Here, we compare three fiber-based diffuse reflection spectroscopy systems that were assembled to create a light-weight, portable, and robust optical spectrometer that could be easily translated for repeated and reliable use in mobile settings. The three systems were built using a broadband light source and a compact, commercially available spectrograph. We tested two different light sources and two spectrographs (manufactured by two different vendors). The assembled systems were characterized by their signal-to-noise ratios, the source-intensity drifts, and detector linearity. We quantified the performance of these instruments in extracting optical properties from diffuse reflectance spectra in tissue-mimicking liquid phantoms with well-controlled optical absorption and scattering coefficients. We show that all assembled systems were able to extract the optical absorption and scattering properties with errors less than 10%, while providing greater than ten-fold decrease in footprint and cost (relative to a previously well-characterized and widely used commercial system). Finally, we demonstrate the use of these small systems to measure optical biomarkers in vivo in a small-animal model cancer therapy study. We show that optical measurements from the simple portable system provide estimates of tumor oxygen saturation similar to those detected using the commercial system in murine tumor models of head and neck cancer.

Diet-induced obesity alters the differentiation potential of stem cells isolated from bone marrow, adipose tissue and infrapatellar fat pad: the effects of free fatty acids.

INTRODUCTION: Obesity is a major risk factor for several musculoskeletal conditions that are characterized by an imbalance of tissue remodeling. Adult stem cells are closely associated with the remodeling and potential repair of several mesodermally derived tissues such as fat, bone and cartilage. We hypothesized that obesity would alter the frequency, proliferation, multipotency and immunophenotype of adult stem cells from a variety of tissues. MATERIALS AND METHODS: Bone marrow-derived mesenchymal stem cells (MSCs), subcutaneous adipose-derived stem cells (sqASCs) and infrapatellar fat pad-derived stem cells (IFP cells) were isolated from lean and high-fat diet-induced obese mice, and their cellular properties were examined. To test the hypothesis that changes in stem cell properties were due to the increased systemic levels of free fatty acids (FFAs), we further investigated the effects of FFAs on lean stem cells in vitro. RESULTS: Obese mice showed a trend toward increased prevalence of MSCs and sqASCs in the stromal tissues. While no significant differences in cell proliferation were observed in vitro, the differentiation potential of all types of stem cells was altered by obesity. MSCs from obese mice demonstrated decreased adipogenic, osteogenic and chondrogenic potential. Obese sqASCs and IFP cells showed increased adipogenic and osteogenic differentiation, but decreased chondrogenic ability. Obese MSCs also showed decreased CD105 and increased platelet-derived growth factor receptor α expression, consistent with decreased chondrogenic potential. FFA treatment of lean stem cells significantly altered their multipotency but did not completely recapitulate the properties of obese stem cells. CONCLUSIONS: These findings support the hypothesis that obesity alters the properties of adult stem cells in a manner that depends on the cell source. These effects may be regulated in part by increased levels of FFAs, but may involve other obesity-associated cytokines. These findings contribute to our understanding of mesenchymal tissue remodeling with obesity, as well as the development of autologous stem cell therapies for obese patients.

Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.

The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS) cells, which once differentiated allow for the enrichment of Nkx2-5(+) cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+) cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological screening and drug development studies.

Radioactive in situ hybridization for detecting diverse gene expression patterns in tissue.

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)(1) that has been working successfully in our lab for many years, especially for adult vertebrate brains(2-5). The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts(6,7). Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches(8,9), in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.