Assessing the ability of human endothelial cells derived from induced-pluripotent stem cells to form functional microvasculature in vivo.

Forming functional blood vessel networks is a major clinical challenge in the fields of tissue engineering and therapeutic angiogenesis. Cell-based strategies to promote neovascularization have been widely explored, but cell sourcing remains a significant limitation. Induced-pluripotent stem cell-derived endothelial cells (iPSC-ECs) are a promising, potentially autologous, alternative cell source. However, it is unclear whether iPSC-ECs form the same robust microvasculature in vivo documented for other EC sources. In this study, we utilized a well-established in vivo model, in which ECs (iPSC-EC or human umbilical vein endothelial cells [HUVEC]) were coinjected with normal human lung fibroblasts (NHLFs) and a fibrin matrix into the dorsal flank of severe combined immunodeficiency mice to assess their ability to form functional microvasculature. Qualitatively, iPSC-ECs were capable of vessel formation and perfusion and demonstrated similar vessel morphologies to HUVECs. However, quantitatively, iPSC-ECs exhibited a two-fold reduction in vessel density and a three-fold reduction in the number of perfused vessels compared with HUVECs. Further analysis revealed the presence of collagen-IV and α-smooth muscle actin were significantly lower around iPSC-EC/NHLF vasculature than in HUVEC/NHLF implants, suggesting reduced vessel maturity. Collectively, these results demonstrate the need for increased iPSC-EC maturation for clinical translation to be realized.

Interferon-γ induces combined pyroptotic angiopathy and APOL1 expression in human kidney disease.

Elevated interferon (IFN) signaling is associated with kidney diseases including COVID-19, HIV, and apolipoprotein-L1 (APOL1) nephropathy, but whether IFNs directly contribute to nephrotoxicity remains unclear. Using human kidney organoids, primary endothelial cells, and patient samples, we demonstrate that IFN-γ induces pyroptotic angiopathy in combination with APOL1 expression. Single-cell RNA sequencing, immunoblotting, and quantitative fluorescence-based assays reveal that IFN-γ-mediated expression of APOL1 is accompanied by pyroptotic endothelial network degradation in organoids. Pharmacological blockade of IFN-γ signaling inhibits APOL1 expression, prevents upregulation of pyroptosis-associated genes, and rescues vascular networks. Multiomic analyses in patients with COVID-19, proteinuric kidney disease, and collapsing glomerulopathy similarly demonstrate increased IFN signaling and pyroptosis-associated gene expression correlating with accelerated renal disease progression. Our results reveal that IFN-γ signaling simultaneously induces endothelial injury and primes renal cells for pyroptosis, suggesting a combinatorial mechanism for APOL1-mediated collapsing glomerulopathy, which can be targeted therapeutically.

Resonant acoustic rheometry for non-contact characterization of viscoelastic biomaterials.

Resonant Acoustic Rheometry (RAR) is a new, non-contact technique to characterize the mechanical properties of soft and viscoelastic biomaterials, such as hydrogels, that are used to mimic the extracellular matrix in tissue engineering. RAR uses a focused ultrasound pulse to generate a microscale perturbation at the sample surface and tracks the ensuing surface wave using pulse-echo ultrasound. The frequency spectrum of the resonant surface waves is analyzed to extract viscoelastic material properties. In this study, RAR was used to characterize fibrin, gelatin, and agarose hydrogels. Single time point measurements of gelled samples with static mechanical properties showed that RAR provided consistent quantitative data and measured intrinsic material characteristics independent of ultrasound parameters. RAR was also used to longitudinally track dynamic changes in viscoelastic properties over the course of fibrin gelation, revealing distinct phase and material property transitions. Application of RAR was verified using finite element modeling and the results were validated against rotational shear rheometry. Importantly, RAR circumvents some limitations of conventional rheology methods and can be performed in a high-throughput manner using conventional labware. Overall, these studies demonstrate that RAR can be a valuable tool to noninvasively quantify the viscoelastic mechanical properties of soft hydrogel biomaterials.

Cross-validation of SARS-CoV-2 responses in kidney organoids and clinical populations.

Kidneys are critical target organs of COVID-19, but susceptibility and responses to infection remain poorly understood. Here, we combine SARS-CoV-2 variants with genome-edited kidney organoids and clinical data to investigate tropism, mechanism, and therapeutics. SARS-CoV-2 specifically infects organoid proximal tubules among diverse cell types. Infections produce replicating virus, apoptosis, and disrupted cell morphology, features of which are revealed in the context of polycystic kidney disease. Cross-validation of gene expression patterns in organoids reflects proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants alpha, beta, gamma, kappa, and delta exhibit comparable levels of infection in organoids. Infection is ameliorated in ACE2-/- organoids and blocked via treatment with de novo-designed spike binder peptides. Collectively, these studies clarify the impact of kidney infection in COVID-19 as reflected in organoids and clinical populations, enabling assessment of viral fitness and emerging therapies.

Cell-mediated matrix stiffening accompanies capillary morphogenesis in ultra-soft amorphous hydrogels.

There is a critical need for biomaterials that support robust neovascularization for a wide-range of clinical applications. Here we report how cells alter tissue-level mechanical properties during capillary morphogenesis using a model of endothelial-stromal cell co-culture within poly(ethylene glycol) (PEG) based hydrogels. After a week of culture, we observed substantial stiffening in hydrogels with very soft initial properties. Endothelial cells or stromal cells alone, however, failed to induce hydrogel stiffening. This stiffening tightly correlated with degree of vessel formation but not with hydrogel compaction or cellular proliferation. Despite a lack of fibrillar architecture within the PEG hydrogels, cell-generated contractile forces were essential for hydrogel stiffening. Upregulation of alpha smooth muscle actin and collagen-1 was also correlated with enhanced vessel formation and hydrogel stiffening. Blocking cell-mediated hydrogel degradation abolished stiffening, demonstrating that matrix metalloproteinase (MMP)-mediated remodeling is required for stiffening to occur. These results highlight the dynamic reciprocity between cells and their mechanical microenvironment during capillary morphogenesis and provide important insights for the rational design of materials for vasculogenic applications.

Viscoelastic characterization of diabetic and non-diabetic human adipose tissue.

BACKGROUND: Obesity-induced chronic inflammation and fibrosis in adipose tissue contributes to the progression of type 2 diabetes mellitus (DM). While fibrosis is known to induce mechanical stiffening of numerous tissue types, it is unknown whether DM is associated with alterations in adipose tissue mechanical properties. OBJECTIVE: The purpose of this study was to investigate whether DM is associated with differences in bulk viscoelastic properties of adipose tissue from diabetic (DM) and non-diabetic (NDM) obese subjects. METHODS: Bulk shear rheology was performed on visceral (VAT) and subcutaneous (SAT) adipose tissue, collected from obese subjects undergoing elective bariatric surgery. Rheology was also performed on the remaining extracellular matrix (ECM) from decellularized VAT (VAT ECM). Linear mixed models were used to assess whether correlations existed between adipose tissue mechanical properties and DM status, sex, age, and body mass index (BMI). RESULTS: DM was not associated with significant differences in adipose tissue viscoelastic properties for any of the tissue types investigated. Tissue type dependent differences were however detected, with VAT having significantly lower shear storage and loss moduli than SAT and VAT ECM independent of DM status. CONCLUSION: Although DM is typically associated with adipose tissue fibrosis, it is not associated with differences in macroscopic adipose tissue mechanical properties.

Deciphering the relative roles of matrix metalloproteinase- and plasmin-mediated matrix degradation during capillary morphogenesis using engineered hydrogels.

Extracellular matrix (ECM) remodeling is essential for the process of capillary morphogenesis. Here we employed synthetic poly(ethylene glycol) (PEG) hydrogels engineered with proteolytic specificity to either matrix metalloproteinases (MMPs), plasmin, or both to investigate the relative contributions of MMP- and plasmin-mediated ECM remodeling to vessel formation in a 3D-model of capillary self-assembly analogous to vasculogenesis. We first demonstrated a role for both MMP- and plasmin-mediated mechanisms of ECM remodeling in an endothelial-fibroblast co-culture model of vasculogenesis in fibrin hydrogels using inhibitors of MMPs and plasmin. When this co-culture model was employed in engineered PEG hydrogels with selective protease sensitivity, we observed robust capillary morphogenesis only in MMP-sensitive matrices. Fibroblast spreading in plasmin-selective hydrogels confirmed this difference was due to protease preference by endothelial cells, not due to limitations of the matrix itself. In hydrogels engineered with crosslinks that were dually susceptible to MMPs and plasmin, capillary morphogenesis was unchanged. These findings highlight the critical importance of MMP-mediated degradation during vasculogenesis and provide strong evidence to justify the preferential selection of MMP-degradable peptide crosslinkers in synthetic hydrogels used to study vascular morphogenesis and promote vascularization. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2507-2516, 2019.

Controlled delivery of basic fibroblast growth factor (bFGF) using acoustic droplet vaporization stimulates endothelial network formation.

The challenge of translating pro-angiogenic growth factors for therapeutic purposes has stimulated a myriad of biomaterials-based, delivery approaches. Many techniques rely on incorporating a growth factor into a hydrogel. The kinetics of release can be tuned based on the physiochemical properties of the growth factor and scaffold. We have developed an acoustically-responsive scaffold (ARS), whereby release of a growth factor is non-invasively and spatiotemporally controlled in an on-demand manner using focused ultrasound. An ARS consists of a fibrin matrix doped with a growth factor-loaded, sonosensitive emulsion. In this study, we used an ARS to investigate the impact of basic fibroblast growth factor (bFGF) release on endothelial tubule formation. The co-culture model of angiogenic sprouting consisted of endothelial cell-coated microbeads and dispersed fibroblasts. bFGF release correlated with the acoustic pressure applied while sprout length correlated with both the volume of bFGF-loaded emulsion in the ARS and acoustic pressure. Minimal bFGF release and sprouting were observed in the absence of ultrasound exposure. Staggering the release of bFGF via multiple ultrasound exposures did not affect sprouting. Additionally, sprouting did not display a dependence on the distance between each microbead and the ARS. Overall, these results highlight the potential of using ultrasound to control regenerative processes via the controlled delivery of a growth factor. STATEMENT OF SIGNIFICANCE: Due to the ineffectiveness of conventional routes of administration, implantable hydrogels are often used as matrices to deliver growth factors (GFs). Spatial control of release is typically realized using anisotropic constructs while temporal control is obtained by modifying matrix properties and GF-scaffold interactions. In this study, we demonstrate how focused ultrasound can be used to non-invasively and spatiotemporally control release of basic fibroblast growth factor (bFGF), in an on-demand manner, from a composite hydrogel. The acoustically-responsive scaffold (ARS) consists of a bFGF-loaded, monodispersed double emulsion embedded within a fibrin matrix. We demonstrate how controlled release of bFGF can stimulate endothelial network formation. These results may be of interest to groups working on controlled release strategies for GFs, especially in the context of stimulating angiogenesis.

Nonadhesive Alginate Hydrogels Support Growth of Pluripotent Stem Cell-Derived Intestinal Organoids.

Human intestinal organoids (HIOs) represent a powerful system to study human development and are promising candidates for clinical translation as drug-screening tools or engineered tissue. Experimental control and clinical use of HIOs is limited by growth in expensive and poorly defined tumor-cell-derived extracellular matrices, prompting investigation of synthetic ECM-mimetics for HIO culture. Since HIOs possess an inner epithelium and outer mesenchyme, we hypothesized that adhesive cues provided by the matrix may be dispensable for HIO culture. Here, we demonstrate that alginate, a minimally supportive hydrogel with no inherent cell instructive properties, supports HIO growth in vitro and leads to HIO epithelial differentiation that is virtually indistinguishable from Matrigel-grown HIOs. In addition, alginate-grown HIOs mature to a similar degree as Matrigel-grown HIOs when transplanted in vivo, both resembling human fetal intestine. This work demonstrates that purely mechanical support from a simple-to-use and inexpensive hydrogel is sufficient to promote HIO survival and development.

Sprouting angiogenesis induces significant mechanical heterogeneities and ECM stiffening across length scales in fibrin hydrogels.

Matrix stiffness is a well-established instructive cue in two-dimensional cell cultures. Its roles in morphogenesis in 3-dimensional (3D) cultures, and the converse effects of cells on the mechanics of their surrounding microenvironment, have been more elusive given the absence of suitable methods to quantify stiffness on a length-scale relevant for individual cell-extracellular matrix (ECM) interactions. In this study, we applied traditional bulk rheology and laser tweezers-based active microrheology to probe mechanics across length scales during the complex multicellular process of capillary morphogenesis in 3D, and further characterized the relative contributions of neovessels and supportive stromal cells to dynamic changes in stiffness over time. Our data show local ECM stiffness was highly heterogeneous around sprouting capillaries, and the variation progressively increased with time. Both endothelial cells and stromal support cells progressively stiffened the ECM, with the changes in bulk properties dominated by the latter. Interestingly, regions with high micro-stiffness did not necessarily correlate with remodeled regions of high ECM density as shown by confocal reflectance microscopy. Collectively, these findings, especially the large spatiotemporal variations in local stiffness around cells during morphogenesis in soft 3D fibrin gels, underscore that characterizing ECM mechanics across length scales. provides an opportunity to attain a deeper mechanobiological understanding of the microenvironment's roles in cell fate and tissue patterning.

In Situ Transfection by Controlled Release of Lipoplexes Using Acoustic Droplet Vaporization.

Localized delivery of nucleic acids to target sites (e.g., diseased tissue) is critical for safe and efficacious gene therapy. An ultrasound-based technique termed acoustic droplet vaporization (ADV) has been used to spatiotemporally control the release of therapeutic small molecules and proteins contained within sonosensitive emulsions. Here, ADV is used to control the release of lipoplex-containing plasmid DNA encoding an enhanced green fluorescent protein reporter-from a sonosensitive emulsion. Focused ultrasound (3.5 MHz, mechanical index (MI) ≥ 1.5) generates robust release of fluorescein (i.e., surrogate payload) and lipoplex from the emulsion. In situ release of the lipoplex from the emulsion using ADV (MI = 1.5, 30 cycles) yields a 55% release efficiency, resulting in 43% transfection efficiency and 95% viability with C3H/10T1/2 cells. Without exposure to ultrasound, the release and transfection efficiencies are 5% and 7%, respectively, with 99% viability. Lipoplex released by ADV retains its bioactivity while the ADV process does not yield any measureable sonoporative enhancement of transfection. Co-encapsulation of Ficoll PM 400 within the lipoplex-loaded emulsion, and its subsequent release using ADV, yield higher transfection efficiency than the lipoplex alone. The results demonstrate that ADV can have utility in the spatiotemporal control of gene delivery.