Explore the vast range of titles available.

The extracellular matrix (ECM) plays a vital role in stem cell differentiation to endothelial cells in vivo and is also important for the specification of endothelial cells in vitro. Individual ECM components have previously been shown to support endothelial differentiation; here, we use a Design of Experiments approach to optimize ECM composition to more effectively drive endothelial differentiation. We found that a combination of Collagen I, Collagen IV, and Laminin 411 could induce endothelial differentiation well beyond that found with Matrigel, the most commonly used differentiation substrate for endothelial cells. We also show that the addition of vascular endothelial growth factor (VEGF) during differentiation improves outcomes and that transforming growth factor beta (TGFβ) inhibits specification. The optimized ECM formulation (EO) was subsequently used to create bioprinted constructs, demonstrating its ability to spatially define endothelial differentiation in 3D environments. Our results build our mechanistic knowledge of the signaling axes that regulate differentiation in response to ECM stimulation with practical implications for the vascularization of engineered tissues.

The plasticity and immunomodulatory capacity of mesenchymal stem cells (MSCs) have spurred clinical use in recent years. However, clinical outcomes vary and many ascribe inconsistency to the tissue source of MSCs. Yet unconsidered is the extent of heterogeneity of individual MSCs from a given tissue source with respect to differentiation potential and immune regulatory function. Here we use single-cell RNA-seq to assess the transcriptional diversity of murine mesenchymal stem cells derived from bone marrow. We found genes associated with MSC multipotency were expressed at a high level and with consistency between individual cells. However, genes associated with osteogenic, chondrogenic, adipogenic, neurogenic and vascular smooth muscle differentiation were expressed at widely varying levels between individual cells. Further, certain genes associated with immunomodulation were also inconsistent between individual cells. Differences could not be ascribed to cycles of proliferation, culture bias or other cellular process, which might alter transcript expression in a regular or cyclic pattern. These results support and extend the concept of lineage priming of MSCs and emphasize caution for in vivo or clinical use of MSCs, even when immunomodulation is the goal, since multiple mesodermal (and even perhaps ectodermal) outcomes are a possibility. Purification might enable shifting of the probability of a certain outcome, but is unlikely to remove multilineage potential altogether.

Differentiation of pluripotent stem cells to cardiomyocytes is influenced by culture conditions including the extracellular matrices or similar synthetic scaffolds on which they are grown. However, the molecular mechanisms that link the scaffold with differentiation outcomes are not fully known. Here, we determined by immunofluorescence staining and mass spectrometry approaches that extracellular matrix (ECM) engagement by mouse pluripotent stem cells activates critical components of canonical wingless/integrated (Wnt) signaling pathways via kinases of the focal adhesion to drive cardiomyogenesis. These kinases were found to be differentially activated depending on type of ECM engaged. These outcomes begin to explain how varied ECM composition of in vivo tissues with development and in vitro model systems gives rise to different mature cell types, having broad practical applicability for the design of engineered tissues.

Multivalency can drive high-avidity binding of ligand-functionalized nanoparticles to cells with high target receptor expression, but it can also contribute to off-target binding to low-expression non-target cells. We explored how ligand affinity and liposome valency shape the resulting binding performance index (BPI), defined as the product of the proportion of liposome-bound target cells and that of non-bound non-target cells. Designed ankyrin repeat proteins (DARPins) spanning a wide range of HER2-binding affinities were tethered onto PEGylated liposomes at varying concentrations. BPI was initially evaluated in mixed-cell suspensions of HER2 SKBR3 (target) cells and HER2 T47D (non-target) cells, with the highest BPI (> 0.8) observed for high-valency liposomes displaying high-affinity DARPins. To further map the BPI landscape, we measured particle binding to HEK293T cells transiently transfected with HER2-EGFP, leveraging the inherent transfection heterogeneity to generate continuous binding response curves as a function of HER2 expression. HER2 (target) and HER2 (non-target) populations were defined by a HER2 threshold, which was varied across the range of HER2 expression to determine maximum BPI values (> 0.85) and corresponding HER2 threshold optima (HER2 ). BPI generally tracks with traditional binding selectivity, but BPI is more sensitive to off-target effects or poor on-target binding and thus may better assess particle performance. We further demonstrate that HER2 can be rationally increased or decreased by adjusting DARPin valency and affinity (separately or synergistically) to lower or higher values, respectively. The approach outlined here enables rapid testing and optimization of ligand parameters for nanoparticle binding toward a given therapeutic target.

The extracellular matrix (ECM) impacts stem cell differentiation, but identifying formulations supportive of differentiation is challenging in 3D models. Prior efforts involving combinatorial ECM arrays seemed intuitively advantageous. We propose an alternative that suggests reducing sample size and technological burden can be beneficial and accessible when coupled to design of experiments approaches. We predict optimized ECM formulations could augment differentiation of cardiomyocytes derived in vitro . We employed native chemical ligation to polymerize 3D poly (ethylene glycol) hydrogels under mild conditions while entrapping various combinations of ECM and murine induced pluripotent stem cells. Systematic optimization for cardiomyocyte differentiation yielded a predicted solution of 61%, 24% and 15% of collagen type I, laminin-111 and fibronectin, respectively. This solution was confirmed by increased numbers of cardiac troponin T, α-myosin heavy chain and α-sarcomeric actinin-expressing cells relative to suboptimum solutions. Cardiomyocytes of composites exhibited connexin43 expression, appropriate contractile kinetics and intracellular calcium handling. Further, adding a modulator of adhesion, thrombospondin-1, abrogated cardiomyocyte differentiation. Thus, the integrated biomaterial platform statistically identified an ECM formulation best supportive of cardiomyocyte differentiation. In future, this formulation could be coupled with biochemical stimulation to improve functional maturation of cardiomyocytes derived in vitro or transplanted in vivo .

Background/Objectives: Hydrophobic semi-solid or liquid biodegradable polymers have shown unique advantages as injectable matrices for sustained release of a wide range of drugs. Here we report the design, synthesis, and characterization of a new low-melt liquid copolymer based on poly(ε-caprolactone) (PCL) and establish its utility as a versatile delivery platform. Methods: The copolymer, mPA20, consisting of short PCL blocks connected via acid-labile acetal linkages, was synthesized using a one-pot reaction and its properties were comprehensively characterized. Results: mPA20 is an amorphous, injectable liquid at physiological temperature and can undergo pH-sensitive hydrolytic degradation. mPA20 bearing methacrylate end groups can be photo-crosslinked into solid matrices with tunable mechanical properties. A hydrophobic fluorophore, Nile Red (NR), was solubilized in mPA20 without any solvent. Sustained release of NR into aqueous medium was achieved using mPA20, either as an injectable liquid depot or a photo-crosslinked solid matrix. Further, mPA20 self-emulsified in water to form nanodroplets, which were subsequently photo-crosslinked into nanogels. Both the nanodroplets and nanogels mediated efficient intracellular delivery of NR with no cytotoxicity. Conclusions: mPA20, a new photo-crosslinkable, hydrophobic liquid copolymer with pH-sensitive degradability, is highly adaptable as either an injectable or implantable depot or nanoscale carrier for the controlled release and intracellular delivery of poorly soluble drugs.

Solid organ fabrication is an ultimate goal of Regenerative Medicine. Since the introduction of Tissue Engineering in 1993, functional biomaterials, stem cells, tunable microenvironments, and high-resolution imaging technologies have significantly advanced efforts to regenerate in vitro culture or tissue platforms. Relatively simple flat or tubular organs are already in (pre)clinical trials and a few commercial products are in market. The road to more complex, high demand, solid organs including heart, kidney and lung will require substantive technical advancement. Here, we consider two emerging technologies for solid organ fabrication. One is decellularization of cadaveric organs followed by repopulation with terminally differentiated or progenitor cells. The other is 3D bioprinting to deposit cell-laden bio-inks to attain complex tissue architecture. We reviewed the development and evolution of the two technologies and evaluated relative strengths needed to produce solid organs, with special emphasis on the heart and other tissues of the cardiovascular system.

Background Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have attracted significant interest for use in disease modeling, drug discovery and potential therapeutic applications. However, conventional hiPSC-CM cryopreservation protocols largely use dimethyl sulfoxide (DMSO) as the cryoprotectant (CPA), which is linked with a loss of post-thaw recovery and function for various cell types and is not ideal for therapeutic protocols. Additionally, the effect of freezing parameters such as cooling rate and nucleation temperature on post-thaw recovery of hiPSC-CMs has not been explored. Methods hiPSC-CMs were generated by Wnt pathway inhibition, followed by sodium l-lactate purification. Subsequently, biophysical characterization of the cells was performed. A differential evolution (DE) algorithm was utilized to determine the optimal composition of a mixture of a sugar, sugar alcohol and amino acid to replace DMSO as the CPA. The hiPSC-CMs were subjected to controlled-rate freezing at different cooling rates and nucleation temperatures. The optimum freezing parameters were identified by post-thaw recoveries and the partitioning ratio obtained from low temperature Raman spectroscopy studies. The post-thaw osmotic behavior of hiPSC-CMs was studied by measuring diameter of cells resuspended in the isotonic culture medium over time. Immunocytochemistry and calcium transient studies were performed to evaluate post-thaw function. Results hiPSC-CMs were found to be slightly larger than hiPSCs and exhibited a large osmotically inactive volume. The best-performing DMSO-free solutions enabled post-thaw recoveries over 90%, which was significantly greater than DMSO (69.4 ± 6.4%). A rapid cooling rate of 5 °C/min and a low nucleation temperature of -8 °C was found to be optimal for hiPSC-CMs. hiPSC-CMs displayed anomalous osmotic behavior post-thaw, dropping sharply in volume after resuspension. Post-thaw function was preserved when hiPSC-CMs were frozen with the best-performing DMSO-free CPA or DMSO and the cells displayed similar cardiac markers pre-freeze and post-thaw. Conclusions It was shown that a CPA cocktail of naturally-occurring osmolytes could effectively replace DMSO for preserving hiPSC-CMs while preserving morphology and function. Understanding the anomalous osmotic behavior and managing the excessive dehydration of hiPSC-CMs could be crucial to improve post-thaw outcomes. Effective DMSO-free cryopreservation would accelerate the development of drug discovery and therapeutic applications of hiPSC-CMs.

Background/Objectives: Focused ultrasound has advantages as an external stimulus for drug delivery as it is non-invasive, has high precision and can penetrate deep into tissues. Here, we report a gold-plated alginate (ALG) hydrogel system that retains highly water-soluble small-molecule fluorescein for sharp off/on release after ultrasound exposure. Methods: The ALG is crosslinked into beads with calcium chloride and layered with a polycation to adjust the surface charge for the adsorption of catalytic platinum nanoparticles (Pt NPs). The coated bead is subject to electroless plating, forming a gold shell. Ultrasound is applied to the gold-plated ALG beads and the release of fluorescein with or without ultrasound stimulation is quantified. Results: Polyethylenimine (PEI), not poly-L-lysine (PLL), is able to facilitate Pt NP adsorption. Gold shell thickness is proportional to the duration of electroless plating and can be controlled. Gold-plated ALG beads are impermeable to the fluorescein cargo and have nearly zero leakage. Exposure to focused ultrasound initiated the release of fluorescein with full release achieved after 72 h. Conclusions: The gold-plated ALG hydrogel is a new material platform that can retain highly water-soluble molecules with a sharp off/on release initiated by focused ultrasound.

Cell fusion generates hybrid cells with unique traits. To understand the transcriptional and signaling alterations after fusion, we analyzed a published single-cell RNA-sequencing dataset of fused murine cardiomyocytes (mHL1) and mesenchymal stromal/stem cells (mMSC). Our analysis showed that fused cells exhibit a transcriptional trajectory suggesting a rapid change that stabilizes over time. We observed asymmetric plasticity. Initially, at Day 1, fusion hybrids resembled mMSCs (mesenchymal reprogramming), but by Day 3, their gene expression shifted to resemble mHL1 cells (myogenic reprogramming). Our analysis also identified distinct transcriptional subpopulations, including a subset enriched for tenascin (extracellular matrix remodeling), accompanied by dynamic changes in cell adhesion and intercellular communication. We also saw a significant shift in signaling pathways over time. At Day 1, Wnt and Melanogenesis (regenerative/antioxidant) signaling were downregulated. By Day 3, stress resistance and cellular adaptation pathways became enriched. Gene regulatory network analysis revealed key changes in master regulators; genes associated with chromatin remodeling ( Hmga2 ), circadian rhythm ( Arntl ), and mesenchymal identity ( Prrx1 ) became more active by Day 3. Collectively, our findings demonstrate that cell fusion is a dynamic reprogramming process, where evolving gene regulatory and signaling networks generate novel hybrid cell states, creating cellular diversity.