Metabolomics Identifies Metabolic Markers of Maturation in Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Cardiovascular disease is a leading cause of death worldwide. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold immense clinical potential and recent studies have enabled generation of virtually pure hPSC-CMs with high efficiency in chemically defined and xeno-free conditions. Despite these advances, hPSC-CMs exhibit an immature phenotype and are arrhythmogenic in vivo, necessitating development of strategies to mature these cells. hPSC-CMs undergo significant metabolic alterations during differentiation and maturation. A detailed analysis of the metabolic changes accompanying maturation of hPSC-CMs may prove useful in identifying new strategies to expedite hPSC-CM maturation and also may provide biomarkers for testing or validating hPSC-CM maturation. In this study we identified global metabolic changes which take place during long-term culture and maturation of hPSC-CMs derived from three different hPSC lines. We have identified several metabolic pathways, including phospholipid metabolism and pantothenate and Coenzyme A metabolism, which showed significant enrichment upon maturation in addition to fatty acid oxidation and metabolism. We also identified increases in glycerophosphocholine and the glycerophosphocholine:phosphocholine ratio as potential metabolic biomarkers of maturation. These biomarkers were also affected in a similar manner during murine heart development in vivo. These results support that hPSC-CM maturation is associated with extensive metabolic changes in metabolic network utilization and understanding the roles of these metabolic changes has the potential to develop novel approaches to monitor and expedite hPSC-CM maturation

Tubing-Electrospinning: A One-Step Process for Fabricating Fibrous Matrices with Spatial, Chemical, and Mechanical Gradients

Guiding newly generated tissues in a gradient pattern, thereby precisely mimicking inherent tissue morphology and subsequently arranging the intimate networks between adjacent tissues, is essential to raise the technical levels of tissue engineering and facilitate its transition into the clinic. In this study, a straightforward electrospinning method (the tubing-electrospinning technique) was developed to create fibrous matrices readily with diverse gradient patterns and to induce patterned cellular responses. Gradient fibrous matrices can be produced simply by installing a series of polymer-containing lengths of tubing into an electrospinning circuit and sequentially processing polymers without a time lag. The loading of polymer samples with different characteristics, including concentration, wettability, and mechanical properties, into the tubing system enabled unique features in fibrous matrices, such as longitudinal gradients in fiber density, surface properties, and mechanical stiffness. The resulting fibrous gradients were shown to arrange cellular migration and residence in a gradient manner, thereby offering efficient cues to mediate patterned tissue formation. The one-step process using tubing-electrospinning apparatus can be used without significant modifications regardless of the type of fibrous gradient. Hence, the tubing-electrospinning system can serve as a platform that can be readily used by a wide-range of users to induce patterned tissue formation in a gradient manner, which will ultimately improve the functionality of tissue engineering scaffolds

Highly Moldable Electrospun Clay-Like Fluffy Nanofibers for Three-Dimensional Scaffolds

The development of three-dimensional polymeric systems capable of mimicking the extracellular matrix is critical for advancing tissue engineering. To achieve these objectives, three-dimensional fibrous scaffolds with “clay”-like properties were successfully developed by coaxially electrospinning polystyrene (PS) and poly­(ε-caprolactone) (PCL) and selective leaching. As PS is known to be nonbiodegradable and vulnerable to mechanical stress, PS layers present at the outer surface were removed using a “selective leaching” process. The fibrous PCL scaffolds that remained after the leaching step exhibited highly advantageous characteristics as a tissue engineering scaffold, including moldability (i.e., clay-like), flexibility, and three-dimensional structure (i.e., cotton-like). More so, the “clay-like” PCL fibrous scaffolds could be shaped into any desired form, and the microenvironment within the clay scaffolds was highly favorable for cell expansion both in vitro and in vivo. These “electrospun-clay” scaffolds overcome the current limitations of conventional electrospun, sheet-like scaffolds, which are structurally inflexible. Therefore, this work extends the scope of electrospun fibrous scaffolds toward a variety of tissue engineering applications

Engineered human pluripotent stem cell-derived natural killer cells with PD-L1 responsive immunological memory for enhanced immunotherapeutic efficacy

Adoptive chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have shown promise in treating various cancers. However, limited immunological memory and access to sufficient numbers of allogenic donor cells have hindered their broader preclinical and clinical applications. Here, we first assess eight different CAR constructs that use an anti-PD-L1 nanobody and/or universal anti-fluorescein (FITC) single-chain variable fragment (scFv) to enhance antigen-specific proliferation and anti-tumor cytotoxicity of NK-92 cells against heterogenous solid tumors. We next genetically engineer human pluripotent stem cells (hPSCs) with optimized CARs and differentiate them into functional dual CAR-NK cells. The tumor microenvironment responsive anti-PD-L1 CAR effectively promoted hPSC-NK cell proliferation and cytotoxicity through antigen-dependent activation of phosphorylated STAT3 (pSTAT3) and pSTAT5 signaling pathways via an intracellular truncated IL-2 receptor β-chain (ΔIL-2Rβ) and STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif. Anti-tumor activities of PD-L1-induced memory-like hPSC-NK cells were further boosted by administering a FITC-folate bi-specific adapter that bridges between a programmable anti-FITC CAR and folate receptor alpha-expressing breast tumor cells. Collectively, our hPSC CAR-NK engineering platform is modular and could constitute a realistic strategy to manufacture off-the-shelf CAR-NK cells with immunological memory-like phenotype for targeted immunotherapy

Sticky “Delivering-From” Strategies Using Viral Vectors for Efficient Human Neural Stem Cell Infection by Bioinspired Catecholamines

Controlled release of biosuprastructures, such as viruses, from surfaces has been a challenging task in providing efficient ex vivo gene delivery. Conventional controlled viral release approaches have demonstrated low viral immobilization and burst release, inhibiting delivery efficiency. Here, a highly powerful substrate-mediated viral delivery system was designed by combining two key components that have demonstrated great potential in the fields of gene therapy and surface chemistry, respectively: adeno-associated viral (AAV) vectors and adhesive catecholamine surfaces. The introduction of a nanoscale thin coating of catecholamines, poly­(norepinephrine) (pNE) or poly­(dopamine) (pDA) to provide AAV adhesion followed by human neural stem cell (hNSC) culture on sticky solid surfaces exhibited unprecedented results: approximately 90% loading vs 25% (AAV_bare surface), no burst release, sustained release at constant rates, approximately 70% infection vs 20% (AAV_bare surface), and rapid internalization. Importantly, the sticky catecholamine-mediated AAV delivery system successfully induced a physiological response from hNSCs, cellular proliferation by a single-shot of AAV encoding fibroblast growth factor-2 (FGF-2), which is typically achieved by multiple treatments with expensive FGF-2 proteins. By combining the adhesive material-independent surface functionalization characters of pNE and pDA, this new sticky “delivering-from” gene delivery platform will make a significant contribution to numerous fields, including tissue engineering, gene therapy, and stem cell therapy

Car-neutrophil Mediated Delivery of Tumor-microenvironment Responsive Nanodrugs for Glioblastoma Chemo-immunotherapy

Glioblastoma (GBM) is one of the most aggressive and lethal solid tumors in human. While efficacious therapeutics, such as emerging chimeric antigen receptor (CAR)-T cells and chemotherapeutics, have been developed to treat various cancers, their effectiveness in GBM treatment has been hindered largely by the blood-brain barrier and blood-brain-tumor barriers. Human neutrophils effectively cross physiological barriers and display effector immunity against pathogens but the short lifespan and resistance to genome editing of primary neutrophils have limited their broad application in immunotherapy. Here we genetically engineer human pluripotent stem cells with CRISPR/Cas9-mediated gene knock-in to express various anti-GBM CAR constructs with T-specific CD3ζ or neutrophil-specific γ-signaling domains. CAR-neutrophils with the best anti-tumor activity are produced to specifically and noninvasively deliver and release tumor microenvironment-responsive nanodrugs to target GBM without the need to induce additional inflammation at the tumor sites. This combinatory chemo-immunotherapy exhibits superior and specific anti-GBM activities, reduces off-target drug delivery and prolongs lifespan in female tumor-bearing mice. Together, this biomimetic CAR-neutrophil drug delivery system is a safe, potent and versatile platform for treating GBM and possibly other devastating diseases