Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be differentiated into cardiomyocytes (hESC-CMs and iPSC-CMs, respectively), which hold great promise for cardiac regenerative medicine and disease modeling efforts. However, the most widely employed differentiation protocols require undefined substrates that are derived from xenogeneic (animal) products, contaminating resultant hESC- and iPSC-CM cultures with xenogeneic proteins and limiting their clinical applicability. Additionally, typical hESC- and iPSC-CM protocols produce CMs that are significantly contaminated by non-CMs and that are immature, requiring lengthy maturation procedures. In this review, we will summarize recent studies that have investigated the ability of purified extracellular matrix (ECM) proteins to support hESC- and iPSC-CM differentiation, with a focus on commercially available ECM proteins and coatings to make such protocols widely available to researchers. The most promising of the substrates reviewed here include laminin-521 with laminin-221 together or Synthemax (a synthetic vitronectin-based peptide coating), which both resulted in highly pure CM cultures. Future efforts are needed to determine whether combinations of specific purified ECM proteins or derived peptides could further improve CM maturation and culture times, and significantly improve hESC- and iPSC-CM differentiation protocols.

Purpose: This study aimed to assess the dynamics of tumor-specific uptake, retention, and blood–tumor barrier penetration of our unique IL-13 receptor α-2 × CD3 bispecific T-cell engager (BTE) following systemic administration in mice with intracranial glioblastoma (GBM) xenografts. Experimental Design: In vitro, BTE binding and accumulation were evaluated in glioma neurospheres. In in vivo studies, the BTEs labeled with either iodine-124 using residualizing chemistry or conjugated to Cy5 were used for longitudinal tracking in patient-derived xenograft models of GBM using PET/CT and confocal microscopy. The survival analysis in mice bearing intracranial GBM tumors was conducted to validate the findings from imaging studies. Results: In vitro, the BTE demonstrated target-specific binding and accumulation in IL-13 receptor α-2–expressing glioma spheres. In vivo, PET/CT imaging revealed that the BTE reached the tumor site within 3 hours after injection, achieving up to 4.8% ID/g, with sustained tumor retention for up to 24 hours, significantly higher than background levels in surrounding normal brain tissue. Confocal microscopy confirmed BTE presence in the tumor bed extravascular space with evidence of T cell–mediated BTE transport across the blood–tumor barrier. Despite its short plasma half-life, the BTE remained in the tumor microenvironment for at least 24 hours. Mice bearing GBM6 brain tumors treated with BTE for 3 to 4 days apart via the intravenous route showed a significant survival advantage over the control group. Conclusions: Our findings provide critical insights into the pharmacokinetics of BTE molecules in GBM. They demonstrate effective penetration and prolonged intratumoral retention following a single systemic dose, supporting further exploration of BTE treatment regimens for translation to clinical settings.

Extracellular vesicles (EV) have garnered significant attention in the fields of drug delivery, imaging, and immunotherapy. There is a need in methods to enhance release of EVs from cells. We found that at high labeling concentrations (100µM), indocarbocyanine lipids DiD and DiR that are commonly used for labeling cells, nanoparticles and EVs, promoted shedding of cell membrane tetraspanins with concomitant release of EVs in the medium. To further investigate this phenomenon, we screened a library of lipids and liposomal formulations for the release of membrane marker CD63 from THP-1 cells, and membrane red nanolantern (RNL) from 4T1 cells. We found a strong dependency of the EV release on lipid structure. In general, lipids that had a cyanine headgroup were more efficient than PEGylated phospholipids, neutral and cationic liposomes, with some lipids enhancing the release of CD63 up to 4-fold, and of RNL up to 8-fold, over vehicle treated control. A side-by-side comparison of cyanine lipid derivatives and corresponding precursor lipids confirmed that the cyanine headgroup significantly promoted shedding of RNL. Mutation of an exosome biogenesis regulator UNC13D did not hinder the release. Lipid-released EV could be modified with anti-interleukin 13 receptor alpha 2 antibody and targeted to glioma cells, suggesting potential utility in drug delivery. Furthermore, the impact of extraneously added lipids on cell membrane integrity should be carefully considered in cell labeling and drug delivery applications.