Engineered extracellular matrices facilitate brain organoids from human pluripotent stem cells.

OBJECTIVE: Brain organoids are miniaturized in vitro brain models generated from pluripotent stem cells, which resemble full-sized brain more closely than conventional two-dimensional cell cultures. Although brain organoids mimic the human brain's cell-to-cell network interactions, they generally fail to faithfully recapitulate cell-to-matrix interactions. Here, an engineered framework, called an engineered extracellular matrix (EECM), was developed to provide support and cell-to-matrix interactions to developing brain organoids. METHODS: We generated brain organoids using EECMs comprised of human fibrillar fibronectin supported by a highly porous polymer scaffold. The resultant brain organoids were characterized by immunofluorescence microscopy, transcriptomics, and proteomics of the cerebrospinal fluid (CSF) compartment. RESULTS: The interstitial matrix-mimicking EECM enhanced neurogenesis, glial maturation, and neuronal diversity from human embryonic stem cells versus conventional protein matrix (Matrigel). Additionally, EECMs supported long-term culture, which promoted large-volume organoids containing over 250 µL of CSF. Proteomics analysis of the CSF found it superseded previous brain organoids in protein diversity, as indicated by 280 proteins spanning 500 gene ontology pathways shared with adult CSF. INTERPRETATION: Engineered EECM matrices represent a major advancement in neural engineering as they have the potential to significantly enhance the structural, cellular, and functional diversity that can be achieved in advanced brain models.

Effects of iron modulation on mesenchymal stem cell-induced drug resistance in estrogen receptor-positive breast cancer.

Patients with estrogen receptor-positive (ER+) breast cancer, the most common subtype, remain at risk for lethal metastatic disease years after diagnosis. Recurrence arises partly because tumor cells in bone marrow become resistant to estrogen-targeted therapy. Here, we utilized a co-culture model of bone marrow mesenchymal stem cells (MSCs) and ER+ breast cancer cells to recapitulate interactions of cancer cells in bone marrow niches. ER+ breast cancer cells in direct contact with MSCs acquire cancer stem-like (CSC) phenotypes with increased resistance to standard antiestrogenic drugs. We confirmed that co-culture with MSCs increased labile iron in breast cancer cells, a phenotype associated with CSCs and disease progression. Clinically approved iron chelators and in-house lysosomal iron-targeting compounds restored sensitivity to antiestrogenic therapy. These findings establish iron modulation as a mechanism to reverse MSC-induced drug resistance and suggest iron modulation in combination with estrogen-targeted therapy as a promising, translatable strategy to treat ER+ breast cancer.

SERS and Fluorescence-Active Multimodal Tessellated Scaffolds for Three-Dimensional Bioimaging.

With the ever-increasing use of 3D cell models toward studying bio-nano interactions and offering alternatives to traditional 2D in vitro and in vivo experiments, methods to image biological tissue in real time and with high spatial resolution have become a must. A suitable technique therefore is surface-enhanced Raman scattering (SERS)-based microscopy, which additionally features reduced photocytotoxicity and improved light penetration. However, optimization of imaging and postprocessing parameters is still required. Herein we present a method to monitor cell proliferation over time in 3D, using multifunctional 3D-printed scaffolds composed of biologically inert poly(lactic-co-glycolic acid) (PLGA) as the base material, in which fluorescent labels and SERS-active gold nanoparticles (AuNPs) can be embedded. The combination of imaging techniques allows optimization of SERS imaging parameters for cell monitoring. The scaffolds provide anchoring points for cell adhesion, so that cell growth can be observed in a suspended 3D matrix, with multiple reference points for confocal fluorescence and SERS imaging. By prelabeling cells with SERS-encoded AuNPs and fluorophores, cell proliferation and migration can be simultaneously monitored through confocal Raman and fluorescence microscopy. These scaffolds provide a simple method to follow cell dynamics in 4D, with minimal disturbance to the tissue model.

Mesoporous Silica Nanoparticle-Supported Lipid Bilayers (Protocells) for Active Targeting and Delivery to Individual Leukemia Cells.

Many nanocarrier cancer therapeutics currently under development, as well as those used in the clinical setting, rely upon the enhanced permeability and retention (EPR) effect to passively accumulate in the tumor microenvironment and kill cancer cells. In leukemia, where leukemogenic stem cells and their progeny circulate within the peripheral blood or bone marrow, the EPR effect may not be operative. Thus, for leukemia therapeutics, it is essential to target and bind individual circulating cells. Here, we investigate mesoporous silica nanoparticle (MSN)-supported lipid bilayers (protocells), an emerging class of nanocarriers, and establish the synthesis conditions and lipid bilayer composition needed to achieve highly monodisperse protocells that remain stable in complex media as assessed in vitro by dynamic light scattering and cryo-electron microscopy and ex ovo by direct imaging within a chick chorioallantoic membrane (CAM) model. We show that for vesicle fusion conditions where the lipid surface area exceeds the external surface area of the MSN and the ionic strength exceeds 20 mM, we form monosized protocells (polydispersity index <0.1) on MSN cores with varying size, shape, and pore size, whose conformal zwitterionic supported lipid bilayer confers excellent stability as judged by circulation in the CAM and minimal opsonization in vivo in a mouse model. Having established protocell formulations that are stable colloids, we further modified them with anti-EGFR antibodies as targeting agents and reverified their monodispersity and stability. Then, using intravital imaging in the CAM, we directly observed in real time the progression of selective targeting of individual leukemia cells (using the established REH leukemia cell line transduced with EGFR) and delivery of a model cargo. Overall, we have established the effectiveness of the protocell platform for individual cell targeting and delivery needed for leukemia and other disseminated disease.