Process engineering of natural killer cell-based immunotherapy.

Cell therapy offers the potential for curative treatment of cancers. Although T cells have been the predominantly used cell type, natural killer (NK) cells have attracted great attention owing to their ability to kill cancer cells and because they are naturally suitable for allogeneic applications. Upon stimulation by cytokines or activation by a target cell, NK cells proliferate and expand their population. These cytotoxic NK cells can be cryopreserved and used as an off-the-shelf medicine. The production process for NK cells thus differs from that of autologous cell therapies. We briefly outline key biological features of NK cells, review the manufacturing technologies for protein biologics, and discuss their adaptation for developing robust NK cell biomanufacturing processes.

Differential effects of macrophage subtypes on SARS-CoV-2 infection in a human pluripotent stem cell-derived model.

Dysfunctional immune responses contribute critically to the progression of Coronavirus Disease-2019 (COVID-19), with macrophages as one of the main cell types involved. It is urgent to understand the interactions among permissive cells, macrophages, and the SARS-CoV-2 virus, thereby offering important insights into effective therapeutic strategies. Here, we establish a lung and macrophage co-culture system derived from human pluripotent stem cells (hPSCs), modeling the host-pathogen interaction in SARS-CoV-2 infection. We find that both classically polarized macrophages (M1) and alternatively polarized macrophages (M2) have inhibitory effects on SARS-CoV-2 infection. However, M1 and non-activated (M0) macrophages, but not M2 macrophages, significantly up-regulate inflammatory factors upon viral infection. Moreover, M1 macrophages suppress the growth and enhance apoptosis of lung cells. Inhibition of viral entry using an ACE2 blocking antibody substantially enhances the activity of M2 macrophages. Our studies indicate differential immune response patterns in distinct macrophage phenotypes, which could lead to a range of COVID-19 disease severity.

Evaluating the Role of IL-1ß in Transmigration of Triple Negative Breast Cancer Cells Across the Brain Endothelium.

INTRODUCTION: In vivo, breast cancer cells spend on average 3-7 days adhered to the endothelial cells inside the vascular lumen before entering the brain. IL-1ß is one of the highly upregulated molecules in brain-seeking triple negative breast cancer (TNBC) cells. In this study, the effect of IL-1ß on the blood-brain barrier (BBB) and astrocytes and its role in transmigration of TNBC cells were evaluated. METHODS: The effect of IL-1ß on transendothelial electrical resistance, gene and protein expression of human induced pluripotent stem cell-derived brain-specific microvascular endothelial-like cells (iBMECs) was studied. Transport of IL-1ß across the iBMEC layer was investigated and the effect of IL-1ß treatment of astrocytes on their cytokine and chemokine secretome was evaluated with a cytokine membrane array. Using BBB-on-a-chip devices, transmigration of MDA-MB-231 cells and their brain-seeking variant (231BR) across the iBMECs was studied, and the effect of an IL-1ß neutralizing antibody on TNBC cell transmigration was investigated. RESULTS: We showed that IL-1ß reduces BBB integrity and induces endothelial-to-mesenchymal transition in iBMECs. IL-1ß crosses the iBMEC layer and induces secretion of multiple chemokines by astrocytes, which can enhance TNBC cell transmigration across the BBB. Transmigration assays in a BBB-on-a-chip device showed that 231BR cells have a higher rate of transmigration across the iBMECs compared to MDA-MB-231 cells, and IL-1ß pretreatment of BBB-on-a-chip devices increases the number of transmigrated MDA-MB-231 cells. Finally, we demonstrated that neutralizing IL-1ß reduces the rate of 231BR cell transmigration. CONCLUSION: IL-1ß plays a significant role in transmigration of brain-seeking TNBC cells across the BBB. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00710-y.

The mini player with diverse functions: extracellular vesicles in cell biology, disease, and therapeutics.

Extracellular vesicles (EVs) are tiny biological nanovesicles ranging from approximately 30-1000 nm in diameter that are released into the extracellular matrix of most cell types and in biofluids. The classification of EVs includes exosomes, microvesicles, and apoptotic bodies, dependent on various factors such as size, markers, and biogenesis pathways. The transition of EV relevance from that of being assumed as a trash bag to be a key player in critical physiological and pathological conditions has been revolutionary in many ways. EVs have been recently revealed to play a crucial role in stem cell biology and cancer progression via intercellular communication, contributing to organ development and the progression of cancer. This review focuses on the significant research progress made so far in the role of the crosstalk between EVs and stem cells and their niche, and cellular communication among different germ layers in developmental biology. In addition, it discusses the role of EVs in cancer progression and their application as therapeutic agents or drug delivery vehicles. All such discoveries have been facilitated by tremendous technological advancements in EV-associated research, especially the microfluidics systems. Their pros and cons in the context of characterization of EVs are also extensively discussed in this review. This review also deliberates the role of EVs in normal cell processes and disease conditions, and their application as a diagnostic and therapeutic tool. Finally, we propose future perspectives for EV-related research in stem cell and cancer biology.

Exosomes: Small vesicles with big roles in cancer, vaccine development, and therapeutics.

Cancer is a deadly disease that is globally and consistently one of the leading causes of mortality every year. Despite the availability of chemotherapy, radiotherapy, immunotherapy, and surgery, a cure for cancer has not been attained. Recently, exosomes have gained significant attention due to the therapeutic potential of their various components including proteins, lipids, nucleic acids, miRNAs, and lncRNAs. Exosomes constitute a set of tiny extracellular vesicles with an approximate diameter of 30-100 nm. They are released from different cells and are present in biofluids including blood, cerebrospinal fluid (CSF), and urine. They perform crucial multifaceted functions in the malignant progression of cancer via autocrine, paracrine, and endocrine communications. The ability of exosomes to carry different cargoes including drug and molecular information to recipient cells make them a novel tool for cancer therapeutics. In this review, we discuss the major components of exosomes and their role in cancer progression. We also review important literature about the potential role of exosomes as vaccines and delivery carriers in the context of cancer therapeutics.

Chemically defined human vascular laminins for biologically relevant culture of hiPSC-derived brain microvascular endothelial cells.

BACKGROUND: In recent years, differentiation of human induced pluripotent stem cells (hiPSCs) into brain-specific microvascular endothelial cells (iBMECs) has frequently been used to model the blood-brain barrier (BBB). However, there are limitations in the use of iBMECs for in vitro studies, such as transendothelial electrical resistance (TEER) instability, weak junctional expression of VE-cadherin, and lack of proper fluid shear stress response. In vivo, the basement membrane (BM) composition of the BBB evolves throughout development, and laminins become the dominant component of the mature vascular BM. However, laminin isoforms of the endothelial BM have not been used for culture of differentiated iBMECs. The main goal of this study is to investigate the effect of different laminin isoforms of the endothelial BM on iBMEC functionality and to determine whether better recapitulation of the physiological BM in vitro can address the aforementioned limitations of iBMECs. METHODS: Using a previously reported method, hiPSCs were differentiated into iBMECs. The influence of main laminins of the endothelial BM, LN 411 and LN 511, on iBMEC functionality was studied and compared to a collagen IV and fibronectin mixture (CN IV-FN). Quantitative RT-PCR, immunocytochemistry, and TEER measurement were utilized to assess gene and protein expression and barrier properties of iBMECs on different extracellular matrices. Single-channel microfluidic devices were used to study the effect of shear stress on iBMECs. RESULTS: LN 511, but not LN 411, improved iBMEC barrier properties and resulted in more sustained TEER stability. Immunocytochemistry showed improved junctional protein expression compared to iBMECs cultured on CN IV-FN. iBMECs cultured on LN 511 showed a reduction of stress fibers, indicating resting endothelial phenotype, whereas gene expression analysis revealed upregulation of multiple genes involved in endothelial activation in iBMECs on CN IV-FN. Finally, culturing iBMECs on LN 511 enhanced physiological responses to shear stress, including morphological changes and enhanced junctional protein association. CONCLUSION: LN 511 improves the functionality and long-term barrier stability of iBMECs. Our findings suggest that incorporation of physiologically relevant LN 511 in iBMEC culture would be beneficial for disease modeling applications and BBB-on-a-chip platforms that accommodate fluid flow.

An isogenic hiPSC-derived BBB-on-a-chip.

The blood-brain barrier (BBB) is composed of brain microvascular endothelial cells (BMECs) that regulate brain homeostasis, and astrocytes within the brain are involved in the maintenance of the BBB or modulation of its integrity in disease states via secreted factors. A major challenge in modeling the normal or diseased BBB is that conventional in vitro models lack either the physiological complexity of the BBB or key functional features such as formation of a sufficiently tight barrier. In this study, we utilized human induced pluripotent stem cell (hiPSC)-derived BMECs in a BBB-on-a-chip device that supports flow and coculture with an astrocyte-laden 3D hydrogel. The BMECs are separated from the hydrogel by a porous membrane with either 0.4 or 8.0 µm pore size, making the device suitable for studying the transport of molecules or cells, respectively, across the BBB. In addition, all cells seeded in the device are differentiated from the same hiPSC line, which could enable genetic and rare disease modeling. Formation of a confluent BMEC barrier was confirmed by immunocytochemistry of tight junction proteins and measurement of fluorescein permeability. Integrity of the barrier was further assessed by performing impedance spectroscopy in the device. Finally, the ability of this device to recapitulate a disease model of BBB disruption was demonstrated, with apical addition of TGF-ß1 leading to transendothelial electrical resistance reduction and indicators of astrocyte activation. These results demonstrate the utility of the fabricated device for a broad range of applications such as drug screening and mechanistic studies of BBB disruption.