Reversibly-bonded microfluidic devices for stable cell culture and rapid, gentle cell extraction.

Microfluidic chips have emerged as significant tools in cell culture due to their capacity for supporting cells to adopt more physiologically relevant morphologies in 3D compared with traditional cell culture in 2D. Currently, irreversible bonding methods, where chips cannot be detached from their substrates without destroying the structure, are commonly used in fabrication, making it challenging to conduct further analysis on cells that have been cultured on-chip. Although some reversible bonding techniques have been developed, they are either restricted to certain materials such as glass, or require complex processing procedures. Here, we demonstrate a simple and reversible polydimethylsiloxane (PDMS)-polystyrene (PS) bonding technique that allows devices to withstand extended operations while pressurized, and supports long-term stable cell cultures. More importantly, it allows rapid and gentle live cell extraction for downstream manipulation and characterization after long-term on-chip culturing, and even further subculturing. Our new approach could greatly facilitate microfluidic chip-based cell and tissue cultures, overcoming current analytical limitations and opening up new avenues for downstream uses of on-chip cultures, including 3D-engineered tissue structures for biomedical applications.

SARS-CoV-2 infection activates inflammatory macrophages in vascular immune organoids.

SARS-CoV-2 provokes devastating tissue damage by cytokine release syndrome and leads to multi-organ failure. Modeling the process of immune cell activation and subsequent tissue damage is a significant task. Organoids from human tissues advanced our understanding of SARS-CoV-2 infection mechanisms though, they are missing crucial components: immune cells and endothelial cells. This study aims to generate organoids with these components. We established vascular immune organoids from human pluripotent stem cells and examined the effect of SARS-CoV-2 infection. We demonstrated that infections activated inflammatory macrophages. Notably, the upregulation of interferon signaling supports macrophages' role in cytokine release syndrome. We propose vascular immune organoids are a useful platform to model and discover factors that ameliorate SARS-CoV-2-mediated cytokine release syndrome.

Organoids in COVID-19: can we break the glass ceiling?

COVID-19 emerged in September 2020 as a disease caused by the virus SARS-CoV-2. The disease presented as pneumonia at first but later was shown to cause multisystem infections and long-term complications. Many efforts have been put into discovering the exact pathogenesis of the disease. In this review, we aim to discuss an emerging tool in disease modeling, organoids, in the investigation of COVID-19. This review will introduce some methods and breakthroughs achieved by organoids and the limitations of this system.

Unveiling the immune system aging in single-cell resolution.

This commentary investigates the findings presented in the article by Yang et al. in 2023, published in the Journal of Leukocyte Biology. This commentary first summarizes the spatial-temporal dynamics of regulatory T cells derived from mice (Tabula Muris Senis) of different ages (3, 18, and 24 mo) at different anatomic niches like lymph nodes and bone marrow. We also reported possible combinations of receptor-ligand interactions among T follicular regulatory cells, T follicular helper cells, and germinal center B cells, such as the calmodulin/Fas axis and PSGL-1/L-selectin axis. Then, we have elaborated on the significance of understanding aging regulatory T cells and offered some possible future research directions for Yang et al., contributing to a critical analysis of their recent study. Building on these foundations, further investigations and studies can be conducted to delve deeper into the mechanisms by which regulatory T cells influence health upon aging, potentially unveiling novel therapeutic targets to ameliorate age-related pathogenicity.

PD-L1 regulates inflammatory programs of macrophages from human pluripotent stem cells.

Programmed death ligand 1 (PD-L1) serves as a pivotal immune checkpoint in both the innate and adaptive immune systems. PD-L1 is expressed in macrophages in response to IFNγ. We examined whether PD-L1 might regulate macrophage development. We established PD-L1 KO (CD274 -/- ) human pluripotent stem cells and differentiated them into macrophages and observed a 60% reduction in CD11B+CD45+ macrophages in CD274 -/- ; this was orthogonally verified, with the PD-L1 inhibitor BMS-1166 reducing macrophages to the same fold. Single-cell RNA sequencing further confirmed the down-regulation of the macrophage-defining transcription factors SPI1 and MAFB Furthermore, CD274 -/- macrophages reduced the level of inflammatory signals such as NF-κB and TNF, and chemokine secretion of the CXCL and CCL families. Anti-inflammatory TGF-ß was up-regulated. Finally, we identified that CD274 -/- macrophages significantly down-regulated interferon-stimulated genes despite the presence of IFNγ in the differentiation media. These data suggest that PD-L1 regulates inflammatory programs of macrophages from human pluripotent stem cells.

Integrating spatial and single-cell transcriptomics data using deep generative models with SpatialScope.

The rapid emergence of spatial transcriptomics (ST) technologies is revolutionizing our understanding of tissue spatial architecture and biology. Although current ST methods, whether based on next-generation sequencing (seq-based approaches) or fluorescence in situ hybridization (image-based approaches), offer valuable insights, they face limitations either in cellular resolution or transcriptome-wide profiling. To address these limitations, we present SpatialScope, a unified approach integrating scRNA-seq reference data and ST data using deep generative models. With innovation in model and algorithm designs, SpatialScope not only enhances seq-based ST data to achieve single-cell resolution, but also accurately infers transcriptome-wide expression levels for image-based ST data. We demonstrate SpatialScope's utility through simulation studies and real data analysis from both seq-based and image-based ST approaches. SpatialScope provides spatial characterization of tissue structures at transcriptome-wide single-cell resolution, facilitating downstream analysis, including detecting cellular communication through ligand-receptor interactions, localizing cellular subtypes, and identifying spatially differentially expressed genes.

Immune-epigenetic crosstalk in haematological malignancies.

Haematological malignancies comprise a diverse set of lymphoid and myeloid neoplasms which can arise during any stage of haematopoiesis in the bone marrow. Accumulating evidence suggests that chronic inflammation generated by inflammatory cytokines secreted by tumour and the tumour-associated cells within the bone marrow microenvironment initiates signalling pathways in malignant cells, resulting in activation of master transcription factors including Smads, STAT3, and NF-κB which confer cancer stem cell phenotypes and drive disease progression. Deciphering the molecular mechanisms for how immune cells interact with malignant cells to induce such epigenetic modifications, specifically DNA methylation, histone modification, expression of miRNAs and lnRNAs to perturbate haematopoiesis could provide new avenues for developing novel targeted therapies for haematological malignancies. Here, the complex positive and negative feedback loops involved in inflammatory cytokine-induced cancer stem cell generation and drug resistance are reviewed to highlight the clinical importance of immune-epigenetic crosstalk in haematological malignancies.

ZEB2 and MEIS1 independently contribute to hematopoiesis via early hematopoietic enhancer activation.

Cell differentiation is achieved by acquiring a cell type-specific transcriptional program and epigenetic landscape. While the cell type-specific patterning of enhancers has been shown to precede cell fate decisions, it remains unclear how regulators of these enhancers are induced to initiate cell specification and how they appropriately restrict cells that differentiate. Here, using embryonic stem cell-derived hematopoietic cell differentiation cultures, we show the activation of some hematopoietic enhancers during arterialization of hemogenic endothelium, a prerequisite for hematopoiesis. We further reveal that ZEB2, a factor involved in the transcriptional regulation of arterial endothelial cells, and a hematopoietic regulator MEIS1 are independently required for activating these enhancers. Concomitantly, ZEB2 or MEIS1 deficiency impaired hematopoietic cell development. These results suggest that multiple regulators expressed from an earlier developmental stage non-redundantly contribute to the establishment of hematopoietic enhancer landscape, thereby restricting cell differentiation despite the unrestricted expression of these regulators to hematopoietic cells.

IPSC-derived CAR-NK cells for cancer immunotherapy.

Adoptive cell therapies (ACT) based on chimeric antigen receptor (CAR)-modified immune cells have made great progress with six CAR-T cell products approved by the U.S. FDA for hematological malignancies. Compared with CAR-T cells, CAR-NK cells have attracted increasing attention owing to their multiple killing mechanisms, higher safety profile, and broad sources. Induced pluripotent stem cell (iPSC)-derived NK (iPSC-NK) cells possess a mature phenotype and potent cytolytic activity, and can provide a homogeneous population of CAR-NK cells that can be expanded to clinical scale. Thus, iPSC-derived CAR-NK (CAR-iNK) cells could be used as a standardized and "off-the-shelf" product for cancer immunotherapy. In this review, we summarize the current status of the manufacturing techniques, genetic modification strategies, preclinical and clinical evidence of CAR-iNK cells, and discuss the challenges and future prospects of CAR-iNK cell therapy as a novel cellular immunotherapy in cancer.

Ex vivo expansion of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are multipotent progenitor cells that can differentiate into various mature blood cells and immune cells, thus reconstituting hematopoiesis. By taking advantage of the tremendous potential of HSCs, varied hereditary and hematologic diseases are promised to be alleviated or cured. To solve the contradiction between the growing demand for HSCs in disease treatment and the low population of HSCs in both cord blood and bone marrow, ex vivo HSC expansion along with multiple protocols has been investigated for harvesting adequate HSCs over the past two decades. This review surveys the state-of-the-art techniques for ex vivo HSC self-renewal and provides a concise summary of the effects of diverse intrinsic and extrinsic factors on the expansion of HSCs. The remaining challenges and emerging opportunities in the field of HSC expansion are also presented.

T, NK, then macrophages: Recent advances and challenges in adaptive immunotherapy from human pluripotent stem cells.

Adaptive cellular immunotherapy, especially chimeric antigen receptor-T (CAR-T) cell therapy, has advanced the treatment of hematological malignancy. However, major limitations still remain in the source of cells comes from the patients themselves. The use of human pluripotent stem cells to differentiate into immune cells, such as T cells, NK cells, and macrophages, then arm with chimeric antigen receptor (CAR) to enhance tumor killing has gained major attention. It is expected to solve the low number of immune cells recovery from patients, long waiting periods, and ethical issues(reprogramming somatic cells to produce induced pluripotent stem cells (iPS cells) avoids the ethical issues unique to embryonic stem cells (Lo and Parham, 2009). However, there are still major challenges to be further solved. This review summarizes the progress, challenges, and future direction in human pluripotent stem cell-based immunotherapy.

Generation of Natural Killer Cells from Human Expanded Potential Stem Cells.

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular products for immunotherapy. Described here is a two-phase protocol that uses a serum-free commercial medium and a cocktail of cytokines (interleukin [IL]-3, IL-7, IL-15, stem cell factor [SCF], and FMS-like tyrosine kinase 3 ligand [Ftl3L]) to differentiate human expanded potential stem cells (hEPSCs) into cells that possess NK cell properties in vitro with both 3-dimensional (3D) and 2-dimensional (2D) culture technology. Following this protocol, CD3-CD56+ or CD45+CD56+ NK cells are consistently generated. When cocultured with tumor targets for 3 h, the differentiated products display mild cytotoxicity as compared to an IL-2-independent permanent cell line, NK92mi cells. The protocol preserves the complexity of the differentiation microenvironment by the generation of 3D structures, thus facilitating the study of the spatial relationships between immune cells and their niches. Meanwhile, the 2D culture system enables the routine phenotypical validation of cell differentiation without harming the delicate differentiation niche.

Give Them Vasculature and Immune Cells: How to Fill the Gap of Organoids.

Valid and relevant models are critical for research to have biological relevance or to proceed in the right path. As well-established two-dimensional cell cultures lack niches and cues and rodent models differ in species, three-dimensional organoids emerged as a powerful platform for research. Cultured in vitro from stem cells, organoids are heterogeneous in cells and closely resemble the in vivo settings. Organoids also recapitulate the unique human features if cultured from a human source and are subjected to genetic modification. However, one type of organoid possesses only a limited selection of cells. In particular, the absence of vasculature and immune cells restricts the organoids from nutrition, cues, or critical interactions, undermining the validity of organoids as physiological or pathological models. To fill the current gap, there is an urgent need to provide organoids with vasculature and immune cells. In this paper, we review the methods to generate physiological and pathological organoid models and summarize ways to vascularize or immunize them. Our discussion continues with some advantages and disadvantages of each method and some emerging solutions to current problems.

The prospect of genetically engineering natural killer cells for cancer immunotherapy.

The use of natural killer (NK) cells in cancer immunotherapy demonstrates promising potential, yet its efficacy is often limited due to the loss of tumor-killing capacity and lack of specificity in vivo. Here, we review current approaches to confer enhanced tumor-killing capacity and specificity by genetic engineering. Increasing sensitivity to cytokines and protecting NK cells from the immune checkpoint endowed sustainability of NK cells in the tumor microenvironment. Transducing chimeric antigen receptor (CAR) in NK cells successfully targeted both hematologic and solid tumors in preclinical models. The use of human pluripotent stem cells as an expandable and genetically amenable platform offers a stable source of engineered NK cells for cancer immunotherapy. We highlight that CAR-NK cells from human pluripotent stem cells are a promising approach for cancer immunotherapy.

Combating challenges in CAR-T cells with engineering immunology.

Chimeric antigen receptors (CAR) T cells (CAR-T) mark a significant step towards producing safe and effective personal anticancer treatments. CAR-T strategies engineers the T cells from the patients to allow specific binding to a tumour-specific antigen. CAR-Ts are a second-wave offensive strategy to clear out remaining chemotherapy-resistant tumour cells. Though showing practical antitumor abilities in multiple haematological malignancies and solid tumour cancers, the issues of antigen escape, tumour infiltration/penetration, and toxicity side effects limit the usage of prolonged CAR-T therapies. However, engineering immunology has exploited human stem cell-based CAR-T therapies and the development of CAR-M (macrophage) therapies to combat the disadvantages of conventional CAR-T therapies. In this review, we will highlight the challenges of CAR-T therapies and combat them with engineering immunology for cancer immunotherapy.

The Tumor Microenvironment Reprograms Immune Cells.

Tumor tissue comprises a highly complex network of diverse cell types. The tumor microenvironment (TME) can be mainly subdivided into cancer cells and stromal cell compartments, the latter include different types of immune cells, fibroblasts, endothelial cells, and pericytes. Tumor cells reprogram immune cells and other stromal cells in the TME to constrain their antitumor capacity by creating an immunosuppressive milieu and metabolism competition. Moreover, the reprogramming effect on immune cells is localized not only in the tumor but also at the systemic level. With wide application of single-cell sequencing technology, tumor-specific characteristics of immune cells and other stromal cells in the TME have been dissected. In this review, we mainly focus on how tumor cells reprogram immune cells both within the TME and peripheral blood. This information can further help us to improve the efficiency of current immunotherapy as well as bring up new ideas to combat cancer.

Locked in a pro-inflammatory state.

Macrophages absorbing cells infected with viable SARS-CoV-2 particles fail to transition into an anti-inflammatory state, potentially contributing to a damaging immune reaction linked to severe forms of COVID-19.

Deciphering Innate Immune Cell-Tumor Microenvironment Crosstalk at a Single-Cell Level.

The tumor microenvironment encompasses various innate immune cells which regulate tumor progression. Exploiting innate immune cells is a new frontier of cancer immunotherapy. However, the classical surface markers for cell-type classification cannot always well-conclude the phenotype, which will further hinge our understanding. The innate immune cells include dendritic cells, monocytes/macrophages, natural killer cells, and innate lymphoid cells. They play important roles in tumor growth and survival, in some cases promoting cancer, in other cases negating cancer. The precise characterization of innate immune cells at the single-cell level will boost the potential of cancer immunotherapy. With the development of single-cell RNA sequencing technology, the transcriptome of each cell in the tumor microenvironment can be dissected at a single-cell level, which paves a way for a better understanding of the cell type and its functions. Here, we summarize the subtypes and functions of innate immune cells in the tumor microenvironment based on recent literature on single-cell technology. We provide updates on recent achievements and prospects for how to exploit novel functions of tumor-associated innate immune cells and target them for cancer immunotherapy.

Off-the-Shelf Chimeric Antigen Receptor Immune Cells from Human Pluripotent Stem Cells.

Autologous chimeric antigen receptor (CAR) T cells have expanded the scope and therapeutic potential of anti-cancer therapy. Nevertheless, autologous CAR-T therapy has been challenging due to labor some manufacturing processes for every patient, and the cost due to the complexity of the process. Moreover, T cell dysfunction results from the immunosuppressive tumor microenvironment in certain patients. Considering technical challenges in autologous donors, the development of safe and efficient allogeneic CAR-T therapy will address these issues. Since the advent of the generation of immune cells from pluripotent stem cells (PSCs), numerous studies focus on the off-the-shelf generation of CAR-immune cells derived from the universal donor PSCs, which simplifies the manufacturing process and standardizes CAR-T products. In this review, we will discuss advances in the generation of immune cells from PSCs, together with the potential and perspectives of CAR-T, CAR-macrophages, and CAR-natural killer (NK) cells in cancer treatment. The combination of PSC-derived immune cells and CAR engineering will pave the way for developing next-generation cancer immunotherapy.