Noncanonical Wnt signaling maintains hematopoietic stem cells in the niche.

Wnt signaling is involved in self-renewal and maintenance of hematopoietic stem cells (HSCs); however, the particular role of noncanonical Wnt signaling in regulating HSCs in vivo is largely unknown. Here, we show Flamingo (Fmi) and Frizzled (Fz) 8, members of noncanonical Wnt signaling, both express in and functionally maintain quiescent long-term HSCs. Fmi regulates Fz8 distribution at the interface between HSCs and N-cadherin(+) osteoblasts (N-cad(+)OBs that enrich osteoprogenitors) in the niche. We further found that N-cad(+)OBs predominantly express noncanonical Wnt ligands and inhibitors of canonical Wnt signaling under homeostasis. Under stress, noncanonical Wnt signaling is attenuated and canonical Wnt signaling is enhanced in activation of HSCs. Mechanistically, noncanonical Wnt signaling mediated by Fz8 suppresses the Ca(2+)-NFAT- IFNγ pathway, directly or indirectly through the CDC42-CK1α complex and also antagonizes canonical Wnt signaling in HSCs. Taken together, our findings demonstrate that noncanonical Wnt signaling maintains quiescent long-term HSCs through Fmi and Fz8 interaction in the niche.

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.

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.

Haematopoietic stem and progenitor cells from human pluripotent stem cells.

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders.

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.

The Single-Cell Level Perspective of the Tumor Microenvironment and Its Remodeling by CAR-T Cells.

The tumor microenvironment (TME) is a complex milieu consisting of lymphoid cells, myeloid cells, fibroblasts, and multiple molecules, which play a key role in tumor progression and immunotherapy. TME is characterized by immune-suppressive features, which release anti-inflammatory cytokines such as IL-4 and TGFß to skew the T cells to a Th2 state as well to polarize tumor-associated macrophages (TAMs) to an anti-inflammatory phenotype to curb the immunotherapy. Considering the heterogeneity of the TME and its role in determining response to chimeric antigen receptor (CAR)-T cells, delineating TME at a single-cell level will provide useful information for cancer treatment. First, we discuss cellular and molecular features that curb the response to CAR-T cells, for example, high expression of immune checkpoint molecules (PD-1, LAG3) and anti-inflammatory cytokines (IL-4, TGFb) that block CAR-T cell function. Then, we summarize how newly invented single-cell technologies such as spatial multi-omics would benefit the understanding of cancer immunotherapy. Finally, we will further describe recent attempts of CAR-T to remodel TME by arming the CAR-T with anti-PD-1 single-chain variants or Th1 triggering cytokines (such as IL-7, IL-12) to remodel TME into a pro-inflammatory state. Herein, we review the single-cell-level signatures of TME and the strategies of CAR-T to remodel TME.

Biomimetic aorta-gonad-Mesonephros-on-a-Chip to study human developmental hematopoiesis.

A fundamental limitation in the derivation of hematopoietic stem and progenitor cells is the imprecise understanding of human developmental hematopoiesis. Herein we established a multilayer microfluidic Aorta-Gonad-Mesonephros (AGM)-on-a-chip to emulate developmental hematopoiesis from pluripotent stem cells. The device consists of two layers of microchannels separated by a semipermeable membrane, which allows the co-culture of human hemogenic endothelial (HE) cells and stromal cells in a physiological relevant spatial arrangement to replicate the structure of the AGM. HE cells derived from human induced pluripotent stem cells (hiPSCs) were cultured on a layer of mesenchymal stromal cells in the top channel while vascular endothelial cells were co-cultured on the bottom side of the membrane within the microfluidic device. We show that this AGM-on-a-chip efficiently derives endothelial-to-hematopoietic transition (EHT) from hiPSCs compared with regular suspension culture. The presence of mesenchymal stroma and endothelial cells renders functional HPCs in vitro. We propose that the AGM-on-a-chip could serve as a platform to dissect the cellular and molecular mechanisms of human developmental hematopoiesis.

Maternal imprinting at the H19-Igf2 locus maintains adult haematopoietic stem cell quiescence.

The epigenetic regulation of imprinted genes by monoallelic DNA methylation of either maternal or paternal alleles is critical for embryonic growth and development. Imprinted genes were recently shown to be expressed in mammalian adult stem cells to support self-renewal of neural and lung stem cells; however, a role for imprinting per se in adult stem cells remains elusive. Here we show upregulation of growth-restricting imprinted genes, including in the H19-Igf2 locus, in long-term haematopoietic stem cells and their downregulation upon haematopoietic stem cell activation and proliferation. A differentially methylated region upstream of H19 (H19-DMR), serving as the imprinting control region, determines the reciprocal expression of H19 from the maternal allele and Igf2 from the paternal allele. In addition, H19 serves as a source of miR-675, which restricts Igf1r expression. We demonstrate that conditional deletion of the maternal but not the paternal H19-DMR reduces adult haematopoietic stem cell quiescence, a state required for long-term maintenance of haematopoietic stem cells, and compromises haematopoietic stem cell function. Maternal-specific H19-DMR deletion results in activation of the Igf2-Igfr1 pathway, as shown by the translocation of phosphorylated FoxO3 (an inactive form) from nucleus to cytoplasm and the release of FoxO3-mediated cell cycle arrest, thus leading to increased activation, proliferation and eventual exhaustion of haematopoietic stem cells. Mechanistically, maternal-specific H19-DMR deletion leads to Igf2 upregulation and increased translation of Igf1r, which is normally suppressed by H19-derived miR-675. Similarly, genetic inactivation of Igf1r partly rescues the H19-DMR deletion phenotype. Our work establishes a new role for this unique form of epigenetic control at the H19-Igf2 locus in maintaining adult stem cells.

Cooperation between both Wnt/{beta}-catenin and PTEN/PI3K/Akt signaling promotes primitive hematopoietic stem cell self-renewal and expansion.

Although self-renewal is the central property of stem cells, the underlying mechanism remains inadequately defined. Using a hematopoietic stem and progenitor cell (HSPC)-specific conditional induction line, we generated a compound genetic model bearing both Pten deletion and ß-catenin activation. These double mutant mice exhibit a novel phenotype, including expansion of phenotypic long-term hematopoietic stem cells (LT-HSCs) without extensive differentiation. Unexpectedly, constitutive activation of ß-catenin alone results in apoptosis of HSCs. However, together, the Wnt/ß-catenin and PTEN/PI3k/Akt pathways interact to drive phenotypic LT-HSC expansion by inducing proliferation while simultaneously inhibiting apoptosis and blocking differentiation, demonstrating the necessity of complementary cooperation between the two pathways in promoting self-renewal. Mechanistically, ß-catenin activation reduces multiple differentiation-inducing transcription factors, blocking differentiation partially through up-regulation of Inhibitor of differentiation 2 (Id2). In double mutants, loss of Pten enhances the HSC anti-apoptotic factor Mcl-1. All of these contribute in a complementary way to HSC self-renewal and expansion. While permanent, genetic alteration of both pathways in double mutant mice leads to expansion of phenotypic HSCs, these HSCs cannot function due to blocked differentiation. We developed a pharmacological approach to expand normal, functional HSCs in culture using factors that reversibly activate both Wnt/ß-catenin and PI3K/Akt signaling simultaneously. We show for the first time that activation of either single pathway is insufficient to expand primitive HSCs, but in combination, both pathways drive self-renewal and expansion of HSCs with long-term functional capacity.

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.

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.

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.

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.

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.

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.

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.

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.

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.