iPSC-derived megakaryocytes and platelets accelerate wound healing and angiogenesis.

BACKGROUND: Platelet-rich plasma (PRP), which is prepared by concentrating platelets in autologous blood, shows efficacy in chronic skin wounds via multiple growth factors. However, it exhibits heterogeneity across patients, leading to unstable therapeutic efficacy. Human induced pluripotent stem cell (iPSC)-derived megakaryocytes and platelets (iMPs) are capable of providing a stable supply, holding promise as materials for novel platelet concentrate-based therapies. In this context, we evaluated the effect of iMPs on wound healing and validated lyophilization for clinical applications. METHODS: The growth factors released by activated iMPs were measured. The effect of the administration of iMPs on human fibroblasts and human umbilical vein endothelial cells (HUVECs) was investigated in vitro. iMPs were applied to dorsal skin defects of diabetic mice to assess the wound closure rate and quantify collagen deposition and angiogenesis. Following the storage of freeze-dried iMPs (FD-iMPs) for three months, the stability of growth factors and their efficacy in animal models were determined. RESULT: Multiple growth factors that promote wound healing were detected in activated iMPs. iMPs specifically released FGF2 and exhibited a superior enhancement of HUVEC proliferation compared to PRP. Moreover, an RNA-seq analysis revealed that iMPs induce polarization to stalk cells and enhance ANGPTL4 gene expression in HUVECs. Animal studies demonstrated that iMPs promoted wound closure and angiogenesis in chronic wounds caused by diabetes. We also confirmed the long-term stability of growth factors in FD-iMPs and their comparable effects to those of original iMPs in the animal model. CONCLUSION: Our study demonstrates that iMPs promote angiogenesis and wound healing through the activation of vascular endothelial cells. iMPs exhibited more effectiveness than PRP, an effect attributed to the exclusive presence of specific factors including FGF2. Lyophilization enabled the long-term maintenance of the composition of the growth factors and efficacy of the iMPs, therefore contributing to stable supply for clinical application. These findings suggest that iMPs provide a novel treatment for chronic wounds.

Retrotransposons in Werner syndrome-derived macrophages trigger type I interferon-dependent inflammation in an atherosclerosis model.

The underlying mechanisms of atherosclerosis, the second leading cause of death among Werner syndrome (WS) patients, are not fully understood. Here, we establish an in vitro co-culture system using macrophages (iMφs), vascular endothelial cells (iVECs), and vascular smooth muscle cells (iVSMCs) derived from induced pluripotent stem cells. In co-culture, WS-iMφs induces endothelial dysfunction in WS-iVECs and characteristics of the synthetic phenotype in WS-iVSMCs. Transcriptomics and open chromatin analysis reveal accelerated activation of type I interferon signaling and reduced chromatin accessibility of several transcriptional binding sites required for cellular homeostasis in WS-iMφs. Furthermore, the H3K9me3 levels show an inverse correlation with retrotransposable elements, and retrotransposable element-derived double-stranded RNA activates the DExH-box helicase 58 (DHX58)-dependent cytoplasmic RNA sensing pathway in WS-iMφs. Conversely, silencing type I interferon signaling in WS-iMφs rescues cell proliferation and suppresses cellular senescence and inflammation. These findings suggest that Mφ-specific inhibition of type I interferon signaling could be targeted to treat atherosclerosis in WS patients.

A let-7 microRNA-RALB axis links the immune properties of iPSC-derived megakaryocytes with platelet producibility.

We recently achieved the first-in-human transfusion of induced pluripotent stem cell-derived platelets (iPSC-PLTs) as an alternative to standard transfusions, which are dependent on donors and therefore variable in supply. However, heterogeneity characterized by thrombopoiesis-biased or immune-biased megakaryocytes (MKs) continues to pose a bottleneck against the standardization of iPSC-PLT manufacturing. To address this problem, here we employ microRNA (miRNA) switch biotechnology to distinguish subpopulations of imMKCLs, the MK cell lines producing iPSC-PLTs. Upon miRNA switch-based screening, we find imMKCLs with lower let-7 activity exhibit an immune-skewed transcriptional signature. Notably, the low activity of let-7a-5p results in the upregulation of RAS like proto-oncogene B (RALB) expression, which is crucial for the lineage determination of immune-biased imMKCL subpopulations and leads to the activation of interferon-dependent signaling. The dysregulation of immune properties/subpopulations, along with the secretion of inflammatory cytokines, contributes to a decline in the quality of the whole imMKCL population.

Silencing of p53 and CDKN1A establishes sustainable immortalized megakaryocyte progenitor cells from human iPSCs.

Platelet transfusions are critical for severe thrombocytopenia but depend on blood donors. The shortage of donors and the potential of universal HLA-null platelet products have stimulated research on the ex vivo differentiation of human pluripotent stem cells (hPSCs) to platelets. We recently established expandable immortalized megakaryocyte cell lines (imMKCLs) from hPSCs by transducing MYC, BMI1, and BCL-XL (MBX). imMKCLs can act as cryopreservable master cells to supply platelet concentrates. However, the proliferation rates of the imMKCLs vary with the starting hPSC clone. In this study, we reveal from the gene expression profiles of several MKCL clones that the proliferation arrest is correlated with the expression levels of specific cyclin-dependent kinase inhibitors. Silencing CDKN1A and p53 with the overexpression of MBX was effective at stably inducing imMKCLs that generate functional platelets irrespective of the hPSC clone. Collectively, this improvement in generating imMKCLs should contribute to platelet industrialization and platelet biology.

T helper 2-driven immune dysfunction in chronic arsenic-exposed individuals and its link to the features of allergic asthma.

Limited information is available regarding the effects of arsenic exposure on immune function. We have recently reported that chronic exposure to As was associated asthma, as determined by spirometry and respiratory symptoms. Because T helper 2 (Th2)-driven immune responses are implicated in the pathogenesis of allergic diseases, including asthma, we studied the associations of serum Th1 and Th2 mediators with the As exposure markers and the features of asthma among individuals exposed to As. A total of 553 blood samples were selected from the same study subjects recruited in our previous asthma study. Serum levels of Th1 and Th2 cytokines were analyzed by immunoassay. Subjects' arsenic exposure levels (drinking water, hair and nail arsenic concentrations) were determined by inductively coupled plasma mass spectroscopy. Arsenic exposure levels of the subjects showed significant positive associations with serum Th2-mediators- interleukin (IL)-4, IL-5, IL-13, and eotaxin without any significant changes in Th1 mediators- interferon-γ and tumor necrosis factor-α. The ratios of Th2 to Th1 mediators were significantly increased with increasing exposure to As. Notably, most of the Th2 mediators were positively associated with serum levels of total immunoglobulin E and eotaxin. The serum levels of Th2 mediators were significantly higher in the subjects with asthma than those without asthma. The results of our study suggest that the exacerbated Th2-driven immune responses are involved in the increased susceptibility to allergic asthma among individuals chronically exposed to As.

Higher risk of hyperglycemia with greater susceptibility in females in chronic arsenic-exposed individuals in Bangladesh.

Arsenic (As) toxicity and diabetes mellitus (DM) are emerging public health concerns worldwide. Although exposure to high levels of As has been associated with DM, whether there is also an association between low and moderate As exposure and DM remains unclear. We explored the dose-dependent association between As exposure levels and hyperglycemia, with special consideration of the impact of demographic variables, in 641 subjects from rural Bangladesh. The total study participants were divided into three groups depending on their levels of exposure to As in drinking water (low, moderate and high exposure groups). Prevalence of hyperglycemia, including impaired glucose tolerance (IGT) and DM was significantly associated with the subjects' drinking water arsenic levels. Almost all exposure metrics (As levels in the subjects' drinking water, hair and nails) showed dose-dependent associations with the risk of hyperglycemia, IGT and DM. Among the variables considered, sex, age, and BMI were found to be associated with higher risk of hyperglycemia, IGT and DM. In sex-stratified analyses, As exposure showed a clearer pattern of dose-dependent risk for hyperglycemia in females than males. Finally, drinking water containing low-to-moderate levels of As (50.01-150 µg/L) was found to confer a greater risk of hyperglycemia than safe drinking water (As ≤10 µg/L). Thus the results suggested that As exposure was dose-dependently associated with hyperglycemia, especially in females.

Generation of tumor antigen-specific murine CD8+ T cells with enhanced anti-tumor activity via highly efficient CRISPR/Cas9 genome editing.

Immunotherapies have led to the successful development of novel therapies for cancer. However, there is increasing concern regarding the adverse effects caused by non-tumor-specific immune responses. Here, we report an effective strategy to generate high-avidity tumor-antigen-specific CTLs, using Cas9/single-guide RNA (sgRNA) ribonucleoprotein (RNP) delivery. As a proof-of-principle demonstration, we selected the gp100 melanoma-associated tumor antigen, and cloned the gp100-specific high-avidity TCR from gp100-immunized mice. To enable rapid structural dissection of the TCR, we developed a 3D protein structure modeling system for the TCR/antigen-major histocompatibility complex (pMHC) interaction. Combining these technologies, we efficiently generated gp100-specific PD-1(-) CD8+ T cells, and demonstrated that the genetically engineered CD8+ T cells have high avidity against melanoma cells both in vitro and in vivo. Our methodology offers computational prediction of the TCR response, and enables efficient generation of tumor antigen-specific CD8+ T cells that can neutralize tumor-induced immune suppression leading to a potentially powerful cancer therapeutic.

Chronic exposure to arsenic, LINE-1 hypomethylation, and blood pressure: a cross-sectional study in Bangladesh.

BACKGROUND: Chronic exposure to arsenic is associated with cancer and hypertension. Growing evidence suggests that altered methylation in long interspersed nuclear element-1 (LINE-1) is involved in many types of disorders, including cardiovascular disease. Here we evaluated the association between arsenic exposure and LINE-1 methylation levels, especially in relation to blood pressure (BP). METHODS: A total of 236 subjects (175 from arsenic-endemic areas and 61 from a non-endemic area) in rural Bangladesh were recruited. The subjects' arsenic exposure levels (i.e., drinking water, hair and nail arsenic concentrations) were measured by inductively coupled plasma mass spectroscopy. The subjects' LINE-1 methylation levels were determined by pyrosequencing. RESULTS: The average LINE-1 methylation levels of the subjects living in the arsenic-endemic areas were significantly (p < 0.01) lower than those of the subjects living in the non-endemic area. In a sex-stratified analysis, the arsenic exposure levels in female but not male subjects showed a significant inverse association with LINE-1 methylation levels before (water arsenic: p < 0.01, hair arsenic: p < 0.05, nail arsenic: p < 0.001) and after (water arsenic: p < 0.01, hair arsenic: p < 0.05, nail arsenic: p < 0.001) adjustment for age, body mass index and smoking. Analyses examining interactions among arsenic levels, BP and LINE-1 methylation showed that arsenic-related elevated levels of BP were associated with LINE-1 hypomethylation. CONCLUSIONS: Our findings demonstrated that chronic exposure to arsenic was inversely associated with LINE-1 methylation levels in blood leukocyte DNA and this was more pronounced in females than males; in addition, the decreased levels of LINE-1 methylation might be involved in the arsenic-induced elevation of BP.