Abstract
Thrombosis leads to platelet activation and subsequent degradation; therefore, replenishment of platelets from hematopoietic stem/progenitor cells (HSPCs) is needed to maintain the ...physiological level of circulating platelets. Platelet-derived microparticles (PMPs) are protein- and RNA-containing vesicles released from activated platelets. We hypothesized that factors carried by PMPs might influence the production of platelets from HSPCs, in a positive feedback fashion. Here we show that, during mouse acute liver injury, the density of megakaryocyte in the bone marrow increases following an increase in circulating PMPs, but without thrombopoietin (TPO) upregulation. In vitro, PMPs are internalized by HSPCs and drive them toward a megakaryocytic fate. Mechanistically, miR-1915-3p, a miRNA highly enriched in PMPs, is transported to target cells and suppresses the expression levels of Rho GTPase family member B, thereby inducing megakaryopoiesis. In addition, direct injection of PMPs into irradiated mice increases the number of megakaryocytes and platelets without affecting TPO levels. In conclusion, our data reveal that PMPs have a role in promoting megakaryocytic differentiation and platelet production.
Intrahepatic cholangiocarcinoma (ICC) is the second most common liver malignancy. ICC typically features remarkable cellular heterogeneity and a dense stromal reaction. Therefore, a comprehensive ...understanding of cellular diversity and the interplay between malignant cells and niche cells is essential to elucidate the mechanisms driving ICC progression and to develop therapeutic approaches.
Herein, we performed single-cell RNA sequencing (scRNA-seq) analysis on unselected viable cells from 8 human ICCs and adjacent samples to elucidate the comprehensive transcriptomic landscape and intercellular communication network. Additionally, we applied a negative selection strategy to enrich fibroblast populations in 2 other ICC samples to investigate fibroblast diversity. The results of the analyses were validated using multiplex immunofluorescence staining, bulk transcriptomic datasets, and functional in vitro and in vivo experiments.
We sequenced a total of 56,871 single cells derived from human ICC and adjacent tissues and identified diverse tumor, immune, and stromal cells. Malignant cells displayed a high degree of inter-tumor heterogeneity. Moreover, tumor-infiltrating CD4 regulatory T cells exhibited highly immunosuppressive characteristics. We identified 6 distinct fibroblast subsets, of which the majority were CD146-positive vascular cancer-associated fibroblasts (vCAFs), with highly expressed microvasculature signatures and high levels of interleukin (IL)-6. Functional assays indicated that IL-6 secreted by vCAFs induced significant epigenetic alterations in ICC cells, particularly upregulating enhancer of zeste homolog 2 (EZH2) and thereby enhancing malignancy. Furthermore, ICC cell-derived exosomal miR-9-5p elicited high expression of IL-6 in vCAFs to promote tumor progression.
Our single-cell transcriptomic dataset delineates the inter-tumor heterogeneity of human ICCs, underlining the importance of intercellular crosstalk between ICC cells and vCAFs, and revealing potential therapeutic targets.
Intrahepatic cholangiocarcinoma is an aggressive and chemoresistant malignancy. Better understanding the complex transcriptional architecture and intercellular crosstalk of these tumors will help in the development of more effective therapies. Herein, we have identified important interactions between cancer cells and cancer-associated fibroblasts in the tumor stroma, which could have therapeutic implications.
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•Single cell transcriptomic datasets are a valuable resource to dissect cellular diversity and intercellular crosstalk of human ICCs.•Malignant cells displayed remarkable inter-tumor heterogeneity and Tregs revealed highly immunosuppressive characteristics.•Six distinct fibroblast subsets were defined in ICCs and adjacent tissues.•CD146+ vCAFs, comprising most of the fibroblasts, had tight interactions with malignant cells through IL-6/IL-6R axis.•Tumor exosomal miR-9-5p elicited IL-6 expression in vCAFs, contributing to ICC progression via upregulation of EZH2.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Endodermal stem/progenitor cells have diverse potential applications in research and regenerative medicine, so a readily available source could have widespread uses. Here we describe derivation of ...human induced endodermal progenitor cells (hiEndoPCs) from gastrointestinal epithelial cells using a cocktail of defined small molecules along with support from tissue-specific mesenchymal feeders. The hiEndoPCs show clonal expansion in culture and give rise to hepatocytes, pancreatic endocrine cells, and intestinal epithelial cells when treated with defined soluble molecules directing differentiation. The hiEndoPC-derived hepatocytes are able to rescue liver failure in Fah−/−Rag2−/− mice after transplantation, and, unlike hESCs, transplanted hiEndoPCs do not give rise to teratomas. Since human gastric epithelial cells are readily available from donors of many ages, this conversion strategy can generate clonally expandable cell populations with a variety of potential applications, including personalized drug screening and therapeutic strategies for liver failure and diabetes.
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•Small molecules convert human gastric epithelial cells into endodermal progenitors•Induced endodermal progenitors expand rapidly and express stem cell markers•Signaling induction differentiates the induced progenitors into endodermal cells•Transplantation of derived hepatocytes into mice can rescue liver failure
Wang et al. show that a set of defined small molecules plus mesenchymal feeder cells can covert human gastric epithelial cells into induced endodermal progenitors with multilineage differentiation capacity. As gastric epithelial cells are readily available after surgical/biopsy procedures, these cells could provide an accessible source for various applications.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abnormal activation of the Sonic hedgehog (SHH) pathway has been described in a wide variety of human cancers and in cancer stem cells (CSCs), however, the role of SHH pathway in gastric CSCs has not ...been reported. In this study, we investigated the possibility that abnormal activation of the SHH pathway maintained the characteristics of gastric CSCs. First, we identified cancer stem-like cells (CSLCs) from human gastric cancer cell lines (HGC-27, MGC-803 and MKN-45) using tumorsphere culture. Compared with adherent cells, the floating tumorsphere cells had more self-renewing capacity and chemoresistance. The cells expressing CSCs markers (CD44, CD24 and CD133) were also significantly more in tumorsphere cells than in adherent cells. More importantly, in vivo xenograft studies showed that tumors could be generated with 2 x 10⁴ tumorsphere cells, which was 100-fold less than those required for tumors seeding by adherent cells. Next, RT-PCR and Western blot showed that the expression levels of Ptch and Gli1 (SHH pathway target genes) were significantly higher in tumorsphere cells than in adherent cells. The results of quantitative real-time PCR were similar to those of RT-PCR and Western blot. Further analysis revealed that SHH pathway blocked by cyclopamine or 5E1 caused a higher reduction in self-renewing capacity of HGC-27 tumorsphere cells than that of adherent cells. We also found that SHH pathway blocking strongly enhanced the efficacy of chemotherapeutic drugs in HGC-27 tumorsphere cells in vitro and in vivo but had no significant effect in adherent cells. Finally, we isolated the tumorspheres from gastric cancer specimen, these cells also had chemoresistance and tumorigenic capacity, and SHH pathway maintained the gastric CSLCs characteristics of tumorsphere cells from primary tumor samples. In conclusion, our data suggested that SHH pathway was essential for maintenance of CSLCs in human gastric cancer.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Stem cells have emerged as a potential therapy for a range of neural insults, but their application in Alzheimer's disease (AD) is still limited and the mechanisms underlying the cognitive benefits ...of stem cells remain to be elucidated. Here, the effects of clinical‐grade human umbilical cord‐derived mesenchymal stem cells (hUC‐MSCs) on the recovery of cognitive ability in SAMP8 mice, a senescence‐accelerated mouse model of AD is explored. A functional assay identifies that the core functional factor hepatocyte growth factor (HGF) secreted from hUC‐MSCs plays critical roles in hUC‐MSC‐modulated recovery of damaged neural cells by down‐regulating hyperphosphorylated tau, reversing spine loss, and promoting synaptic plasticity in an AD cell model. Mechanistically, structural and functional recovery, as well as cognitive enhancements elicited by exposure to hUC‐MSCs, are at least partially mediated by HGF in the AD hippocampus through the activation of the cMet‐AKT‐GSK3β signaling pathway. Taken together, these data strongly implicate HGF in mediating hUC‐MSC‐induced improvements in functional recovery in AD models.
This study explores the effects of clinical‐grade hUC‐MSCs on the recovery of cognitive ability of Alzheimer's disease (AD). All results uncover that HGF secreted from hUC‐MSCs, mediates the beneficial effects of hUC‐MSCs on functional recovery of damaged neural cells by downregulating hyperphosphorylated tau, improving neurofibrillary tangles, reversing spine loss, and promoting synaptic plasticity in AD hippocampus through the cMet‐AKT‐GSK3β signaling pathway.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Human hematopoietic organoids have a wide application value for modeling human bone marrow diseases, such as acute hematopoietic radiation injury. However, the manufacturing of human hematopoietic ...organoids is an unaddressed challenge because of the complexity of hematopoietic tissues.
To manufacture hematopoietic organoids, we obtained CD34
hematopoietic stem and progenitor cells (HSPCs) from human embryonic stem cells (hESCs) using stepwise induction and immunomagnetic bead-sorting. We then mixed these CD34
HSPCs with niche-related cells in Gelatin-methacryloyl (GelMA) to form a three-dimensional (3D) hematopoietic organoid. Additionally, we investigated the effects of radiation damage and response to granulocyte colony-stimulating factor (G-CSF) in hematopoietic organoids.
The GelMA hydrogel maintained the undifferentiated state of hESCs-derived HSPCs by reducing intracellular reactive oxygen species (ROS) levels. The established hematopoietic organoids in GelMA with niche-related cells were composed of HSPCs and multilineage blood cells and demonstrated the adherence of hematopoietic cells to niche cells. Notably, these hematopoietic organoids exhibited radiation-induced hematopoietic cell injury effect, including increased intracellular ROS levels, γ-H2AX positive cell percentages, and hematopoietic cell apoptosis percentages. Moreover, G-CSF supplementation in the culture medium significantly improved the survival of HSPCs and enhanced myeloid cell regeneration in these hematopoietic organoids after radiation.
These findings substantiate the successful manufacture of a preliminary 3D hematopoietic organoid from hESCs-derived HSPCs, which was utilized for modeling hematopoietic radiation injury and assessing the radiation-mitigating effects of G-CSF in vitro. Our study provides opportunities to further aid in the standard and scalable production of hematopoietic organoids for disease modeling and drug testing.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
With the expansion of the aging population, age-associated sarcopenia (AAS) has become a severe clinical disease of the elderly and a key challenge for healthy aging. Regrettably, no approved ...therapies currently exist for treating AAS. In this study, clinical-grade human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were administrated to two classic mouse models (SAMP8 mice and D-galactose-induced aging mice), and their effects on skeletal muscle mass and function were investigated by behavioral tests, immunostaining, and western blotting. Core data results showed that hUC-MSCs significantly restored skeletal muscle strength and performance in both mouse models via mechanisms including raising the expression of crucial extracellular matrix proteins, activating satellite cells, enhancing autophagy, and impeding cellular aging. For the first time, the study comprehensively evaluates and demonstrates the preclinical efficacy of clinical-grade hUC-MSCs for AAS in two mouse models, which not only provides a novel model for AAS, but also highlights a promising strategy to improve and treat AAS and other age-associated muscle diseases. This study comprehensively evaluates the preclinical efficacy of clinical-grade hUC-MSCs in treating age-associated sarcopenia (AAS), and demonstrates that hUC-MSCs restore skeletal muscle strength and performance in two AAS mouse models via raising the expression of extracellular matrix proteins, activating satellite cells, enhancing autophagy, and impeding cellular aging, which highlights a promising strategy for AAS and other age-associated muscle diseases.
Alzheimer's disease (AD) is an incurable neurodegenerative disease and many types of stem cells have been used in AD therapy with some favorable effects. In this study, we investigated the potential ...therapeutical effects of human dental pulp stem cells (hDPSCs) on AD cellular model which established by okadaic acid (OA)‐induced damage to human neuroblastoma cell line, SH‐SY5Y, in vitro for 24 h. After confirmed the AD cellular model, the cells were co‐culture with hDPSCs by transwell co‐culture system till 24 h for treatment. Then the cytomorphology of the hDPSCs‐treated cells were found to restore gradually with re‐elongation of retracted dendrites. Meanwhile, Cell Counting Kit‐8 assay and Hoechst 33258 staining showed that hDPSCs caused significant increase in the viability and decrease in apoptosis of the model cells, respectively. Observation of DiI labeling also exhibited the prolongation dendrites in hDPSCs‐treated cells which were obviously different from the retraction dendrites in AD model cells. Furthermore, specific staining of α‐tubulin and F‐actin demonstrated that the hDPSCs‐treated cells had the morphology of restored neurons, with elongated dendrites, densely arranged microfilaments, and thickened microtubular fibrils. In addition, results from western blotting revealed that phosphorylation at Ser 396 of Tau protein was significantly suppressed by adding of hDPSCs. These results indicate that hDPSCs may promote regeneration of damaged neuron cells in vitro model of AD and may serve as a useful cell source for treatment of AD.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Generation of insulin-producing cells from human pluripotent stem cells (hPSCs) in vitro would be useful for drug discovery and cell therapy in diabetes. Three-dimensional (3D) culture is important ...for the acquisition of mature insulin-producing cells from hPSCs, but the mechanism by which it promotes β cell maturation is poorly understood.
We established a stepwise method to induce high-efficiency differentiation of human embryonic stem cells (hESCs) into mature monohormonal pancreatic endocrine cells (PECs), with the last maturation stage in 3D culture. To comprehensively compare two-dimensional (2D) and 3D cultures, we examined gene expression, pancreas-specific markers, and functional characteristics in 2D culture-induced PECs and 3D culture-induced PECs. The mechanisms were considered from the perspectives of cell-cell and cell-extracellular matrix interactions which are fundamentally different between 2D and 3D cultures.
The expression of the pancreatic endocrine-specific transcription factors PDX1, NKX6.1, NGN3, ISL1, and PAX6 and the hormones INS, GCG, and SST was significantly increased in 3D culture-induced PECs. 3D culture yielded monohormonal endocrine cells, while 2D culture-induced PECs co-expressed INS and GCG or INS and SST or even expressed all three hormones. We found that focal adhesion kinase (FAK) phosphorylation was significantly downregulated in 3D culture-induced PECs, and treatment with the selective FAK inhibitor PF-228 improved the expression of β cell-specific transcription factors in 2D culture-induced PECs. We further demonstrated that 3D culture may promote endocrine commitment by limiting FAK-dependent activation of the SMAD2/3 pathway. Moreover, the expression of the gap junction protein Connexin 36 was much higher in 3D culture-induced PECs than in 2D culture-induced PECs, and inhibition of the FAK pathway in 2D culture increased Connexin 36 expression.
We developed a strategy to induce differentiation of monohormonal mature PECs from hPSCs and found limited FAK-dependent activation of the SMAD2/3 pathway and unregulated expression of Connexin 36 in 3D culture-induced PECs. This study has important implications for the generation of mature, functional β cells for drug discovery and cell transplantation therapy for diabetes and sheds new light on the signaling events that regulate endocrine specification.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Red blood cells (RBCs) and platelets derived from stem cells are possible solutions to the increasing demand for blood transfusion. Based on the availability of stem cells, their relatively defined ...differentiation mechanisms, and the massive exploration of induction systems, the generation of RBCs or platelets in vitro from cord blood hematopoietic stem/progenitor cells (CB‐HSPCs) has potential for clinical applications. However, information on the clinical translation of stem cell‐derived RBCs and platelets in the literature and at the ClinicalTrials.gov website is very limited. The only clinical trial on cultured RBCs, which aimed to assess the lifespan of RBCs cultured in vivo, was reported by Luc Douay and colleagues. Of note, the cultured RBCs they used were derived from autologous peripheral blood HSPCs, and no cultured platelets have been applied clinically to date. However, CB‐HSPC‐derived megakaryocytes, platelet precursors, have been used in the treatment of thrombocytopenia. A successful phase I trial was reported, followed by phase II and III clinical trials conducted in China. In this review, the gap between the many basic studies and limited clinical trials on stem cell‐derived RBCs and platelets is summarized. The possible reasons and solutions for this gap are discussed. Further technological improvements for blood cell expansion and maturation ex vivo and the establishment of biological standards for stem cell derivatives might help to facilitate the therapeutic applications of cultured RBCs and platelets derived from CB‐HSPCs in the near future.
We review the progress in producing red blood cells (RBCs) and platelets from human cord blood hematopoietic stem/progenitor cells (HSPCs) and relatively limited reports on the clinical trials with cultured RBCs and megakaryocytic progenitors (MPs). Technical improvement together with the establishment of biological standards for stem cell derivations might help to achieve therapeutic applications of cultured RBCs and platelets. EPCs, erythroid progenitor cells.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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