Tissue-derived Cells Research Articles

Single-cell RNA sequencing reveals the role of cell heterogeneity in the sex difference in primary hyperparathyroidism.

To explore the difference in parathyroid tissue-derived cells between male and female PHPT patients. Resected parathyroid tissues were collected from PHPT patients of both sexes. Single cells were isolated and sequenced for RNA expression profiles. The cell sequencing data were annotated by cell type, followed by population analysis, functional analysis, pathway analysis, cell communication analysis, differential gene expression analysis, and pseudotime trajectory analysis. The subcluster analyses were also performed in the parathyroid cells. No substantial difference in the cell population, function, or communication is found between the two sexes. The interferon-a response, oxidative phosphorylation, and reactive oxygen species pathways are up-regulated in females than in male patients, mainly contributed by fibroblast cells, endothelial cells, parathyroid cells, and myeloid cells, which also have significantly more up-regulated pathways and cellular interactions than the other three cell types. The subcluster analysis of parathyroid cells identified five subpopulations: SPARCL1-OC and ISG15-OC are predominant in females, while more S100A13-PCC and PTHLH-OC are found in males. The cellular functions are also elevated in females compared with males. Cells from female patients show a higher expression level of parathyroid hormone (PTH) but a lower expression level of parathyroid hormone-like hormone (PTHLH). The cell pseudotime trajectory and pathway analyses show that the oxyphil cells may be more mature and functionally active than the chief cells in both sexes. These findings suggest that the sex difference in PHPT may be caused by the differentially expressed genes and activated pathways in different cell types in the parathyroid tissue. The heterogeneity of parathyroid cell subpopulations, especially in oxyphil cells, may be associated with the sex differences in PHPT pathogenesis.

Safety and Efficacy of Autologous Stem Cell Treatment for Facetogenic Chronic Back Pain.

Chronic back pain due to facet joint syndrome is a common and debilitating condition. Advances in regenerative medicine have shown that autologous unmodified adipose tissue-derived regenerative cells (ADRC) provide several beneficial effects. These regenerative cells can differentiate into various tissues and exhibit a strong anti-inflammatory potential. ADRCs can be obtained from a small amount of fatty tissue derived from the patient's abdominal fat. We report long-term results of 37 patients (age 31-78 years, mean 62.5) suffering from "Facet Joint Syndrome" The pathology was confirmed by clinical, radiological examinations and fluoroscopically guided test injections. Then, liposuction was performed. An amount of 50-100 cc of fat was harvested. To recover stem cells from adipose tissue, we use the CE-certified Transpose RT™ system from InGeneron GmbH. The cells were then injected under fluoroscopic control in the periarticular fat. Follow-up examinations were performed at 1 week, 1 year, and 5 years. Every patient reported improved VAS pain at any follow-up (1 week, 1 year, and 5 years) with ADRCs compared to the baseline. Our observational data indicate that facet joint syndrome patients treated with unmodified adipose tissue-derived regenerative cells experience improved the quality of life in the long term.

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue.

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central roles in the pathogenesis of rheumatoid arthritis. It is important to understand the functions of both cell populations to reveal the mechanisms underlying pathological progression and remission in inflammatory arthritis. In general, in vitro experimental conditions should mimic the in vivo environment as much as possible. Primary tissue-derived cells have been used in experiments characterizing synovial fibroblasts in arthritis. In contrast, in experiments investigating the biological functions of macrophages in inflammatory arthritis, cell lines, bone marrow-derived macrophages, and blood monocyte-derived macrophages have been used. However, it is unclear whether such macrophages actually reflect the functions of tissue-resident macrophages. To obtain resident macrophages, previous protocols were modified to isolate and expand both primary macrophages and fibroblasts from synovial tissue in an inflammatory arthritis mouse model. These primary synovial cells may be useful for in vitro analysis of inflammatory arthritis.

Structural Analysis of MALAT1 long non-coding RNA in cells and in evolution.

Although not canonically polyadenylated, the long non-coding RNA MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) is stabilized by a highly conserved 76 nucleotide triple helix structure on its 3' end. The entire MALAT1 transcript is over 8,000 nucleotides long in humans. The strongest structural conservation signal in MALAT1 (as measured by co-variation of base-pairs) is in the triple helix structure. Primary sequence analysis of co-variation alone does not reveal the degree of structural conservation of the entire full-length transcript, however. Furthermore, RNA structure is often context dependent; RNA binding proteins that are differentially expressed in different cell types may alter structure. We investigate here the in cell and cell free structures of the full-length human and green monkey (Chlorocebus sabaeus) MALAT1 transcripts in multiple tissue-derived cell lines using SHAPE chemical probing. Our data reveal levels of uniform structural conservation in different cell lines, in cells and cell free, and even between species, despite significant differences in primary sequence. The uniformity of the structural conservation across the entire transcript suggests that, despite seeing co-variation signals only in the triple helix junction of the lncRNA, the rest of the transcript's structure is remarkably conserved at least in primates and across multiple cell types and conditions.

Better isolation, proliferation and differentiation of human adipose-derived mesenchymal stem cells using human serum.

Mesenchymal stem cells have many applications in medicine. Attention to the proliferation and differentiation of stem cell differentiation is an important issue. The aim of this study was to investigate the possibility of optimal isolation, proliferation, and differentiation of adipose tissue-derived mesenchymal stem cells (ADSCs) using human serum. Human serum (HS) was obtained from the venous blood of eight healthy individuals. The rate of proliferation and differentiation of ADSCs and expression of surface markers was assessed by flow cytometry. Bone differentiation was assessed using Alizarin Red staining. Data were analyzed using statistical software. Over time, HS showed more proliferation than fetal bovine serum (FBS) -enriched cells (p <0.05). Differentiation of ADSCs cells ls in HS-enriched medium is faster and more pronounced than differentiation in the control group. The expression of surface markers in the medium containing HS was the same as the medium containing FBS where the expression levels of CD105 and CD95 were found to be positive and the expression of CD34 and CD45 was negative. Due to the better proliferation of adipose tissue-derived mesenchymal cells in the medium containing HS than FBS, it is suggested that human serum be used in future clinical studies. Also, HS is healthier, safer, more accessible, and more affordable than FBS.

PTX3 from vascular endothelial cells contributes to trastuzumab-induced cardiac complications.

Trastuzumab, the first humanized monoclonal antibody that targets human epidermal growth factor receptor 2 (ERBB2/HER2), is currently used as a first-line treatment for HER2 (+) tumours. However, trastuzumab increases the risk of cardiac complications without affecting myocardial structure, suggesting a distinct mechanism of cardiotoxicity. We used medium from trastuzumab-treated human umbilical vein endothelial cells (HUVECs) to treat CCC-HEH-2 cells, the human embryonic cardiac tissue-derived cell lines, and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to assess the crosstalk between vascular endothelial cells (VECs) and cardiomyocytes. Protein mass spectrometry analysis was used to identify the key factors from VECs that regulate the function of cardiomyocytes. We applied RNA-sequencing to clarify the mechanism, by which PTX3 causes cardiac dysfunction. We used an anti-human/rat HER2 (neu) monoclonal antibody to generate a rat model that was used to evaluate the effects of trastuzumab on cardiac structure and function and the rescue effects of lapatinib on trastuzumab-induced cardiac side effects. Medium from trastuzumab-treated HUVECs apparently impaired the contractility of CCC-HEH-2 cells and iPSC-CMs. PTX3 from VECs caused defective cardiomyocyte contractility and cardiac dysfunction in mice, phenocopying trastuzumab treatment. PTX3 affected calcium homoeostasis in cardiomyocytes, which led to defective contractile properties. EGFR/STAT3 signalling in VECs contributed to the increased expression and release of PTX3. Notably, lapatinib, a dual inhibitor of EGFR/HER2, could rescue the cardiac complications caused by trastuzumab by blocking the release of PTX3. We identified a distinct mode of cardiotoxicity, wherein the activation of EGFR/STAT3 signalling by trastuzumab in VECs promotes PTX3 excretion, which contributes to the impaired contractility of cardiomyocytes by inhibiting cellular calcium signalling. We confirmed that lapatinib could be a feasible preventive agent against trastuzumab-induced cardiac complications and provided the rationale for the combined application of lapatinib and trastuzumab in cancer therapy.

Periodontal Tissue Regeneration by Transplantation of Autologous Adipose Tissue-Derived Multi-Lineage Progenitor Cells With Carbonate Apatite

We have developed an autologous transplantation method using adipose tissue-derived multi-lineage progenitor cells (ADMPCs) as a method of periodontal tissue regeneration that can be adapted to severe periodontal disease. Our previous clinical study confirmed the safety of autologous transplantation of ADMPCs and demonstrated its usefulness in the treatment of severe periodontal disease. However, in the same clinical study, we found that the fibrin gel used as the scaffold material might have caused gingival recession and impaired tissue regeneration in some patients. Carbonate apatite has a high space-making capacity and has been approved in Japan for periodontal tissue regeneration. In this study, we selected carbonate apatite as a candidate scaffold material for ADMPCs and conducted an in vitro examination of its effect on the cellular function of ADMPCs. We further performed autologous ADMPC transplantation with carbonate apatite as the scaffold material in a model of one-wall bone defects in beagles and then analyzed the effect on periodontal tissue regeneration. The findings showed that carbonate apatite did not affect the cell morphology of ADMPCs and that it promoted proliferation. Moreover, no effect on secretor factor transcription was found. The results of the in vivo analysis confirmed the space-making capacity of carbonate apatite, and the acquisition of significant new attachment was observed in the group involving ADMPC transplantation with carbonate apatite compared with the group involving carbonate apatite application alone. Our results demonstrate the usefulness of carbonate apatite as a scaffold material for ADMPC transplantation.

Neuroprotection for Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Early to intermediate AMD is characterized by the accumulation of lipid- and protein-rich drusen. Late stages of the disease are characterized by the development of choroidal neovascularization, termed "exudative" or "neovascular AMD," or retinal pigment epithelium (RPE) cell and photoreceptor death, termed "geographic atrophy" (GA) in advanced nonexudative AMD. Although we have effective treatments for exudative AMD in the form of anti-VEGF agents, they have no role for patients with GA. Neuroprotection strategies have emerged as a possible way to slow photoreceptor degeneration and vision loss in patients with GA. These approaches include reduction of oxidative stress, modulation of the visual cycle, reduction of toxic molecules, inhibition of pathologic protein activity, prevention of cellular apoptosis or programmed necrosis (necroptosis), inhibition of inflammation, direct activation of neurotrophic factors, delivery of umbilical tissue-derived cells, and RPE replacement. Despite active investigation in this area and significant promise based on preclinical studies, many clinical studies have not yielded successful results. We discuss selected past and current neuroprotection trials for AMD, highlight the lessons learned from these past studies, and discuss our perspective regarding remaining questions that must be answered before neuroprotection can be successfully applied in the field of AMD research.

In Vitro Infection Dynamics of Wuxiang Virus in Different Cell Lines.

Wuxiang virus (WUXV) is a newly discovered Bunyavirales transmitted by sandflies. It is found to infect humans and chickens and can cause neurological symptoms and even death in mice. However, the susceptibility of different hosts and tissue-derived cells to this virus is unclear. In this study, we examined cells derived from murine (BHK-21, N2A), human (HEK-293T, SH-SY5Y), dog (MDCK), pig (PK-15), monkey (Vero), and chicken (DF1), which were inoculated with WUXV at 0.05 MOI, and monitored for monolayer cytopathic effect (CPE). Culture supernatants and cells were collected from 0 to 96 h post-infection, cell viability was determined by trypan blue staining, numbers of infectious virus particles were quantified using plaque tests, and viral nucleic acid contents were determined by RT-qPCR. The presence of WUXV N antigen in infected cells was detected by Western blotting (WB). In response to virus infection, BHK-21, MDCK, and PK-15 cells were characterized by a clear CPE, and we observed reductions in the proportion of viable cells after 96 h. By contrast, no significant CPEs were observed in the other cell lines. We detected increases in viral titers, viral nucleic acid content, and N antigen expression in BHK-21, MDCK, PK-15, HEK-293T, N2A, SH-SY5Y, and DF1 cells post-infection. Vero cells showed no CPE, and the findings for other tests were negative. In conclusion, we tested the susceptibility of different cell lines to WUXV, enhanced our current understanding of WUXV biology at the cellular level, and laid the foundations for further investigation of the underlying virus infection mechanisms.

M1-like, but not M0- or M2-like, macrophages, reduce RSV infection of primary bronchial epithelial cells in a media-dependent fashion.

Respiratory syncytial virus (RSV) is a common childhood infection that in young infants can progress into severe bronchiolitis and pneumonia. Disease pathogenesis results from both viral mediated and host immune processes of which alveolar macrophages play an important part. Here, we investigated the role of different types of alveolar macrophages on RSV infection using an in vitro co-culture model involving primary tissue-derived human bronchial epithelial cells (HBECs) and human blood monocyte-derived M0-like, M1-like, or M2-like macrophages. It was hypothesized that the in vitro model would recapitulate previous in vivo findings of a protective effect of macrophages against RSV infection. It was found that macrophages maintained their phenotype for the 72-hour co-culture time period and the bronchial epithelial cells were unaffected by the macrophage media. HBEC infection with RSV was decreased by M1-like macrophages but enhanced by M0- or M2-like macrophages. The medium used during the co-culture also impacted the outcome of the infection. This work demonstrates that alveolar macrophage phenotypes may have differential roles during epithelial RSV infection, and demonstrates that an in vitro co-culture model could be used to further investigate the roles of macrophages during bronchial viral infection.

Genomic screening reveals ubiquitin-like modifier activating enzyme 1 as a potent and druggable target in c-MYC-high triple negative breast cancer models.

Triple negative breast cancer (TNBC) accounts for over 30% of all breast cancer (BC)-related deaths, despite accounting for only 10% to 15% of total BC cases. Targeted therapy development has largely stalled in TNBC, underlined by a lack of traditionally druggable addictions like receptor tyrosine kinases (RTKs). Here, through full genome CRISPR/Cas9 screening of TNBC models, we have uncovered the sensitivity of TNBCs to the depletion of the ubiquitin-like modifier activating enzyme 1 (UBA1). Targeting UBA1 with the first-in-class UBA1 inhibitor TAK-243 induced unresolvable endoplasmic reticulum (ER)-stress and activating transcription factor 4 (ATF4)-mediated upregulation of proapoptotic NOXA, leading to cell death. c-MYC expression correlates with TAK-243 sensitivity and cooperates with TAK-243 to induce a stress response and cell death. Importantly, there was an order of magnitude greater sensitivity of TNBC lines to TAK-243 compared to normal tissue-derived cells. In five patient derived xenograft models (PDXs) of TNBC, TAK-243 therapy led to tumor inhibition or frank tumor regression. Moreover, in an intracardiac metastatic model of TNBC, TAK-243 markedly reduced metastatic burden, indicating UBA1 is a potential new target in TNBC expressing high levels of c-MYC.

Revealing the characteristics of ZIKV infection through tissue-specific transcriptome sequencing analysis

BackgroundRecently, Zika virus (ZIKV) re-emerged in India and was potentially associated with microcephaly. However, the molecular mechanisms underlying ZIKV pathogenesis remain to be explored.ResultsHerein, we performed a comprehensive RNA-sequencing analysis on ZIKV-infected JEG-3, U-251 MG, and HK-2 cells versus corresponding uninfected controls. Combined with a series of functional analyses, including gene annotation, pathway enrichment, and protein–protein interaction (PPI) network analysis, we defined the molecular characteristics induced by ZIKV infection in different tissues and invasion time points. Data showed that ZIKV infection and replication in each susceptible organ commonly stimulated interferon production and down-regulated metabolic-related processes. Also, tissue-specific immune responses or biological processes (BPs) were induced after ZIKV infection, including GnRH signaling pathway in JEG-3 cells, MAPK signaling pathway in U-251 MG cells, and PPAR signaling pathway in HK-2 cells. Of note, ZIKV infection induced delayed antiviral interferon responses in the placenta-derived cell lines, which potentially explains the molecular mechanism by which ZIKV replicates rapidly in the placenta and subsequential vertical transmission occurs.ConclusionsTogether, these data may provide a systemic insight into the pathogenesis of ZIKV infection in distinct human tissue-derived cell lines, which is likely to help develop prophylactic and therapeutic strategies against ZIKV infection.

(Not) Home alone: Antigen presenting cell - T Cell communication in barrier tissues.

Priming of T cells by antigen presenting cells (APCs) is essential for T cell fate decisions, enabling T cells to migrate to specific tissues to exert their effector functions. Previously, these interactions were mainly explored using blood-derived cells or animal models. With great advances in single cell RNA-sequencing techniques enabling analysis of tissue-derived cells, it has become clear that subsets of APCs are responsible for priming and modulating heterogeneous T cell effector responses in different tissues. This composition of APCs and T cells in tissues is essential for maintaining homeostasis and is known to be skewed in infection and inflammation, leading to pathological T cell responses. This review highlights the commonalities and differences of T cell priming and subsequent effector function in multiple barrier tissues such as the skin, intestine and female reproductive tract. Further, we provide an overview of how this process is altered during tissue-specific infections which are known to cause chronic inflammation and how this knowledge could be harnessed to modify T cell responses in barrier tissue.

Knee meniscus regeneration using autogenous injection of uncultured adipose tissue-derived regenerative cells.

IntroductionThe low healing potential of mature menisci necessitates traditional surgical removal (meniscectomy) to eliminate acute or chronic degenerative tears. However, removal of meniscal tissue is main factor causing osteoarthritis. Adipose tissue-derived regenerative cells (ADRCs), a heterogeneous cell population that includes multipotent adipose-derived stem cells and other progenitor cells, were easily isolated in large amounts from autologous adipose tissue, and same-day processing without culture or expansion was possible. This study investigated the regenerative potential of autologous ADRCs for use in meniscus defects.MethodsIn 10- to 12-week-old male SD rat partial meniscectomy model, an atelocollagen sponge scaffold without or with ADRCs (5.0 × 105 cells) was injected into each meniscus defect. Reconstructed menisci were subjected to histologic, and dynamic mechanical analyses.ResultsAfter 12 weeks, areas of regenerated meniscal tissue in the atelocollagen sponge scaffold in rats with ADRCs (64.54 ± 0.52%, P < 0.05, n = 10) were larger than in those without injection (57.96 ± 0.45%). ADRCs were shown capable of differentiating chondrocyte-like cells and meniscal tissue components such as type II collagen. Higher elastic moduli and lower fluid permeability of regenerated meniscal tissue demonstrated a favorable structure-function relationship required for native menisci, most likely in association with micron-scale porosity, with the lowest level for tissue integrity possibly reproducible.ConclusionsThis is the first report of meniscus regeneration induced by injection of ADRCs. The results indicate that ADRCs will be useful in future clinical cell-based therapy strategies, including as a cell source for reconstruction of damaged knee menisci.

Isolation of Lymphocytes from Human Skin and Murine Tissues: A Rapid and Epitope-Preserving Approach

Tissue-resident immune cells have been shown to play an important role in skin health and disease. However, owing to limited access to human skin samples and time-consuming, technically demanding protocols, the characterization of tissue-derived cells remains challenging. For this reason, blood-derived leukocytes are frequently used as a surrogate specimen, although they do not necessarily reflect local immune responses in the skin. Therefore, we aimed to establish a rapid protocol to isolate a sufficient number of viable immune cells from 4-mm skin biopsies that can be directly used for a deeper characterization such as comprehensive phenotyping and functional studies of T cells. In this optimized protocol, only two enzymes, type IV collagenase and DNase I, were used to achieve both the highest possible cellular yield and marker preservation of leukocytes stained for multicolor flow cytometry. We further report that the optimized protocol may be used in the same manner for murine skin and mucosa. In summary, this study allows a rapid acquisition of lymphocytes from human or mouse skin suitable for comprehensive analysis of lymphocyte subpopulations, for disease surveillance, and for identification of potential therapeutic targets or other downstream applications.

Establishing and characterising a new cell line from Calliphora vicina (diptera: calliphoridae) fly embryonic tissues

Insect cell lines represent a promising and expanding field as they have several research applications including biotechnology, virology, immunity, toxicology, cell signalling mechanisms and evolution. They constitute a powerful tool having a direct impact on human and veterinary medicine and agriculture. Although more than 1000 cell lines have currently been established from various insect species, Calliphora vicina-derived fly cell lines are lacking. This study was aimed at establishing a new C. vicina embryonic tissue-derived cell line. Adult flies were collected and embryonated eggs were mechanically homogenised and seeded in four types of culture media (L15, Grace's insect medium, Grace's/L15 and DMEM). Cell growth and morphological characteristics were recorded and cytogenetic and molecular patterns were determined. The CV-062020-PPB cell line was established and was shown to have optimal growth in Grace's/L15 medium. CV-062020-PPB cell monolayers that had been sub-cultured over 16 times consisted of firmly adhering cells having different morphologies; a fibroblast-like shape dominated and the karyotype had a 12-chromosome diploid number. RAPD-PCR analysis of the CV-062020-PPB cell line revealed a high similarity index and strong intraspecific relationship with C. vicina adult flies and a weaker relationship with the Lutzomyia longipalpis-derived cell line (Lulo). The CV-062020-PPB cell line constitutes the first cell line obtained from C. vicina embryonic tissues and represents an important basic and applied research tool.

Effect of Crohn's disease mesenteric mesenchymal stem cells and their extracellular vesicles on T-cell immunosuppressive capacity.

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal intestinal tract and has characteristic hypertrophic adipose changes observed in the mesentery. To better understand the role of the mesentery in the pathophysiology of Crohn's disease (CD), we evaluated the immunomodulatory potential of mesenchymal stem cells (MSCs) and their secreted extracellular vesicles (EVs) derived from Crohn's patients. MSCs and EVs were isolated from the mesentery and subcutaneous tissues of CD patients and healthy individuals subcutaneous tissues, and were analysed for differentiation, cytokine expression, self‐renewal and proliferation. The varying capacity of these tissue‐derived MSCs and EVs to attenuate T‐cell activation was measured in in vitro and an in vivo murine model. RNA sequencing of inflamed Crohn's disease mesentery tissue revealed an enrichment of T‐cell activation compared to non‐inflamed subcutaneous tissue. MSCs and MSC‐derived EVs isolated from Crohn's mesentery lose their ability to attenuate DSS‐induced colitis compared to subcutaneous tissue‐derived cell or EV therapy. We found that treatment with subcutaneous isolated MSCs and their EV product compared to Crohn's mesentery MSCs or EVs, the inhibition of T‐cell proliferation and IFN‐γ, IL‐17a production increased, suggesting a non‐inflamed microenvironment allows for T‐cell inhibition by MSCs/EVs. Our results demonstrate that Crohn's patient‐derived diseased mesentery tissue MSCs lose their immunosuppressive capacity in the treatment of colitis by distinct regulation of pathogenic T‐cell responses and/or T‐cell infiltration into the colon.

Adipose tissue-derived Muse cells promote autophagy and oxidative stress tolerance in human epidermal melanocytes.

To investigate the effect of human adipose tissue-derived multilineage-differentiating stress-enduring (Muse) cells on the oxidative stress injury of human epidermal melanocytes (HEMs) in vitro. HEMs were treated with H2O2 to establish an oxidative stress injury model and then were co-cultured with adipose tissue-derived Muse cells. Immunohistochemistry, flow cytometry and Western blotting were used to assess changes in autophagy flux, apoptosis, expression of melanin synthesis related proteins and proliferation of melanocytes. Our findings demonstrate that co-culture with Muse cells significantly increased the tolerance of HEMs to oxidative stress, enhanced autophagy flux and reduced apoptosis. The expression of proteins related to the formation of melanin increased as did cell proliferation. Treatment with the autophagy inhibitor, 3-methyladenine (3MA), partially counteracted the improvement of oxidative stress tolerance in melanocytes elicited by co-culture with Muse cells. Muse cells promote autophagy and oxidative stress tolerance of melanocytes.

Iris-derived induced pluripotent stem cells that express GFP in all somatic cells of mice and differentiate into functional retinal neurons

When regenerated tissue is generated from induced pluripotent stem cells (iPSCs), it is necessary to track and identify the transplanted cells. Fluorescently-labeled iPSCs synthesize a fluorescent substance that is easily tracked. However, the expressed protein should not affect the original genome sequence or pluripotency. To solve this problem, we created a cell tool for basic research on iPSCs. Iris tissue-derived cells from GFP fluorescence-expressing mice (GFP-DBA/2 mice) were reprogrammed to generate GFP mouse iris-derived iPSCs (M-iris GFP iPSCs). M-iris GFP iPSCs expressed cell markers characteristic of iPSCs and showed pluripotency in differentiating into the three germ layers. In addition, when expressing GFP, the cells differentiated into functional recoverin- and calbindin-positive cells. Thus, this cell line will facilitate future studies on iPSCs.

Interleukin-33 (IL-33): A critical review of its biology and the mechanisms involved in its release as a potent extracellular cytokine.

Interleukin-33 (IL-33), a member of the IL-1 family, is an alarmin cytokine with crucial roles in tissue homeostasis and repair, type 2 immunity, allergic and non-allergic inflammation, viral infection, and cancer. IL-33 is abundant in the nuclei of tissue-derived cells, including endothelial cells from blood vessels, epithelial cells from barrier tissues, and fibroblastic stromal cells from various tissues. IL-33 is released upon cell damage or tissue injury and activates Myd88-dependent signaling pathways in cells expressing the ST2 (IL-1RL1) receptor. Analysis of patient samples and studies in murine models support an important role of IL-33/ST2 signaling in allergic inflammation in different tissues (lung, nasopharynx, skin) and diseases (asthma, chronic rhinosinusitis, allergic rhinitis, atopic dermatitis). IL33 and IL1RL1/ST2 are among the most highly replicated susceptibility loci for asthma. However, the IL-33/ST2 pathway is also important in non-allergic inflammation. Indeed, targets of IL-33 include immune cells involved in both type 2 and type 1 immunity and regulatory responses, such as group 2 innate lymphoid cells (ILC2s), mast cells, regulatory T cells (Tregs), Th2 cells, basophils, eosinophils, macrophages, dendritic cells (DCs), neutrophils, Th1 cells, CD8 T cells, NK and iNKT cells. In the main part of this review, we discuss the basic biology of the IL-33 protein (molecular characteristics, nuclear localization, cellular sources in vivo), and its mechanisms of release, and bioactive forms in various contexts. Importantly, we alert the scientific community to the problems of specificity of IL-33 reagents, we explain why studies without specificity controls with IL-33-deficient cells are misleading to the field and lead to unnecessary controversy, and we make recommendations to generate reliable results. In the final part, we review the genetic and environmental regulation of IL-33 in allergic airway inflammation and asthma, and we highlight recent studies showing clinical efficacy of anti-IL-33 antibodies in asthma and chronic obstructive pulmonary disease (COPD).