Marker Expression
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Roos Houtsma ◽  
Nisha K. van der Meer ◽  
Kees Meijer ◽  
Linde Morsink ◽  
Shanna M. Hogeling ◽  

Acute myeloid leukemia (AML) often presents as an oligoclonal disease whereby multiple genetically distinct subclones can co-exist within patients. Differences in signaling and drug sensitivity of such subclones complicates treatment and warrants tools to identify them and track disease progression. We previously identified over 50 AML-specific plasma membrane (PM) proteins and seven of these (CD82, CD97, FLT3, IL1RAP, TIM3, CD25 and CD123) were implemented in routine diagnostics in patients with AML (n=256) and MDS (n=33). We developed a pipeline termed CombiFlow in which expression data of multiple PM markers is merged, allowing a Principle Component-based analyses to identify distinctive marker expression profiles and to generate single cell tSNE landscapes to longitudinally track clonal evolution. Positivity for one or more of the markers after 2 courses of intensive chemotherapy predicted a shorter relapse-free survival supporting a role of these markers in measurable residual disease (MRD) detection. CombiFlow also allowed the tracking of clonal evolution in paired diagnosis and relapse samples (n=12). Extending the panel to 36 AML-specific markers further refined the CombiFlow pipeline. In conclusion, CombiFlow provides a valuable tool in the diagnosis, MRD detection, clonal tracking, and the understanding of clonal heterogeneity in AML.

2021 ◽  
Renata Skovronova ◽  
Cristina Grange ◽  
Veronica Dimuccio ◽  
Maria Chiara Deregibus ◽  
Giovanni Camussi ◽  

Extracellular vesicles released by mesenchymal stromal cells (MSC EVs) are a promising resource for regenerative medicine. In particular, small MSC EVs represent the active EV fraction for therapeutic applications. A bulk analysis is applied to characterize MSC EVs identity and purity, coupled with the assessment of single EV morphology, size and integrity using electron microscopy. We here applied different orthogonal methods to provide a quantitative analysis of size and surface marker expression in medium/large and small fractions, namely 10k and 100k fractions, of MSC EVs obtained by sequential ultracentrifugations. Bone marrow, adipose tissue, and umbilical cord MSC EVs were compared, in naive and apoptotic conditions. The 100k EV size <100 nm, as detected by electron microscopy, was confirmed by super-resolution microscopy and ExoView. Quantitative single vesicle imaging using super-resolution microscopy revealed heterogeneous patterns of tetraspanin expressions, being all MSC EV fractions single, double and triple positive, in variable proportions, for CD63, CD81 and CD9. Moreover, ExoView analysis allowed a comparative multiplex screening of single MSC EV tetraspanin and mesenchymal marker levels. Finally, a semiquantitative bead based cytofluorimetric analysis showed the segregation of immunological and pro-coagulative markers on the 10k MSC EV fraction. Apoptotic MSC EVs were released in higher number, without significant differences from the naive fractions in surface marker expression. These results indicate that a consistent profile of MSC EV fractions among the different MSC sources, and a safer profile of the 100k MSC EV population for clinical application. Finally, our study identified suitable applications for different EV analytical techniques.

Cartilage ◽  
2021 ◽  
pp. 194760352110424
Elizabeth Vinod ◽  
Kawin Padmaja ◽  
Abel Livingston ◽  
Jithu Varghese James ◽  
Soosai Manickam Amirtham ◽  

Purpose Chondrocytes, isolated from articular cartilage, are routinely utilized in cell-based therapeutics for the treatment of cartilage pathologies. However, restoration of the biological tissue faces hindrance due to the formation of primarily fibrocartilaginous repair tissue. Chondroprogenitors have been reported to display superiority in terms of their chondrogenic potential and lesser proclivity for hypertrophy. In line with our recent results, comparing chondroprogenitors and chondrocytes, we undertook isolation of progenitors from the general pool of chondrocytes, based on surface marker expression, namely, CD166, CD34, and CD146, to eliminate off-target differentiation and generate cells of stronger chondrogenic potential. This study aimed to compare chondrocytes, chondroprogenitors, CD34−CD166+CD146+ sorted chondrocytes, and CD34−CD166+CD146− sorted chondrocytes. Methods Chondrocytes obtained from 3 human osteoarthritic knee joints were subjected to sorting, to isolate CD166+ and CD34− subsets, and then were further sorted to obtain CD146+ and CD146− cells. Chondrocytes and fibronectin adhesion-derived chondroprogenitors served as controls. Assessment parameters included reverse transcriptase polymerase chain reaction for markers of chondrogenesis and hypertrophy, trilineage differentiation, and total GAG/DNA content. Results Based on gene expression analysis, CD34−CD166+CD146+ sorted chondrocytes and chondroprogenitors displayed comparability and significantly higher chondrogenesis with a lower tendency for hypertrophy when compared to chondrocytes and CD34−CD166+CD146− sorted chondrocytes. The findings were also reiterated in multilineage potential differentiation with the 146+ subset and chondroprogenitors displaying lower calcification and chondroprogenitors displaying higher total GAG/DNA content compared to chondrocytes and 146− cells. Conclusion This unique progenitor-like population based on CD34−CD166+CD146+ sorting from chondrocytes exhibits efficient potential for cartilage repair and merits further evaluation for its therapeutic application.

2021 ◽  
Rui Li ◽  
Dize Li ◽  
Huanan Wang ◽  
Kaiwen Chen ◽  
Si Wang ◽  

Abstract Objectives: Bone defects caused by diseases and trauma are usually accompanied by inflammation, and the implantation of biomaterials as a common repair method has also been found to cause inflammatory reactions, which affect bone metabolism and new bone formation. This study investigated whether exosomes from adipose-derived stem cells (ADSC-Exos) plays an immunomodulatory role in traumatic bone defects and elucidated the underlying mechanisms.Methods:ADSC-Exos were loaded by a biomaterial named gelatine nanoparticles (GNPs), physical and chemical properties were analyzed by zeta potential, surface topography and rheology. A rat model of skull defect was used for our in vivo studies, micro-CT and histological staining were used to analyse histological changes in the bone defect area. RT-qPCR and western blotting were performed to verify that ADSC-Exos could regulate M1/M2 macrophage polarization. MicroRNA (miRNA) array analysis was conducted to determine the miRNA expression profiles of ADSC-Exos. After macrophages were treated with a miR-451a mimic, miR-451a inhibitor and ISO-1, the relative expression of genes and proteins was measured by RT-qPCR and western blotting.Results: In vivo, micro-CT and histological staining showed that exosome-loaded GNPs(GNP-Exos) hydrogel, with good biocompatibility and strong mechanical adaptability,exhibited immunomodulatory effect mainly by regulating macrophage immunity and promoting bone tissue healing. Immunofluorescence further indicated that ADSC-Exos reduced M1 marker (iNOS) expression and increased M2 marker (CD206) expression. Moreover, in vitro studies, western blotting and RT-qPCR showed that ADSC-Exos inhibited M1 macrophage marker expression and upregulated M2 macrophage marker expression. MiR-451a was enriched in ADSC-Exos and targeted macrophage migration inhibitory factor (MIF). Macrophages treated with the miR-451a mimic showed lower expression of M1 markers. In contrast, miR-451a inhibitor treatment upregulated the expression of M1 markers and downregulated the expression of M2 markers, while ISO-1 (a MIF inhibitor) treatment upregulated miR-451a expression and downregulated M1 macrophage marker expression.Conclusion: GNP-Exos can effectively regulate bone immune metabolism and futher promote bone healing partly through immune regulation of miR-451a,which may provide a therapeutic direction for bone repair.

2021 ◽  
Patricia Kaaijk ◽  
Veronica Olivo Pimentel ◽  
Maarten E. Emmelot ◽  
Martien Poelen ◽  
Alper Cevirgel ◽  

Background: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to considerable morbidity/mortality worldwide, but most infections, especially among children, have a mild course. However, it remains largely unknown whether infected children develop cellular immune memory. Methods: To determine whether a memory T cell response is being developed as an indicator for long-term immune protection, we performed a longitudinal assessment of the SARS-CoV-2-specific T cell response by IFN-γ ELISPOT and activation marker expression analyses of peripheral blood samples from children and adults with mild-to-moderate COVID-19. Results: Upon stimulation of PBMCs with heat-inactivated SARS-CoV-2 or overlapping peptides of spike (S-SARS-CoV-2) and nucleocapsid proteins, we found S-SARS-CoV-2-specific IFN-ɣ T cell responses in most infected children (83%) and all adults (100%) that were absent in unexposed controls. Frequencies of SARS-CoV-2-specific T cells were higher in infected adults, especially in those with moderate symptoms, compared to infected children. The S-SARS-CoV-2 IFN-ɣ T cell response correlated with S1-SARS-CoV-2-specific serum IgM, IgG, and IgA antibody concentrations. Predominantly, effector memory CD4+ T cells of a Th1 phenotype were activated upon exposure to SARS-CoV-2 antigens, which persisted for 4-8 weeks after symptom onset. We detected very low frequencies of SARS-CoV-2-reactive CD8+ T cells in these individuals. Conclusions: Our data indicate that an antigen-specific memory CD4+ T cell response is induced in children and adults with mild SARS-CoV-2 infection. T cell immunity induced after mild COVID-19 could contribute to protection against re-infection.

Metabolites ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 626
Wan Hua ◽  
Sarantos Kostidis ◽  
Oleg Mayboroda ◽  
Martin Giera ◽  
Marten Hornsveld ◽  

The cytokine transforming growth factor-β (TGF-β) can induce normal breast epithelial cells to take on a mesenchymal phenotype, termed epithelial-to-mesenchymal transition (EMT). While the transcriptional and proteomic changes during TGF-β-induced EMT have been described, the metabolic rewiring that occurs in epithelial cells undergoing EMT is not well understood. Here, we quantitively analyzed the TGF-β-induced metabolic reprogramming during EMT of non-transformed NMuMG mouse mammary gland epithelial cells using nuclear magnetic resonance (NMR) spectroscopy. We found that TGF-β elevates glycolytic and tricarboxylic acid (TCA)-cycle activity and increases glutaminolysis. Additionally, TGF-β affects the hexosamine pathway, arginine-proline metabolism, the cellular redox state, and strongly affects choline metabolism during EMT. TGF-β was found to induce phosphocholine production. A kinase inhibitor RSM-93A that inhibits choline kinase α (CHKα) mitigated TGF-β-induced changes associated with EMT, i.e., increased filamentous (F)-actin stress fiber formation and N-Cadherin mesenchymal marker expression.

2021 ◽  
Vol 71 (1) ◽  
Yongzhong Mao ◽  
Ling Jiang

Abstract Background Endothelial to mesenchymal transition (EndMT) participates in the progression of atherosclerosis (AS). MiR-200c-3p has been implicated in EndMT. However, the functional role of miR-200c-3p in AS remains largely unknown. Here, we demonstrated the critical role of miR-200c-3p in regulating EndMT in AS. Methods ApoE−/− mice were fed with high-fat diet to establish AS mouse model, and human umbilical vein endothelial cells (HUVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to mimic AS cell model. The expression of miR-200c-3p, SMAD7 and YAP in ApoE−/− mice and HUVECs was detected by quantitative real-time PCR. Rhodamine phalloidin staining and Western blot were performed to observe cell morphology and EndMT marker expression of HUVECs. Luciferase reporter assay and Co-Immunoprecipitation were performed to verify the relationship among miR-200c-3p, SMAD7, and YAP. Results MiR-200c-3p was highly expressed, and SMAD7 and YAP were down-regulated in the aortic tissues of ApoE−/− mice and ox-LDL-treated HUVECs. MiR-200c-3p overexpression promoted the transformation of ox-LDL-treated HUVECs from cobblestone-like epithelial phenotype to a spindle-like mesenchymal phenotype. Meanwhile, miR-200c-3p up-regulation repressed the expression of endothelial markers CD31 and vWF and promoted the expression of mesenchymal markers α-SMA and vimentin in the ox-LDL-treated HUVECs. MiR-200c-3p inhibited SMAD7 and YAP expression by interacting with 3′ untranslated region of SMAD7. Moreover, miR-200c-3p promoted EndMT in ox-LDL-treated HUVECs by inhibiting SMAD7/YAP pathway. Conclusion This work demonstrated that MiR-200c-3p promoted ox-LDL-induced EndMT in HUVECs through SMAD7/YAP pathway, which may be important for the onset of atherosclerosis.

2021 ◽  
Na Wang ◽  
Siran Wu ◽  
Jing Zhao ◽  
Min Chen ◽  
Jiaoxia Zeng ◽  

Abstract Purpose Intestinal metaplasia (IM) is a precancerous lesion that increases the risk of subsequent gastric cancer (GC) development. Previously, miR-1 has been shown to play an essential role in the initiation of bile acid (BA)-induced IM. The objective of the present study was to investigate the mechanism underlying miR-1 inhibition by BA in gastric cells. Methods Ingenuity pathway analysis (IPA) was used to identify molecules acting upstream of miR-1. The effects of deoxycholic acid (DCA), FXR and SNAI2 on the expression of intestinal markers were assessed using quantitative real-time PCR (qRT-PCR) and Western blotting. The expression level of major molecules was detected by immunohistochemistry (IHC) in tissue microarrays. The transcriptional regulation of miR-1 was verified using luciferase reporter and chromatin immunoprecipitation (ChIP) assays. Results We found that BA treatment caused aberrant expression of FXR and intestinal markers in gastric cells. Augmented FXR led to transcriptional activation of SNAI2, which in turn suppressed the miR-1 promoter. Moreover, we found that compared with normal tissues, the expression levels of both FXR and SNAI2 were increased and positively correlated with each other in IM tissues. Additionally, their expression showed an inverse correlation with that of miR-1 in IM tissues. Conclusions Our findings indicate that FXR may be responsible for a series of molecular changes in gastric cells after BA treatment, and that the FXR/SNAI2/miR-1 axis exhibits a crucial role in BA-induced progression of IM. Blocking the FXR-oriented axis may provide a promising approach for IM or even GC treatment.

Phuong T. M. Dam ◽  
Van T. Hoang ◽  
Hue Thi Hong Bui ◽  
Le Minh Hang ◽  
Duc M. Hoang ◽  

Abstract Background We have observed an increased expression of negative markers in some clinical-grade, xeno- and serum-free cultured adipose-derived mesenchymal stem/stromal cell (ADMSC) samples. It gave rise to concern that xeno- and serum-free conditions might have unexpected effects on human ADMSCs. This study aims to test this hypothesis for two xeno- and serum-free media, PowerStem MSC1 media (PS) and StemMACS MSC Expansion Media (SM), that support the in vitro expansion of ADMSCs. Methods We investigated the expression of negative markers in 42 clinical-grade ADMSC samples expanded in PS. Next, we cultured ADMSCs from seven donors in PS and SM and examined their growth and colony-forming ability, surface marker expression, differentiation, cell cycle and senescence, as well as genetic stability of two passages representing an early and late passage for therapeutic MSCs. Results 15 of 42 clinical-grade PS-expanded ADMSC samples showed an increased expression of negative markers ranging from 2.73% to 34.24%, which positively correlated with the age of donors. This rise of negative markers was related to an upregulation of Human Leukocyte Antigen – DR (HLA-DR). In addition, the PS-cultured cells presented decreased growth ability, lower frequencies of cells in S/G2/M phases, and increased ß-galactosidase activity in passage 7 suggesting their senescent feature compared to those grown in SM. Although MSCs of both PS and SM cultures were capable of multilineage differentiation, the PS-cultured cells demonstrated chromosomal abnormalities in passage 7 compared to the normal karyotype of their SM counterparts. Conclusions These findings suggest that the SM media is more suitable for the expansion of therapeutic ADMSCs than PS. The study also hints a change of ADMSC features at more advanced passages and with increased donor’s age. Thus, it emphasizes the necessity to cover these aspects in the quality control of therapeutic MSC products. Graphical abstract

2021 ◽  
Xiaofei Wang ◽  
Rui Zhang ◽  
Bryce D Lindaman ◽  
Caitlin N Leeper ◽  
Adam G Schrum ◽  

Vasoactive intestinal peptide (VIP) is a neuropeptide capable of downregulating innate immune responses in antigen presenting cells (APCs) by suppressing their pro-inflammatory cytokine secretion and cell surface marker expression. Though VIP's bioactivity could possibly be leveraged as a treatment for autoimmune disorders and transplant tolerance, drug delivery innovation is required to overcome its intrinsically limited cellular delivery capacity due to its short in vivo lifetime. One option is to employ peptide amphiphiles (PAs) which are lipidated peptides capable of self-assembling into micelles in water that can enhance cellular association. With this approach in mind, a series of triblock VIP amphiphiles (VIPAs) has been synthesized to explore the influence of block arrangement and hydrophobicity on micelle biocompatibility and bioactivity. VIPA formulation has been found to influence the shape, size, and surface charge of VIPA micelles (VIPAMs) as well as their cytotoxicity and immunomodulatory effects. Specifically, the enclosed work provides strong evidence that cylindrical VIPAMs with aspect ratios of 1.5 - 150 and moderate positive surface charge are able to potentiate the bioactivity of VIP limiting TNF-a; secretion and MHC II and CD86 surface expression on APCs. With this criteria, we have identified PalmK-(EK)4-VIP as our lead formulation, which showed comparable or enhanced anti-inflammatory effects relative to the unmodified VIP at all dosages evaluated. Additionally, the relationships between peptide block location and lipid block size provide further information on the chemistry-structure-function relationships of peptide amphiphile micelles for the delivery of VIP as well as potentially for other peptides more broadly.

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