Biological and molecular profile of fracture non‐union tissue: A systematic review and an update on current insights

Fracture non‐union represents a common complication, seen in 5%–10% of all acute fractures. Despite the enhancement in scientific understanding and treatment methods, rates of fracture non‐union remain largely unchanged over the years. This systematic review investigates the biological, molecular and genetic profiles of both (i) non‐union tissue and (ii) non–union‐related tissues, and the genetic predisposition to fracture non‐union. This is crucially important as it could facilitate earlier identification and targeted treatment of high‐risk patients, along with improving our understanding on pathophysiology of fracture non‐union. Since this is an update on our previous systematic review, we searched the literature indexed in PubMed Medline; Ovid Medline; Embase; Scopus; Google Scholar; and the Cochrane Library using Medical Subject Heading (MeSH) or Title/Abstract words (non‐union(s), non‐union(s), human, tissue, bone morphogenic protein(s) (BMPs) and MSCs) from August 2014 (date of our previous publication) to 2 October 2021 for non‐union tissue studies, whereas no date restrictions imposed on non–union‐related tissue studies. Inclusion criteria of this systematic review are human studies investigating the characteristics and properties of non‐union tissue and non–union‐related tissues, available in full‐text English language. Limitations of this systematic review are exclusion of animal studies, the heterogeneity in the definition of non‐union and timing of tissue harvest seen in the included studies, and the search term MSC which may result in the exclusion of studies using historical terms such as ‘osteoprogenitors’ and ‘skeletal stem cells’. A total of 24 studies (non‐union tissue: n = 10; non–union‐related tissues: n = 14) met the inclusion criteria. Soft tissue interposition, bony sclerosis of fracture ends and complete obliteration of medullary canal are commonest macroscopic appearances of non‐unions. Non‐union tissue colour and surrounding fluid are two important characteristics that could be used clinically to distinguish between septic and aseptic non‐unions. Atrophic non‐unions had a predominance of endochondral bone formation and lower cellular density, when compared against hypertrophic non‐unions. Vascular tissues were present in both atrophic and hypertrophic non‐unions, with no difference in vessel density between the two. Studies have found non‐union tissue to contain biologically active MSCs with potential for osteoblastic, chondrogenic and adipogenic differentiation. Proliferative capacity of non‐union tissue MSCs was comparable to that of bone marrow MSCs. Rates of cell senescence of non‐union tissue remain inconclusive and require further investigation. There was a lower BMP expression in non‐union site and absent in the extracellular matrix, with no difference observed between atrophic and hypertrophic non‐unions. The reduced BMP‐7 gene expression and elevated levels of its inhibitors (Chordin, Noggin and Gremlin) could potentially explain impaired bone healing observed in non‐union MSCs. Expression of Dkk‐1 in osteogenic medium was higher in non‐union MSCs. Numerous genetic polymorphisms associated with fracture non‐union have been identified, with some involving the BMP and MMP pathways. Further research is required on determining the sensitivity and specificity of molecular and genetic profiling of relevant tissues as a potential screening biomarker for fracture non‐unions.     

Influence of age on stem cells depends on the sex of the bone marrow donor

Ageing is often accompanied by an increase in bone marrow fat together with reduced bone volume and diseases of the bone such as osteoporosis. As mesenchymal stem cells (MSCs) are capable of forming bone, cartilage and fat tissue, studying these cells is of great importance to understand the underlying mechanisms behind age‐related bone diseases. However, inter‐donor variation has been found when handling MSCs. Therefore, the aim of this study was to investigate the effects of donor age and sex by comparing in vitro characteristics of human bone marrow‐derived MSCs (hBMSCs) from a large donor cohort (n = 175). For this, hBMSCs were analysed for CFU‐F capacity, proliferation, differentiation capacity and surface antigen expression under standardized culture conditions. The results demonstrated a significantly reduced CFU‐F number for hBMSCs of female compared to male donors. Furthermore, there was a significant decrease in the proliferation rate, adipogenic differentiation potential and cell surface expression of SSEA‐4, CD146 and CD274 of hBMSCs with an increase in donor age. Interestingly, all these findings were exclusive to hBMSCs from female donors. Further research should focus on postmenopausal‐related effects on hBMSCs, as the results imply a functional loss and immunophenotypic change of hBMSCs particularly in aged women.     

Heterogeneous expression of ACE2 and TMPRRS2 in mesenchymal stromal cells

The outbreak of COVID‐19 has become a serious public health emergency. The virus targets cells by binding the ACE2 receptor. After infection, the virus triggers in some humans an immune storm containing the release of proinflammatory cytokines and chemokines followed by multiple organ failure. Several vaccines are enrolled, but an effective treatment is still missing. Mesenchymal stem cells (MSCs) have shown to secrete immunomodulatory factors that suppress this cytokine storm. Therefore, MSCs have been suggested as a potential treatment option for COVID‐19. We report here that the ACE2 expression is minimal or nonexistent in MSC derived from three different human tissue sources (adipose tissue, umbilical cord Wharton`s jelly and bone marrow). In contrast, TMPRSS2 that is implicated in SARS‐CoV‐2 entry has been detected in all MSC samples. These results are of particular importance for future MSC‐based cell therapies to treat severe cases after COVID‐19 infection.     

The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

The manufacturing of viable and functional β‐cell spheroids is required for diabetes cell therapy and drug testing. Mesenchymal stromal/stem cells (MSCs) are known to improve β‐cell viability and functionality. We therefore investigated the aggregation behavior of three different β‐cell lines (rat insulinoma‐1 cell line [INS‐1], mouse insulinoma‐6 cell line [MIN6], and a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells [1.1B4]), two MSC types, and mixtures of β‐cells and MSCs under different conditions. We screened several static systems to produce uniform β‐cell and MSC spheroids, finding cell‐repellent plates the most suitable. The three different β‐cell lines differed in their aggregation behavior, spheroid size, and growth in the same static environment. We found no major differences in spheroid formation between primary MSCs and an immortalized MSC line, although both differed with regard to the aggregation behavior of the β‐cell lines. All spheroids showed a reduced viability due to mass transfer limitations under static conditions. We therefore investigated three dynamic systems (shaking multi‐well plates, spinner flasks, and shaking flasks). In shaking flasks, there were no β‐cell‐line‐dependent differences in aggregation behavior, resulting in uniform and highly viable spheroids. We found that the aggregation behavior of the β‐cell lines changed in a static coculture with MSCs. The β‐cell/MSC coculture conditions must be refined to avoid a rapid segregation into distinct populations under dynamic conditions.     

An optimized method for obtaining clinical‐grade specific cell subpopulations from human umbilical cord‐derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are heterogeneous populations with broad application prospects in cell therapy, and using specific subpopulations of MSCs can enhance their particular capability under certain conditions and achieve better therapeutic effects. However, no studies have reported how to obtain high‐quality specific MSC subpopulations in vitro culture. Here, for the first time, we established a general operation process for obtaining high‐quality clinical‐grade cell subpopulations from human umbilical cord MSCs (hUC‐MSCs) based on particular markers. We used the MSC‐CD106⁺ subpopulations, whose biological function has been well documented, as an example to explore and optimize the crucial links of primary preparation, pre‐treatment, antibody incubation, flow sorting, quality and function test. After comprehensively evaluating the quality and function of the acquired MSC‐CD106⁺ subpopulations, including in vitro cell viability, apoptosis, proliferation, marker stability, adhesion ability, migration ability, tubule formation ability, immunomodulatory function and in vivo wound healing ability and proangiogenic activity, we defined an important pre‐treatment scheme which might effectively improve the therapeutic efficiency of MSC‐CD106⁺ subpopulations in two critical clinical application scenarios—direct injection after cell sorting and post‐culture injection into bodies. Based on the above, we tried to establish a general five‐step operation procedure for acquiring high‐quality clinical‐grade MSC subpopulations based on specific markers, which cannot only improve their enrichment efficiency and the reliability of preclinical studies, but also provide valuable methodological guidance for the rapid clinical transformation of specific MSC subpopulations.

We established an optimized operation process for obtaining human umbilical cord mesenchymal stem cells (hUC‐MSC) subpopulations with specific markers, which is also suitable for MSCs derived from other tissues. Step One. Preparation of clinical‐grade hUC‐MSCs. The clinical‐grade hUC‐MSCs obtained fully comply with the cGMP guidelines and meet the quality standards of the NIFDC. Step Two. Removal of non‐specific sites and poor‐quality cells without cellular damage. Treatment with MACS was proved as a safe and effective pre‐treatment by verifying multiple core indicators related to subpopulation quality. Step Three. Incubation with the antibodies against specific functional markers. Select the optimum condition of specific antibodies to ensure the purity of subsets obtained by subsequent sorting. Step Four. Flow Sorting and obtaining hUC‐MSC subpopulations with specific markers. This step has higher technical requirements related to flow cytometry. Step Five. Quality test and function research of hUC‐MSC subpopulations with specific markers. Do more research and verification to ensure the efficiency of clinical transformation of the obtained MSC subpopulations. Application Scenes. The clinical‐grade MSC subpopulations obtained through the above‐optimized operation process do not only guarantee the quality of the cells in basic research and preclinical research but also provide safety assurance for clinical application.     

Mesenchymal stroma/stem‐like cells of GARP knockdown inhibits cell proliferation and invasion of mouse colon cancer cells (MC38) through exosomes

Mesenchymal stroma/stem‐like cells (MSCs) have antitumour activity, and MSC‐derived exosomes play a role in the growth, metastasis and invasion of tumour cells. Additionally, glycoprotein A repetition predominant (GARP) promotes oncogenesis in breast cancer. Therefore, GARP is speculated to be a target gene for cancer therapy. We aimed to explore the therapy role of MSC‐derived exosomes targeting GARP in mouse colon cancer cell MC38. We successfully established a GARP knockdown system using three kinds of siRNA‐GARP in MSC cells. Exosomes were isolated from MSC and siGARP‐MSC cells, and verified by the exosome surface protein markers CD9, CD63 and CD81. GARP expression was significantly decreased in siGARP‐MSC exosomes compared with that of MSC exosomes. We found that siGARP‐MSC exosomes inhibited cell proliferation, migration and invasion of MC38 cells, using CCK‐8, colony formation, wound‐healing and Transwell invasion assays. Furthermore, siGARP‐MSC exosomes impeded IL‐6 secretion and partly inactivated JAK1/STAT3 pathway, measured using ELISA and RT‐qPCR. In conclusion, MSC‐derived exosomes targeting GARP are a potential strategy for cancer therapy.     

Human mesenchymal stem cells - current trends and future prospective

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton''s jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.     

Immunosuppressive effect of mesenchymal stem cells on lung and gut CD8+ T cells in lipopolysaccharide‐induced acute lung injury in mice

ObjectivesAcute lung injury (ALI) not only affects pulmonary function but also leads to intestinal dysfunction, which in turn contributes to ALI. Mesenchymal stem cell (MSC) transplantation can be a potential strategy in the treatment of ALI. However, the mechanisms of synergistic regulatory effects by MSCs on the lung and intestine in ALI need more in‐depth study.Materials and methodsWe evaluated the therapeutic effects of MSCs on the murine model of lipopolysaccharide (LPS)‐induced ALI through survival rate, histopathology and bronchoalveolar lavage fluid. Metagenomic sequencing was performed to assess the gut microbiota. The levels of pulmonary and intestinal inflammation and immune response were assessed by analysing cytokine expression and flow cytometry.ResultsMesenchymal stem cells significantly improved the survival rate of mice with ALI, alleviated histopathological lung damage, improved intestinal barrier integrity, and reduced the levels of inflammatory cytokines in the lung and gut. Furthermore, MSCs inhibited the inflammatory response by decreasing the infiltration of CD8⁺ T cells in both small‐intestinal lymphocytes and Peyer''s patches. The gut bacterial community diversity was significantly altered by MSC transplantation. Furthermore, depletion of intestinal bacterial communities with antibiotics resulted in more severe lung and gut damages and mortality, while MSCs significantly alleviated lung injury due to their immunosuppressive effect.ConclusionsThe present research indicates that MSCs attenuate lung and gut injury partly via regulation of the immune response in the lungs and intestines and gut microbiota, providing new insights into the mechanisms underlying the therapeutic effects of MSC treatment for LPS‐induced ALI.     

siRNA‐induced CD44 knockdown suppresses the proliferation and invasion of colorectal cancer stem cells through inhibiting epithelial–mesenchymal transition

CD44 has shown prognostic values and promising therapeutic potential in multiple human cancers; however, the effects of CD44 silencing on biological behaviors of cancer stem cells (CSCs) have not been fully understood in colorectal cancer. To examine the contribution of siRNA‐induced knockdown of CD44 to the biological features of colorectal CSCs, colorectal CSCs HCT116‐CSCs were generated, and CD44 was knocked down in HCT116‐CSCs using siRNA. The proliferation, migration and invasion of HCT116‐CSCs were measured, and apoptosis and cell‐cycle analyses were performed. The sensitivity of HCT116‐CSCs to oxaliplatin was tested, and xenograft tumor growth assay was performed to examine the role of CD44 in HCT116‐CSCs tumorigenesis in vivo. In addition, the expression of epithelial–mesenchymal transition (EMT) markers E‐cadherin, N‐cadherin and vimentin was quantified. siRNA‐induced knockdown of CD44 was found to inhibit the proliferation, migration and invasion, induce apoptosis, promote cell‐cycle arrest at the G1/G0 phase and increase the sensitivity of HCT116‐CSCs to oxaliplatin in HCT116‐CSCs, and knockdown of CD44 suppressed in vivo tumorigenesis and intrapulmonary metastasis of HCT116‐CSCs. Moreover, silencing CD44 resulted in EMT inhibition. Our findings demonstrate that siRNA‐induced CD44 knockdown suppresses the proliferation, invasion and in vivo tumorigenesis and metastasis of colorectal CSCs by inhibiting EMT.     

Oral intake of Lactobacillus plantarum L‐14 extract alleviates TLR2‐ and AMPK‐mediated obesity‐associated disorders in high‐fat‐diet‐induced obese C57BL/6J mice

ObjectivesWhether periodic oral intake of postbiotics positively affects weight regulation and prevents obesity‐associated diseases in vivo is unclear. This study evaluated the action mechanism of Lactobacillus plantarum L‐14 (KTCT13497BP) extract and the effects of its periodic oral intake in a high‐fat‐diet (HFD) mouse model.Materials and methodsMouse pre‐adipocyte 3T3‐L1 cells and human bone marrow mesenchymal stem cells (hBM‐MSC) were treated with L‐14 extract every 2 days during adipogenic differentiation, and the mechanism underlying anti‐adipogenic effects was analysed at cellular and molecular levels. L‐14 extract was orally administrated to HFD‐feeding C57BL/6J mice every 2 days for 7 weeks. White adipose tissue was collected and weighed, and liver and blood serum were analysed. The anti‐adipogenic mechanism of exopolysaccharide (EPS) isolated from L‐14 extract was also analysed using Toll‐like receptor 2 (TLR2) inhibitor C29.ResultsL‐14 extract inhibited 3T3‐L1 and hBM‐MSC differentiation into mature adipocytes by upregulating AMPK signalling pathway in the early stage of adipogenic differentiation. The weight of the HFD + L‐14 group (31.51 ± 1.96 g) was significantly different from that of the HFD group (35.14 ± 3.18 g). L‐14 extract also significantly decreased the serum triacylglycerol/high‐density lipoprotein cholesterol ratio (an insulin resistance marker) and steatohepatitis. In addition, EPS activated the AMPK signalling pathway by interacting with TLR2, consequently inhibiting adipogenesis.ConclusionsEPS from L‐14 extract inhibits adipogenesis via TLR2 and AMPK signalling pathways, and oral intake of L‐14 extract improves obesity and obesity‐associated diseases in vivo. Therefore, EPS can be used to prevent and treat obesity and metabolic disorders.     

Development of a 3D‐printed single‐use separation chamber for use in mRNA‐based vaccine production with magnetic microparticles

Laboratory protocols using magnetic beads have gained importance in the purification of mRNA for vaccines. Here, the produced mRNA hybridizes specifically to oligo(dT)‐functionalized magnetic beads after cell lysis. The mRNA‐loaded magnetic beads can be selectively separated using a magnet. Subsequently, impurities are removed by washing steps and the mRNA is eluted. Magnetic separation is utilized in each step, using different buffers such as the lysis/binding buffer. To reduce the time required for purification of larger amounts of mRNA vaccine for clinical trials, high‐gradient magnetic separation (HGMS) is suitable. Thereby, magnetic beads are selectively retained in a flow‐through separation chamber. To meet the requirements of biopharmaceutical production, a disposable HGMS separation chamber with a certified material (United States Pharmacopeia Class VI) was developed which can be manufactured using 3D printing. Due to the special design, the filter matrix itself is not in contact with the product. The separation chamber was tested with suspensions of oligo(dT)‐functionalized Dynabeads MyOne loaded with synthetic mRNA. At a concentration of c_B = 1.6–2.1 g·L^–1 in lysis/binding buffer, these 1 μm magnetic particles are retained to more than 99.39% at volumetric flows of up to 150 mL·min^–1 with the developed SU‐HGMS separation chamber. When using the separation chamber with volumetric flow rates below 50 mL·min^–1, the retained particle mass is even more than 99.99%.     

Wharton’s jelly‐derived stromal cells and their cell therapy applications in allogeneic haematopoietic stem cell transplantation

For decades, mesenchymal stromal cells (MSCs) have been of great interest in the fields of regenerative medicine, tissue engineering and immunomodulation. Their tremendous potential makes it desirable to cryopreserve and bank MSCs to increase their accessibility and availability. Postnatally derived MSCs seem to be of particular interest because they are harvested after delivery without ethical controversy, they have the capacity to expand at a higher rate than adult‐derived MSCs, in which expansion decreases with ageing, and they have demonstrated immunological and haematological supportive properties similar to those of adult‐derived MSCs. In this review, we focus on MSCs obtained from Wharton''s jelly (the mucous connective tissue of the umbilical cord between the amniotic epithelium and the umbilical vessels). Wharton''s jelly MSCs (WJ‐MSCs) are a good candidate for cellular therapy in haematology, with accumulating data supporting their potential to sustain haematopoietic stem cell engraftment and to modulate alloreactivity such as Graft Versus Host Disease (GVHD). We first present an overview of their in‐vitro properties and the results of preclinical murine models confirming the suitability of WJ‐MSCs for cellular therapy in haematology. Next, we focus on clinical trials and discuss tolerance, efficacy and infusion protocols reported in haematology for GVHD and engraftment.     

Novel long non‐coding RNA CYB561‐5 promotes aerobic glycolysis and tumorigenesis by interacting with basigin in non‐small cell lung cancer

Abnormally expressed long non‐coding RNAs (lncRNAs) have been recognized as potential diagnostic biomarkers or therapeutic targets in non‐small cell lung cancer (NSCLC). The role of the novel lnc‐CYB561‐5 in NSCLC and its specific biological activity remain unknown. In this study, lncRNAs highly expressed in NSCLC tissue samples compared with paired adjacent normal tissue samples and atypical adenomatous hyperplasia were identified by RNA‐seq analysis. Lnc‐CYB561‐5 is highly expressed in human NSCLC and is associated with a poor prognosis in lung adenocarcinoma. In vivo, downregulation of lnc‐CYB561‐5 significantly decreases tumour growth and metastasis. In vitro, lnc‐CYB561‐5 knockdown treatment inhibits cell migration, invasion and proliferation ability, as well as glycolysis rates. In addition, RNA pulldown and RNA immunoprecipitation (RIP) assays show that basigin (Bsg) protein interacts with lnc‐CYB561‐5. Overall, this study demonstrates that lnc‐CYB561‐5 is an oncogene in NSCLC, which is involved in the regulation of cell proliferation and metastasis. Lnc‐CYB561‐5 interacts with Bsg to promote the expression of Hk2 and Pfk1 and further lead to metabolic reprogramming of NSCLC cells.     

MiR‐223‐3p inhibits rTp17‐induced inflammasome activation and pyroptosis by targeting NLRP3

The incidence of syphilis caused by Treponema pallidum subsp pallidum (T pallidum) infection is accompanied by inflammatory injuries of vascular endothelial cells. Studies have revealed that T pallidum infection could induce inflammasome activation and pyroptosis in macrophages. MicroRNA‐223‐3p (miR‐223‐3p) was reported to be a negative regulator in inflammatory diseases. The present study aimed to explore whether miR‐223‐3p regulates T pallidum‐induced inflammasome activation and pyroptosis in vascular endothelial cells, and determine the mechanisms which underlie this process. MiR‐223‐3p levels in syphilis and control samples were determined. The biological function of miR‐223‐3p in the NLRP3 inflammasome and pyroptosis was evaluated in T pallidum‐infected human umbilical vein endothelial cells (HUVECs). We observed a dramatic decrease in miR‐223‐3p levels in syphilis patients (n = 20) when compared to healthy controls (n = 20). Moreover, miR‐223‐3p showed a notable inhibitory effect on recombinant Tp17 (rTP17)‐induced caspase‐1 activation, resulting in decrease in IL‐1β production and pyroptosis, which was accompanied by the release of lactate dehydrogenase (LDH) in HUVECs. Additionally, the dual‐luciferase assay confirmed that NLRP3 is a direct target of miR‐223‐3p. Moreover, NLRP3 overexpression or knockdown largely blocked the effects of miR‐223‐3p on T pallidum‐induced inflammasome activation and pyroptosis in HUVECs. Most importantly, a notable negative correlation was observed between miR‐223‐3p and NLRP3, caspase‐1, and IL‐1β, respectively, in the serum of syphilis patients and healthy controls. Taken together, our results reveal that miR‐223‐3p targets NLRP3 to suppress inflammasome activation and pyroptosis in T pallidum‐infected endothelial cells, implying that miR‐223‐3p could be a potential target for syphilis patients.     

Recent advances in the therapeutic efficacy of hepatocyte growth factor gene‐modified mesenchymal stem cells in multiple disease settings

Mesenchymal stem cell (MSC) therapy is considered a new treatment for a wide range of diseases and injuries, but challenges remain, such as poor survival, homing and engraftment rates, thus limiting the therapeutic efficacy of the transplanted MSCs. Many strategies have been developed to enhance the therapeutic efficacy of MSCs, such as preconditioning, co‐transplantation with graft materials and gene modification. Hepatocyte growth factor (HGF) is secreted by MSCs, which plays an important role in MSC therapy. It has been reported that the modification of the HGF gene is beneficial to the therapeutic efficacy of MSCs, including diseases of the heart, lung, liver, urinary system, bone and skin, lower limb ischaemia and immune‐related diseases. This review focused on studies involving HGF/MSCs both in vitro and in vivo. The characteristics of HGF/MSCs were summarized, and the mechanisms of their improved therapeutic efficacy were analysed. Furthermore, some insights are provided for HGF/MSCs'' clinical application based on our understanding of the HGF gene and MSC therapy.     

Dynamics of NK,CD8 and Tfh cell mediated the production of cytokines and antiviral antibodies in Chinese patients with moderate COVID‐19

Recent studies have demonstrated a marked decrease in peripheral lymphocyte levels in patients with coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Few studies have focused on the changes of NK, T‐ and B‐cell subsets, inflammatory cytokines and virus‐specific antibodies in patients with moderate COVID‐19. A total of 11 RT‐PCR‐confirmed convalescent patients with COVID‐19 and 11 patients with non‐SARS‐CoV‐2 pneumonia (control patients) were enrolled in this study. NK, CD8⁺ T, CD4⁺ T, Tfh‐like and B‐cell subsets were analysed using flow cytometry. Cytokines and SARS‐CoV‐2‐specific antibodies were analysed using an electrochemiluminescence immunoassay. NK cell counts were significantly higher in patients with COVID‐19 than in control patients (P = 0.017). Effector memory CD8⁺ T‐cell counts significantly increased in patients with COVID‐19 during a convalescent period of 1 week (P = 0.041). TIM‐3⁺ Tfh‐like cell and CD226⁺ Tfh‐like cell counts significantly increased (P = 0.027) and decreased (P = 0.022), respectively, during the same period. Moreover, ICOS⁺ Tfh‐like cell counts tended to decrease (P = 0.074). No abnormal increase in cytokine levels was observed. The high expression of NK cells is important in innate immune response against SARS‐CoV‐2. The increase in effector memory CD8⁺ T‐cell counts, the up‐regulation of inhibitory molecules and the down‐regulation of active molecules on CD4⁺ T cells and Tfh‐like cells in patients with COVID‐19 would benefit the maintenance of balanced cellular and humoural immune responses, may prevent the development of severe cases and contribute to the recovery of patients with COVID‐19.     

Characterization of a novel microfluidic platform for the isolation of rare single cells to enable CTC analysis from head and neck squamous cell carcinoma patients

Detailed examination of tumor components is leading‐edge to establish personalized cancer therapy. Accompanying research on cell‐free DNA, the cell count of circulating tumor cells (CTCs) in patient blood is seen as a crucial prognostic factor. The potential of CTC analysis is further not limited to the determination of the overall survival rate but sheds light on understanding inter‐ and intratumoral heterogeneity. In this regard, commercial CTC isolation devices combining an efficient enrichment of rare cells with a droplet deposition of single cells for downstream analysis are highly appreciated. The Liquid biopsy platform CTCelect was developed to realize a fully‐automated enrichment and single cell dispensing of CTCs from whole blood without pre‐processing. We characterized each process step with two different carcinoma cell lines demonstrating up to 87 % enrichment (n = 10) with EpCAM coupled immunomagnetic beads, 73 % optical detection and dispensing efficiency (n = 5). 40 to 56.7 % of cells were recovered after complete isolation from 7.5 ml untreated whole blood (n = 6). In this study, CTCelect enabled automated dispensing of single circulating tumor cells from HNSCC patient samples, qPCR‐based confirmation of tumor‐related biomarkers and immunostaining. Finally, the platform was compared to commercial CTC isolation technologies to highlight advantages and limitations of CTCelect. This system offers new possibilities for single cell screening in cancer diagnostics, individual therapy approaches and real‐time monitoring.     

Magnetic Fe3O4@graphene oxide improves the therapeutic effects of embryonic stem cells on acute liver damage

ObjectiveAcute liver failure is usually associated with inflammation and oxidation of hepatocytes and has high mortality and resource costs. Mesenchymal stem cell (MSCs) has occasionally been reported to have no beneficial effect due to poor transplantation and the survival of implanted cells. Recent studies showed that embryonic stem cell (ESC)‐derived MSCs are an alternative for regenerative medicine. On the other hand, graphene‐based nanostructures have proven useful in biomedicine. In this study, we investigated whether magnetic graphene oxide (MGO) improved the effects of ESC‐MSC conditioned medium (CM) on protecting hepatocytes and stimulating the regeneration of damaged liver cells.Materials and methodsTo provide a rat model of acute liver failure, male rats were injected intraperitoneally with carbon tetrachloride (CCl₄). The rats were randomly divided into six groups, namely control, sham, CCl₄, ESC‐MSC‐CM, MGO and ESC‐MSC‐CM + MGO. In the experimental groups, the rats received, depending on the group, 2 ml/kg body weight CCl₄ and either ESC‐MSC‐CM with 5 × 10⁶ MSCs or 300 μg/kg body weight MGO or both. Symptoms of acute liver failure appeared 4 days after the injection. All groups were compared and analysed both histologically and biochemically 4 days after the injection. Finally, the results of ESC‐MSC‐CM and MSC‐CM were compared.ResultsThe results indicated that the use of MGO enhanced the effect of ESC‐MSC‐CM on reducing necrosis, inflammation, aspartate transaminase, alanine aminotransferase and alkaline phosphatase in the CCl₄‐induced liver failure of the rat model. Also, the expression of vascular endothelial growth factor and matrix metalloproteinase‐9 (MMP‐9) was significantly upregulated after treatment with MGO. Also, the results showed that the ESC‐MSC‐CM has more efficient effective compared to MSC‐CM.ConclusionMagnetic graphene oxide improved the hepatoprotective effects of ESC‐MSC‐CM on acute liver damage, probably by suppressing necrosis, apoptosis and inflammation of hepatocytes.     

Toll‐7 promotes tumour growth and invasion in Drosophila

Objectives Drosophila melanogaster has become an excellent model organism to explore the genetic mechanisms underlying tumour progression. Here, by using well‐established Drosophila tumour models, we identified Toll‐7 as a novel regulator of tumour growth and invasion.Materials and methodsTransgenic flies and genetic epistasis analysis were used. All flies were raised on a standard cornmeal and agar medium at 25°C unless otherwise indicated. Immunostaining and RT‐qPCR were performed by standard procedures. Images were taken by OLYMPUS BX51 microscope and Zeiss LSM 880 confocal microscope. Adobe Photoshop 2020 and Zeiss Zen were used to analyse the images. All results were presented in Scatter plots or Column bar graphs created by GraphPad Prism 8.0.ResultsLoss of Toll‐7 suppresses Ras^V12/lgl ^−/−‐induced tumour growth and invasion, as well as cell polarity disruption‐induced invasive cell migration, whereas expression of a constitutively active allele of Toll‐7 is sufficient to promote tumorous growth and cell migration. In addition, the Egr‐JNK signalling is necessary and sufficient for Toll‐7‐induced invasive cell migration. Mechanistically, Toll‐7 facilitates the endocytosis of Egr, which is known to activate JNK in the early endosomes. Moreover, Toll‐7 activates the EGFR‐Ras signalling, which cooperates with the Egr‐JNK signalling to promote Yki‐mediated cell proliferation and tissue overgrowth. Finally, Toll‐7 is necessary and sufficient for the proper maintenance of EGFR protein level.ConclusionsOur findings characterized Toll‐7 as a proto‐oncogene that promotes tumour growth and invasion in Drosophila, which shed light on the pro‐tumour function of mammalian Toll‐like receptors (TLRs).     

Mesenchymal stem cell‐derived extracellular vesicles reduce senescence and extend health span in mouse models of aging

Aging drives progressive loss of the ability of tissues to recover from stress, partly through loss of somatic stem cell function and increased senescent burden. We demonstrate that bone marrow‐derived mesenchymal stem cells (BM‐MSCs) rapidly senescence and become dysfunctional in culture. Injection of BM‐MSCs from young mice prolonged life span and health span, and conditioned media (CM) from young BM‐MSCs rescued the function of aged stem cells and senescent fibroblasts. Extracellular vesicles (EVs) from young BM‐MSC CM extended life span of Ercc1 ^−/− mice similarly to injection of young BM‐MSCs. Finally, treatment with EVs from MSCs generated from human ES cells reduced senescence in culture and in vivo, and improved health span. Thus, MSC EVs represent an effective and safe approach for conferring the therapeutic effects of adult stem cells, avoiding the risks of tumor development and donor cell rejection. These results demonstrate that MSC‐derived EVs are highly effective senotherapeutics, slowing the progression of aging, and diseases driven by cellular senescence.