Aggregation of human osteoblasts unlocks self-reliant differentiation and constitutes a microenvironment for 3D-co-cultivation with other bone marrow cells.

Skeletal bone function relies on both cells and cellular niches, which, when combined, provide guiding cues for the control of differentiation and remodeling processes. Here, we propose an in vitro 3D model based on human fetal osteoblasts, which eases the study of osteocyte commitment in vitro and thus provides a means to examine the influences of biomaterials, substances or cells on the regulation of these processes. Aggregates were formed from human fetal osteoblasts (hFOB1.19) and cultivated under proliferative, adipo- and osteoinductive conditions. When cultivated under osteoinductive conditions, the vitality of the aggregates was compromised, the expression levels of the mineralization-related gene DMP1 and the amount of calcification and matrix deposition were lower, and the growth of the spheroids stalled. However, within spheres under growth conditions without specific supplements, self-organization processes occur, which promote extracellular calcium deposition, and osteocyte-like cells develop. Long-term cultivated hFOB aggregates were free of necrotic areas. Moreover, hFOB aggregates cultivated under standard proliferative conditions supported the co-cultivation of human monocytes, microvascular endothelial cells and stromal cells. Overall, the model presented here comprises a self-organizing and easily accessible 3D osteoblast model for studying bone marrow formation and in vitro remodeling and thus provides a means to test druggable molecular pathways with the potential to promote life-long bone formation and remodeling.

Direct male development in chromosomally ZZ zebrafish.

The genetics of sex determination varies across taxa, sometimes even within a species. Major domesticated strains of zebrafish (Danio rerio), including AB and TU, lack a strong genetic sex determining locus, but strains more recently derived from nature, like Nadia (NA), possess a ZZ male/ZW female chromosomal sex-determination system. AB fish pass through a juvenile ovary stage, forming oocytes that survive in fish that become females but die in fish that become males. To understand mechanisms of gonad development in NA zebrafish, we studied histology and single cell transcriptomics in developing ZZ and ZW fish. ZW fish developed oocytes by 22 days post-fertilization (dpf) but ZZ fish directly formed testes, avoiding a juvenile ovary phase. Gonads of some ZW and WW fish, however, developed oocytes that died as the gonad became a testis, mimicking AB fish, suggesting that the gynogenetically derived AB strain is chromosomally WW. Single-cell RNA-seq of 19dpf gonads showed similar cell types in ZZ and ZW fish, including germ cells, precursors of gonadal support cells, steroidogenic cells, interstitial/stromal cells, and immune cells, consistent with a bipotential juvenile gonad. In contrast, scRNA-seq of 30dpf gonads revealed that cells in ZZ gonads had transcriptomes characteristic of testicular Sertoli, Leydig, and germ cells while ZW gonads had granulosa cells, theca cells, and developing oocytes. Hematopoietic and vascular cells were similar in both sex genotypes. These results show that juvenile NA zebrafish initially develop a bipotential gonad; that a factor on the NA W chromosome, or fewer than two Z chromosomes, is essential to initiate oocyte development; and without the W factor, or with two Z doses, NA gonads develop directly into testes without passing through the juvenile ovary stage. Sex determination in AB and TU strains mimics NA ZW and WW zebrafish, suggesting loss of the Z chromosome during domestication. Genetic analysis of the NA strain will facilitate our understanding of the evolution of sex determination mechanisms.

Potential of Mac-2-binding protein glycan isomer as a new therapeutic target in pancreatic cancer.

Pancreatic cancer (PC) is a challenging malignancy to treat. Mac-2-binding protein glycan isomer (M2BPGi) is a novel serum marker of liver fibrosis and hepatocellular carcinoma and is secreted by hepatic stellate and stroma cells. Serum M2BPGi levels are upregulated in PC patients. We measured the expression of M2BPGi in the serum of 27 PC patients and determined whether M2BPGi affects the malignant potential of PC cells in vitro. We also examined the effect of M2BP on PC tumor growth and gemcitabine sensitivity in vivo. Serum M2BPGi levels in PC patients were higher compared with those of healthy subjects. M2BPGi extraction in cancer-associated fibroblasts (CAFs) was higher compared with that of PC cells. M2BPGi treatment promoted the proliferation and invasion of PC cells. The suppression of galectin-3, which binds to M2BPGi, did not affect the proliferation-promoting effect of M2BPGi in PC cells. The suppression of M2BP reduced tumor growth and enhanced gemcitabine sensitivity in PC-bearing xenograft mice. CAF-derived M2BPGi promotes the proliferation and invasion of PC cells. Targeting M2BPGi may represent a new therapeutic strategy to circumvent refractory PC.

Second-line therapy for patients with steroid-refractory aGVHD: systematic review and meta-analysis of randomized controlled trials.

Objective: Steroids-refractory (SR) acute graft-versus-host disease (aGVHD) is a life-threatening condition in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the optimal second-line therapy still has not been established. We aimed to perform a systematic review and meta-analysis of randomized controlled trials (RCTs) to compare the efficacy and safety of different second-line therapy regimens. Methods: Literature search in MEDLINE, Embase, Cochrane Library and China Biology Medicine databases were performed to retrieve RCTs comparing the efficacy and safety of different therapy regimens for patients with SR aGVHD. Meta-analysis was conducted with Review Manager version 5.3. The primary outcome is the overall response rate (ORR) at day 28. Pooled relative risk (RR) and 95% confidence interval (CI) were calculated with the Mantel-Haenszel method. Results: Eight eligible RCTs were included, involving 1127 patients with SR aGVHD and a broad range of second-line therapy regimens. Meta-analysis of 3 trials investigating the effects of adding mesenchymal stroma cells (MSCs) to other second-line therapy regimens suggested that the addition of MSCs is associated with significantly improvement in ORR at day 28 (RR = 1.15, 95% CI = 1.01-1.32, P = 0.04), especially in patients with severe (grade III-IV or grade C-D) aGVHD (RR = 1.26, 95% CI = 1.04-1.52, P = 0.02) and patients with multiorgan involved (RR = 1.27, 95% CI = 1.05-1.55, P = 0.01). No significant difference was observed betwwen the MSCs group and control group in consideration of overall survival and serious adverse events. Treatment outcomes of the other trials were comprehensively reviewed, ruxolitinib showed significantly higher ORR and complete response rate at day 28, higher durable overall response at day 56 and longer failure-free survival in comparison with other regimens; inolimomab shows similar 1-year therapy success rate but superior long-term overall survial in comparison with anti-thymocyte globulin, other comparisons did not show significant differences in efficacy. Conclusions: Adding MSCs to other second-line therapy regimens is associated with significantly improved ORR, ruxolitinib showed significantly better efficacy outcomes in comparison with other regimens in patients with SR aGVHD. Further well-designed RCTs and integrated studies are required to determine the optimal treatment. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42022342487.

Classification of colorectal cancer into clinically relevant subtypes based on genes and mesenchymal cells.

BACKGROUND: Most studies on subtype identification of colorectal cancer (CRC) were based on expressions of either genes or immune cells. However, few studies have hitherto used the combination of genes with immune and stroma cells for subtype identification. METHODS: Dataset GSE17536 was obtained from the Gene Expression Omnibus (GEO) database. The xCell algorithm was used to estimate the composition and density of 64 cell types, including immune and stroma cell types. Clustering analysis was then conducted on the top 3000 most variable genes from a total of 20,174 genes for CRC subtype identification. We employed the ensemble method of Similarity network fusion and 112 Consensus Clustering (SNF-CC) for cancer subtype identification. Reactome pathway analysis was conducted to identify the impact of the representative genes on prognosis. The results were validated in independent gene expression data from dataset GSE17537. RESULTS: In this study, we identified 3 clinically relevant subtypes and their representative genes, immune and stroma cells. Moreover, we confirmed the correlation of these subtypes with their clinical characteristics. The representative genes of the subtype with poor prognosis correlated with extracellular matrix structural constituent, while the subtype with good prognosis correlated with Toll-like receptor signaling pathway or chemokine signaling pathway. However, different subtypes were associated with distinct cell subtypes; the subtype with poor prognosis had a high abundance of fibroblasts and endothelial cells; the subtype with median prognosis had a higher abundance of immune cells, such as CD4 + T-cell, Th2 cells and aDC; the subtype with good prognosis had a higher abundance of NKT. CONCLUSION: This study highlights the utility of immune and innate cells, especially during gene analysis, to provide the theoretical basis for personalized treatment in colorectal cancer patients.

MLLT11 Regulates Endometrial Stroma Cell Adhesion, Proliferation and Survival in Ectopic Lesions of Women with Advanced Endometriosis.

MLLT11 is a gene implicated in cell differentiation and the development and progression of human cancers, but whose role in the pathogenesis of endometriosis is still unknown. Using quantitative RT-PCR and immunohistochemistry, we analyzed 37 women with and 33 women without endometriosis for differences in MLLT11 expression. We found that MLLT11 is reduced in the ectopic stroma cells of women with advanced stage endometriosis compared to women without endometriosis. MLLT11 knockdown in control stroma cells resulted in the downregulation of their proliferation accompanied by G1 cell arrest and an increase in the expression of p21 and p27. Furthermore, the knockdown of MLLT11 was associated with increased apoptosis resistance to camptothecin associated with changes in BCL2/BAX signaling. Finally, MLLT11 siRNA knockdown in the control primary stroma cells led to an increase in cell adhesion associated with the transcriptional activation of ACTA2 and TGFB2. We found that the cellular phenotype of MLLT11 knockdown cells resembled the phenotype of the primary endometriosis stroma cells of the lesion, where the levels of MLLT11 are significantly reduced compared to the eutopic stroma cells of women without the disease. Overall, our results indicate that MLLT11 may be a new clinically relevant player in the pathogenesis of endometriosis.

Cancer Stem Cells in Hepatocellular Carcinoma: Intrinsic and Extrinsic Molecular Mechanisms in Stemness Regulation.

Hepatocellular carcinoma (HCC) remains the most predominant type of liver cancer with an extremely poor prognosis due to its late diagnosis and high recurrence rate. One of the culprits for HCC recurrence and metastasis is the existence of cancer stem cells (CSCs), which are a small subset of cancer cells possessing robust stem cell properties within tumors. CSCs play crucial roles in tumor heterogeneity constitution, tumorigenesis, tumor relapse, metastasis, and resistance to anti-cancer therapies. Elucidation of how these CSCs maintain their stemness features is essential for the development of CSCs-based therapy. In this review, we summarize the present knowledge of intrinsic molecules and signaling pathways involved in hepatic CSCs, especially the CSC surface markers and associated signaling in regulating the stemness characteristics and the heterogeneous subpopulations within the CSC pool. In addition, we recapitulate the effects of crucial extrinsic cellular components in the tumor microenvironment, including stromal cells and immune cells, on the modulation of hepatic CSCs. Finally, we synopsize the currently valuable CSCs-targeted therapy strategies based on intervention in these intrinsic and extrinsic molecular mechanisms, in the hope of shedding light on better clinical management of HCC patients.

Targeting therapy and tumor microenvironment remodeling of triple-negative breast cancer by ginsenoside Rg3 based liposomes.

The chemotherapy effect of docetaxel (DTX) against triple-negative breast cancer (TNBC) remains mediocre and limited when encapsulated in conventional cholesterol liposomes, mainly ascribed to poor penetration and immunosuppressive tumor microenvironment (TME) caused by tumor stroma cells, especially cancer-associated fibroblasts (CAFs). Many studies have attempted to address these problems but trapped into the common dilemma of excessively complicated formulation strategies at the expense of druggability as well as clinical translational feasibility. To better address the discrepancy, ginsenoside Rg3 was utilized to substitute cholesterol to develop a multifunctional DTX-loaded Rg3 liposome (Rg3-Lp/DTX). The obtained Rg3-Lp/DTX was proved to be preferentially uptake by 4T1 cells and accumulate more at tumor site via the interaction between the glycosyl moiety of Rg3 exposed on liposome surface and glucose transporter1 (Glut1) overexpressed on tumor cells. After reaching tumor site, Rg3 was shown to reverse the activated CAFs to the resting stage and attenuate the dense stroma barrier by suppressing secretion of TGF-ß from tumor cells and regulating TGF-ß/Smad signaling. Therefore, reduced levels of CAFs and collagens were found in TME after incorporation of Rg3, inducing enhanced penetration of Rg3-Lp/DTX in the tumor and reversed immune system which can detect and neutralize tumor cells. Compared with wooden cholesterol liposomes, the smart and versatile Rg3-Lp/DTX could significantly improve the anti-tumor effect of DTX, providing a promising approach for TNBC therapy with excellent therapeutic efficacy and simple preparation process.

Single-cell transcriptomics profiling the compatibility mechanism of As2O3-indigo naturalis formula based on bone marrow stroma cells.

Compound realgar natural indigo tablet is the only oral arsenic agent widely used in acute promyelocytic leukemia (APL) treatment. However, as a therapeutic drug for diseases of the blood system, the scientific knowledge of As2O3-indigo naturalis formula compatibility has not been studied in bone marrow stromal cells (BMSCs). We chose arsenic trioxide (As2O3: A), tanshinone IIA (T) and indirubin (I) as representative active compounds of realgar, indigo naturalis, and Salvia miltiorrhiza, respectively, to evaluated the pharmaceutical mechanism and the compatibility of ATI (drug combination) using single-cell RNA sequencing (scRNA-seq). The overlapped genes associated with both disease and drug were selected in BMSCs for in-depth analysis. Results show that joint applications of ATI had the strongest therapeutic efficacy in a murine APL model. Lepr-MSCs, OLCs and BMECs were the sensitive cell groups targeted by ATI in the murine APL model. ATI could regulate the related genes of osteogenic differentiation, adipogenic differentiation, and endothelial cell migration in bone marrow mesenchymal lineage cells in murine APL model and improve normal hematopoiesis-related gene expression and poor prognosis of Lepr-MSCs, OLCs and BMECs in mice with leukemia according to scRNA-seq data. The strongest regulatory effects were found in the joint applications of ATI. ATI combination had the potential mechanism to maintain the stability of the hematopoietic microenvironment and promote hematopoiesis to assist in the treatment of APL. This study illustrated the potential mechanism of ATI in regulating BMSCs from the overall perspective of the hematopoietic microenvironment, and broadened the scientific understanding of ATI compatibility in BMSCs.

Aged Callus Skeletal Stem/Progenitor Cells Contain an Inflammatory Osteogenic Population With Increased IRF and NF-κB Pathways and Reduced Osteogenic Potential.

Skeletal stem/progenitor cells (SSPCs) are critical for fracture repair by providing osteo-chondro precursors in the callus, which is impaired in aging. However, the molecular signatures of callus SSPCs during aging are not known. Herein, we performed single-cell RNA sequencing on 11,957 CD45-CD31-Ter119- SSPCs isolated from young and aged mouse calluses. Combining unsupervised clustering, putative makers, and DEGs/pathway analyses, major SSPC clusters were annotated as osteogenic, proliferating, and adipogenic populations. The proliferating cluster had a differentiating potential into osteogenic and adipogenic lineages by trajectory analysis. The osteoblastic/adipogenic/proliferating potential of individual clusters was further evidenced by elevated expression of genes related to osteoblasts, adipocytes, or proliferation. The osteogenic cluster was sub-clustered into house-keeping and inflammatory osteogenic populations that were decreased and increased in aged callus, respectively. The majority of master regulators for the inflammatory osteogenic population belong to IRF and NF-κB families, which was confirmed by immunostaining, RT-qPCR, and Western blot analysis. Furthermore, cells in the inflammatory osteogenic sub-cluster had reduced osteoblast differentiation capacity. In conclusion, we identified 3 major clusters in callus SSPCs, confirming their heterogeneity and, importantly, increased IRF/NF-κB-mediated inflammatory osteogenic population with decreased osteogenic potential in aged cells.

Induction of Collagen I by CXCL10 in Ovarian Theca-Stroma Cells via the JNK Pathway.

Premature ovarian insufficiency (POI) poses a great threat to reproductive-age women. Ovarian fibrogenesis is a basic histologic feature of POI. Ovarian theca-stroma cells are responsible for ovarian fibrosis, but few studies have focused on the ovarian microenvironment. The role and mechanism of chemokines in the development of POI remain unclear. Here, we evaluated C-X-C motif chemokine ligand 10 (CXCL10) in biochemical POI patients, POI patients, and a POI mouse model. CXCL10 levels in serum and follicular fluid were higher in both bPOI and POI patients than in controls. An increased level of CXCL10 was also observed in a POI mouse model. CXCL10 concentrations in serum and follicular fluid were positively associated with follicle-stimulating hormone and negatively associated with antral follicle count. Our study for the first time found that CXCL10 induced COL1A1 and COL1A2 production, two subunits of collagen I in mouse theca-stroma cells by activating the JNK/c-Jun pathway. Inhibition of JNK and c-Jun attenuated the increases of COL1A1 and COL1A2 caused by CXCL10. Moreover, CXCL10 had no effects on hormone synthesis, proliferation, and apoptosis in human luteinized granulosa (hGL) cells. Our findings revealed a potential diagnostic value of CXCL10 in the early stage of POI and shed new insights into the biological function of CXCL10 in ovarian fibrosis.

Long non-coding RNA DHRS4 antisense RNA 1 inhibits ectopic endometrial cell proliferation, migration, and invasion in endometriosis by regulating microRNA-139-5p expression.

Endometriosis is an estrogen-dependent chronic gynecological syndrome. Recent studies have shown that long non-coding RNAs participate in the pathogenesis and development of endometriosis. This study aimed to explore the mechanisms of DHRS4 antisense RNA 1 (DHRS4-AS1) in endometriosis. Dual-luciferase reporter assays were conducted to determine the relationship between DHRS4-AS1, microRNA (miR)-139-5p, and arrestin domain-containing 3 (ARRDC3). Furthermore, the expression of DHRS4-AS1 and miR-139-5p in ectopic endometrial stromal cells (EC-ESCs) and endometriosis tissues was examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Additionally, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry, and Transwell assays were performed to evaluate the proliferation, apoptosis, and migration and invasion of EC-ESCs, respectively. Western blotting and RT-qPCR were further utilized to determine cleaved-Caspase 3, Caspase 3, and matrix metalloproteinase 9 (MMP-9) expression levels. Compared with the EN group, DHRS4-AS1 levels were lower and miR-139-5p levels were higher in EC-ESCs and tissues obtained from patients with endometriosis. Functional assays validated that DHRS4-AS1 targets miR-139-5p, with ARRDC3 being a downstream target of miR-139-5p. Rescue experiments demonstrated that DHRS4-AS1 inhibited EC-ESC proliferation, migration, and invasion, but promoted apoptosis, by targeting miR-139-5p in endometriosis. cleaved-Caspase3 expression level and the cleaved-Caspase 3/Caspase 3 ratio increased, while the expression levels of MMP-9 decreased, after transfection with DHRS4-AS1 overexpression plasmids; however, the effects induced by DHRS4-AS1 overexpression could be partially reversed by co-transfection with the miR-139-5p mimic. The current study demonstrates that the DHRS4-AS1/miR-139-5p/ARRDC3 axis participates in the regulation of EC-ESC function.

Successful Treatment of Noise-Induced Hearing Loss by Mesenchymal Stromal Cells: An RNAseq Analysis of Protective/Repair Pathways.

Mesenchymal stromal cells (MSCs) are an adult derived stem cell-like population that has been shown to mediate repair in a wide range of degenerative disorders. The protective effects of MSCs are mainly mediated by the release of growth factors and cytokines thereby modulating the diseased environment and the immune system. Within the inner ear, MSCs have been shown protective against tissue damage induced by sound and a variety of ototoxins. To better understand the mechanism of action of MSCs in the inner ear, mice were exposed to narrow band noise. After exposure, MSCs derived from human umbilical cord Wharton's jelly were injected into the perilymph. Controls consisted of mice exposed to sound trauma only. Forty-eight hours post-cell delivery, total RNA was extracted from the cochlea and RNAseq performed to evaluate the gene expression induced by the cell therapy. Changes in gene expression were grouped together based on gene ontology classification. A separate cohort of animals was treated in a similar fashion and allowed to survive for 2 weeks post-cell therapy and hearing outcomes determined. Treatment with MSCs after severe sound trauma induced a moderate hearing protective effect. MSC treatment resulted in an up-regulation of genes related to immune modulation, hypoxia response, mitochondrial function and regulation of apoptosis. There was a down-regulation of genes related to synaptic remodeling, calcium homeostasis and the extracellular matrix. Application of MSCs may provide a novel approach to treating sound trauma induced hearing loss and may aid in the identification of novel strategies to protect hearing.

Intrauterine hCG application increases expression of endothelial cell-cell adhesion molecules in human.

Endometrial receptivity is a decisive factor in human reproduction. Human chorionic gonadotropin (hCG) is one of the first embryonic signals that precedes the implantation by trophoblast invasion into the endometrium. Meta-analysis of randomized controlled trials reports a moderate-quality evidence for improved live birth rate for an intrauterine hCG dose ≥ 500 IU. Nevertheless, all hCG endometrial effects are not completely understood. We, therefore, utilized endometrial tissue from 12 patients after estradiol and progesterone treatment with or without intrauterine hCG flushing at the window of implantation (WOI) to analyze cellular composition by measuring marker proteins for stromal, endothelial, epithelial and immune cells. Flow cytometry analysis revealed that significantly more cells expressed the endothelial adhesion molecules VE-cadherin (CD144) and S-Endo-1 (CD146) after intrauterine hCG administration. In contrast, the endothelial marker CD31 and markers involved in vessel formation (VEGFR1 and VEGFR2) remained unchanged in their expression. Similarly, stroma markers (CD73, CD90 and CD105), epithelial markers (Desmocollin-2 and E-Cadherin) and immune cell markers (CD11b, CD45, CD79a and HLA-DR) displayed no alterations in their expression. This finding directs the focus on endothelial adhesion molecules as a potential mechanistically explanation of hCG conveyed increase of embryo implantation and pregnancy rates in women undergoing ART.

Ion Channel Profiling in Prostate Cancer: Toward Cell Population-Specific Screening.

In the last three decades, a growing number of studies have implicated ion channels in all essential processes of prostate carcinogenesis, including cell proliferation, apoptosis, migration, and angiogenesis. The changes in the expression of individual ion channels show a specific profile, making these proteins promising clinical biomarkers that may enable better molecular subtyping of the disease and lead to more rapid and accurate clinical decision-making. Expression profiles and channel function are mainly based on the tumoral tissue itself, in this case, the epithelial cancer cell population. To date, little data on the ion channel profile of the cancerous prostate stroma are available, even though tumor interactions with the microenvironment are crucial in carcinogenesis and each distinct population plays a specific role in tumor progression. In this review, we describe ion channel expression profiles specific for the distinct cell population of the tumor microenvironment (stromal, endothelial, neuronal, and neuroendocrine cell populations) and the technical approaches used for efficient separation and screening of these cell populations.

miR-142-3p Reduces the Size, Migration, and Contractility of Endometrial and Endometriotic Stromal Cells by Targeting Integrin- and Rho GTPase-Related Pathways That Regulate Cytoskeletal Function.

Downregulated microRNA-142-3p signaling contributes to the pathogenesis of endometriosis, an invasive disease where the lining of the uterus grows at ectopic locations, by yet incompletely understood mechanisms. Using bioinformatics and in vitro assays, this study identifies cytoskeletal regulation and integrin signaling as two relevant categories of miR-142-3p targets. qPCR revealed that miR-142-3p upregulation in St-T1b cells downregulates Rho-associated protein kinase 2 (ROCK2), cofilin 2 (CFL2), Ras-related C3 botulinum toxin substrate 1 (RAC1), neural Wiskott-Aldrich syndrome protein (WASL), and integrin α-V (ITGAV). qPCR and Western-blotting showed miR-142-3p effect on WASL and ITGAV was significant also in primary endometriotic stroma cells. Luciferase reporter assays in ST-T1b cells then confirmed direct regulation of ITGAV and WASL. On the functional side, miR-142-3p upregulation significantly reduced ST-T1b cell size, the size of vinculin plaques, migration through fibronectin-coated transwell filters, and the ability of ST-T1b and primary endometriotic stroma cells to contract collagen I gels. These results suggest that miR-142-3p has a strong mechanoregulatory effect on endometrial stroma cells and its external administration reduces the invasive endometrial phenotype. Within the limits of an in vitro investigation, our study provides new mechanistic insights into the pathogenesis of endometriosis and provides a perspective for the development of miR-142-3p based drugs for inhibiting invasive growth of endometriotic cells.

Downregulation of extracellular vesicle microRNA-101 derived from bone marrow mesenchymal stromal cells in myelodysplastic syndrome with disease progression.

To evaluate the mechanism underlying the communication between myeloid malignant and bone marrow (BM) microenvironment cells in disease progression, the current study established BM mesenchymal stromal cells (MSCs) and assessed extracellular vesicle (EV) microRNA (miR) expression in 22 patients with myelodysplastic syndrome (MDS) and 7 patients with acute myeloid leukemia and myelodysplasia-related changes (AML/MRC). Patients with MDS were separated into two categories based on the revised International Prognostic Scoring System (IPSS-R), and EV-miR expression in BM-MSCs was evaluated using a TaqMan low-density array. The selected miRs were evaluated using reverse transcription-quantitative PCR. The current study demonstrated that the expression of BM-MSC-derived EV-miR was heterogenous and based on MDS severity, the expression of EV-miR-101 was lower in high-risk group and patients with AML/MRC compared with the control and low-risk groups. This reversibly correlated with BM blast percentage, with which the cellular miR-101 from BM-MSCs or serum EV-miR-101 expression exhibited no association. Database analyses indicated that miR-101 negatively regulated cell proliferation and epigenetic gene expression. The downregulation of BM-MSC-derived EV-miR-101 may be associated with cell-to-cell communication and may accelerate the malignant process in MDS cells.

Activation and function of receptor tyrosine kinases in human clear cell renal cell carcinomas.

BACKGROUND: The receptor tyrosine kinases (RTKs) play critical roles in the development of cancers. Clear cell renal cell carcinoma (ccRCC) accounts for 75% of the RCC. The previous studies on the RTKs in ccRCCs mainly focused on their gene expressions. The activation and function of the RTKs in ccRCC have not been fully investigated. METHODS: In the present study, we analyzed the phosphorylation patterns of RTKs in human ccRCC patient samples, human ccRCC and papillary RCC cell lines, and other kidney tumor samples using human phospho-RTK arrays. We further established ccRCC patient-derived xenograft models in nude mice and assessed the effects of RTKIs (RTK Inhibitors) on the growth of these cancer cells. Immunofluorescence staining was used to detect the localization of keratin, vimentin and PDGFRß in ccRCCs. RESULTS: We found that the RTK phosphorylation patterns of the ccRCC samples were all very similar, but different from that of the cell lines, other kidney tumor samples, as well as the adjacent normal tissues. 9 RTKs, EGFR1-3, Insulin R, PDGFRß, VEGFR1, VEGFR2, HGFR and M-CSFR were found to be phosphorylated in the ccRCC samples. The adjacent normal tissues, on the other hand, had predominantly only two of the 4 EGFR family members, EGFR and ErbB4, phosphorylated. What's more, the RTK phosphorylation pattern of the xenograft, however, was different from that of the primary tissue samples. Treatment of the xenograft nude mice with corresponding RTK inhibitors effectively inhibited the Erk1/2 signaling pathway as well as the growth of the tumors. In addition, histological staining of the cancer samples revealed that most of the PDGFRß expressing cells were localized in the vimentin-positive periepithelial stroma. CONCLUSIONS: Overall, we have identified a set of RTKs that are characteristically phosphorylated in ccRCCs. The phosphorylation of RTKs in ccRCCs were determined by the growing environments. These phosphorylated/activated RTKs will guide targeting drugs development of more effective therapies in ccRCCs. The synergistical inhibition of RTKIs combination on the ccRCC suggest a novel strategy to use a combination of RTKIs to treat ccRCCs.

Treatment of complex recurrent fistula-in-ano by surgery combined to autologous bone marrow-derived mesenchymal stroma cells and platelet-rich plasma injection.

PURPOSE: We report a case of successful management of complex recurrent cryptoglandular fistula-in-ano by surgery combined with autologous bone marrow-derived mesenchymal stroma cells (MSCs) and platelet-rich plasma (PRP) injection. METHODS: Clinical, radiological, and surgical data of the patient were reviewed, as well as the current literature on complex fistula-in-ano. RESULTS: A 37-year-old man with a recurrent cryptoglandular perianal fistula was addressed to our department. Inflammatory bowel disease was excluded by clinical history, endoscopy, and blood tests. Physical examination and MRI showed an anterior external orifice on the midline, 5 cm from the anal verge, with an internal orifice on the same line. Surgery combined to injection of MSC-PRP solution was successfully performed. MSC-PRP solution was prepared while the patient was under general anesthesia: bone marrow MSCs were obtained by centrifugation of a tibial puncture specimen and PRP from a peripheral whole blood sample of the patient. There were no adverse events post-operatively. Clinical and MRI examination 4 months after treatment confirmed the absence of perianal fistula. More than 2 years after surgery, there has been no recurrence. CONCLUSIONS: Treatment of complex recurrent cryptoglandular fistula-in-ano by surgery combined to autologous bone marrow-derived MSCs and PRP injection seems safe in selected patients, allowing long-term healing. This procedure seems promising but further evaluation by clinical trials is warranted.

Activation of Rev-erbα attenuates lipopolysaccharide-induced inflammatory reactions in human endometrial stroma cells via suppressing TLR4-regulated NF-κB activation.

Perturbation of the circadian rhythm damages the biological characteristics of cells and leads to their dysfunction. Rev-erbα, an important gene in the transcription-translation loop of circadian rhythm, is involved in regulating the balance between pro-inflammation and anti-inflammation. The disruption of this balance in human endometrial stroma cells (hESCs) destroys their biological behavior function in maintaining the menstrual cycle and embryonic implantation. Whether pharmacological modulation of Rev-erbα affects the inflammation of hESCs remains unclear. In this study, we treated hESCs with lipopolysaccharide (LPS) and found that LPS treatment increased the mRNA levels of pro-inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, IL-8, IL-18, and TNFα, and the secretion of IL-6. SR9009, a Rev-erbα agonist, significantly alleviated the LPS-induced production of pro-inflammatory cytokines in hESCs. Meanwhile, knockdown of Rev-erbα increased the expressions of IL-1ß, IL-6, and IL-8, accompanied by an increased mRNA level of the core clock gene Bmal1. Western blot analysis showed that SR9009 inhibited the expression of toll-like receptor 4 (TLR4) and the activation of NF-κB induced by LPS. All these findings suggested that pharmacological activation of Rev-erbα attenuated the LPS-induced inflammatory response of hESCs by suppressing TLR4-regulated NF-κB activation. This study may provide a strategy for preventing inflammation-related endometrial dysfunction and infertility or recurrent implantation failure.