Spatio-temporal metabolokinetics and therapeutic effect of CD106+ mesenchymal stem/stromal cells upon mice with acute lung injury.

Longitudinal investigations have revealed the unique attributes of mesenchymal stem/stromal cells (MSCs) in regenerative medicine. However, the spatio-temporal metabolokinetics and efficacy of MSCs with vascular cell adhesion molecule 1 (also known as CD106) expression in phenotypes and therapeutic effect upon acute lung injury (ALI) mice are largely obscure. For the purpose, we took advantage of the "3IL"-based strategy and Lentivirus-mediated green fluorescent protein (GFP) delivery for the generation of the CD106+ subset (denote as CD106+ -MSCs) from umbilical cord-derived MSCs (denote as NT-MSCs). Therewith, the cellular phenotypes of CD106+ -MSCs including immunophenotypes, multilineage differentiation potential towards adipocytes and osteoblasts were confirmed by flow cytometry and qRT-PCR assay. Meanwhile, multifaceted characteristics of transcriptomic features were analyzed by utilizing the RNA-SEQ and bioinformatics. Furthermore, to compare the therapeutic effects and spatio-temporal dynamics of CD106+ -MSCs, we conducted in vivo fluorescent tracer, hematoxylin and eosin staining, blood smear, blood routine and cytokine detection in mice. Herein, we generated CD106+ -MSCs with GFP expression and confirmed the conservative property of phenotypes. Compared to NT-MSCs with minimal CD106 expression, CD106+ -MSCs manifested consistent distribution and metabolokinetics in vivo but with preferable ameliorative effect upon the pathological appearance and proinflammatory cytokine secretion in ALI mice. Collectively, our data indicated the preferable therapeutic effects of CD106+ -MSCs upon ALI mice, which would benefit the further exploration of the CD106+ subset for pulmonary diseases and investigational new drug application purposes.

Liraglutide treatment attenuates inflammation markers in the cardiac, cerebral and renal microvasculature in streptozotocin-induced diabetic rats.

BACKGROUND: Diabetes mellitus (DM) induces cardiac and cerebral microvascular dysfunction via increased glycation, oxidative stress and endothelial activation. Liraglutide, a glucagon-like peptide-1 analogue, inhibited NOX2 and adhesion molecules in isolated endothelial cells. Here, we have studied how Liraglutide affects advanced glycation, NOX expression and inflammation of the cardiac, cerebral and renal microvasculature in diabetic rats. METHODS: DM was induced in Sprague-Dawley rats (n = 15) via intraperitoneal streptozotocin (STZ) injection (60 mg/kg bodyweight). Ten control rats remained nondiabetic. From day 9 post-STZ injection, Liraglutide (200 µg/kg bodyweight; n = 7) or vehicle (n = 8) was injected subcutaneously daily until termination on day 29. The advanced glycation endproduct N-ε-(carboxymethyl)lysine (CML), NOX2, NOX4, ICAM-1 and VCAM-1 were subsequently immunohistochemically analysed and quantified to compare Liraglutide treatment with placebo. RESULTS: In the heart, Liraglutide treatment significantly reduced the DM-increased scores/cm2 for CML in both ventricles (from 253 ± 53 to 72 ± 12; p = .003) and atria (343 ± 29 to 122 ± 8; p = .0001) and for NOX2, ICAM-1 and VCAM-1, but not for NOX4. Also in the cerebrum and cerebellum of the brain, Liraglutide significantly reduced the scores/cm2 for CML (to 60 ± 7 (p = .0005) and 47 ± 13 (p = .02), respectively), and for NOX2 and NOX4. In the kidney, the DM-induced expression of ICAM-1 and VCAM-1 was decreased in the blood vessels and glomeruli by Liraglutide treatment. Liraglutide did not affect blood glucose levels or bodyweight. CONCLUSIONS: Our study implies that Liraglutide protects the cardiac, cerebral and renal microvasculature against diabetes-induced dysfunction, independent of lowering blood glucose in a type 1 diabetes rat model.

Increased Soluble VCAM-1 and Normal P-Selectin in Cystic Fibrosis: a Cross-Sectional Study.

PURPOSE: As life expectancy in cystic fibrosis (CF) increases, questions regarding its potential impact on cardiovascular health arise. Soluble vascular cell adhesion molecule 1 (sVCAM-1), P-selectin (sP-selectin) are proposed as biomarkers of cardiovascular disease. We aimed to: compare their concentrations in clinically stable CF patients and healthy subjects (HS) and verify whether they independently correlate with CF characteristics. METHODS: Serum sVCAM-1 and sP-selectin levels were measured using ELISA. CF was characterized using: forced expiratory volume in 1 s, exocrine pancreatic and CF-related liver disease status, Pseudomonas aeruginosa colonization, serum high-sensitivity C-reactive protein, and body mass index (BMI). CFTR genotypes were classified as severe (classes I and II) or other. RESULTS: 108 CF patients and 51 healthy subjects volunteered for the study. In the CF group BMI was lower (median [IQR]: 20.5 kg/m2 [18.4-22.2] vs. 21.6 kg/m2 [19.9-23.4], p = 0.02) and hsCRP levels were higher (3.6 mg/L [1.1-7.1] vs. 0.5 mg/dL [0.3-1.0], p < 10-10). While sVCAM-1 concentrations were greater in CF patients (1018 ng/mL [851-1279] vs. 861 ng/mL [806-979], p < 10-4), sP-selectin levels did not differ (155 ng/mL [129-188] vs. 156 ng/mL [144-177], p = 0.48). None of the multivariable regression models was valid for the prediction of sVCAM-1 and sP-selectin in CF. CONCLUSIONS: We found higher sVCAM-1 concentrations in CF patients than in healthy subjects, which were not explained by CF characteristics. Further research is required to check whether sVCAM-1 is a marker of microangiopathy in CF.

Healing of full-thickness articular cartilage defects treated with cultured autologous chondrogenic satellite cells isolated from chondral stem cell niche in rabbits.

BACKGROUND: Healing of articular cartilage has remained in question with the use of conventional treatment modalities such as subchondral drilling and microfracture. As demonstrated in the past, adult stem cells retain promising clonogenicity. Therefore, we conducted this study to elucidate the effects of cultured autologous chondrogenic satellite cells (CACSCs) compared with subchondral drilling (SCD) for the repair of full-thickness articular cartilage defects. MATERIALS AND METHODS: We examined CACSCs isolated from the knee of rabbits using flow cytometry for the expression of stemness and chondrocyte-specific factors. Subsequently, we created a full-thickness cartilage defect model with a diameter of 3 mm and depth of 2 mm on the articular surface of trochlear grooves in the left knee of 24 New Zealand white rabbits. Then we drilled subchondrally through the defect in all animals and stuffed the defects with 10-µg/cm(2) collagen scaffolds. In the treatment group, we instilled CACSCs at 5 × 10(6) cells/mL in the collagen scaffold and collected samples on days 15, 30, and 45. RESULTS: The CACSCs revealed significant expression of CD106, CD44, collagen type 2, and aggrecan. In conjunction with SCD, CACSCs improved healing of the articular cartilage defect, as evidenced by the formation of hyaline-like tissue grossly and histologically. The healed tissue also revealed a significant (P < 0.05) increase in the expression of collagen type 2 and aggrecan (by real-time polymerase chain reaction) during the experiment. CONCLUSIONS: In conjunction with SCD, CACSCs may be considered to improve articular cartilage damage.

CD106/VCAM-1 distinguishes a fibroblast subpopulation with high colony-forming capacity and distinct protein expression from the uterosacral ligament.

Background: Pelvic organ prolapse (POP) is a common degenerative disease in women which may diminish quality of life. Investigating the pathological changes of the uterosacral ligament, including the functional changes of fibroblasts, is critical to understanding the pathophysiology of POP. This study was designed to isolate CD106-positive (CD106+) fibroblasts from the human uterosacral ligament and assess the function and expression of this subpopulation. Methods: We separated CD106+ fibroblasts and CD106 negative (CD106-) fibroblasts by fluorescence-activated cell sorting (FACS) and cultured them for subsequent experiments. Flow cytometric analysis was used to test the sorting efficiency, CD106 expression, and typical mesenchymal stem cell (MSC) phenotype marker expression. A colony-forming unit (CFU) assay was applied to evaluate the colony-forming ability of the fibroblasts. Trilineage differentiation capacities were assessed after in vitro induction. The protein levels of vimentin, fibroblast specific protein-1 (FSP-1), collagen I (COL 1), matrix metallopeptidase-1 (MMP-1), and α-smooth muscle actin (α-SMA) were detected by western blot analysis. The expression of CD106 was verified by flow cytometric analysis and immunohistochemistry (IHC) in the POP and non-POP groups. Results: The CD106+ fibroblasts were isolated with a purity of (93.50±3.91)%. The CD106+ fibroblasts exhibited higher colony-forming capacity than that of CD106- fibroblasts, but neither of them showed adipogenic or osteogenic differentiation similar to that of MSCs. The protein levels of MMP-1 and α-SMA were lower, and the level of COL 1 was higher in the CD106+ fibroblasts than in the CD106- fibroblasts. In addition, we observed a decreased expression of CD106 in the POP group compared with the non-POP group. Conclusions: Our results suggest that CD106+ fibroblasts possess a high colony-forming capacity and distinct protein expression, and this subpopulation is reduced in POP.

Expression of CD44+/CD24-, RAD6 and DDB2 on chemotherapy response in ovarian Cancer: A prospective flow cytometry study.

Backgrounds: Ovarian cancer is the 8th deadliest common cancer in women around the world. Almost all ovarian cancer patients would experience chemoresistance, recurrence, and poor prognosis after cytoreductive surgery and platinum-based chemotherapy. Chemoresistant cancer cells have characteristic expressions of cancer stem cell proteins (CSCs) CD44+/CD24-, RAD6 and DDB2. The increased expression of CD44+/CD24-, RAD6, and decreased DDB2 are believed to be associated with chemoresistance, recurrence, and poor prognosis of the disease. Thus, this study's objective is to analyze the correlation between the expression of CD44+/CD24-, RAD6 and DDB2 with ovarian cancer chemoresistance. Materials and methods: This study was conducted with a prospective cohort of 64 patients who is divided into two groups (32 patients in each group) at the Obstetrics-gynecology and pathology department of Cipto Mangunkusumo, Tarakan, Dharmais, and Fatmawati Hospital. All suspected ovarian cancer patients underwent cytoreductive debulking and histopathological examination. Chemotherapy was given for six series followed by six months of observation. After the observation, we determined the therapy's response with the RECIST Criteria (Response Criteria in Solid Tumors) and then classified the results into chemoresistant or chemosensitive groups. Flow cytometry blood tests were then performed to examine the expression of CD44+/CD24-, RAD6 and DDB2. Results: There was a significant relationship between increased levels of CD44+/CD24-, and RAD6 (p < 0.05) levels with the chemoresistance of ovarian cancer. The logistic regression test showed that the CD44+/CD24- was better marker. Conclusions: These results indicate that CD44+/CD24 and RAD6 expressions are significantly associated with ovarian cancer chemoresistance, and CD44+/CD24- is the better marker to predict ovarian cancer chemoresistance.

Isolation of muscle stem cells from rat skeletal muscles.

Muscle stem cells (MuSCs) are involved in homeostatic maintenance of skeletal muscle and play a central role in muscle regeneration in response to injury. Thus, understanding MuSC autonomous properties is of fundamental importance for studies of muscle degenerative diseases and muscle plasticity. Rat, as an animal model, has been widely used in the skeletal muscle field, however rat MuSC isolation through fluorescence-activated cell sorting has never been described. This work validates a protocol for effective MuSC isolation from rat skeletal muscles. Tibialis anterior was harvested from female rats and digested for isolation of MuSCs. Three protocols, employing different cell surface markers (CD106, CD56, and CD29), were compared for their ability to isolate a highly enriched MuSC population. Cells isolated using only CD106 as a positive marker showed high expression of Pax7, ability to progress through myogenic lineage while in culture, and complete differentiation in serum-deprived conditions. The protocol was further validated in gastrocnemius, diaphragm, and the individual components of the pelvic floor muscle complex (coccygeus, iliocaudalis, and pubocaudalis), proving to be reproducible. CD106 is an efficient marker for reliable isolation of MuSCs from a variety of rat skeletal muscles.

Vascular cell adhesion molecule 1 in patients with severe osteoarthritis of the hip : A prospective cross-sectional study.

BACKGROUND: Osteoarthritis (OA) of the hip is a frequent and debilitating joint disease. Only few clinical risk factors for hip OA are established and clinically applicable biomarkers to identify patients at risk are still lacking. The glycoprotein vascular cell adhesion molecule 1 (VCAM-1) is expressed by chondrocytes and synovial tissue and was a predictive marker for development of severe large joint OA in a previous study. OBJECTIVE: It was tested whether increased serum levels of VCAM-1 are prevalent in patients with severe OA of the hips. METHODS: In this prospective, multicenter, cross-sectional study, risk factors of severe hip OA were investigated in patients scheduled for hip joint arthroplasty and 100 patients were randomly selected for validation of VCAM-1 as a potential biomarker for hip OA. Serum samples were analyzed by an enzyme-linked immunosorbent assay and compared with a sex and age-matched control cohort. RESULTS: The groups were similar in age, gender ratio and prevalence of diabetes. Serum concentrations of VCAM-1 were 8% higher in OA patients compared to controls, without reaching statistical significance (818 ng ml-1, 95% confidence interval, CI 746-891 ng ml-1 versus 759 ng m-1, 95% CI 711-807 ng ml-1; P = 0.4839). CONCLUSION: The results of this study show that serum concentrations of VCAM-1 cannot distinguish patients with severe hip OA from age and sex-matched controls.

Changes in phenotype and differentiation potential of human mesenchymal stem cells aging in vitro.

BACKGROUND: Adult mesenchymal stem cells (MSCs) hold great promise for regenerative medicine because of their self-renewal, multipotency, and trophic and immunosuppressive effects. Due to the rareness and high heterogeneity of freshly isolated MSCs, extensive in-vitro passage is required to expand their populations prior to clinical use; however, senescence usually accompanies and can potentially affect MSC characteristics and functionality. Therefore, a thorough characterization of the variations in phenotype and differentiation potential of in-vitro aging MSCs must be sought. METHODS: Human bone marrow-derived MSCs were passaged in vitro and cultivated with either DMEM-based or αMEM-based expansion media. Cells were prepared for subculture every 10 days up to passage 8 and were analyzed for cell morphology, proliferative capacity, and surface marker expression at the end of each passage. The gene expression profile and adipogenic and osteogenic differentiation capability of MSCs at early (passage 4) and late (passage 8) passages were also evaluated. RESULTS: In-vitro aging MSCs gradually lost the typical fibroblast-like spindle shape, leading to elevated morphological abnormality and inhomogeneity. While the DMEM-based expansion medium better facilitated MSC proliferation in the early passages, the cell population doubling rate reduced over time in both DMEM and αMEM groups. CD146 expression decreased with increasing passage number only when MSCs were cultured under the DMEM-based condition. Senescence also resulted in MSCs with genetic instability, which was further regulated by the medium recipe. Regardless of the expansion condition, MSCs at both passages 4 and 8 could differentiate into adipocyte-like cells whereas osteogenesis of aged MSCs was significantly compromised. For osteogenic induction, use of the αMEM-based expansion medium yielded longer osteogenesis and better quality. CONCLUSIONS: Human MSCs subjected to extensive in-vitro passage can undergo morphological, phenotypic, and genetic changes. These properties are also modulated by the medium composition employed to expand the cell populations. In addition, adipogenic potential may be better preserved over osteogenesis in aged MSCs, suggesting that MSCs at early passages must be used for osteogenic differentiation. The current study presents valuable information for future basic science research and clinical applications leading to the development of novel MSC-based therapeutic strategies for different diseases.

Two novel mechanisms for maintenance of stemness in mesenchymal stem cells: SCRG1/BST1 axis and cell-cell adhesion through N-cadherin.

Mesenchymal stem cells (MSCs) retain the ability to self-renew and differentiate into mesenchymal cells. Therefore, human MSCs are suitable candidates for use in regenerative medicine and cell therapies. Upon activation by tissue damage, MSCs contribute to tissue repair through a multitude of processes such as self-renewal, migration, and differentiation. However, loss of self-renewal and multi-lineage differentiation potential occurs at a high rate during cell doubling. Effective MSC therapies require the establishment of new techniques that preserve MSC multipotency after lengthy cell expansions. Here, two novel mechanisms are described for maintenance of stemness in MSCs via scrapie responsive gene 1 (SCRG1)/bone marrow stromal cell antigen-1 (BST1) ligand-receptor combination and cell-cell adhesion through N-cadherin. These two mechanisms findings provide a valuable tool for regenerative medicine and cell therapeutic methods that require the ex vivo expansion of human MSCs while maintaining native stem cell potential.

CD106 is a novel mediator of bone marrow mesenchymal stem cells via NF-κB in the bone marrow failure of acquired aplastic anemia.

BACKGROUND: Acquired aplastic anemia (AA) is characterized by deficiency or dysfunction of the bone marrow (BM) microenvironment. However, little is known about the impairment of BM-derived mesenchymal stem cells (MSCs) in AA patients. METHODS: We used Illumina HiSeqTM 2000 sequencing, quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry (FCM), and Western blotting to test the expression of CD106 gene (vascular cell adhesion molecule 1 (VCAM1)) and CD106 protein of BM-MSCs. Furthermore, we used hematoxylin and eosin (H&E) and histochemical staining analysis, immunofluorescence, and the formation of capillary-like structures to analyze capillary tube-like formation in vitro; we also used the Matrigel plug assay to test in vivo vasculogenesis, and an assay of colony forming units (CFUs) and colony-forming unit-megakaryocyte (CFU-MK) to detect the support function of MSCs in vitro. The in vivo engraftment of CD34+ cells and MSCs in NOD/SCID mice was tested by FACS and survival assay; the expression of NF-κB was tested by NanoPro analysis and immunofluorescence. NF-κB-regulated CD106 gene (VCAM1) was confirmed by tumor necrosis factor alpha (TNF-α)-stimulated and lipopolysaccharide (LPS)-stimulated MSCs, blockade assay, and immunofluorescence. RESULTS: Here, we report that BM-MSCs from AA patients exhibited downregulation of the CD06 gene (VCAM1) and low expression of CD106 in vitro. Further analysis revealed that CD106+ MSCs from both AA patients and healthy controls had increased potential for in vitro capillary tube-like formation and in vivo vasculogenesis compared with CD106- MSCs, and the results were similar when healthy MSCs were compared with AA MSCs. CD106+ MSCs from both AA patients and healthy controls more strongly supported in vitro growth and in vivo engraftment of CD34+ cells in NOD/SCID mice than CD106- MSCs, and similar results were obtained when healthy MSCs and AA MSCs were compared. The expression of NF-κB was decreased in AA MSCs, and NF-κB regulated the CD106 gene (VCAM1) which supported hematopoiesis. CONCLUSIONS: These results revealed the effect of CD106 and NF-κB in BM failure of AA.

Human umbilical cord mesenchymal stem cells increase interleukin-9 production of CD4+ T cells.

Mesenchymal stem cells (MSC) are able to differentiate into cells of multiple lineage, and additionally act to modulate the immune response. Interleukin (IL)-9 is primarily produced by cluster of differentiation (CD)4+ T cells to regulate the immune response. The present study aimed to investigate the effect of human umbilical cord derived-MSC (UC-MSC) on IL-9 production of human CD4+ T cells. It was demonstrated that the addition of UC-MSC to the culture of CD4+ T cells significantly enhanced IL-9 production by CD4+ T cells. Transwell experiments suggested that UC-MSC promotion of IL-9 production by CD4+ T cells was dependent on cell-cell contact. Upregulated expression of CD106 was observed in UC-MSC co-cultured with CD4+ T cells, and the addition of a blocking antibody of CD106 significantly impaired the ability of UC-MSC to promote IL-9 production by CD4+ T cells. Therefore, the results of the present study demonstrated that UC-MSC promoted the generation of IL-9 producing cells, which may be mediated, in part by CD106. The findings may act to expand understanding and knowledge of the immune modulatory role of UC-MSC.

VCAM-1+ placenta chorionic villi-derived mesenchymal stem cells display potent pro-angiogenic activity.

INTRODUCTION: Mesenchymal stem cells (MSCs) represent a heterogeneous cell population that is promising for regenerative medicine. The present study was designed to assess whether VCAM-1 can be used as a marker of MSC subpopulation with superior angiogenic potential. METHODS: MSCs were isolated from placenta chorionic villi (CV). The VCAM-1(+/-) CV-MSCs population were separated by Flow Cytometry and subjected to a comparative analysis for their angiogenic properties including angiogenic genes expression, vasculo-angiogenic abilities on Matrigel in vitro and in vivo, angiogenic paracrine activities, cytokine array, and therapeutic angiogenesis in vascular ischemic diseases. RESULTS: Angiogenic genes, including HGF, ANG, IL8, IL6, VEGF-A, TGFß, MMP2 and bFGF, were up-regulated in VCAM-1(+)CV-MSCs. Consistently, angiogenic cytokines especially HGF, IL8, angiogenin, angiopoitin-2, µPAR, CXCL1, IL-1ß, IL-1α, CSF2, CSF3, MCP-3, CTACK, and OPG were found to be significantly increased in VCAM-1(+) CV-MSCs. Moreover, VCAM-1(+)CV-MSCs showed remarkable vasculo-angiogenic abilities by angiogenesis analysis with Matrigel in vitro and in vivo and the conditioned medium of VCAM-1(+) CV-MSCs exerted markedly pro-proliferative and pro-migratory effects on endothelial cells compared to VCAM-1(-)CV-MSCs. Finally, transplantation of VCAM-1(+)CV-MSCs into the ischemic hind limb of BALB/c nude mice resulted in a significantly functional improvement in comparison with VCAM-1(-)CV-MSCs transplantation. CONCLUSIONS: VCAM-1(+)CV-MSCs possessed a favorable angiogenic paracrine activity and displayed therapeutic efficacy on hindlimb ischemia. Our results suggested that VCAM-1(+)CV-MSCs may represent an important subpopulation of MSC for efficient therapeutic angiogenesis.

HTLV-1 infects human mesenchymal stromal cell in vitro and modifies their phenotypic characteristics.

The typical characteristics of mesenchymal stem cells (MSCs) can be affected by inflammatory microenvironment; however, the exact contribution of HTLV-1 to MSC dysfunction remains to be elucidated. In this study, we demonstrated that MSC cell surface molecules VCAM-1 and ICAM-1 are upregulated by contact with HTLV-1, and HLA-DR was most highly expressed in MSCs co-cultured with MT2 cells. The expression levels of VCAM-1 and HLA-DR were increased in MSCs cultured in the presence of PBMCs isolated from HTLV-1-infected symptomatic individuals compared with those cultured with cells from asymptomatic infected individuals or healthy subjects. HTLV-1 does not impair the MSC differentiation process into osteocytes and adipocytes. In addition, MSCs were efficiently infected with HTLV-1 in vitro through direct contact with HTLV-1-infected cells; however, cell-free virus particles were not capable of causing infection. In summary, HTLV-1 can alter MSC function, and this mechanism may contribute to the pathogenesis of this viral infection.

Growth factor independence 1 (Gfi1) regulates cell-fate decision of a bipotential granulocytic-monocytic precursor defined by expression of Gfi1 and CD48.

The transcriptional repressor Gfi1 regulates the expression of genes important for survival, proliferation and differentiation of hematopoietic cells. Gfi1 deficient mice are severely neutropenic and accumulate ill-defined CD11b(+)GR1(int) myeloid cells. Here we show that Gfi1 expression levels determine mono- or granulocytic lineage choice in precursor cells. In addition, we identify CD48 as a cell surface marker which enables a better definition of monocytes and granulocytes in mouse bone marrow. Using the CD48/Gr1/Gfi1 marker combination we can show that the CD11b(+)GR1(int) cells accumulating in Gfi1 deficient mice are monocytes and not granulocyte precursors. Expression of CD48, Gr1 and Gfi1 define different bone marrow subpopulations that are either committed to the granulocytic lineage, or bipotential precursors of granulocytes or monocytes. Finally, a comparison of genes differentially expressed between murine Gfi1 high granulocytic precursors and mature granulocytes with gene expression changes from human myeloblasts versus neutrophils show a strong resemblance of human and mouse differentiation pathways. This underlines the value of the markers CD48 and Gfi1 identified here to study human and murine granulo-monocytic differentiation.