Endothelial dysfunction plays an important role in the pathogenesis of pulmonary arterial hypertension (PAH) in sickle cell disease (SCD). A variety of evidence suggests that circulating endothelial progenitor cells (EPCs) play an integral role in vascular repair. We hypothesized that SCD patients with PAH are deficient in EPCs, potentially contributing to endothelial dysfunction and disease progression. The number of circulating CD34+/CD14-/CD106+ EPCs was significantly lower in SCD patients with PAH than without PAH (P=0.025). CD34+/CD14-/CD106+ numbers significantly correlated with tricuspid regurgitation velocity (TRV, r=-0.44, P=0.033) 6-minute walk distance (6MWD, r= 0.72, P=0.001), mean pulmonary artery pressure (mPAP, r= -0.43, P=0.05), and pulmonary vascular resistance (PVR, r=-0.45, P=0.05). Other EPC subsets including CD31+/CD133+/CD146+ were similar between both groups. Numbers of EPCs did not correlate with age, sex, hemoglobin, WBC count, reticulocyte count, lactate dehydrogenase (LDH), iron/ferritin levels, and serum creatinine. These data indicate that subsets of EPC are lower in SCD patients with PAH than in those without PAH. Fewer EPCs in PAH patients may contribute to the pulmonary vascular pathology. Reduced number of EPCs in SCD patients with PAH might not only give potential insight into the pathophysiological mechanisms but also might be useful for identifying suitable therapeutic targets in these patients.

OBJECTIVE

To compare the two sources of adipose and bone marrow derived mesenchymal stem cells (BMSCs and AMSCs) in immune regulation and to evaluate the therapeutic effects of AMSCs on Con A induced hepatitis and the possible mechanism involved in it.

METHODS

We isolated bone marrow and adipose derived mesenchymal stem cells respectively and compared their differences on T lymphocyte activation, proliferation and suppression. We also test the anti-apoptosis ability of AMSCs on LO2 cell line. The effects of intravenous infusion of AMSCs on liver damage were also tested and we detected donor AMSCs in liver of recipient and their effects on the activity of intrahepatic NKT cells.

RESULTS

BMSCs and AMSCs were similar in cell phenotype and the difference existed only in the expression of CD106. The results showed that the capacity of suppressing T cells proliferation and activation was weakened in AMSCs. AMSCs ameliorated liver damage and this effect was time and dose dependent. We detected donor AMSCs in liver of recipient which suggested tissue damage could be a clue for AMSCs migration. We also found AMSCs suppress the activity of intrahepatic NKT cells, but this suppress effects was not restricted in liver only, but the whole body.

CONCLUSIONS

Cell origin and abundance are decisive factors in stem cells applications and with the same premise of AMSCs and BMSCs, adipose tissue is a more promising origin source of stem cells. The immunoregulatory features of MSCs might play an important role in various MSCs cellular therapies.

The present study was undertaken to investigate the immunoregulatory effects of T-614 (3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-o ne) on synovial cells in vitro. Synovial cells were cultured with T-614 in the presence or absence of various cytokines. After incubation, the costimulatory molecule expression on synovial cells and cytokine production in culture supernatants were analyzed by an indirect immunofluorescence method and enzyme-linked immunosorbent assay, respectively. We also examined the effect of T-614 on the function of synovial cells as antigen-presenting cells (APCs). The costimulatory molecules including CD54, CD58, and CD106 were constitutionally expressed on the surface of synovial cells. However, neither CD80 nor CD86 nor CD102 was found on the surface, and these costimulatory molecules could not be induced by any cytokines. T-614 itself did not affect the costimulatory molecule expression and cytokine production of unstimulated synovial cells. The stimulation of synovial cells with interferon-gamma (IFN-gamma), interleukin-1beta, or 12-O-tetradecanoyl phorbol 13-acetate enhanced the expression of costimulatory molecules and the proinflammatory cytokine production of these cells. Both the up-regulated expression of these costimulatory molecules and the enhanced production of proinflammatory cytokines were significantly inhibited by T-614. Autologous T cell proliferation in response to purified protein derivative by IFN-gamma-treated synovial cells was significantly suppressed by T-614. T-614 has considerable immunosuppressive effects on synovial cells by inhibiting the costimulatory molecule expression and cytokine production of these cells and the antigen-specific T cell proliferation mediated by the synovial cells. These results suggest that T-614 plays an important immunoregulatory role in rheumatoid synovial tissues.

BACKGROUND

Mesenchymal progenitor cells interact with immune cells and modulate inflammatory responses. The cellular characteristics required for this modulation are under fervent investigation. Upon interaction with macrophage cells, they can contribute to or suppress an inflammatory response. Current studies have focused on mesenchymal progenitors derived from bone marrow, adipose, and placenta. However, the arterial wall contains many mesenchymal progenitor cells, which during vascular disease progression have the potential to interact with macrophage cells. To examine the consequence of vascular-tissue progenitor cell-macrophage cell interactions in an inflammatory environment, we used a recently established mesenchymal progenitor cell line derived from the mouse aorta.

METHODS

Mouse bone marrow-derived macrophage (MΦ) cells and mouse aorta-derived mesenchymal progenitor (mAo) cells were cultured alone or co-cultured directly and indirectly. Cells were treated with oxidized low-density lipoprotein (ox-LDL) or exposed to the inflammatory mediators lipopolysaccharide (LPS) and interferon-gamma (IFNγ) or both. A Toll-like receptor-4 (TLR4)-deficient macrophage cell line was used to determine the role of the mAo cells. To monitor inflammation, nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFα) secretions were measured.

RESULTS

Mesenchymal progenitor cells isolated from aorta and cloned by high proliferative capacity (mAo) can differentiate into multiple mesenchymal lineages and are positive for several commonly used mouse mesenchymal stem cell markers (that is, CD29, CD44, CD105, CD106, and Sca-1) but are negative for CD73 and ecto-5'-nucleotidase. In co-culture with MΦ cells, they increase MΦ oxidized-LDL uptake by 52.2%. In an inflammatory environment, they synergistically and additively contribute to local production of both NO and IL-6. After exposure to ox-LDL, the inflammatory response of MΦ cells to LPS and LPS/IFNγ is muted. However, when lipid-laden MΦ cells are co-cultured with mAo cell progenitors, the muted response is recovered and the contribution by the mAo cell progenitor is dependent upon cell contact.

CONCLUSIONS

The resident mesenchymal progenitor cell is a potential contributor to vascular inflammation when in contact with inflamed and lipid-laden MΦ cells. This interaction represents an additional target in vascular disease treatment. The potential for resident cells to contribute to the local immune response should be considered when designing therapeutics targeting inflammatory vascular disease.

A monoclonal antibody (mAb), designated mNI-58A, was produced by immunizing mice with the lipopolysaccharide (LPS)-stimulated monocyte-like cell line, U937. The antigen defined by mNI-58A was widely expressed on various lymphoid cells and all cell lines examined except the erythroid cell line, K562. When the reactive patterns between mNI-58A and the mAbs to various human differentiation antigens (CD11a, CD11b, CD11c, CD14, CD16, CD18, CD23, CD28, CD29, CD31, CD43, CD44, CD45RA, CD50, CD54, CD58, CD80, CD102, CD106, HLA-class I and-class II antigen) were compared, that of mNI-58A was found to be similar to those of the leukocyte function-associated antigen-1 (LFA-1) mAbs. Using a competitive immunofluorescence binding assay it was found that the preincubation with one of the CD11a mAbs, 2F12 completely blocked the subsequent binding of mNI-58A. mNI-58A prevented the homotypic cell aggregation of the phorbol myristate acetate (PMA)-activated U937 cells (referred to as PMA-U937) and PMA-activated Epstein-Barr virus (EBV)-transformed B cell lines, B-85 and Mann. mNI-58A markedly induced the spread formation of the PMA-U937 cells following this blocking of the homotypic cell aggregation, whereas 2F12 did not under the same condition. The spread formation induced by mNI-58A was completely blocked by cytochalasin B (CyB), cytochalasin D (CyD), cycloheximide (CHX) or protein kinase C inhibitors, sphingosine and H-7. The U937 cells markedly adhered to the tumor necrosis factor-alpha (TNF-alpha)-stimulated human umbilical vein endothelial cells (HUVECs) and also to the extracellular matrix protein, fibronectin, but mNI-58A did not enhance or block these adhesion process. mNI-58A precipitated two glycoproteins with molecular weight 180 kDa and 95 kDa as determined by SDS-PAGE analysis, which were identical to the LFA-alpha (CD11a) and beta (CD18) chains of leukocyte integrin precipitated by the CD11a mAbs, respectively. Sequential immunoprecipitation studies using the CD11a mAb (2F12) also indicate that mNI-58A recognizes an epitope on the alpha-chain of the LFA-1 molecule. The ability of mNI-58A to block the PMA-U937 cells and to induce the spread formation of these cells suggests that mNI-58A is a novel mAb reacting with an epitope on the alpha-chain of LFA-1 different from those recognized with the existing CD11a mAbs.

Chordomas are thought to be tumors originating from notochord remnants characterized histologically by cohesive cells with epithelial features and by immunohistochemical expression of epithelial markers. To investigate the expression and distribution of cell adhesion molecules in chordomas, we immunohistochemically studied the expression of representative cell adhesion molecules, E-cadherin, P-cadherin, N-cadherin, beta-catenin, CD44, ICAM-1 (CD54), NCAM (CD56), and VCAM-1 (CD106) in 16 tumors from 16 patients (skull base, n=5; cervical, n=2; sacral, n=9) and 3 cases of fetal notochord. Of 16 tumors, 12 (75.0%) expressed membranous immunoreactivity for NCAM, 10 (62.5%) for VCAM-1, 9 (56.3%) for CD44, 8 (50.0%) for N-cadherin, 6 (37.5%) for beta-catenin, 4 (25%) for ICAM-1, and 1 (6.3%) for P-cadherin. Nuclear staining for E-cadherin was recognized in 11 (68.8%) tumors, and membranous staining for E-cadherin in 3 (18.8%); none of the tumors showed both nuclear and membranous staining. Intranuclear accumulation of beta-catenin was not found in any chordoma. One fetal notochord case showed immunoreactivity for N-cadherin, E-cadherin (some cells showed staining in both cytoplasm and nuclei), CD44 and beta-catenin. These results indicate that chordomas frequently express immunoreactivity for multiple adhesion molecules including VCAM, CD44 and N-cadherin, as well as for NCAM and E-cadherin, as previously reported. These molecules may participate in producing the cellular cohesion evident in tumor morphological structure. Although the precise underlying mechanisms remain to be elucidated, the high frequency of nuclear expression of E-cadherin (11 of 16 cases) may be diagnostically useful.

The aim of this study was to determine whether the inflammatory milieu and/or hypoxia induces the dedifferentiation of synovial cells into mesenchymal stem-like cells, which may contribute to the tumor-like growth of synovial cells. Expression of mesenchymal stem cell markers (CD24, CD44, CD90, CD106, CD146 and Stro-1) was compared among cultured fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), bone marrow mesenchymal stem cells (BM MSCs) and normal dermal fibroblasts. After the cells were stimulated with pro-inflammatory cytokines for 3 days under hypoxia or normoxia, the stem cell markers were analyzed by FACS. CD44 and CD90 were expressed constitutively in all four cell types. Only the BM MSCs strongly expressed CD146. The expression of stem cell markers was similar between FLSs from RA and those from OA patients. In addition, the expression levels in FLSs were similar to those in normal dermal fibroblasts. The stimulation of FLSs and dermal fibroblasts with IL-1β or a mixture of cytokines under hypoxia did not induce a marked change in the expression of stem cell markers. These results indirectly suggest that the pro-inflammatory milieu may be not sufficient to induce the dedifferentiation of FLSs in arthritic joints.

BACKGROUND

Neutrophils participate in acute vascular rejection (AVR) of organ xenografts. Induced antibodies (Abs), including anti-Galalpha1,3Gal (alpha-Gal) Abs, have been suggested to cause AVR. We investigated the adhesion of naive human neutrophils to porcine aortic endothelial cells (PAECs) stimulated with anti-alpha-Gal Abs under conditions of flow. In addition, the ability of human neutrophils to adhere to human and porcine endothelium under static and flow conditions was evaluated.

RESULTS

In a flow-adhesion assay, a significant increase in adhesion of human neutrophils to PAECs, but not to human aortic endothelial cells (HAECs), was detected 6 hours after anti-alpha-Gal Ab-binding. After Ab stimulation, PAECs expressed CD62E and increased levels of CD106, indicating an activated endothelial cell (EC) phenotype. In a migration assay, supernatants from Ab-stimulated PAECs induced migration of human neutrophils, which was partially blocked by anti-porcine (p) interleukin (IL)-8 Abs and an antagonist to platelet-activating factor (PAF). In static and flow-adhesion assays, no difference in adhesion of human neutrophils to unstimulated or tumor necrosis factor (TNF)-alpha-stimulated HAECs and PAECs could be detected.

CONCLUSIONS

Our data suggest that anti-alpha-Gal Abs play an important role in the initiation of AVR by mediating adhesion and recruitment of neutrophils within an organ xenograft. In contrast with previous investigations, our data argues against a differential recognition of PAECs and HAECs by human neutrophils. Thus, to prevent AVR and accomplish long-term xenograft survival, it will be important to remove anti-alpha-Gal Abs before and after pig-to-human transplantation.

BACKGROUND

Early placental development is associated with complex regulatory mechanisms, and molecular communication problems that arise during the developmental process are dangerous for continuation of the pregnancy. As studies on the process of invasion and migration of trophoblast cells have shown the importance of cell-cell and cell-matrix interactions, we examined the effects of adhesion molecules on the mechanism(s) of spontaneous abortions and compared them to elective abortion materials using histopathological and immunohistochemical methods. To the best of our knowledge, this is the first study to investigate adhesion molecules in spontaneous abortions.

METHODS

Curettage materials from abortions were examined retrospectively in the Department of Pathology, Zonguldak Karaelmas University School of Medicine, Zonguldak, Turkey. CD31/PECAM-1 (endothelial cell marker), CD44v (variant 3), E-cadherin, CD54/ICAM-1, and CD106/VCAM-1 expression profiles were evaluated by immunohistochemistry, and cellular localization was determined under light microscopy. The results of spontaneous abortions were compared to those of elective abortions.

RESULTS

The staining percentages of CD31, CD44, CD106, and E-cadherin decreased in cases of spontaneous abortion, but CD54 (ICAM-1) expression increased. Statistically significant differences were detected between spontaneous and elective abortion materials with regard to cytotrophoblasts (CTs), syncytiotrophoblasts (STs), and extravillous trophoblasts (EVTs) with the anti-CD31 antibody (p=0.0001). In addition, CD54 (p=0.007 and p=0.002) and E-cadherin (p=0.002 and p=0.02) expression in CTs and STs, respectively, were significantly different. Furthermore, CD44 expression (p=0.003) in decidual (D) cells and CD106 (p=0.0001) expression in vessels of endometrial (E) and villous tissues were also significantly different.

CONCLUSIONS

Decreased CD31 expression in CTs that invade the spiral arterioles and mimic E cells in spontaneous abortion cases suggests that CD31/PECAM-1 is an important molecule in uteroplacental adequacy. Moreover, diminished expression of CD44 in D cells caused impaired stroma-villous connections. Enhancement of ICAM-1 in placental and invading STs may be useful as a diagnostic marker for patients who may have a tendency to have spontaneous abortions. A down-regulation of E-cadherin was observed, which may be responsible for impaired CT differentiation and loss of the pregnancy. Furthermore, decreased VCAM-1 expression in spontaneous abortions may be consistent with the importance of VCAM-1 in trophoblast-endothelial cell interactions. Many adhesion molecules are known to be effective in the normal development of a pregnancy, and the analysis of adhesion molecules in spontaneous abortions will provide useful information for clarifying the physiopathology of spontaneous abortions.

OBJECTIVE

To appreciate the evolution of serum angiogenic and/or adhesion molecules levels during a long term follow-up of rheumatoid arthritis (RA) patients.

METHODS

Serum levels of 5 soluble adhesion/angiogenesis glycoproteins (VEGF, CD31, CD54, CD62E, CD106) were measured in Elisa in samples collected over 6 years in a cohort of 43 RA patients with monitored clinical parameters of disease activity and severity.

RESULTS

RA patients had significantly higher levels (p < 0.0001) of sCD106 (VCAM-1) than control subjects. Conversely, the levels of soluble VEGF, CD31, CD54 and CD62E were normal or lower than normal. No statistically significant time effect was noted. No effect either was noted as related to the therapeutic agents taken by the patients.

CONCLUSIONS

The sustained elevated serum levels of sCD106 observed here imply that this molecule might be related to the chronicity and progression of RA.

BACKGROUND

Follicular lymphoma, the neoplastic counterpart of germinal center B cells, typically recapitulates a follicular architecture. Several observations point to the crucial role of the cellular microenvironment in the development and/or progression of follicular lymphoma cells in vivo. The aim of our study was to characterize the spontaneous apoptosis of follicular lymphoma cells in vitro, and the modulation of this apoptosis by follicular dendritic cells.

METHODS

We used a cell line derived from follicular dendritic cells to model the functional interactions of these cells and lymphoma cells in co-culture. Follicular lymphoma cells were isolated from tissue biopsies. Apoptosis was quantified by flow cytometry and apoptotic pathways were investigated by western blotting.

RESULTS

The spontaneous apoptosis of follicular lymphoma cells in vitro involves the activation of caspases-3 and -8 but not of caspase-9, occurs despite persistent high levels of BCL-2 and MCL-1, and is associated with down-regulation of c-FLIP(L). Spontaneous apoptosis of follicular lymphoma cells is partially prevented by co-culture with the follicular dendritic cells, which prevents activation of caspase-8, caspase-3 and induces an upregulation of c-FLIP(L). Using neutralizing antibodies, we demonstrated that interactions involving CD54 (ICAM-1), CD106 (VCAM-1) and CD40 are implicated in this biological process.

CONCLUSIONS

Follicular dendritic cells constitute a useful tool to study the functional interactions between follicular lymphoma cells and follicular dendritic cells in vitro. Understanding the molecular mechanisms involved in these protective interactions may lead to the identification of therapeutic agents that might suppress the survival and growth of follicular lymphoma cells.

The culture of synovial fibroblasts (SFs) is one of the most effective tools for investigating the pathology and physiology of synovial tissues and should prove useful for identifying the importance of SFs in disease as well as for the development of novel therapeutic approaches for several chronic joint diseases, such as rheumatoid arthritis. However, thus far, a detailed protocol for the primary culture and isolation of murine SFs has not been established. Therefore, the present study describes an easy and convenient method for isolating and culturing SFs from C57BL/6 mice. This protocol can be divided into 4 stages: Isolation of synovial tissues, isolation of SFs, seeding of SFs for growth in culture and purity analysis of SFs using the four cell markers, vimentin, cluster of differentiation 90.2 (CD90.2; Thy-1.2), intracellular adhesion molecule 1 (CD54) and vascular cell adhesion molecule 1 (CD106). This method is efficient and a purified population of SFs can be obtained 10 days after the initiation of culture.

Human periodontal ligament stem cells (PDLSCs) are a unique population of mesenchymal stem cells (MSCs) that demonstrate the capacity to generate cementum- and periodontal ligament-like structures in vivo. As such, PDLSCs represent a promising cell-based therapy in reconstructive dentistry for the treatment of periodontal disease. The present chapter describes two methods for isolating PDLSCs from human PDL tissue including traditional plastic adherence, and immunomagnetic selection based on the expression of MSC-associated surface markers STRO-1 antigen, CD146 (MUC-18), CD29 (Integrin β-1), CD44, and CD106 (VCAM-1). Although no single antibody demonstrates specificity for MSCs, isolation based on expression of individual markers results in homogenous preparations of PDLSCs. Methods to further characterize the immunophenotype and multipotent capacity of PDLSCs to differentiate into adipocytes, osteoblast-, and cementoblast-like cells in vitro, and cementum- and periodontal ligament-like tissues in vivo, are also described.

The role of C-X-C chemokine receptor type 4 (CXCR4) in umbilical mesenchymal stem cells (UMSCs) as therapy for liver disease is ill understood. The aim of the study was to evaluate rat UMSCs (rUMSCs) on CXCR4 expression and homing to injured liver tissue. rUMSCs were isolated from umbilical cords of pregnant rats. Acute liver failure (ALF) models were developed using D-galactosamine. CXCR4 expression induction by serum from rats with ALF (LFS), cytokines, growth factors, and LPS was analyzed. CXCR4 expression was analyzed by RT-PCR, western blot, and flow cytometry. rUMSCs were labeled with carboxyfluorescein and pretreated with LFS to induce CXCR4 expression and were transplanted into ALF rats. Animals were sacrificed 48 h and 1 week after transplantation. Liver-homing rUMSCs were observed under fluorescence microscopy. rUMSCs were successfully isolated, expressing CD90 and CD106, but not CD34 and CD45. mRNA and protein expressions of CXCR4 were strongly up-regulated by LFS and by the mixture of cytokines, stem cell factor, and LPS (CM). Expression of cell surface CXCR4 on rUMSCs in groups treated with LFS (42.37 ± 1.60 %) and CM (40.17 ± 1.78 %) was higher than that in the untreated control group (9.67 ± 1.06 %) (both P < 0.001). At 48 h after transplantation, more rUMSCs pretreated with LFS appeared in the portal area, and migrated to the liver parenchyma after 1 week. LFS strongly induced the surface expression of CXCR4 on rUMSCs. Increasing CXCR4 expression on rUMSCs may enhance their homing ability to injured liver tissue, and may eventually be used for treating liver diseases.

Osteoarthritis (OA) affects a large number of patients; however, human umbilical cord stem cells exhibit therapeutic potential for treating OA. The aim of the present study was to explore the interaction between human umbilical cord stem cells and degenerated chondrocytes, and the therapeutic potential of human umbilical cord stem cells on degenerated chondrocytes. Human umbilical cord‑derived mesenchymal stem cells (hUC‑MSCs) were harvested from human umbilical cords, and flow cytometry was used to analyze the surface antigen markers, in addition, chondrogenic, osteogenic and adipogenic differentiation on the cells was investigated. OA cells at P3 were cocultured with hUC‑MSCs in a separated co‑culture system, and reverse transcription‑polymerase chain reaction and western blot were used to evaluate the mRNA, and protein expression of collagen type II (Col2), SRY‑box 9 (sox‑9) and aggrecan. The level of inflammatory cytokines, tumor necrosis factor‑α, interleukin (IL)‑1β, IL‑6, IL‑10, were analyzed by ELISA in the supernatant. hUC‑MSCs grow in a fibroblastic shape with stable proliferation. hUC‑MSCs expressed cluster of differentiation 44 (CD44), CD73, CD90, CD105; while did not express CD34, CD45, CD106, CD133. After multi‑induction, hUC‑MSCs were able to differatiate into adipogenic, osteogenic and chondrogenic lineage. hUC‑MSCs inhibited the expression of matrix metalloproteinase‑13, collagen type X α1 chain and cyclooxygenase‑2 in OA chondrocytes, and enhanced the proliferation of OA chondrocytes, while OA chondrocytes stimulated the production of Col2, sox‑9 and aggrecan and promoted hUC‑MSCs differentiate into chondrocytes. Flow cytometry analysis demonstrated hUC‑MSCs have a predominant expression of stem cell markers, while the hematopoietic and endothelial markers were absent. Osteogenic, chondrogenic and adipogenic differentiation was observed in certain induction conditions. hUC‑MSCs improved the proliferation of OA chondrocytes and downregulated the expression of inflammatory cytokines, while OA chondrocytes promoted MSCs to differentiate into chondrocytes. Taken together, the co‑culture of hUC‑MSCs and OA chondrocytes may provide a therapeutic potential in OA treatment.

We studied the effects of long-term (20-day) simulated microgravity (clinostatic exposure) and osteogenic differentiation stimuli on cultured mesenchymal stromal precursor cells isolated from human bone marrow. Clinostatic exposure significantly reduced proliferative activity of mesenchymal stem cells in comparison with the static and dynamic control, increased the number of large flat cells in the culture, and stimulated migration activity of cells. Phenotypic studies of surface antigens (CD90, CD54, CD106, CD105, CD34, CD45, class 1 HLA) during clinostatic exposure of mesenchymal stem cell cultures showed differences in their expression between experimental and control groups. Studies of osteogenesis of precursor cell showed that cell differentiation potential can be directed towards osteogenesis by a combination of clinostatic exposure and differentiation stimuli. The results confirm gravity sensitivity of human bone marrow precursor cells and open new vistas for understanding of the mechanisms of bone tissue loss in humans under conditions of space mission.

The emerging field of tissue engineering and regenerative medicine is a multidisciplinary science that is based on the combination of a reliable source of stem cells, biomaterial scaffolds, and cytokine growth factors. Adult mesenchymal stem cells are considered important cells for applications in this field, and adipose tissue has revealed to be an excellent source of them. Indeed, adipose-derived stem cells (ASCs) can be easily isolated from the stromal vascular fraction (SVF) of adipose tissue. During the isolation and propagation of murine ASCs, we observed the appearance of a spontaneously immortalized cell clone, named m17.ASC. This clone has been propagated for more than 180 passages and stably expresses a variety of stemness markers, such as Sca-1, c-kit/CD117, CD44, CD106, islet-1, nestin, and nucleostemin. Furthermore, these cells can be induced to differentiate toward osteogenic, chondrogenic, adipogenic, and cardiogenic phenotypes. m17.ASC clone displays a normal karyotype and stable telomeres; it neither proliferates when plated in soft agar nor gives rise to tumors when injected subcutaneously in NOD/SCID-γ (null) mice. The analysis of gene expression highlighted transcriptional traits of SVF cells. m17.ASCs were genetically modified by lentiviral vectors carrying green fluorescent protein (GFP) as a marker transgene and efficiently engrafted in the liver, when injected in the spleen of NOD/SCID-γ (null) monocrotaline-treated mice. These results suggest that this non-tumorigenic spontaneously immortalized ASC line may represent a useful tool (cell model) for studying the differentiation mechanisms involved in tissue repair as well as a model for pharmacological/toxicological studies.

Valve interstitial cells (VICs) are crucial in the development of calcific aortic valve disease. The purpose of the present investigation was to compare the phenotype, differentiation potential and stem cell-like properties of cells from calcified and healthy aortic valves. VICs were isolated from human healthy and calcified aortic valves. Calcification was induced with osteogenic medium. Unlike VICs from healthy valves, VICs from calcified valves cultured without osteogenic medium stained positively for calcium deposits with Alizarin Red confirming their calcific phenotype. Stimulation of VICs from calcified valves with osteogenic medium increased calcification (p = 0.02), but not significantly different from healthy VICs. When stimulated with myofibroblastic medium, VICs from calcified valves had lower expression of myofibroblastic markers, measured by flow cytometry and RT-qPCR, compared to healthy VICs. Contraction of collagen gel (a measure of myofibroblastic activity) was attenuated in cells from calcified valves (p = 0.04). Moreover, VICs from calcified valves, unlike cells from healthy valves had lower potential to differentiate into adipogenic pathway and lower expression of stem cell-associated markers CD106 (p = 0.04) and aldehyde dehydrogenase (p = 0.04). In conclusion, VICs from calcified aortic have reduced multipotency compared to cells from healthy valves, which should be considered when investigating possible medical treatments of aortic valve calcification.

Current methods for freezing mesenchymal stromal cells (MSCs) result in poor post-thaw function, which limits the clinical utility of these cells. This investigation develops a novel approach to preserve MSCs using combinations of sugars, sugar alcohols, and small-molecule additives. MSCs frozen using these solutions exhibit improved post-thaw attachment and a more normal alignment of the actin cytoskeleton compared to cells exposed to dimethylsulfoxide (DMSO). Osteogenic and chondrogenic differentiation assays show that cells retain their mesenchymal lineage properties. Genomic analysis indicates that the different freezing media evaluated have different effects on the levels of DNA hydroxymethylation, which are a principal epigenetic mark and a key step in the demethylation of CpG doublets. RNA sequencing and quantitative real time-polymerase chain reaction validation demonstrate that transcripts for distinct classes of cytoprotective genes, as well as genes related to extracellular matrix structure and growth factor/receptor signaling are upregulated in experimental freezing solutions compared to DMSO. For example, the osmotic regulator galanin, the antiapoptotic marker B cell lymphoma 2, as well as the cell surface adhesion molecules CD106 (vascular cell adhesion molecule 1) and CD54 (intracellular adhesion molecule 1) are all elevated in DMSO-free solutions. These studies validate the concept that DMSO-free solutions improve post-thaw biological functions and are viable alternatives for freezing MSCs. These novel solutions promote expression of cytoprotective genes, modulate the CpG epigenome, and retain the differentiation ability of MSCs, suggesting that osmolyte-based freezing solutions may provide a new paradigm for therapeutic cell preservation.

The adenoidal epithelial crypt is a potential site of antigen transport from pharyngeal lumen to adenoidal tissue. The base of the crypt is consistently infiltrated with leucocytes, forming a reticular lymphoepithelial structure. To evaluate mechanisms that possibly mediate leucocyte infiltration, expressions of leucocyte adhesion molecules, such as platelet endothelial cell adhesion molecule-1 (PECAM-1) (CD31), vascular cell adhesion molecule-1 (VCAM-1) (CD106) and intercellular adhesion molecule-1 (ICAM-1) (CD54), were studied in the adenoidal epithelial crypt. Epithelial cells in the outer opening of the adenoidal crypt were positive for VCAM-1, whereas epithelial cells at the base of the crypt were positive for PECAM-1. Isolated ICAM-1-expressing cells were found throughout the epithelial crypt. Double immunofluorescence staining revealed that the epithelial cells positive for PECAM-1 or VCAM-1 were positive for cytokeratin. The expression of PECAM-1 in the base and VCAM-1 at the orifice of the adenoidal epithelial crypt implies that the base and the orifice of the crypt have a distinct ability to recruit leucocytes. Epithelial cells expressing PECAM-1 may have a role in the formation of the reticular lymphoepithelial structure in the epithelial crypt.

The applicability of stem cells from the human endometrium and fallopian tube for regeneration is a fascinating area of research because of the role of these cells in dynamic tissue remodelling and their cyclical regenerative property during the menstrual cycle and pregnancy. Nevertheless, studies on the identity of biomarkers of these stem cells are limited and need to be extended. The present study has aimed at exploring the tissue-specific biomarkers of stem cells derived from the human endometrium and fallopian tube compared with those from bone marrow. Cells were isolated from human endometrium and fallopian tubes and characterized for biomarkers, including CD34, CD133, CD117, CD90, CD105, CD73, nestin, CD29, CD44, CD31, CD54, CD166, CD106, CD49d, CD45, ABCG2, SSEA4, OCT4, SOX2, CD140b and CD146, by flowcytometry. Both endometrium and fallopian tube sources exhibited positivity over a wide range of markers, as did bone marrow. In particular, they exhibited pluripotency, perivascular and mesenchymal stem cell markers and cell adhesion molecules, thereby suggesting their relevance in tissue repair and regeneration. Overall, the results of this study provide evidence for the presence of stem cells in the human endometrium and fallopian tube, which could thus represent additional stem cell sources for regenerative medicine.

To investigate the biological character of human adipose-derived adult stem cells (hADAS cells) when cultured in vitro and the relationship between hADAS cell's replication activity and the donor's age factor, and to assess the stem cells as a new source for tissue engineering. hADAS cells are isolated from human adipose tissue of different age groups (from adolescents to olds: <20 years old, 21-40 years old, 41-60 years old and >61 years old groups). The protein markers (CD29, CD34, CD44, CD45, CD49d, HLA-DR, CD106) of hADAS cells were detected by flow cytometry (FCM) to identify the stem cell, and the cell cycle was examined for P20 hADAS cells to evaluate the safety of the subculture in vitro. The generative activity of hADAS cells in different age groups was also examined by MTT method. The formula "TD = t x log2/logNt - logN0" was used to get the time doubling (TD) of the cells. The results showed that the cells kept heredity stabilization by chromosome analysis for at least 20 passages. The TD of these cells increased progressively by ageing, and the TD of the <20 years old group was lower than that of the >61 years old group (statistical analysis of variance (ANOVA), P=0.002, P<0.05). These findings suggested that a higher level of hADAS cells replication activity was found in the younger donators, and they represent novel and valuable seed cells for studies of tissue engineering.

Various types of endogenous stem cells (SCs) participate in wound healing in the skin at different anatomical locations. SCs need to be identified through multiple markers, and this is usually performed using flow cytometry. However, immunohistological identification of endogenous stem cells in the skin at different anatomical locations by co-staining multiple SC markers has been seldom explored. We examined the immunohistological localization of four major types of SCs in wounded skin by co-staining for their multiple markers. Hematopoietic SCs were co-stained for Sca1 and CD45; mesenchymal SCs for Sca1, CD29, and CD106; adipose SCs for CD34, CD90, and CD105; and endothelial progenitor cells and their differentiated counterparts were co-stained for CD34, Tie2, and von Willebrand factor. We found Sca1(+)CD45(+) SCs in the epidermis, dermis and hypodermis of wounded skin. Sca1(+)CD29(+) and Sca1(+)CD106(+) mesenchymal SCs, CD34(+)CD105(+), CD34(+)CD90(+), and CD90(+)CD105(+) adipose SCs, as well as CD34(+)Tie2(+) endothelial progenitor cells were also located in the epidermis, dermis, and hypodermis. This study demonstrates the feasibility of using immunohistological staining to determine the location of SCs in wounded skin and the intracellular distribution of their molecular markers.

Sjögren's syndrome (SS) is a systemic autoimmune disease that is characterized by focal lymphocytic infiltration into exocrine organs such as salivary and lacrimal glands, resulting in dry mouth and eyes, and other systemic injuries. There is no curative clinical therapy for SS, and stem cell therapy has shown great potential in this area. The mesenchymal stem cells (MSCs) in the salivary glands of healthy individuals and in patients with SS have not been extensively studied. The aim of this study was to elucidate the characteristics of MSCs from the labial glands of healthy controls and of those from patients with SS to elucidate the related pathogenesis and to uncover potential avenues for novel clinical interventions. Labial glands from patients with SS and healthy subjects were obtained, and MSCs were isolated and cultured by using the tissue adherent method. The MSC characteristics of the cultured cells were confirmed by using morphology, proliferation, colony forming-unit (CFU) efficiency, and multipotentiality, including osteogenic, adipogenic, and salivary gland differentiation. The MSCs from the healthy controls and SS patients expressed characteristic MSC markers, including CD29, CD44, CD73, CD90, and CD105; they were negative for CD34, CD45, and CD106, and also negative for the salivary gland epithelium markers (CD49f and CD117). Labial gland MSCs from both groups were capable of osteogenic and adipogenic differentiation. The CFU efficiency and adipogenic differentiation potential of MSCs were significantly lower in the SS group compared with the healthy controls. Cells from both groups could also be induced into salivary gland-like cells. Real-time polymerase chain reaction and immunofluorescence staining showed that the gene and protein expression of AMY1, AQP5, and ZO-1 in cells from the SS group was lower than that in cells from the healthy group. Thus, MSCs from the labial glands in patients with SS could lack certain characteristics and functions, especially related to salivary secretion. These preliminary data provided insights that could lead to the development of novel therapeutic strategies for the treatment of SS.