Normal human melanocytes are interspersed singly among keratinocytes along the basement membrane of the epidermis, whereas melanoma cells readily adhere to each other during invasion of the dermis or distant organs. The tumorigenic and metastatic phenotype of melanoma cells often correlates with increased expression of cell-cell and cell-matrix adhesion receptors. Mel-CAM (MCAM, MUC 18, CD146) is a cell-cell adhesion receptor highly expressed by melanoma cells but not normal melanocytes. We show here that inhibition of Mel-CAM expression in metastatic melanoma cells using genetic suppressor elements of Mel-CAM cDNA leads to inhibition of adhesion between melanoma cells and to downregulation of the tumorigenic phenotype. Growth was not inhibited in genetic suppressor elements-transduced melanoma cells cultured in monolayers but was inhibited when cells were maintained anchorage-independently in soft agar and greatly reduced in immunodeficient mice. A three-dimensional epidermal skin equivalent model demonstrated that Mel-CAM allows melanoma cells to separate from the epidermis and invade the basement membrane zone and dermis. However, melanoma cells with little or no Mel-CAM were poorly invasive, possibly due to their loss of gap junctional communication. These results suggest the multifunctional role of a melanoma-associated cell-cell adhesion receptor in tumor progression.

c-Met is responsible for cell motility and tumour spreading. c-Met expression and signal transducers reflecting c-Met functionality were investigated in breast carcinomas, in correlation with patient outcome and tumour vasculature. Tissue microarrays of 930 breast carcinomas were constructed, categorised according to patients' follow-up (4- to 10-year follow-up; median, 6.5 years). Standardised immunocytochemical procedures were performed using anti-c-Met, -PI3K, -FAK, -JAK, and -CD146, -FYN and an automated autostainer (Ventana). High-throughput densitometry measuring the extent of immunoprecipitates was assessed by image analysis (SAMBA). c-Met overexpression correlated with poor survival along with PI3K and FAK reflecting c-Met functionality and CD146 and FYN expression in endothelial cells. Automated quantification of immunocytochemical precipitates using image analysis was shown to provide an objective means of measuring cellular proteins that are potentially relevant for current practice in pathological diagnosis and for specific therapy combining inhibitors of both c-Met and downstream transducer pathways, and of tumour angiogenesis.

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are widely used to decrease cholesterol synthesis and are well established to reduce vascular diseases. Recently, it has been proposed that statins mobilize endothelial progenitor cells from bone marrow during the first four weeks, which could help to prevent vascular diseases. However, in humans there are few data concerning the long term effects of statin treatment on these endothelial progenitor cells. We investigated whether endothelial progenitor cells can be detected and characterized in patients receiving long term statin therapy. Mononuclear cells from patients receiving or not receiving statin therapy were assessed for progenitor cell content by flow cytometry and were cultured in specific conditions to determine the number and the type of progenitors. Our results showed there were significantly more CD34(+), CD34(+)/CD144(+) circulating progenitor cells in the statin(pos) group than in the statin(neg) group. In culture two types of endothelial progenitor cells were detected. Early endothelial progenitor cells gave colonies at day 5 comprising elongated cells whereas late endothelial progenitor cells generated cobblestone-like colonies with strong proliferation capacities. The number of circulating early endothelial progenitor cells was significantly higher in the statin(neg) group, while only late endothelial progenitor cells were detected in the statin(pos) group. Moreover, cells from cobblestones clearly had an endothelial phenotype CD31(+), VEGF-R2(+), CD34(+), CD146(+) in contrast to cells from colonies from early endothelial progenitor cells, which were VEGF-R2(low), CD34(-). These results strongly suggest that long term statin treatment specifically maintains late endothelial progenitor cells in circulation with a CD34(+)/CD144(+) phenotype.

BACKGROUND

CD146, a transmembrane immunoglobulin mainly expressed at the intercellular junction of endothelial cells, is involved in cell-cell cohesion, paracellular permeability, monocyte transmigration and angiogenesis. CD146 exists as 2 isoforms, short (sh) and long (lg), but which isoform is involved remains undefined.

OBJECTIVE

The recently described role of CD146 in angiogenesis prompted us to investigate which isoform was involved in this process in human late endothelial progenitors (EPCs), with the objective of increasing their proangiogenic potential.

RESULTS

Immunofluorescence experiments showed that, in subconfluent EPCs, shCD146 was localized in the nucleus and at the migrating edges of the membrane, whereas lgCD146 was intracellular. In confluent cells, shCD146 was redistributed at the apical membrane and lgCD146 was directed toward the junction. In contrast to lgCD146, shCD146 was overexpressed in EPCs as compared to mature endothelial cells and upregulated by vascular endothelial growth factor and SDF-1 (stromal cell-derived factor 1). Study of the properties of both isoforms in vitro provided evidence that shCD146 was involved in EPC adhesion to activated endothelium, migration, and proliferation, with a paracrine secretion of interleukin-8 or angiopoietin 2, whereas lgCD146 was implicated in stabilization of capillary-like structures in Matrigel and transendothelial permeability. In an animal model of hindlimb ischemia, transplantation of shCD146-modified EPCs selectively promoted both EPC engraftment and blood flow.

CONCLUSIONS

Altogether, these findings establish that CD146 isoforms display distinct functions in vessels regeneration. Selective improvement of therapeutic angiogenesis by shCD146 overexpression suggests a potential interest of shCD146-transduced EPCs for the treatment of peripheral ischemic disease.

The LX-2 cell line has characteristics of hepatic stellate cells (HSCs), which are considered pericytes of the hepatic microcirculatory system. Recent studies have suggested that HSCs might have mesenchymal origin. We have performed an extensive characterization of the LX-2 cells and have compared their features with those of mesenchymal cells. Our data show that LX-2 cells have a phenotype resembling activated HSCs as well as bone marrow-derived mesenchymal stem cells (BM-MSCs). Our immunophenotypic analysis showed that LX-2 cells are positive for activated HSC markers (αSMA, GFAP, nestin and CD271) and classical mesenchymal makers (CD105, CD44, CD29, CD13, CD90, HLA class-I, CD73, CD49e, CD166 and CD146) but negative for the endothelial marker CD31 and endothelial progenitor cell marker CD133 as well as hematopoietic markers (CD45 and CD34). LX-2 cells also express the same transcripts found in immortalized and primary BM-MSCs (vimentin, annexin 5, collagen 1A, NG2 and CD140b), although at different levels. We show that LX-2 cells are capable to differentiate into multilineage mesenchymal cells in vitro and can stimulate new blood vessel formation in vivo. LX-2 cells appear not to possess tumorigenic potential. Thus, the LX-2 cell line behaves as a multipotent cell line with similarity to BM-MSCs. This line should be useful for further studies to elucidate liver regeneration mechanisms and be the foundation for development of hepatic cell-based therapies.

BACKGROUND

Spontaneous in vitro transition of undifferentiated spermatogonia into the pluripotent cell state has been achieved using neonatal and adult mouse testis tissue. In an effort to establish an analogous source of human patient-specific pluripotent stem cells, several research groups have described the derivation of embryonic stem cell-like cells from primary cultures of human testis. These cells are characterized in all studies as growing in compact colonies, expressing pluripotency-associated markers and possessing multilineage differentiation capabilities in vitro, but only one study claimed their ability to induce teratomas. This controversy initiated a debate about the pluripotent state and origin of human testis-derived ES-like cells (htES-like cells).

METHODS

htES-like cell colonies were obtained from primary testicular cultures of three individuals and selectively expanded using culture conditions known to support the propagation of blastocyst-derived human embryonic stem cells (ESCs), mouse epiblast stem cells and 'naïve' human ESCs. The stem cell properties of htES-like cells were subsequently assessed by testing the expression of ESC-specific markers, differentiation abilities in vitro and in vivo, and microarray profiling.

RESULTS

The expression of pluripotency-associated markers in htES-like cells and their differentiation abilities differed significantly from those of ESCs. Gene expression microarray analysis revealed that htES-like cells possess a transcriptome distinct from human ESCs and fibroblasts, but closely resembling the transcriptome of mesenchymal stem cells (MSCs). The similarity to MSCs was confirmed by detection of SSEA4/CD146 expressing cells within htES-like colonies and efficient in vitro differentiation toward three mesodermal lineages (adipogenic, osteogenic, chondrogenic).

CONCLUSIONS

Taken together, these results indicate that htES-like cells, in contrast to pluripotent stem cells derived from adult mouse testis, are not pluripotent and most likely not of germ cell but of mesenchymal origin.

We recently reported that mouse bone marrow stromal cells, also known as bone marrow (BM)-derived mesenchymal stem cells (MSCs), seeded onto a scaffold and implanted in vivo, led to an ectopic bone deposition by host cells. This MSCs capacity was critically dependent on their commitment level, being present only in MSCs cultured in presence of fibroblast growth factor-2. Taking advantage of a chimeric mouse model, in this study we show that seeded MSCs trigger a cascade of events resulting in the mobilization of macrophages, the induction of their functional switch from a proinflammatory to a proresolving phenotype, and the subsequent formation of a bone regenerative niche through the recruitment, within the first 2 weeks of implantation, of endothelial progenitors and of cells with an osteogenic potential (CD146+CD105+), both of them derived from the BM. Moreover, we demonstrated that, in an inflammatory environment, MSCs secrete a large amount of prostaglandin E2 playing a key role in the macrophage phenotype switch.

OBJECTIVE

Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen.

METHODS

Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity.

RESULTS

Cell preparation yielded the following cell counts per gram of liver tissue: 2.0 ± 0.4 × 10(7) hepatocytes, 1.8 ± 0.5 × 10(6 )Kupffer cells, 4.3 ± 1.9 × 10(5) liver sinusoidal endothelial cells, and 3.2 ± 0.5 × 10(5) stellate cells. Hepatocytes were identified by albumin (95.5 ± 1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5 ± 1.2%) and exhibited phagocytic activity, as determined with 1 μm latex beads. Endothelial cells were CD146(+) (97.8 ± 1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1 ± 1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence.

CONCLUSIONS

Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.

Mesenchymal stromal cells derived from the human amnion (hAMSC) currently play an important role in stem cell research, as they are multipotent cells that can be isolated using noninvasive methods and are immunologically tolerated in vivo. The objective of this study was to evaluate their endothelial differentiation potential with regard to a possible therapeutic use in vascular diseases. hAMSC were isolated from human term placentas and cultured in Dulbecco's modified Eagle's medium (DMEM) (non-induced hAMSC) or endothelial growth medium (EGM-2) (induced hAMSC). Induced hAMSC changed their fibroblast-like toward an endothelial-like morphology, and were able to take up acetylated low-density lipoprotein and form endothelial-like networks in the Matrigel assay. However, they did not express the mature endothelial cell markers von Willebrand factor and vascular endothelial-cadherin. Gene expression analysis revealed that induced hAMSC significantly downregulated pro-angiogenic genes such as tenascin C, Tie-2, vascular endothelial growth factor A (VEGF-A), CD146, and fibroblast growth factor 2 (FGF-2), whereas they significantly upregulated anti-angiogenic genes such as serpinF1, sprouty1, and angioarrestin. Analysis of protein expression confirmed the downregulation of FGF-2 and Tie-2 (27%±8% and 13%±1% of non-induced cells, respectively) and upregulation of the anti-angiogenic protein endostatin (226%±4%). Conditioned media collected from hAMSC enhanced viability of endothelial cells and had a stabilizing effect on endothelial network formation as shown by lactate dehydrogenase and Matrigel assay, respectively. In summary, endothelial induced hAMSC acquired some angiogenic properties but resisted undergoing a complete differentiation into mature endothelial cells by upregulation of anti-angiogenic factors. Nevertheless, they had a survival-enhancing effect on endothelial cells that might be useful in a variety of cell therapy or tissue-engineering approaches.

Mesenchymal stromal cells (MSC) have been intensively studied for innovative therapeutic applications. MSC in vitro are characterized by plastic-adherent proliferation, their specific immunophenotype and multipotency, whereas MSC progenitors in vivo are described as perivascular cells. Whether MSC progenitors acquire in vitro MSC characteristics upon in vitro culture is still unclear. This question can be experimentally accessed by analyzing changes in cellular properties that occur during the early in vitro culture phase, the MSC derivation phase. Here, we examined dynamics in morphology, proliferation, and expression of surface markers used for MSC characterization (such as CD34, CD105, CD146, and CD271) in tight kinetics during the MSC derivation phase of adipose tissue-derived MSC (AT-MSC). Using multiparametric flow cytometry, we identified 3 major ex vivo stromal vascular cell subsets: CD34+ CD146-CD271(+/-) adventitial stromal cell-like cells (AdSC), CD34- CD146+ CD271(+/-) pericyte-like cells (PC), and CD34+ CD31+ CD146+ endothelial cells. Of these subsets, only AdSC, but not PC gave rise to MSC under MSC culture conditions. At day 4 of culture, AdSC became fibroblastoid and upregulated CD105, CD146, and CD271. Following this phenotypic transition, AdSC commenced proliferation and downregulated CD34. In our study, we demonstrate that AdSC are more clonogenic AT-MSC progenitors than PC. Moreover, we, for the first time have dissected the phenotypic transitions from MSC progenitors to in vitro MSC during the MSC derivation phase using multiparametric flow cytometry. Hence, we propose a model describing how de novo acquisition of the typical MSC morphology by AdSC is accompanied by concerted regulation of surface marker expression upon in vitro culture.

Given the highly heterogeneous character of brain malignancies and the associated implication for its proper diagnosis and treatment, finding biomarkers that better characterize this disease from a molecular standpoint is imperative. In this study, we evaluated CD146 as a potential molecular target for diagnosis and targeted therapy of glioblastoma multiforme (GBM), the most common and lethal brain malignancy. YY146, an anti-CD146 monoclonal antibody, was generated and radiolabeled for noninvasive positron-emission tomography (PET) imaging of orthotopic GBM models. (64)Cu-labeled YY146 preferentially accumulated in the tumors of mice bearing U87MG xenografts, which allowed the acquisition of high-contrast PET images of small tumor nodules (∼ 2 mm). Additionally, we found that tumor uptake correlated with the levels of CD146 expression in a highly specific manner. We also explored the potential therapeutic effects of YY146 on the cancer stem cell (CSC) and epithelial-to-mesenchymal (EMT) properties of U87MG cells, demonstrating that YY146 can mitigate those aggressive phenotypes. Using YY146 as the primary antibody, we performed histological studies of World Health Organization (WHO) grades I through IV primary gliomas. The positive correlation found between CD146-positive staining and high tumor grade (χ(2) = 9.028; P = 0.029) concurred with the GBM data available in The Cancer Genome Atlas (TCGA) and validated the clinical value of YY146. In addition, we demonstrate that YY146 can be used to detect CD146 in various cancer cell lines and human resected tumor tissues of multiple other tumor types (gastric, ovarian, liver, and lung), indicating a broad applicability of YY146 in solid tumors.

Angiogenesis is a rate-limiting factor for numerous human diseases. Angiogenic vessels and also the endothelium of certain organs such as the lung display molecular addresses that can be exploited for the selective delivery of gene therapeutics. Lentiviral vectors (LVs) are powerful tools for stable gene delivery but their integration and expression in undesired cell types poses a serious safety concern. We have developed a dual-targeted LV that can specifically target primary endothelial cells (ECs). Cell selectivity is achieved during entry, using a modified Sindbis virus envelope, and during transcription, with an EC-specific promoter. We evaluated four surface markers for EC targeting and seven promoter sequences from genes preferentially expressed in ECs. The efficiency and specificity of the double targeted vector were assayed in a panel of human primary cultures and tumor cell lines. A vector targeted to CD146, an endothelial adhesion molecule, and carrying a derivative of the EC tyrosine kinase Tie2 promoter, increased specificity of transduction up to 50 times and was also effective at selectively transducing ECs in a mixed coculture with human fibroblasts. The vector presented here is a potentially powerful tool that could be used in a variety of human diseases.

The interaction of tumor cells with the tumor vasculature is mainly studied for its role in tumor angiogenesis and intravascular metastasis of circulating tumor cells. In addition, a specific interaction of tumor cells with the abluminal surfaces of vessels, or angiotropism, may promote the migration of angiotropic tumor cells along the abluminal vascular surfaces in a pericytic location. This process has been termed extravascular migratory metastasis. The abluminal vascular surface may also provide a vascular niche inducing or sustaining stemness to angiotropic tumor cells. This pilot study investigated if angiotropic melanoma cells might represent a subset population with pericytic and embryonic or stem cell properties. Through microarray analysis, we showed that the interaction between melanoma cells and the abluminal surface of endothelial cells triggers significant differential expression of several genes. The most significantly differentially expressed genes have demonstrated properties linked to cancer cell migration (CCL2, ICAM1 and IL6), cancer progression (CCL2, ICAM1, SELE, TRAF1, IL6, SERPINB2 and CXCL6), epithelial to mesenchymal transition (CCL2 and IL6), embryonic/stem cell properties (CCL2, PDGFB, EVX1 and CFDP1) and pericytic recruitment (PDGFB). In addition, bioinformatics-based analysis of the differentially expressed genes has shown that the most significantly enriched functional groups included development, cell movement, cancer, and embryonic development. Finally, the investigation of pericyte/mesenchymal stem cells markers via immunostaining of human melanoma samples revealed expression of PDGFRB, NG2 and CD146 by angiotropic melanoma cells. Taken together, these preliminary data are supportive of the "pericytic mimicry" by angiotropic melanoma cells, and suggest that the interaction between melanoma cells and the abluminal vascular surface induce differential expression of genes linked to cancer migration and embryonic/stem cell properties.

Circulating tumor cells (CTCs) can be enumerated using CellSearch, but not all breast cancer subtypes, specifically those with epithelial-mesenchymal transition (EMT) characteristics, sufficiently express the enrichment (EpCAM) and selection (CK8/18/19) markers used in this method. While CD146 can detect EpCAM-negative CTCs, we here evaluated the value of various cytokeratins and CD49f to detect CK8/18/19-negative CTCs. The tested cytokeratins provided no substantial benefit, but adding CD49f to CK8/18/19 as a selection marker resulted in improved recovery of normal-like cell lines. Combined staining of CK8/18/19 and CD49f after CD146/EpCAM enrichment is likely to further improve CTC detection in breast cancer.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells(1-8); therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation(6,9,10). Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers & also their differentiation capacity. Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,(11-13) i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 & CD44), hematopoietic/endothelial Markers (CD34, CD45 & CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) & Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE & dentin sialophosphoprotein; DSPP).(14).

OBJECTIVE

Although clinical applications using mesenchymal stromal cells (MSCs) are becoming more frequent, procedures for their in vitro culture are far from standardized. Growth factors such as FGF-2 are frequently added during expansion to improve population growth and differentiation characteristics. However, up to now its influence on surface marker distribution of MSCs has been close to unknown. The purpose of this study was therefore to analyse effects of FGF-2 supplementation on pre-selection of MSC subpopulations.

METHODS

Mesenchymal stromal cells were harvested from bone marrow of six patients and expanded in alpha-MEM or DMEM-LG. Starting in passage 2, 10 ng/ml FGF-2 was administered and non-supplemented media were used as controls. Growth indices were calculated from P0 to P4. After P4, fluorescence cytometry for common MSC surface markers was performed and standard chondrogenic, adipogenic and osteogenic differentiation protocols were applied.

RESULTS

Cell population growth indices were higher for those in FGF-2 supplemented media. Significant differences in surface marker distribution were observed for CD13, CD14, CD49, CD90, CD340 and STRO-1 depending on respective culture conditions. FGF-2 suppressed CD146 expression in both alpha-MEM and DMEM-LG. No differences in adipogenic and osteogenic differentiation potential could be observed, while FGF-2 significantly improved chondrogenic differentiation in DMEM-LG.

CONCLUSIONS

While holding the benefit of improving MSC chondrogenic differentiation potential, FGF-2 pre-selects certain MSC subtypes. Our data clearly show that expansion culture conditions have a significant effect on distribution of a number of MSC surface markers.

Modified vascular smooth muscle cells of the kidney afferent arterioles have recently been shown to serve as progenitors for glomerular epithelial cells in response to glomerular injury. To determine whether such cells of renin lineage (CoRL) serve as progenitors for other cells in kidney disease characterized by both glomerular and tubulointerstitial injury, permanent genetic cell fate mapping of adult CoRL using Ren1cCreER × Rs-tdTomato-R reporter mice was performed. TdTomato-labeled CoRL were almost completely restricted to the juxtaglomerular compartment in healthy kidneys. Following 2 wk of antibody-mediated focal segmental glomerulosclerosis (FSGS) or 16 wk of ⅚ nephrectomy-induced chronic kidney diseases, tdTomato-mapped CoRL were identified in both interstitial and glomerular compartments. In the interstitium, PDGFβ receptor (R)-expressing cells significantly increased, and a portion of these expressed tdTomato. This was accompanied by a decrease in native pericyte number, but an increase in the number of tdTomato cells that coexpressed the pericyte markers PDGFβ-R and NG2. These cells surrounded vessels and coexpressed the pericyte markers CD73 and CD146, but not the endothelial marker ERG. Within glomeruli of reporter mice with the ⅚ nephrectomy model, a subset of labeled CoRL migrated to the glomerular tuft and coexpressed podocin and synaptopodin. By contrast, labeled CoRL were not detected in glomerular or interstitial compartments following uninephrectomy. These observations indicate that in addition to supplying new adult podocytes to glomeruli, CoRL have the capacity to become new adult pericytes in the setting of interstitial disease. We conclude that CoRL have the potential to function as progenitors for multiple adult cell types in kidney disease.

OBJECTIVE

Does post-menopausal endometrium contain mesenchymal stem/stromal cells (MSC) that have adult stem cell properties and can be prospectively isolated from a biopsy?

CONCLUSIONS

Perivascular W5C5(+) cells isolated from post-menopausal endometrial biopsies displayed characteristic MSC properties of clonogenicity, multipotency and surface phenotype irrespective of whether the women were or were not pre-treated with estrogen to regenerate the endometrium.

BACKGROUND

Recently MSCs have been identified in human premenopausal endometrium, and can be prospectively isolated using a single marker, W5C5/SUSD2.

METHODS

Endometrial tissue of both the functional and basal layers, from 17 premenopausal (pre-MP) women, 19 post-menopausal (post-MP) women without hormonal treatment and 15 post-menopausal women on estrogen replacement therapy (post-MP+ E2), was collected through a prospective phase IV clinical trial over 2 years.

METHODS

Post-menopausal women <65 years of age were treated with or without E2 for 6-8 weeks prior to tissue collection. Serum E2 levels were determined by estradiol immunoenzymatic assay. Endometrial tissue was obtained from women by biopsy (curettage) just prior to the hysterectomy. The effect of E2 on endometrial thickness and glandular and luminal epithelial height was determined using image analysis. Endometrial tissue was dissociated into single cell suspensions and MSC properties were examined in freshly isolated and short-term cultured, magnetic bead-purified W5C5(+) cells. MSC properties were assessed using clonogenicity, serial cloning, mesodermal differentiation in adipogenic, chondrogenic, osteogenic and myogenic induction culture media, and surface phenotype analysis by flow cytometry. Estrogen receptor α expression in W5C5(+) cells was examined using dual colour immunofluorescence. Vascularity was analysed using CD34 and alpha smooth muscle actin immunostaining and subsequent image analysis.

RESULTS

A small population of stromal cells with MSC properties was purified with the W5C5 antibody from post-menopausal endometrium, whether atrophic from low circulating estrogen or regenerated from systemic estrogen treatment, similar to premenopausal endometrium. The MSC derived from post-menopausal endometrium treated with or without E2 fulfilled the minimum MSC criteria: clonogenicity, surface phenotype (CD29(+), CD44(+), CD73(+), CD105(+), CD140b(+), CD146(+)) and multipotency. The post-menopausal endometrial MSCs also showed comparable properties to premenopausal eMSC with respect to self-renewal in vitro and W5C5 expression. The W5C5(+) cells were located perivascularly as expected and did not express estrogen receptor α.

CONCLUSIONS

The properties of the MSC derived from post-menopausal endometrium were evaluated in vitro and their in vivo tissue reconstitution capacity has not been established as it has for premenopausal endometrial MSC.

CONCLUSIONS

The endometrium is an accessible source of MSC obtainable with minimum morbidity that could be used for future clinical applications as a cell-based therapy. This study shows that menopausal women can access their endometrial MSC by a simple biopsy for use in autologous therapies, particularly if their endometrium has been regenerated by short-term E2 treatment, provided they have an intact uterus and are not contraindicated for short-term E2 treatment. Endometrial MSC in post-menopausal women possess key MSC properties and are a promising source of MSC independent of a woman's age.

BACKGROUND

This study was supported by the National Health and Medical Research Council (NHMRC) of Australia grant (1021126) (C.E.G., A.R.) and Senior Research Fellowship (1042298) (C.E.G.), Australian Gynaecological Endoscopic Society grant (A.R.) , Monash International Postgraduate Research Scholarship (DU), Australian Stem Cell Centre, South East Melbourne Alliance for Regenerative Therapies and Australian Stem Cell Centre top up scholarships (DU) and Victorian Government's Operational Infrastructure Support Program. Competing interests: AR receives Preceptorship fees from AMS, advisory board fees and sponsored study from Astellas, and conducts investigator led studies sponsored by AMS and Boston Scientific for other projects.

BACKGROUND

CTNRN12610000563066.

BACKGROUND

Periodontal ligament (PDL) contributes to maintaining homeostasis in periodontal tissues by supplying stem/progenitor cells. It has long been suggested that PDL stem cells/progenitors are located around blood vessels. Recently mesenchymal stem cells (MSCs) have been isolated and cultured from PDL in vitro, although the location of the stem cells in PDL is unclear. The purpose of this study is to test the characteristics of human PDL stem cells (PDLSCs) and examine their similarity to related vascular cell types, such as pericytes and endothelial cells.

METHODS

PDLSCs were obtained from healthy extracted teeth using the collagenase/dispase enzyme digestion method. MSC and pericyte characteristics of PDLSCs were examined by cell surface marker expression using flow cytometry. The expression of pericyte markers was tested using immunohistochemistry. Pericyte-like functions of PDLSCs were examined in co-culture of PDLSCs and umbilical vein endothelial cells on a gel matrix.

RESULTS

Cultured PDLSCs were positive for both MSC markers and pericyte markers, including cluster of differentiation 146 (CD146), neural/glial antigen 2 (NG2), and CD140b. When pericyte marker expression was explored in rat periodontal tissue sections, CD146- and NG2-positive signals were observed in the perivascular area of the PDL. Further, when the cells were cultured with human umbilical cord endothelial cells under conditions for forming capillary-like structures in vitro, PDLSCs localized adjacent to endothelial cells and contributed to the stability of the capillary-like structure.

CONCLUSIONS

PDLSCs possess pericyte-like characteristics and may localize as pericytes in the PDL. These data provide useful information for stem cell biology in periodontal research and stem cell-based periodontal therapy.

BACKGROUND

The human anterior cruciate ligament (hACL) and medial collateral ligament (hMCL) of the knee joint are frequently injured, especially in athletic settings. It has been known that, while injuries to the MCL typically heal with conservative treatment, ACL injuries usually do not heal. As adult stem cells repair injured tissues through proliferation and differentiation, we hypothesized that the hACL and hMCL contain stem cells exhibiting unique properties that could be responsible for the differential healing capacity of the two ligaments.

METHODS

To test the above hypothesis, we derived ligament stem cells from normal hACL and hMCL samples from the same adult donors using tissue culture techniques and characterized their properties using immunocytochemistry, RT-PCR, and flow cytometry.

RESULTS

We found that both hACL stem cells (hACL-SCs) and hMCL stem cells (hMCL-SCs) formed colonies in culture and expressed stem cell markers nucleostemin and stage-specific embryonic antigen-4 (SSEA-4). Moreover, both hACL-SCs and hMCL-SCs expressed CD surface markers for mesenchymal stem cells, including CD44 and CD90, but not those markers for vascular cells, CD31, CD34, CD45, and CD146. However, hACL-SCs differed from hMCL-SCs in that the size and number of hACL-SC colonies in culture were much smaller and grew more slowly than hMCL-SC colonies. Moreover, fewer hACL-SCs in cell colonies expressed stem cell markers STRO-1 and octamer-binding transcription factor-4 (Oct-4) than hMCL-SCs. Finally, hACL-SCs had less multi-differentiation potential than hMCL-SCs, evidenced by differing extents of adipogenesis, chondrogenesis, and osteogenesis in the respective induction media.

CONCLUSIONS

This study shows for the first time that hACL-SCs are intrinsically different from hMCL-SCs. We suggest that the differences in their properties contribute to the known disparity in healing capabilities between the two ligaments.

Human dental pulp stem cells (DPSCs) can be isolated from inflamed pulp derived from carious teeth with symptomatic irreversible pulpitis (I-DPSCs), which possess stemness and multidifferentiation potentials similar to DPSCs from healthy pulp. Since macrophages-essential cell players of the pulpal innate immunity-can regulate pulpal inflammation and repair, the authors investigated the immunomodulatory effects of DPSCs/I-DPSCs on macrophage functions and their underlying mechanisms. Similar to DPSCs, I-DPSCs were capable of colony-forming efficiency and adipogenic and osteo/dentinogenic differentiation under in vitro induction conditions. I-DPSCs also expressed a similar phenotypic profile of mesenchymal stem cell markers, except a relatively higher level of CD146 as compared with DPSCs. Coculture of DPSCs or I-DPSCs with differentiated THP-1 cells, the human monocyte cell line, markedly suppressed tumor necrosis factor α (TNF-α) secretion in response to stimulation with lipopolysaccharides (LPS) and/or nigericin. However, unlike TNF-α, the secreted level of interleukin 1β was not affected by coculture with DPSCs or I-DPSCs. Furthermore, DPSC/I-DPSC-mediated inhibition of TNF-α secretion by macrophages was abolished by pretreatment with 1-methyl-D-tryptophan, a specific inhibitor of indoleamine-pyrrole 2,3-dioxygenase (IDO), but not by NSC-398, a specific inhibitor of COX-2, suggesting IDO as a mediator. Interestingly, IDO expression was significantly augmented in macrophages and mesenchymal stromal cells in inflamed human pulp tissues. Collectively, these findings show that I-DPSCs, similar to DPSCs, possess stem cell properties and suppress macrophage functions via the TNF-α/IDO axis, thereby providing a physiologically relevant context for their innate immunomodulatory activity in the dental pulp and their capability for pulp repair.

Background aims: Intrahepatic cholangiocarcinoma (ICC) is the second most common liver malignancy. ICC typically features a remarkably cellular heterogeneity and dense stromal reaction. Therefore, a comprehensive understanding of cellular diversity and the interplay between malignant cells and niche cells is essential for elucidating the mechanisms driving ICC progression and developing therapeutic approaches.

Methods: Herein, we performed single-cell RNA sequencing (scRNA-seq) analysis on unselected viable cells from eight human ICC and adjacent samples to elucidate the comprehensive transcriptomic landscape and intercellular communication network. Additionally, we applied a negative selection strategy to enrich fibroblast populations in two other ICC samples to investigate fibroblast diversity. The results of the analyses were validated using multiplex immunofluorescence staining, bulk transcriptomic datasets, and functional in vitro and in vivo experiments.

Results: We sequenced a total of 56,871 single cells derived from human ICC and adjacent tissues and identified diverse tumor, immune, and stromal cells. Malignant cells displayed a high degree of inter-tumor heterogeneity. Moreover, tumor-infiltrating CD4 regulatory T cells (Tregs) exhibited highly immunosuppressive characteristics. We identified six distinct fibroblast subsets, of which the majority were CD146 positive vascular cancer-associated fibroblasts (vCAFs), with highly expressed microvasculature signatures and high levels of IL-6. Functional assays indicated that IL-6 secreted by vCAFs induced significant epigenetic alterations in ICC cells, particularly upregulating enhancer of zeste homolog 2 (EZH2) and thereby enhancing malignancy. Furthermore, ICC cell-derived exosomal miR-9-5p elicited a high expression of IL-6 in vCAFs to promote tumor progression.

Conclusions: Our study provides a transcriptomic landscape of human ICC at single-cell resolution and intercellular crosstalk between ICC cells and vCAFs, suggesting potential therapeutic targets.

Keywords: CD146; Cancer-associated fibroblasts; Intrahepatic cholangiocarcinoma; Single-cell RNA sequencing; Tumor heterogeneity.

OBJECTIVE

Bone marrow (BM) mesenchymal stromal/stem cells (MSC) are therapeutic tools in regenerative medicine and oncology. MSC isolation is often performed starting from a separation step based on research-grade 1.077 g/mL density gradient media (DGM). However, MSC clinical application should require the introduction of good manufacturing practice (GMP) reagents. We took advantage of two novel GMP DGM with densities of 1.077 and 1.073 g/mL (Ficoll-Paque PREMIUM and Ficoll-Paque PREMIUM 1.073, respectively) to test whether these reagents could isolate MSC efficiently while simultaneously comparing their performance.

METHODS

BM samples were processed using either 1.077 or 1.073 g/mL GMP DGM. BM mononucleated cell (MNC) fractions were analyzed for viability, immunophenotype, clonogenic potential, ex vivo expansion and differentiation potential.

RESULTS

No differences were noticed in cell recovery and viability between the groups. Fluorescence-activated cell-sorting (FACS) analyzes on freshly isolated cells indicated that the 1.073 g/mL GMP DGM more efficiently depleted the CD45(+) fraction in comparison with 1.077 GMP DGM. Moreover, in the 1.073 group, fibroblastic colony-forming units (CFU-F) were 1.5 times higher and the final MSC yield 1.8 times increased after four passages. Both reagents isolated MSC with the expected phenotype; however, 1.073-isolated MSC showed a higher expression of CD90, CD146 and GD2. Additionally, MSC from both groups were capable of fully differentiating into bone, adipose cells and cartilage.

CONCLUSIONS

Both GMP DGM enriched MSC from BM samples, suggesting that these reagents would be suitable for clinical-grade expansions. In addition, the density of 1.073 g/mL provides a significant advantage over 1.077 g/mL GMP DGM, impacting the quantity of MSC obtained and reducing the ex vivo expansion time for optimized cell-based clinical applications.

BACKGROUND

While vascular and immune abnormalities are common in juvenile and adult dermatomyositis (DM), the molecular changes that contribute to these abnormalities are not clear. Therefore, we investigated pathways that facilitate new blood vessel formation and dendritic cell migration in dermatomyositis.

METHODS

Muscle biopsies from subjects with DM (9 children and 6 adults) and non-myositis controls (6 children and 7 adults) were investigated by immunohistochemistry using antibodies that recognize existing (anti-CD146) and newly formed blood vessels (anti-alphaVbeta3) and mature dendritic cells (anti-DC-LAMP). Blood vessel quantification was performed by digitalized image analysis. Additional muscle biopsies from subjects with adult DM and non-myositis controls were used for global gene expression profiling experiments.

RESULTS

A significant increase in neovascularization was found in muscle biopsies of DM patients; neovascularization (alphaVbeta3 positive capillaries and vessels per muscle fiber) was much higher in juvenile than in adult DM patients (control vs juvenile DM: Mean +/- SE: 0.06 +/- 0.01 vs 0.6 +/- 0.05; p < 0.0001 and control vs adult DM: Mean +/- SE: 0.60 +/- 0.1 vs 0.75 +/- 0.1; p = 0.051). Gene expression analysis demonstrated that genes that participate not only in angiogenesis but also in leukocyte trafficking and the complement cascade were highly up regulated in DM muscle in comparison to age matched controls. DC-LAMP positive dendritic cells were highly enriched at perivascular inflammatory sites in juvenile and adult DM patients along with molecules that facilitate dendritic cell transmigration and reverse transmigration (CD142 and CD31).

CONCLUSIONS

These results suggest active neovascularization and endothelial cell activation in both juvenile and adult DM. It is likely that close association of monocytes with endothelial cells initiate rapid dendritic cell maturation and an autoimmune response in DM.