Subpopulation Of Stromal Cells Research Articles

Abstract 3590: Investigation of exosome-encapsulated microRNA secretion in breast cancer

Abstract Intercellular communication between breast tumour cells and stromal cells plays a fundamental role in cancer initiation and progression. Mesenchymal Stem Cells(MSCs) are multipotent cells with the proven ability to home to the site of breast tumours and integrate into the tumour architecture. The mechanisms of MSC interaction with breast cancer cells are poorly defined. Exosomes are microvesicles secreted by cells that have the capacity to transport genetic material including microRNAs(miRNAs), which are important regulators of gene expression. However, the miRNAs selectively packaged into exosomes, and their true role in the tumour microenvironment, is poorly understood. Aim: Investigate the presence of MSCs in primary breast tumours, and identify exosome-encapsulated miRNAs secreted by breast cancer cells and MSCs in vitro. Methods: Stromal cells were isolated from primary breast tumours and characterized based on MSC-associated cell surface antigens. Tumour stromal cells were cultured in appropriate conditions to investigate potential for differentiation into osteoblasts or adipocytes, followed by Von Kossa staining of calcium deposition, and Oil Red O staining of fat droplets respectively. MSCs isolated from healthy volunteers and breast cancer cell lines were cultured in exosome-depleted media for 48 hrs, and secreted exosomes isolated using ultracentrifugation. Transmission Electron Microscopy(TEM) and Western Blot were performed to confirm the presence of exosomes in the purified fraction. Extracted miRNA was subjected to global miRNA expression analysis using the Exiqon miRCURY™ LNA Array. Results: A subpopulation of tumour stromal cells expressed the full panel of surface markers associated with MSCs. This subpopulation also had the capacity to differentiate into osteoblasts and adipocytes, demonstrated by calcium deposition and formation of fat droplets. Exosomes were successfully isolated from conditioned media of breast cancer cell lines and MSCs. TEM revealed microvesicles with the appropriate size of 40-100nm. Western Blot confirmed the presence of the exosome-associated protein CD63. miRNA array analysis identified 413 miRNAs encapsulated in MSC-secreted exosomes from a panel of 2089 analysed, while each of the breast cancer cell lines secreted from 381 to 394 miRNAs in exosomes. Of these, 287 were common to all four breast cancer cell lines. Interestingly, a subset of these miRNAs are known to target genes associated with cell cycle regulation and apoptosis (e.g. miR-15a, miR-204, miR-221, miR-223). A panel of the miRNAs also appeared to have differential expression with reference to epithelial subtype. Conclusion: MSCs are present in primary breast tumours, and elucidating their interaction with breast cancer cells will be fundamental to understanding their role in the tumour microenvironment. Bidirectional transfer of exosome-encapsulated miRNAs may play an important role in this intercellular communication. Citation Format: Claire Glynn, Sonja Khan, Cathy Brougham, Cillian Clancy, Doireann Joyce, Peter Dockery, Michael J. Kerin, Roisin M. Dwyer. Investigation of exosome-encapsulated microRNA secretion in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3590. doi:10.1158/1538-7445.AM2014-3590

Adipogenic differentiation potential of rat adipose tissue-derived subpopulations of stromal cells

Adipose-derived stromal cells (ASCs) are mostly isolated by enzymatic digestion, centrifugation and adherent growth resulting in a very heterogeneous cell population. Therefore, other cell types in the cell culture can comprise the differentiation and proliferation potential of the ASC population. Recent studies indicated that an antibody-aided isolation of distinct ASC subpopulations provides advantages over the conventional method of ASC isolation. The aim of this study was to investigate the adipogenic differentiation potential of CD29-, CD71-, CD73- and CD90-selected ASCs invitro. The stromal vascular fraction (SVF) was obtained from rat adipose tissue by enzymatic digestion and centrifugation. Subsequently, CD29(+)-, CD71(+)-, CD73(+)- and CD90(+) cells were isolated by magnetic activated cell sorting (MACS), seeded into culture plates and differentiated into the adipogenic lineage. ASCs isolated by adherent growth only served as controls. Adipogenic differentiation was assessed by Oil Red O staining and quantification of the adiponectin and leptin concentrations in the cell culture supernatants. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by the Scheffe's post hoc procedure. The results showed that different subpopulations with different adipogenic differentiation potentials can be isolated by the MACS procedure. The highest adipogenic differentiation potential was determined in the CD29-selected ASC population followed by the unsorted ASC population. The CD71-, CD73- and CD90-selected cells exhibited significantly the lowest adipogenic differentiation potential. In conclusion, the CD29-selected ASCs and the unsorted ASCs exhibited a similar adipogenic differentiation potential. Therefore, we do not see a clear advantage in the application of an anti-CD29-based isolation of ASCs over the conventional technique using adherent growth. However, the research on isolation/purification methods of adipogenic ASCs should continue in order to make this stem cell source even more attractive for future adipose tissue engineering applications.

Network of telocytes in the temporomandibular joint disc of rats

The phenotypes of the temporomandibular joint (TMJ) disc cells range from fibroblasts to chondrocytes. There are relatively few reported studies using transmission electron microscopy (TEM) to determine the ultrastructural features of these cells. It was hypothesized that at least a subpopulation of TMJ stromal cells could be represented by the telocytes, cells with telopodes. In this regard a TEM study was performed on rat TMJ samples. Collagen-embedded networks were found built-up by cells with telopodes with subplasmalemmal caveolae, moderate content in matrix secretory organelles and well-represented intermediate filaments. Appositions of cell bodies were found. Prolongations of such cells were closely related to nerves and microvessels. Our study indicates that the TMJ disc attachment seems equipped with telocytes capable of stromal signaling. However, further studies are needed to assess whether the telocytes belong to a renewed cell population derived from circulating precursors.

Enrichment and Characterization of Two Subgroups of Committed Osteogenic Cells in the Mouse Endosteal Bone Marrow with Expression Levels of CD24

Primary osteogenic cells have been known to reside within the CD45-CD31-Ter119-Sca-1- cell fraction, particularly in the CD51+ subpopulation. However, detailed determination of the frequency of osteogenic cells within this Sca-1- cell population remains yet to be determined. In addition, it is not clear that other cell surface markers can be used to further sub-fractionate this Sca-1-CD51+ osteogenic cell population and to define their developmental stages. In this report, both Sca-1-CD24med and Sca-1- CD24-/lo cells have been shown to be two small subsets of the Sca-1-CD51+ cell fraction. These two cell fractions show subtle difference in the expression level of osteogenic marker genes such as Osx and Opn, and in vitro proliferate rate. All these observations suggest that they may be at different developmental stages of osteogenesis. The Sca-1-CD24med cell fraction is enriched for the more mature osteolineage cells than the Sca-1-CD24-/lo counterpart. In contrast, most of the Sca-1-CD24hi and Sca-1+CD24-/lo cells do not contain CFU-ALP nor express osteogenic gene markers. The high proliferation ability and osteo-adipogenic differentiation potentials confirm that the Sca-1+CD24-/lo cells are the multipotential mesenchymal stromal cells. The determination of individual stromal cell subpopulations will lead to a better understanding in the hierarchical organization of these osteolineage cells.

Functionally and Phenotypically Distinct Subpopulations of Marrow Stromal Cells Are Fibroblast in Origin and Induce Different Fates in Peripheral Blood Monocytes

Marrow stromal cells constitute a heterogeneous population of cells, typically isolated after expansion in culture. In vivo, stromal cells often exist in close proximity or in direct contact with monocyte-derived macrophages, yet their interaction with monocytes is largely unexplored. In this report, isolated CD146(+) and CD146(-) stromal cells, as well as immortalized cell lines representative of each (designated HS27a and HS5, respectively), were shown by global DNase I hypersensitive site mapping and principal coordinate analysis to have a lineage association with marrow fibroblasts. Gene expression profiles generated for the CD146(+) and CD146(-) cell lines indicate significant differences in their respective transcriptomes, which translates into differences in secreted factors. Consequently, the conditioned media (CM) from these two populations induce different fates in peripheral blood monocytes. Monocytes incubated in CD146(+) CM acquire a tissue macrophage phenotype, whereas monocytes incubated in CM from CD146(-) cells express markers associated with pre-dendritic cells. Importantly, when CD14(+) monocytes are cultured in contact with the CD146(+) cells, the combined cell populations, assayed as a unit, show increased levels of transcripts associated with organismal development and hematopoietic regulation. In contrast, the gene expression profile from cocultures of monocytes and CD146(-) cells does not differ from that obtained when monocytes are cultured with CD146(-) CM. These in vitro results show that the CD146(+) marrow stromal cells together with monocytes increase the expression of genes relevant to hematopoietic regulation. In vivo relevance of these data is suggested by immunohistochemistry of marrow biopsies showing juxtaposed CD146(+) cells and CD68(+) cells associated with these upregulated proteins.

F11R Is a Novel Monocyte Prognostic Biomarker for Malignant Glioma

ObjectiveBrain tumors (gliomas) contain large populations of infiltrating macrophages and recruited microglia, which in experimental murine glioma models promote tumor formation and progression. Among the barriers to understanding the contributions of these stromal elements to high-grade glioma (glioblastoma; GBM) biology is the relative paucity of tools to characterize infiltrating macrophages and resident microglia. In this study, we leveraged multiple RNA analysis platforms to identify new monocyte markers relevant to GBM patient outcome.MethodsHigh-confidence lists of mouse resident microglia- and bone marrow-derived macrophage-specific transcripts were generated using converging RNA-seq and microarray technologies and validated using qRT-PCR and flow cytometry. Expression of select cell surface markers was analyzed in brain-infiltrating macrophages and resident microglia in an induced GBM mouse model, while allogeneic bone marrow transplantation was performed to trace the origins of infiltrating and resident macrophages. Glioma tissue microarrays were examined by immunohistochemistry, and the Gene Expression Omnibus (GEO) database was queried to determine the prognostic value of identified microglia biomarkers in human GBM.ResultsWe generated a unique catalog of differentially-expressed bone marrow-derived monocyte and resident microglia transcripts, and demonstrated that brain-infiltrating macrophages acquire F11R expression in GBM and following bone-marrow transplantation. Moreover, mononuclear cell F11R expression positively correlates with human high-grade glioma and additionally serves as a biomarker for GBM patient survival, regardless of GBM molecular subtype.SignificanceThese studies establish F11R as a novel monocyte prognostic marker for GBM critical for defining a subpopulation of stromal cells for future potential therapeutic intervention.

A slowly proliferating subpopulation in human umbilical cord mesenchymal stem cells in culture

In this report, a slow-growing subpopulation of human umbilical cord mesenchymal stromal cells (MSCs) was identified. These cells were around 5 μm in diameter and their relative gravity was between 1.031 and 1.043 g/ml. In sharp contrast to the parent MSCs, they expressed highly CD271 and poorly the receptor for platelet-derived growth factor. Quantitative PCR with the identification of the products by DNA sequencing proved that these cells expressed Nanog at a higher level than cells from the other subpopulation (approximately 30-fold), which was further confirmed by western blotting. Furthermore, they did not grow at clonal density and depletion of these cells from the population had little effect on the colony formation of the parent MSCs. The results here indicate that a subpopulation of cells with special biological features exist in human cord MSCs in culture.

Neonatal mesenchymal-like cells adapt to surrounding cells

Hematopoietic cord blood (CB) transplantations are performed to treat patients with life-threatening diseases. Besides endothelial cells, the neonatal multipotent stromal cell subpopulations CDSCs (CB-derived stromal cells) and USSCs (unrestricted somatic stromal cells) are like bone marrow (BM) SCs interesting candidates for clinical applications if detailed knowledge is available. Clonal USSC compared to CDSC and BMSC lines differ in their developmental origin reflected by a distinct HOX expression. About 20 (out of 39) HOX genes are expressed in CDSCs (HOX+), whereas native USSCs reveal no HOX gene expression (HOX-). Moreover, USSCs display a lineage-specific absence of the adipogenic differentiation potential. As the specific HOX code can be ascribed to topographic bodysites it may be important to match the HOX code of transplanted cells to the tissue of interest. Herein co-culture experiments were performed, presenting a novel approach to modulate the differentiation potency of USSCs towards HOX positive stromal cells. After co-culturing native USSCs with CDSCs and BMSCs, USSCs adapt a positive HOX code and gain the adipogenic differentiation capacity. These results present for the first time modulation of a lineage-specific differentiation potential by co-culture. Finally, USSCs can be claimed as potential candidates to substitute unique progenitor cell populations in clinical approaches.

EphB2 isolates a human marrow stromal cell subpopulation with enhanced ability to contribute to the resident intestinal cellular pool

To identify human bone marrow stromal cell (BMSC) subsets with enhanced ability to engraft/contribute to the resident intestinal cellular pool, we transplanted clonally derived BMSCs into fetal sheep. Analysis at 75 d post-transplantation showed 2 of the 6 clones engrafting the intestine at 4- to 5-fold higher levels (5.03±0.089 and 5.04±0.15%, respectively) than the other clones (P<0.01), correlating with the percentage of donor-derived Musashi-1(+) (12.01-14.17 vs. 1.2-3.8%; P<0.01) or leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5)(+) cells within the intestinal stem cell (ISC) region. Phenotypic and transcriptome analysis determined that the clones with enhanced intestinal contribution expressed high levels of Ephrin type B receptor 2 (EphB2). Intestinal explants demonstrated proliferation of the engrafted cells and ability to generate crypt-like structures in vitro still expressing EphB2. Additional transplants based on BMSC EphB2 expression demonstrated that, at 7 d post-transplant, the EphB2(high) BMSCs engrafted in the ISC region at levels of 2.1 ± 0.2%, while control EphB2(low) BMSCs engrafted at 0.3 ± 0.1% (P<0.01). Therefore we identified a marker for isolating and culturing an expandable subpopulation of BMSCs with enhanced intestinal homing and contribution to the ISC region.

Differential Expression of Surface Markers in Mouse Bone Marrow Mesenchymal Stromal Cell Subpopulations with Distinct Lineage Commitment

Bone marrow mesenchymal stromal cells (BM MSCs) represent a heterogeneous population of progenitors with potential for generation of skeletal tissues. However the identity of BM MSC subpopulations is poorly defined mainly due to the absence of specific markers allowing in situ localization of those cells and isolation of pure cell types. Here, we aimed at characterization of surface markers in mouse BM MSCs and in their subsets with distinct differentiation potential. Using conditionally immortalized BM MSCs we performed a screening with 176 antibodies and high-throughput flow cytometry, and found 33 markers expressed in MSCs, and among them 3 were novel for MSCs and 13 have not been reported for MSCs from mice. Furthermore, we obtained clonally derived MSC subpopulations and identified bipotential progenitors capable for osteo- and adipogenic differentiation, as well as monopotential osteogenic and adipogenic clones, and thus confirmed heterogeneity of MSCs. We found that expression of CD200 was characteristic for the clones with osteogenic potential, whereas SSEA4 marked adipogenic progenitors lacking osteogenic capacity, and CD140a was expressed in adipogenic cells independently of their efficiency for osteogenesis. We confirmed our observations in cell sorting experiments and further investigated the expression of those markers during the course of differentiation. Thus, our findings provide to our knowledge the most comprehensive characterization of surface antigens expression in mouse BM MSCs to date, and suggest CD200, SSEA4 and CD140a as markers differentially expressed in distinct types of MSC progenitors.

Mesenspheres of neural crest-derived cells enriched from bone marrow stromal cell subpopulation

Neural crest-derived cells (NCCs) can be used for cell replacement therapy of neurodegenerative diseases and nerve injury, and it is of significance to open readily accessible tissue sources for NCCs due to their insufficient supply. In this study, we aimed to examine the possibility of enriching NCCs from bone marrow stromal cell (BMSC) subpopulation. The epidermal growth factor/fibroblast growth factor-2 (EGF/FGF2)-responsive BMSC subpopulation (BMSC-C2) was isolated from rat bone marrow by repetitive two-step condition culture. The BMSC-C2 subpopulation showed a long-term proliferative capacity and high cell growth rate, and possessed a significant sphere-forming ability. The mesenspheres derived from BMSC-C2 subpopulation were self-renewable and could express NCC markers, such as CD29, CD44, nestin, CD133 and p75NTR. In particular, the mesenspheres could be induced to differentiate into neuron- and glia-like cells in vitro. Collectively, our results might provide a basis for in-depth studies of recruiting postmigratory NCCs from bone marrow and various neural crest-derived tissues.

Fibroblast Activation Protein in Remodeling Tissues

Tissue remodeling is critical during development and wound healing. It also characterizes a number of pathologic conditions, including chronic inflammation, fibrosis and cancer. It is well appreciated that reactive stromal cells play critical roles in these settings. However, understanding of the mechanisms involved in the differentiation of reactive stromal cells and their biologic activities has been hampered by the fact that they are generated from diverse progenitors, and by their phenotypic and function heterogeneity. Furthermore, molecular markers that are expressed by all reactive stromal cells and that distinguish them from all other cell types have been lacking. Fibroblast activation protein (FAP) is a serine protease that was originally discovered as a cell surface protein expressed on astrocytomas and sarcomas. Over the last two decades, FAP has attracted increasing attention as a selective marker of carcinoma-associated fibroblasts (CAFs) and more broadly, of activated fibroblasts in tissues undergoing remodeling of their extracellular matrix (ECM) due to chronic inflammation, fibrosis or wound healing. Herein we review the evidence that FAP is indeed a robust and selective marker for reactive mesenchymal stromal cells associated with pathophysiologic tissue remodeling. We also review recent insights obtained using FAP as a tool to define the relationship between subpopulations of reactive stromal cells in various settings of tissue remodeling. Furthermore, we review recent genetic and pharmacologic data indicating that FAP and FAP-expressing cells play important roles in such conditions. Finally, we discuss the potential risks and therapeutic benefits of targeting FAP and FAP-expressing cells, as well as approaches to do so.

Selection of p53-Deficient Stromal Cells in the Tumor Microenvironment

The tumor microenvironment is considered pro-oncogenic for reasons that, among others, involve the stimulation of cancer cell growth. However, little is known regarding how the tumor microenvironment affects the proliferation of stromal cells that coexist with cancer cells in the tumors. In the present study, we show that cancer cells trigger a paracrine response in normal fibroblasts that is not consistently mitogenic but may also become antimitogenic, inhibiting fibroblasts' proliferation in vitro. In vivo experiments on xenografts of MDA-MB-231 breast and A549 lung cancer cells in SCID mice or primary Notch-induced breast cancers that develop in transgenic mice showed that stromal cells frequently undergo senescence, confirming the presence of stress-inducing conditions within the tumor's microenvironment. These antimitogenic responses of the stromal cells were less pronounced in fibroblasts that are p53-deficient, suggesting that p53 plays a role in the execution of these mitogenic stress responses. In addition, they provide a biological basis for the selection of certain stromal cell subpopulations within malignant tumors exemplified by the p53-deficient stromal cells. Indeed, reconstitution of A549 human lung tumors in mice with admixed wild-type and p53-deficient stromal fibroblasts indicates that the selection of p53-deficient stromal cells is more intense when the number of cancer cells, and therefore the intensity of the stress response-inducing signals, is higher. These findings possess implications related to the effects of the cancer cells in the stromal cells and provide hints for the mechanism(s) underlining the transition of the normal stroma to cancer-associated stroma.

In vitro and in vivo interactions between glioma and marrow-isolated adult multilineage inducible (MIAMI) cells

The prognosis of patients with malignant glioma remains extremely poor despite surgery and improvements in radio- and chemo-therapies. We recently showed that marrow-isolated adult mutilineage inducible (MIAMI) cells, a subpopulation of human mesenchymal stromal cells (MSCs), can serve as cellular carriers of drug-loaded nanoparticles to brain tumors. However, the safety of MIAMI cells as cellular treatment vectors in glioma therapy must be evaluated, in particular their effect on glioma growth and their fate in a tumor environment. In this study, we showed that MIAMI cells were able to specifically migrate toward the orthotopic U87MG tumor model and did not influence its growth. In this model, MIAMI cells did not give rise to cells resembling endothelial cells, pericytes, cancer-associated fibroblasts (CAFs), or astrocytes. Despite these encouraging results, the effects of MIAMI cells may be glioma-dependent. MIAMI cells did not migrate toward the orthotopic Lab1 GB and they can induce the proliferation of other glioma cell lines in vitro. Furthermore, a fraction of MIAMI cells was found to be in a state of proliferation in the U87MG tumor environment. These findings indicate that the use of MIAMI cells as cellular treatment vectors for malignant tumors must be controlled. These cells may be used as “suicide vectors”: vectors for killing not only tumor cells but themselves.

Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion.

Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described. We have identified a cell non-autonomous role of Rb, using three-dimensional cultures, where depletion of Rb in stromal fibroblasts enhances invasive potential of transformed epithelia. In part, this is mediated by upregulation of keratinocyte growth factor (KGF), which is produced by the depleted fibroblasts. KGF drives invasion of epithelial cells through induction of MMP1 expression in an AKT- and Ets2-dependent manner. Our data identify that stromal fibroblasts can alter the invasive behaviour of the epithelium, and we show that altered expression of KGF can mediate these functions.

Paracrine Interactions between Mesenchymal Stem Cells Affect Substrate Driven Differentiation toward Tendon and Bone Phenotypes

We investigated substrate dependent paracrine signaling between subpopulations of bone marrow stromal cells (BMSCs) that may affect the formation, or perhaps malformation, of the regenerating tendon to bone enthesis. Polyacrylamide substrates approximating the elastic modulus of tendon granulation tissue and the osteoid of healing bone (10–90 kPa) were functionalized with whole length fibronectin (Fn), type-I collagen (Col), or a mixed ligand solution (Fn/Col), and BMSCs were cultured in growth media alone or media supplemented with soluble Col or Fn. More rigid substrates with a narrow mechanical gradient (70–90 kPa) robustly induced osteogenic cell differentiation when functionalized with either Col or Fn. On broader mechanical gradient substrates (with a linear elastic modulus gradient from 10–90 kPa), cell differentiation was markedly osteogenic on subregions of Fn functionalized substrates above 20 kPa, but osteogenic activity was inhibited on all subregions of Col substrates. Osteogenic behavior was not observed when cells were cultured on Fn substrates if Col was present either in the media or on the substrate (Fn/Col). Tenogenic differentiation markers were observed only on Col substrates with moderate rigidity (∼30–50 kPa). Tenogenic differentiation was unaltered by soluble or substrate bound Fn. Co-culture of narrow gradient subsections revealed that any inclusion of tenogenic substrates (30–50 kPa, Col), caused otherwise osteogenic substrates to not develop markers of osteogenic differentiation, while increasing cell proliferation. These apparently paracrine effects could be mediated by bone morphogenetic protein-2 (BMP-2), as first confirmed by gene-level expression of BMP-2 and the transcription factor Smad8, and verified by BMP-2 media supplementation at levels similar to observed cell-secreted concentrations, which arrested osteogenic differentiation in 14 day cultures. Thus, cell instructive biomaterials with engineered mechanical and biochemical properties represent potentially powerful tools for directing BMSC differentiation to tendon and bone, however paracrine signals from tenogenic cells may delay osteogenesis at the healing enthesis.

Perivascular Human Endometrial Mesenchymal Stem Cells Express Pathways Relevant to Self-Renewal, Lineage Specification, and Functional Phenotype1

Human endometrium regenerates on a cyclic basis from candidate stem/progenitors whose genetic programs are yet to be determined. A subpopulation of endometrial stromal cells, displaying key properties of mesenchymal stem cells (MSCs), has been characterized. The endometrial MSC (eMSC) is likely the precursor of the endometrial stromal fibroblast. The goal of this study was to determine the transcriptome and signaling pathways in the eMSC to understand its functional phenotype. Endometrial stromal cells from oocyte donors (n = 20) and patients undergoing benign gynecologic surgery (n = 7) were fluorescence-activated cell sorted into MCAM (CD146)(+)/PDGFRB(+) (eMSC), MCAM (CD146)(-)/PDGFRB(+) (fibroblast), and MCAM (CD146)(+)/PDGFRB(-) (endothelial) populations. The eMSC population contained clonogenic cells with a mesenchymal phenotype differentiating into adipocytes when cultured in adipogenic medium. Gene expression profiling using Affymetrix Human Gene 1.0 ST arrays revealed 762 and 1518 significantly differentially expressed genes in eMSCs vs. stromal fibroblasts and eMSCs vs. endothelial cells, respectively. By principal component and hierarchical clustering analyses, eMSCs clustered with fibroblasts and distinctly from endothelial cells. Endometrial MSCs expressed pericyte markers and were localized by immunofluorescence to the perivascular space of endometrial small vessels. Endometrial MSCs also expressed genes involved in angiogenesis/vasculogenesis, steroid hormone/hypoxia responses, inflammation, immunomodulation, cell communication, and proteolysis/inhibition, and exhibited increased Notch, TGFB, IGF, Hedgehog, and G-protein-coupled receptor signaling pathways, characteristic of adult tissue MSC self-renewal and multipotency. Overall, the data support the eMSC as a clonogenic, multipotent pericyte that displays pathways of self-renewal and lineage specification, the potential to respond to conditions during endometrial desquamation and regeneration, and a genetic program predictive of its differentiated lineage, the stromal fibroblast.

The composition of the mesenchymal stromal cell compartment in human bone marrow changes during development and aging

Life-long hematopoiesis depends on the support of mesenchymal stromal cells within the bone marrow. Therefore, changes in the hematopoietic compartment that occur during development and aging probably correlate with variation in the composition of the stromal cell microenvironment. Mesenchymal stromal cells are a heterogeneous cell population and various subtypes may have different functions. In accordance with others, we show that CD271 and CD146 define distinct colony-forming-unit-fibroblast containing mesenchymal stromal cell subpopulations. In addition, analysis of 86 bone marrow samples revealed that the distribution of CD271(bright)CD146(-) and CD271(bright)CD146(+) subsets correlates with donor age. The main subset in adults was CD271(bright)CD146(-), whereas the CD271(bright)CD146(+) population was dominant in pediatric and fetal bone marrow. A third subpopulation of CD271(-)CD146(+) cells contained colony-forming-unit-fibroblasts in fetal samples only. These changes in composition of the mesenchymal stromal cell compartment during development and aging suggest a dynamic system, in which these subpopulations may have different functions.

Interleukin‐8 derived from local tissue‐resident stromal cells promotes tumor cell invasion

The aim of this study is to evaluate the role of adipose tissue resident stromal cells on tumor cell invasion. Our data show that a subpopulation of adipose tissue derived stromal cells expressing Nestin, NG2, α-smooth muscle actin and PDGFR-α migrate toward the cancer cells. Microarray analysis revealed the upregulation of IL-8 in the migrated cells. We demonstrated that stromal cell derived IL-8 promote the invasion and the anchorage-independent growth of cancer cells. We conclude that human breast cancer cells attract a subpopulation of stromal cells that secrete IL-8 to promote tumor cell invasion in a paracrine fashion.

Cell-based quantification of molecular biomarkers in histopathology specimens

To investigate the use of a computer-assisted technology for objective, cell-based quantification of molecular biomarkers in specified cell types in histopathology specimens, with the aim of advancing current visual estimation and pixel-level (rather than cell-based) quantification methods. Tissue specimens were multiplex-immunostained to reveal cell structures, cell type markers, and analytes, and imaged with multispectral microscopy. The image data were processed with novel software that automatically delineates and types each cell in the field, measures morphological features, and quantifies analytes in different subcellular compartments of specified cells.The methodology was validated with the use of cell blocks composed of differentially labelled cultured cells mixed in known proportions, and evaluated on human breast carcinoma specimens for quantifying human epidermal growth factor receptor 2, estrogen receptor, progesterone receptor, Ki67, phospho-extracellular signal-related kinase, and phospho-S6. Automated cell-level analyses closely matched human assessments, but, predictably, differed from pixel-level analyses of the same images. Our method reveals the type, distribution, morphology and biomarker state of each cell in the field, and allows multiple biomarkers to be quantified over specified cell types, regardless of their abundance. It is ideal for studying specimens from patients in clinical trials of targeted therapeutic agents, for investigating minority stromal cell subpopulations, and for phenotypic characterization to personalize therapy and prognosis.