Plastic-adherent Cells Research Articles

Single-cell analysis of K562 cells: An imatinib-resistant subpopulation is adherent and has upregulated expression of BCR-ABL mRNA and protein

In chronic myeloid leukemia (CML) cells from different stages of maturation may have differential expression of BCR-ABL at both messenger RNA (mRNA) and protein level. However, the significance of such differential expression to clinical disease behavior is unknown. Using the CML-derived, BCR-ABL expressing cell line, K562, distinct plastic-adherent (K562/Adh) and nonadherent (K562/NonAdh) subpopulations were established and then analyzed both as single cells and as bulk cell populations. BCR-ABL mRNA was upregulated in K562/Adh compared with K562/NonAdh cells in both single cell and bulk population analyses (p < 0.0001). Similarly, phosphorylation of BCR protein was upregulated in K562/Adh, compared with K562/NonAdh cells (63.42% vs. 23.1%; p = 0.007), and these two K562 subpopulations were found to express significantly different microRNA species. Furthermore, treatment with the BCR-ABL tyrosine kinase inhibitor, imatinib, reduced cell viability more rapidly in K562/NonAdh compared with K562/Adh cells (p < 0.005) both at single and bulk cell levels. This discovery of an adherent subpopulation of K562 cells with increased BCR-ABL mRNA, increased phosphorylated BCR protein expression, differential microRNA expression, and increased imatinib resistance suggests that a similar subpopulation of cells can also mediate clinical resistance to imatinib during treatment of patients with CML.

CD146 expression on mesenchymal stem cells is associated with their vascular smooth muscle commitment

Bone marrow mesenchymal stem cells (MSCs) are plastic adherent cells that can differentiate into various tissue lineages, including osteoblasts, adipocytes and chondrocytes. However, this progenitor property is not shared by all cells within the MSC population. In addition, MSCs vary in their proliferation capacity and expression of markers. Because of heterogeneity of CD146 expression in the MSC population, we compared CD146−/Low and CD146High cells under clonal conditions and after sorting of the non-clonal cell population to determine whether this expression is associated with specific functions. CD146−/Low and CD146High bone marrow MSCs did not differ in colony-forming unit-fibroblast number, osteogenic, adipogenic and chondrogenic differentiation or in vitro haematopoietic-supportive activity. However, CD146−/Low clones proliferated slightly but significantly faster than did CD146High clones. In addition, a strong expression of CD146 molecule was associated with a commitment to a vascular smooth muscle cell (VSMC) lineage characterized by a strong up-regulation of calponin-1 and SM22α expression and an ability to contract collagen matrix. Thus, within a bone marrow MSC population, certain subpopulations characterized by high expression of CD146, are committed towards a VSMC lineage.

THU0030 Evidence for good intrinsic mesenchymal stem cell activity in gelatinous heberden’s nodes in osteoarthritis at clinical presentation

BackgroundHeberden’s nodes (HN) are common pre-radiographic features of generalised osteoarthritis (GOA) and they may present as acute cystic lesions before eventually leading to florid new bone formation [1].ObjectivesTo examine if...

Simple lipoaspirate washing using a coffee filter

Background: Lipoaspirate contains noxious substances derived from liposuction. Therefore, extensive washing is recommended before the lipoaspirate is processed further for culture or fat grafting. Washing a small amount of lipoaspirate may not pose a problem, but washing a large volume of lipoaspirate may be cumbersome, time consuming, and requires a lot of phosphate buffered saline (PBS). Objective: To introduce a simple method for lipoaspirate washing using fine-mesh stainless-steel tea or coffee filter, a small tea spoon, and a porcelain bowl. Methods: The filter was used to collect the adipose tissue fragments. Further washing of the fragments was achieved by soaking the adipose tissue containing filter in a PBS containing porcelain bowl and stirring using a small tea spoon to transfer the contaminating materials to the PBS. Enzymatic processing to dissociate the cells from the tissue and primary cultures was conducted as usual in MesenCult. Results: Using the equipment mentioned above, the adipose tissue fragments were readily separated from the blood, free lipids, anesthetics, and other noxious material in the liquid portion. This simple method saves time and PBS compared with previously described methods. Further enzymatic processing produced sufficient cells to be cultured, and culture results showed plastic adherent cells on day 2 that became confluent on day 6. Conclusion: Lipoaspirate washing using a fine mesh stainless steel filter is time saving and produced cells that grow well in MesenCult. Keywords: Cell, culture, filter, lipoaspirate

Further Proof for an Unpopular Concept: A Single Cell From Bone Marrow Can Serve as a Stem Cell for Both Hematopoiesis and Osteogenesis

In science, unpopular concepts must be proven time and again. The article by Hofmann et al. in this issue of Molecular Therapy provides further proof for the unpopular idea that a single cell from bone marrow can serve as a stem cell for both hematopoiesis and osteogenesis.1 Moreover, Hofmann et al. establish that this single cell is actually an old friend: the long-term repopulating hematopoietic stem cell (LTR-HSC). These observations also suggest, but do not conclusively prove, the interesting idea that the same LTR-HSC can provide both the HSC and the microenvironmental niche required for the HSC to self-replicate and differentiate. Such a dual function can easily explain why an LTR-HSC is a long-term repopulating cell. The study provides new evidence for a refreshingly simple concept that probably should have occurred to us earlier.

Comparison of fracture site callus with iliac crest bone marrow as the source of plastic-adherent cells

Background: Red marrow has been described as the main source of mesenchymal stem cells although its aspiration and isolation from bone marrow was reported to have significant donor site morbidity. Since secondary bone healing occurs through formation of callus as the result of proliferation and differentiation of mesenchymal stem cells, callus may become alternative source for mesenchymal stem cells. In this study, we compared the number of plastic-adherent cells from fracture site callus and bone marrow of iliac crest after two and four weeks of culture. Methods: Sixteen New Zealand rabbits were fracturized at the femoral shaft. Then, these rabbits were taken care. After two weeks of fracturization, 3 mL iliac crest bone marrow aspiration and callus extraction of eight rabbits were cultured (group I). The other eight rabbits were treated equally after four weeks of fracturization (group II). Simultaneously, the cultures were observed after one and two weeks. Four weeks later, they were harvested. Cells were counted using Neubauer hemocytometer. The average number of cells between the sources and groups were statistically analyzed using the unpaired t-test. Results: In group I, there were 2.6±0.1 x 104 cells in the culture of iliac crest bone marrow aspirate and 2.5 ± 0.1 x 104 cells in culture of callus extract from fracture site (p=0.34). In group II, there were 2.7 ± 0.1 x 104 cells and 2.1 ± 0.1 x 104 cells, respectively (p &lt; 0.001). Conclusion: Fracture site callus at the second week post-fracturization may be potential as source of plastic-adherent cells compared with iliac crest bone marrow.

The Signal Peptide of the Tumor-shared Antigen Midkine Hosts CD4+ T Cell Epitopes

The CD4 T cell response to the tumor antigen Midkine was unknown. Most of the T cell response to Midkine relies on T cell epitopes contained in its signal peptide. The signal peptide of Midkine is accessible to HLA class II pathway for CD4 T cell presentation. It is a new function for signal peptides to contribute to tumor-specific CD4 T cell response. Because of the key role of CD4 T cell response in immunity to tumors, we investigated the CD4(+) T cell response to the recently identified tumor antigen Midkine (MDK). By weekly stimulations of T lymphocytes harvested from seven HLA-DR-typed healthy donors, we derived CD4(+) T cell lines specific for eight MDK peptides. Most of the T cell lines reacted with the peptides 9-23 and 14-28, located in and overlapping the MDK signal peptide, respectively. Accordingly, the MDK signal peptide appeared to be rich in good binders to common HLA-DR molecules. The peptide 9-23-specific T cell lines were specifically stimulated by autologous dendritic cells loaded with lysates of MDK-transfected cells or with lysates of tumor cells naturally expressing the MDK protein. One T cell line was stimulated by HLA-compatible MDK-transfected tumor cells. By contrast, the peptide 14-28-specific T cell lines were not stimulated in any of these conditions. Our data demonstrate that CD4(+) T cell epitopes present in the signal peptide can be accessible to recognition by CD4(+) T cells and may therefore contribute to tumor immunity, whereas a peptide overlapping the junction between the signal peptide and the mature protein is not.

Comparative Analysis of Mesenchymal Stromal Cells Biological Properties

The stromal progenitors of mesodermal cells, mesenchymal stromal cells (MSCs), are a heterogeneous population of plastic adherent fibroblast-like cells with extensive proliferative capacity and differentiation potential. Human MSCs have now been isolated from various tissues including bone marrow, muscle, skin, and adipose tissue, the latter being one of the most suitable cell sources for cell therapy, because of its easy accessibility, minimal morbidity, and abundance of cells. Bone marrow and subcutaneous or visceral adipose tissue samples were collected, digested with collagenase if needed, and seeded in Iscove's medium containing 5% human platelet lysate. Nonadherent cells were removed after 2-3 days and the medium was replaced twice a week. Confluent adherent cells were detached, expanded, and analyzed for several biological properties such as morphology, immunophenotype, growth rate, senescence, clonogenicity, differentiation capacity, immunosuppression, and secretion of angiogenic factors. The results show significant differences between lines derived from subcutaneous fat compared to those derived from visceral fat, such as the higher proliferation rate of the first and the strong induction of angiogenesis of the latter. We are convinced that the identification of the peculiarities of MSCs isolated from different tissues will lead to their more accurate use in cell therapy.

Isolation, characterization and the multi‐lineage differentiation potential of rabbit bone marrow‐derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are recognized by their plastic adherent ability, fibroblastic-like appearance, expression of specific surface protein markers, and are defined by their ability to undergo multi-lineage differentiation. Although rabbit bone marrow-derived MSCs (rbMSCs) have been used extensively in previous studies especially in translational research, these cells have neither been defined morphologically and ultrastructurally, nor been compared with their counterparts in humans in their multi-lineage differentiation ability. A study was therefore conducted to define the morphology, surface marker proteins, ultrastructure and multi-lineage differentiation ability of rbMSCs. Herein, the primary rbMSC cultures of three adult New Zealand white rabbits (at least 4 months old) were used for three independent experiments. rbMSCs were isolated using the gradient-centrifugation method, an established technique for human MSCs (hMSCs) isolation. Cells were characterized by phase contrast microscopy observation, transmission electron microscopy analysis, reverse transcriptase-polymerase chain reaction (PCR) analysis, immunocytochemistry staining, flow cytometry, alamarBlue(®) assay, histological staining and quantitative (q)PCR analysis. The isolated plastic adherent cells were in fibroblastic spindle-shape and possessed eccentric, irregular-shaped nuclei as well as rich inner cytoplasmic zones similar to that of hMSCs. The rbMSCs expressed CD29, CD44, CD73, CD81, CD90 and CD166, but were negative (or dim positive) for CD34, CD45, CD117 and HLD-DR. Despite having similar morphology and phenotypic expression, rbMSCs possessed significantly larger cell size but had a lower proliferation rate as compared with hMSCs. Using established protocols to differentiate hMSCs, rbMSCs underwent osteogenic, adipogenic and chondrogenic differentiation. Interestingly, differentiated rbMSCs demonstrated higher levels of osteogenic (Runx2) and chondrogenic (Sox9) gene expressions than that of hMSCs (P < 0.05). There was, however, no difference in the adipogenic (Pparγ) expressions between these cell types (P > 0.05). rbMSCs possess similar morphological characteristics to hMSCs, but have a higher potential for osteogenic and chondrogenic differentiation, despite having a lower cell proliferation rate than hMSCs. The characteristics reported here may be used as a comprehensive set of criteria to define or characterize rbMSCs.

Mesenchymal stromal cells: misconceptions and evolving concepts

Nearly half a century has passed since the publication of the first articles describing plastic-adherent cells from bone marrow, referred to initially as colony-forming unit fibroblasts, then marrow stromal cells, mesenchymal stem cells and most recently multipotent mesenchymal stromal cells (MSCs). As expected, our understanding of the nature and biologic functions of MSCs has undergone major paradigm shifts over this time. Despite significant advances made in deciphering their complex biology and therapeutic potential in both experimental animal models and human clinical trials, numerous misconceptions regarding the nature and function of MSCs have persisted in the field. Continued propagation of these misconceptions in some cases may significantly impede the advancement of MSC-based therapies in clinical medicine. We have identified six prevalent misconceptions about MSCs that we believe affect the field, and we attempt to rectify them based on current available data.

Immunological characteristics of mesenchymal stem cells

Although bone marrow is the main source, mesenchymal stem cells have already been isolated from various other tissues, such as the liver, pancreas, adipose tissue, peripheral blood and dental pulp. These plastic adherent cells are morphologically similar to fibroblasts and have a high proliferative potential. This special group of cells possesses two essential characteristics: self-renewal and differentiation, with appropriate stimuli, into various cell types. Mesenchymal stem cells are considered immunologically privileged, since they do not express costimulatory molecules, required for complete T cell activation, on their surface. Several studies have shown that these cells exert an immunosuppressive effect on cells from both innate and acquired immunity systems. Mesenchymal stem cells can regulate the immune response in vitro by inhibiting the maturation of dendritic cells, as well as by suppressing the proliferation and function of T and B lymphocytes and natural killer cells. These special properties of mesenchymal stem cells make them a promising strategy in the treatment of immune mediated disorders, such as graft-versus-host disease and autoimmune diseases, as well as in regenerative medicine. The understanding of immune regulation mechanisms of mesenchymal stem cells, and also those involved in the differentiation of these cells in various lineages is primordial for their successful and safe application in different areas of medicine.

Shared Cell Surface Marker Expression in Mesenchymal Stem Cells and Adult Sarcomas

Advanced adult soft-tissue sarcomas (STSs) are rare tumors with a dismal prognosis and limited systemic treatment options. STSs may originate from mesenchymal stem cells (MSCs); the latter have mainly been isolated from adult bone marrow as plastic-adherent cells with differentiation capacity into mesenchymal tissues. Recently, a panel of antibodies has been established that allows for the prospective isolation of primary MSCs with high selectivity. Similar to cancer stem cells in other malignancies, sarcoma stem cells may bear immunophenotypic similarity with the corresponding precursor, that is, MSCs. We therefore set out to establish the expression pattern of MSC markers in sarcoma cell lines and primary tumor samples by flow cytometry. In addition, fibroblasts from different sources were examined. The results document a significant amount of MSC markers shared by sarcoma cells. The expression pattern includes uniformly expressed markers, as well as MSC markers that only stained subpopulations of sarcoma cells. Expression of W5C5, W8B2 (tissue nonspecific alkaline phosphatase [TNAP]), CD344 (frizzled-4), and CD271 marked subpopulations displaying increased proliferation potential. Moreover, CD271+ cells displayed in vitro doxorubicin resistance and an increased capacity to form spheres under serum-free conditions. Interestingly, another set of antigens, including the bona fide progenitor cell markers CD117 and CD133, were not expressed. Comparative expression patterns of novel MSC markers in sarcoma cells, as well as fibroblasts and MSCs, are presented. Our data suggest a hierarchical cytoarchitecture of the most common adult type sarcomas and introduce W5C5, TNAP, CD344, and CD271 as potential sarcoma progenitor cell markers.

Do the Fibroblasts Contained in Early Passage MSC Population Adversely Affect the Characteristics of Stem Cell Population Obtained from Human Placenta?

Mesenchymal stem cells (MSCs) have been obtained from various human tissues by harvesting plastic adherent fibroblast-like cell population. For potential use in regeneration medicine, early passage MSC population is preferred to avoid cell senescence. The early passage adherent cell population contains MSCs as well as fibroblasts, however, the significance of the contained fibroblasts has not been well investigated. Thus, we investigated the stem cell characteristics of the early passage MSC population with and without fibroblasts depletion. We obtained adherent cell populations from full term placenta at passage 2∼3 and divided them into two subpopulations: fibroblasts depleted (popFD) and non-depleted population (popFND) using magnetic cell sorting method. The two subpopulations were compared in terms of cell morphology, potential for long term culture, colony forming ability, and tri-lineage differentiation for adipogenic, chondrogenic, and osteogenic differentiation. The percentage of fibroblasts contained in the early passage MSC population was 5.3% (2.9∼8.4). Both the popFD and popFND was spindle shaped from early passages and maintained long term culture up to 20∼22 passages. CFU-F assay showed no difference between the subpopulations. Overall, tri-lineage differentiation showed a tendency of better differentiation potential of popFND than popFD. We confirmed that fibroblasts are contained in early population of placenta-derived MSCs obtained by current method. This study revealed that the contained fibroblasts in early passage MSC population do not adversely affect the properties of MSCs in terms of cell morphology, potential for long term culture, colony forming ability, and tri-lineage differentiation.

DNA Methylation Plasticity of Human Adipose-Derived Stem Cells in Lineage Commitment

Adult stem cells have an enormous potential for clinical use in regenerative medicine that avoids many of the drawbacks characteristic of embryonic stem cells and induced pluripotent stem cells. In this context, easily obtainable human adipose-derived stem cells offer an interesting option for future strategies in regenerative medicine. However, little is known about their repertoire of differentiation capacities, how closely they resemble the target primary tissues, and the potential safety issues associated with their use. DNA methylation is one of the most widely recognized epigenetic factors involved in cellular identity, prompting us to consider how the analyses of 27,578 CpG sites in the genome of these cells under different conditions reflect their different natural history. We show that human adipose-derived stem cells generate myogenic and osteogenic lineages that share much of the DNA methylation landscape characteristic of primary myocytes and osteocytes. Most important, adult stem cells and in vitro-generated myocytes and osteocytes display a significantly different DNA methylome from that observed in transformed cells from these tissue types, such as rhabdomyosarcoma and osteosarcoma. These results suggest that the plasticity of the DNA methylation patterns plays an important role in lineage commitment of adult stem cells and that it could be used for clinical purposes as a biomarker of efficient and safely differentiated cells.

Isolation, culture and characterization of caprine mesenchymal stem cells derived from amniotic fluid

Amniotic fluid (AF) represents heterologous cell types and a specific group of these cells show high growth rate and multipotent characteristics. The aim of the present study was to culture and fully characterize the putative stem cell population isolated from caprine mesenchymal stem cells. Plastic adherent fibroblastoid cell population could be successfully isolated from the caprine amniotic fluid. In vitro expanded caprine amniotic fluid derived mesenchymal stem cells (cAF-MSCs) showed high proliferation ratio with a doubling time of 33.1h and stained positive for alkaline phosphatase. Relative transcript abundance of CD-73, CD-90 and CD-105 surface markers were analyzed by SYBR green based real time PCR and their respective proteins were localized through immunocytochemistry, however cAF-MSCs were found negative for haematopoietic marker CD-34. When exposed to corresponding induction condition, cAF-MSCs differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through von Kossa, Oil Red O and Alcian blue staining respectively. Furthermore, these cells were found positive for undifferentiated embryonic stem cell markers like Oct-4, Nanog, Sox-2, SSEA-1 and SSEA-4 which accentuate their pluripotent property. In conclusion, caprine amniotic fluid represents a promising source of mesenchymal stem cells with high proliferative and differentiation potential and these cells offer their scope for multiple regenerative therapies.

Maintenance and characterization of multipotent mesenchymal stem cells isolated from canine umbilical cord matrix by collagenase digestion

Multipotent mesenchymal stem cells have been considered as a novel clinical approach for cell therapy and regenerative medicine. In this study, mesenchymal stem cells (MSCs) were successfully isolated from canine umbilical cord matrix (cUCM; also referred to as Wharton’s Jelly) by collagenase digestion and further characterized for multipotent properties associated with MSCs. Our cUCM-derived MSCs (cUCM-MSCs) were plastic adherent, spindle-shaped and fibroblast-like cells, maintaining expression of pluripotency markers such as Oct3/4, Nanog, Sox-2 and SSEA-4 as well as normal chromosomal number during a long-term proliferative culture. The cells expressed MSCs-specific surface markers, including CD44, CD90, CD105, and CD184, but did not CD29, CD33, CD34, and CD45. More importantly, cUCM-MSCs could differentiate into mesodermal (adipocyte, osteocyte and chondrocyte) and ectodermal (neuronal cell) cell lineages. These results imply that collagenase digestion would be a highly effective way to isolate multipotent MSCs in abundant amounts.

Increased anti-melanoma efficacy of hu14.18K322A by combining with αCD40 +CpG (74.1)

Abstract Targeted monoclonal antibodies (mAb) can be used therapeutically for tumors with identifiable antigens such as disialoganglioside GD2, expressed on neuroblastoma and melanoma. αGD2 mAbs have been clinically successful in patients with neuroblastoma. An important mechanism of mAb therapy is Antibody Dependent Cellular Cytotoxicity (ADCC). Combinatorial therapeutic strategies can dramatically increase the anti-tumor response elicited by mAbs. We combined a novel αGD2 mAb, hu14.18K322A, with an immunostimulatory regimen of agonist CD40 mAb and class B CpG-ODN 1826. This combination immunotherapy produced a synergistic anti-tumor response in a syngeneic model of melanoma with minimal tumor burden, resulting in long-term cure of mice. NK depletion in B6 mice showed NK cells were required for the anti-tumor effect. Anti-tumor responses were also observed in tumor-bearing SCID/beige mice; thus NK cell cytotoxicity was not essential. Interrogation of the myeloid population in the peritoneal cavity showed an increase in neutrophils and monocytes with high Fc receptor expression after treatment with αCD40 +CpG. Furthermore, plastic adherent peritoneal cells inhibited tumor cells in vitro in an antibody-dependent manner. These data highlight the importance of myeloid cells as potential effectors in immunotherapy regimens utilizing tumor-specific mAb and suggest further studies are needed to investigate the therapeutic potential of activated myeloid cells.

Mesenchymal stromal cells and fibroblasts: a case of mistaken identity?

The plastic-adherent fibroblast-looking cells that can be isolated and culture-expanded from bone marrow and many other tissues are widely known as mesenchymal stromal cells (MSC). In addition to their fibroblast-like morphology, they are characterized by a panel of cell-surface markers and their potential to differentiate into bone, fat and cartilage. Based on their intriguing immunomodulatory and regenerative properties, MSC are being investigated as cellular therapeutics for a variety of clinical indications. However, many questions regarding the true identity and functionality of these cells in vivo remain unanswered. Fibroblasts, known for a much longer time but still poorly characterized, are also considered to be a ubiquitous stromal element of almost all tissues and are believed to play a role in tissue homeostasis. Despite the presence of MSC and fibroblasts in almost all tissues, similar morphology and other shared characteristics, the exact relationship between MSC and fibroblasts has remained undetermined. In this review, based on recent and old, but often neglected, literature it is suggested that ex vivo culture-expanded MSC and fibroblasts are indistinguishable by morphology, cell-surface markers, differentiation potential and immunologic properties.

Abstract 1254: FAK activity regulates αvβ5 integrin and osteopontin expression to control breast and ovarian cancer anchorage-independent growth

Abstract Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that controls cell motility and survival downstream of integrin receptors. FAK expression and activity are elevated during breast and ovarian cancer progression and this is associated with a poor clinical prognosis. Small molecule FAK inhibitors (FAK-I) block breast and ovarian tumor progression through a novel mechanism that is associated with the apoptosis under anchorage-deprived conditions. Metastatic cells can grow as anchorage-independent spheroids and we hypothesize that a spheroid micro-environment remains dependent on integrin- and FAK-associated signaling for survival. Here we show that intraperitoneal (ip) injection of murine ID8 ovarian carcinoma cells into mice, recovery of ascites-associated tumor cells (ID8ip) and growth ex vivo, yielded tumor cells with elevated levels of phosphorylated FAK (Y397 and Y576 phosphorylation), αvβ5 integrin, and osteopontin (OPN) matrix protein expression compared to parental ID8 cells. Analyses of paraffin-embedded breast and ovarian tumor sections revealed significantly higher pY397 FAK (p&amp;lt;0.05), β5 integrin (p&amp;lt;0.0001), and OPN expression (p&amp;lt;0.005) in stage II-III compared to stage I tumors. Treatment of ovarian ID8ip, human ovarian HEY carcinoma, or human MDA-MB-231 breast carcinoma cells with FAK-I inhibited cell growth, prevented FAK Y397 phosphorylation, reduced β5 integrin and OPN levels independent of effects on β1 or β3 integrins or changes in matrix proteins such as fibronectin. FAK-I impairs anchorage independent cell growth and orthotopic tumor growth but not growth of cells in plastic-adherent cell culture. Stable knockdown of either FAK or β5 integrin in HEY cells prevented anchorage-independent growth. Re-expression of wildtype or kinase-dead FAK within HEY cells verified the importance of FAK activity in promoting β5 integrin and OPN expression associated with enhanced tumor growth. Ongoing studies are focused on determining the molecular connections of FAK to the regulation of αvβ5 and OPN expression. Together, our studies support a novel role for a signaling loop involving OPN and αvβ5 integrin leading to FAK activation and reinforcement of OPN and β5 integrin expression within the spheroid and tumor micro-environment enhancing breast and ovarian cancer cell survival and tumor progression. Supported by Susan G. Komen for the Cure (KG111237) and NIH grant CA102310 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1254. doi:1538-7445.AM2012-1254

PCL Nanopillars Versus Nanofibers: A Contrast in Progenitor Cell Morphology, Proliferation, and Fate Determination

The role of nanotopography on the long-term response of progenitor cells is explored using polycaprolactone (PCL) nanopillar and nanofiber surfaces seeded with plastic-adherent rat multipotent mesenchymal stromal cells (MSCs). After 4 weeks in culture under normal expansion media conditions, MSCs cultured on nanofibers exhibit better adherence, increased proliferation, and maintain increasingly dense fibroblast-like morphologies. In contrast, MSCs seeded on nanopillar surfaces display lowered adherence, reduced proliferation, and adopt highly elongated cellular morphologies. Immunofluorescent staining of MSCs on PCL nanopillars reveals the presence of two bone marker proteins, osteopontin and osteocalcin, providing evidence for surface induced differentiation into osteoblast-like cells. Unlike the nanopillar topography, MSCs cultured on nanofiber and smooth PCL surfaces did not appear to undergo osteogenesis. Observed differences in cellular response to the PCL nanotopographies offer strategies to direct progenitor cell populations solely based upon submicron surface modifications. This study provides a foundation for future work exploring variations in PCL nanopillar topography with the goal of optimizing adherence and osteogenic response of MSCs.