Extensive Proliferative Potential Research Articles

Exploiting a living biobank to delineate mechanisms underlying disease-specific chromosome instability.

Chromosome instability (CIN) is a cancer hallmark that drives tumour heterogeneity, phenotypic adaptation, drug resistance and poor prognosis. High-grade serous ovarian cancer (HGSOC), one of the most chromosomally unstable tumour types, has a 5-year survival rate of only ~30% - largely due to late diagnosis and rapid development of drug resistance, e.g., via CIN-driven ABCB1 translocations. However, CIN is also a cell cycle vulnerability that can be exploited to specifically target tumour cells, illustrated by the success of PARP inhibitors to target homologous recombination deficiency (HRD). However, a lack of appropriate models with ongoing CIN has been a barrier to fully exploiting disease-specific CIN mechanisms. This barrier is now being overcome with the development of patient-derived cell cultures and organoids. In this review, we describe our progress building a Living Biobank of over 120 patient-derived ovarian cancer models (OCMs), predominantly from HGSOC. OCMs are highly purified tumour fractions with extensive proliferative potential that can be analysed at early passage. OCMs have diverse karyotypes, display intra- and inter-patient heterogeneity and mitotic abnormality rates far higher than established cell lines. OCMs encompass a broad-spectrum of HGSOC hallmarks, including a range of p53 alterations and BRCA1/2 mutations, and display drug resistance mechanisms seen in the clinic, e.g., ABCB1 translocations and BRCA2 reversion. OCMs are amenable to functional analysis, drug-sensitivity profiling, and multi-omics, including single-cell next-generation sequencing, and thus represent a platform for delineating HGSOC-specific CIN mechanisms. In turn, our vision is that this understanding will inform the design of new therapeutic strategies.

Generation of an Immortalized Human Adipose-Derived Mesenchymal Stromal Cell Line Suitable for Wound Healing Therapy.

Adipose-derived mesenchymal stromal cells (ADSC) are a promising source for cellular therapy of chronic wounds. However, the limited life span during in vitro expansion impedes their extensive use in clinical applications and basic research. We hypothesize that by introduction of an ectopic expression of telomerase into ADSC, the cells’ lifespans could be significantly extended. To test this hypothesis, we aimed at engineering an immortalized human ADSC line using a lentiviral transduction with human telomerase (hTERT). ADSC were transduced with a third-generation lentiviral system and a hTERT codifying plasmid (pLV-hTERT-IRES-hygro). A population characterized by increased hTERT expression, extensive proliferative potential and remarkable (potent) multilineage differentiation capacity was selected. The properties for wound healing of this immortalized ADSC line were assessed after 17 passages. Their secretome induced the proliferation and migration of keratinocytes, dermal fibroblasts, and endothelial cells similarly to untransduced ADSC. Moreover, they sustained the complete re-epithelialization of a full thickness wound performed on a skin organotypic model. In summary, the engineered immortalized ADSC maintain the beneficial properties of parent cells and could represent a valuable and suitable tool for wound healing in particular, and for skin regenerative therapy in general.

Roles of Cartilage-Resident Stem/Progenitor Cells in Cartilage Physiology, Development, Repair and Osteoarthritis.

Osteoarthritis (OA) is a degenerative disease that causes irreversible destruction of articular cartilage for which there is no effective treatment at present. Although articular cartilage lacks intrinsic reparative capacity, numerous studies have confirmed the existence of cartilage-resident stem/progenitor cells (CSPCs) in the superficial zone (SFZ) of articular cartilage. CSPCs are characterized by the expression of mesenchymal stromal cell (MSC)-related surface markers, multilineage differentiation ability, colony formation ability, and migration ability in response to injury. In contrast to MSCs and chondrocytes, CSPCs exhibit extensive proliferative and chondrogenic potential with no signs of hypertrophic differentiation, highlighting them as suitable cell sources for cartilage repair. In this review, we focus on the organizational distribution, markers, cytological features and roles of CSPCs in cartilage development, homeostasis and repair, and the application potential of CSPCs in cartilage repair and OA therapies.

Progenitor Cells in Healthy and Osteoarthritic Human Cartilage Have Extensive Culture Expansion Capacity while Retaining Chondrogenic Properties.

Articular cartilage-derived progenitor cells (ACPCs) are a potential new cell source for cartilage repair. This study aims to characterize endogenous ACPCs from healthy and osteoarthritic (OA) cartilage, evaluate their potential for cartilage regeneration, and compare this to cartilage formation by chondrocytes. ACPCs were isolated from full-thickness healthy and OA human cartilage and separated from the total cell population by clonal growth after differential adhesion to fibronectin. ACPCs were characterized by growth kinetics, multilineage differentiation, and surface marker expression. Chondrogenic redifferentiation of ACPCs was compared with chondrocytes in pellet cultures. Pellets were assessed for cartilage-like matrix production by (immuno)histochemistry, quantitative analyses for glycosaminoglycans and DNA content, and expression of chondrogenic and hypertrophic genes. Healthy and OA ACPCs were successfully differentiated toward the adipogenic and chondrogenic lineage, but failed to produce calcified matrix when exposed to osteogenic induction media. Both ACPC populations met the criteria for cell surface marker expression of mesenchymal stromal cells (MSCs). Healthy ACPCs cultured in pellets deposited extracellular matrix containing proteoglycans and type II collagen, devoid of type I collagen. Gene expression of hypertrophic marker type X collagen was lower in healthy ACPC pellets compared with OA pellets. This study provides further insight into the ACPC population in healthy and OA human articular cartilage. ACPCs show similarities to MSCs, yet do not produce calcified matrix under well-established osteogenic culture conditions. Due to extensive proliferative potential and chondrogenic capacity, ACPCs show potential for cartilage regeneration and possibly for clinical application, as a promising alternative to MSCs or chondrocytes.

Multiple Roles for Cholinergic Signaling from the Perspective of Stem Cell Function.

Stem cells have extensive proliferative potential and the ability to differentiate into one or more mature cell types. The mechanisms by which stem cells accomplish self-renewal provide fundamental insight into the origin and design of multicellular organisms. These pathways allow the repair of damage and extend organismal life beyond that of component cells, and they probably preceded the evolution of complex metazoans. Understanding the true nature of stem cells can only come from discovering how they are regulated. The concept that stem cells are controlled by particular microenvironments, also known as niches, has been widely accepted. Technical advances now allow characterization of the zones that maintain and control stem cell activity in several organs, including the brain, skin, and gut. Cholinergic neurons release acetylcholine (ACh) that mediates chemical transmission via ACh receptors such as nicotinic and muscarinic receptors. Although the cholinergic system is composed of organized nerve cells, the system is also involved in mammalian non-neuronal cells, including stem cells, embryonic stem cells, epithelial cells, and endothelial cells. Thus, cholinergic signaling plays a pivotal role in controlling their behaviors. Studies regarding this signal are beginning to unify our understanding of stem cell regulation at the cellular and molecular levels, and they are expected to advance efforts to control stem cells therapeutically. The present article reviews recent findings about cholinergic signaling that is essential to control stem cell function in a cholinergic niche.

Potential Use of Human Periapical Cyst-Mesenchymal Stem Cells (hPCy-MSCs) as a Novel Stem Cell Source for Regenerative Medicine Applications.

Mesenchymal stem cells (MSCs) are attracting growing interest by the scientific community due to their huge regenerative potential. Thus, the plasticity of MSCs strongly suggests the utilization of these cells for regenerative medicine applications. The main issue about the clinical use of MSCs is related to the complex way to obtain them from healthy tissues; this topic has encouraged scientists to search for novel and more advantageous sources of these cells in easily accessible tissues. The oral cavity hosts several cell populations expressing mesenchymal stem cell like-features, furthermore, the access to oral and dental tissues is simple and isolation of cells is very efficient. Thus, oral-derived stem cells are highly attractive for clinical purposes. In this context, human periapical cyst mesenchymal stem cells (hPCy-MSCs) exhibit characteristics similar to other dental-derived MSCs, including their extensive proliferative potential, cell surface marker profile and the ability to differentiate into various cell types such as osteoblasts, adipocytes and neurons. Importantly, hPCy-MSCs are easily collected from the surgically removed periapical cysts; this reusing of biological waste guarantees a smart source of stem cells without any impact on the surrounding healthy tissues. In this review, we report the most interesting research topics related to hPCy-MSCs with a newsworthy discussion about the future insights. This newly discovered cell population exhibits interesting and valuable potentialities that could be of high impact in the future regenerative medicine applications.

Leukemia Inhibitory Factor Promotes Aggressiveness of Chordoma.

Chordomas are rare tumors of the spine and skull base that are locally destructive and resistant to chemotherapy and radiation therapy, with a poor prognosis and limited therapeutic options. Chordoma patients have a long life expectancy with high mortality from the disease. Cancer stem cells, which are known to exist in chordomas, have extensive proliferative and self-renewal potential and are responsible for maintaining tumor heterogeneity along with chemotherapy and radiotherapy resistance. Leukemia inhibitory factor (LIF) has multiple functions in stem cell biology, the immune response, and cancer, and is potentially a key molecule that allows cancer stem cells to self-renew. The purpose of this study was to determine whether LIF increases the aggressive traits of chordoma cells and leads to a poor prognosis in patients. Chordoma cell lines were treated with LIF, and functional tests were done. Twenty skull base chordoma samples were checked for levels of LIF and a correlation with clinicopathological features. The whole transcriptome microarray was used to observe changes in gene expression. We observed increased migration, invasion, tumorosphere formation, colony formation, epithelial–mesenchymal transition, and chemoresistance accompanied by a dramatic elevation in inflammatory gene networks and pathways in chordomas. The expression of LIF was associated with tumor size and a poorer overall survival. Microarray and quantitative real-time polymerase chain reaction assessments suggest that LIF can facilitate tumor-promoting inflammation. Results indicate that LIF plays a role in maintaining cancer stem cells in chordomas.

Проліферативна активність мезенхімальних стовбурових клітин щура за впливу культурального середовища

Mesenchymal stem cells (MSCs) are bone marrow populating cells, different from hematopoietic stem cells, which possess an extensive proliferative potential and ability to differentiate into various cell types, including: osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes and neurons. MSCs play a key role in the maintenance of bone marrow homeostasis and regulate the maturation of both hematopoietic and non-hematopoietic cells. With published data we are known about the effectiveness of using stem cells in degenerative processes in the organs. Therefore, we can argue about expediency of use cell technologies for the treatment degenerative pathology’s. MSCs seem to represent a future powerful tool in regenerative medicine, therefore their research are particularly important in clinical research. Goal of the work . Investigate the proliferative activity of MSCs depending on the type used by culture media and to establish the ability of mesenchymal stem cells to differentiate into kardiomyogenic direction under the influence of exogenous factors. Materials and methods . In studies using MSCs, obtained from 12-day not linear rats. We compared the impact of three different culture media on proliferative activity derived cells. We investigated the ability of MSCs to differentiate into kardiomyogenic direction under the influence of 5 - azacytidine. Results of research and discussion. Was investigated influence of three different composition culture medium on proliferative activity of MSCs. Was defined the better culture medium. Proved that mesenchymal stem cells are able of aimed differentiation at kardiomyogenic under the direction of transcription factor 5 azacytidine. The data confirmed the morphological assessment of culture and research at the molecular level. Conclusions. Cultivation the rat mesenchymal stem cells in DMEM F12 - D8437 and ECT - 20% with the addition of antibiotic-antimycotics 10 ml / cm3 provide the highest proliferative activity. Mesenchymal stem cells are capable of aimed cardiomyogenic differentiation under the direction of transcription factor 5 azacytidine.

Mesenchymal Fetal Stem Cells (FMSC) from Amniotic Fluid (AF): Expansion and Phenotypic Characterization

Introduction. Mesenchymal stem cells (MSCs) are multipotent precursors able to differentiate into bone, cartilage and fat. In humans, the MCS were isolated from bone marrow, cord blood, peripheral blood, adipose tissue and the amniotic fluid (AF) in the second trimester of pregnancy (FA-MSC). The FA-MCS have immunosuppressive properties, extensive proliferative potential and self-renewal, and express the fetal stem transcription factor Oct4. The purpose of this work was to isolate and characterize cells isolated from human FA as alternative source of multipotent MSC for regenerative medicine and transplantation.Materials and Methods. Expansion. The FA cells (extracted from the first three milliliters of FA collected during diagnostic amniocentesis and can not be used for prenatal diagnosis), were cultured in serum free medium specific to the MSC (STEM CELL). Phenotypic analysis. On the FA-expanded MSC the presence of mesenchymal markers CD106, CD146 and CD105, and non-mesenchymal markers CD45 and CD34 have been evaluated by flow cytometry. The fetal origin of the FA-expanded MSC was verified by QF-PCR comparing FA-MSC DNA and maternal mononuclear DNA. Stemness analysis. The stemness, potentialbefore and after expansion, was evaluated by expression analysis(RT-PCR) of Oct4 and Nanog gene. cDNA from adult peripheral blood and cDNA from chorionic villus at 10 weeks of gestation were used as control.The specific primers for the analysis of Oct4 and Nanog genes were designed in the laboratory on the basis of the gene maps (GENE ATLAS).Results and Conclusions. After 8 days of culture FA cells have produced CFU-F colonies. At 15 days of culture started the proliferation of fibroblasts, and at 28 days of culture the fibroblasts have reached 80% confluency.Phenotypicanalysis. The flow cytometry assay allowed cultures immunophenotypic characterization. The flow cytometry analysis of the FA-MSC showed the presence of mesenchymal markers CD146, and CD105 and the absence of hematopoietic/endothelial markers CD34 and CD45. The QF-PCR confirmed the fetal origin of the expanded FA-MSC. Using medium serum-free adherence method, it has been developed a reproducible protocol for the isolation and expansion of AF-MSC. Phenotypic analysis results, confirmed the nature of the mesenchymal cells expanded AF. The presence of expression of Nanog (a homeobox playing a key role in embryonic development and in maintaining the pluripotency of stem cells), and Oct4 (a transcription factor involved in stem cells pluripotency and self-renewal maintenance) confirmed the stamness persistence of AF-MSCs after expansion.The expanded AF-MSCs could be used in different therapeutic fields: bone marrow transplantation in adults, transplant "in utero" and gene therapy for congenital blood diseases, treatment of chronic inflammatory bowel disease. The use of AF-MSCs also can open new perspectives for research in regenerative medicine because of their possible de-differentiation by "ex vivo" procedures. DisclosuresNo relevant conflicts of interest to declare.

Molecular Identity of Human Outer Radial Glia during Cortical Development

Radial glia, the neural stem cells of the neocortex, are located in two niches: the ventricular zone and outer subventricular zone. Although outer subventricular zone radial glia may generate the majority of human cortical neurons, their molecular features remain elusive. By analyzing gene expression across single cells, we find that outer radial glia preferentially express genes related to extracellular matrix formation, migration, and stemness, including TNC, PTPRZ1, FAM107A, HOPX, and LIFR. Using dynamic imaging, immunostaining, and clonal analysis, we relate these molecular features to distinctive behaviors of outer radial glia, demonstrate the necessity of STAT3 signaling for their cell cycle progression, and establish their extensive proliferative potential. These results suggest that outer radial glia directly support the subventricular niche through local production of growth factors, potentiation of growth factor signals by extracellular matrix proteins, and activation of self-renewal pathways, thereby enabling the developmental and evolutionary expansion of the human neocortex.

P17.82 * ITGA6 IS INVOLVED IN RADIORESISTANCE OF GLIOBLASTOMA STEM CELLS

BACKGROUND: In the last decade, the cancer stem cell hypothesis has gained progressively agreement towards researchers. Cancer stem cells (CSCs) or tumour initiating cells (TICs) are considered to be responsible for tumor initiation, propagation and therapeutic resistance. In glioblastoma multiforme (GBM), the most frequent and malignant primary brain tumour in adults, TICs are related with chemo- and radioresistance. Integrins are cell surface receptors involved in the crosstalk of tumoral cells with their extracellular matrix (ECM). In particular integrin α6 (ITGA6) is known to be extremely important in embryonic, hematopoietic and neural stem cells. In brain it is involved in maintaining adhesion to the ventricular zone and ensures proper cell division. Besides, recently it has been proposed as a functional marker of TICs. Given the raising evidence of the ECM and perivascular niche role in the maintenance and guidance of TICs, we focused on ITGA6 to determine whether ITGA6 expression is associated with radiation-resistance of TICs in glioblastoma patients. METHODS: Four different GBM cultures obtained from post-surgical specimens were growth in two different growth conditions: a differentiating FBS-containing media and an FBS-free media supplemented with bFGF and EGF which is considered to be selective for TICs. Self-renewal capability and proliferative potential has been evaluated. Tumour initiating capability has been assessed through injection of low number of cells in immunocompromised mice. ITGA6 expression has been analyzed at RNA and protein levels. TICs have been sorted in order to get a population enriched for ITGA6high cells and dose-response to ionizing radiation (IR) has been evaluated and compared to ITGA6low cells by clonogenic assays. RESULTS: TICs have shown noticeable self-renewal capability, extensive proliferative potential, and a remarkable radioresistance compared to the same primary cultures maintained under differentiated conditions. In TICs, ITGA6 RNA and protein levels were statistically significant higher than in differentiated glioblastoma cells. In addition, this expression is decreasing consistently when TICs were cultured under differentiating conditions. Response to radiotherapy assessed by the clonogenic surviving fraction showed that ITGA6high cells are remarkably more radioresistant than ITGA6low cells (p < 0.001). In addition, ITGA6high cells showed a higher α/β ratio than ITGA6low cells (30 Gy vs. 6 Gy, respectively). In conclusion, these results suggest that ITGA6 is involved in the radioresistant phenotype of glioblastoma stem cells.

Experimental research on evaluating differentiation ability of adipose- derived mesenchymal stem cells into hepatocyte- like cells in vitro

Mesenchymal stem cells (MSCs) have many properties that suggest considerable potential utility in cellular therapy of many disorders. It was proved that MSCs have characteristics including extensive proliferative potential and the ability to undergo multilineage differentiation. In previous publications, it was demonstrated that hADSCs (human adipose-derived stem cells) could alleviate liver fibrosis in CCl4 induced - liver fibrosis mouse model by differentiating into hepatocytes replacing damaged hepatocytes or inducing proliferation of endogenous cells in liver for functioning. In this experiment, hADSCs isolated from Vietnamese women’s adipose tissue were used to evaluate ability of differentiation into hepatocyte-like cells in vitro. After differentiation, properties of hADSCs-derived hepatocyte-like cells were determined by changes of morphology, hepatocyte-specific gene expression (α-fetoprotein, albumin, cytokeratin 18, cytokeratin 19) and functionally tested by cytoplasmic accumulation of glycogen. The results showed that hADSCs differentiated into hepatocyte-like cells in vitro successfully by changes of morphology, increase in gene expression and ability of cytoplasmic glycogen accumulation. This study is the fundamental experiment for further experiments on using transplantation of hADSCs as treatment methods of liver disorders.

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.

Endothelial Progenitor Cells in HIV-Positive Patients

Endothelial Progenitor Cells in HIV-Positive Patients

The germinative epithelium of sheep vibrissae and wool follicles has extensive proliferative potential but is dependent on the dermal papilla

Aim:To investigate the growth potential of keratinocytes derived from the germinative epithelium (GE) of ovine hair follicles. Stem cells from the outer root sheath (ORS) of hair follicles migrate to the GE in the lower follicle where they proliferate and differentiate to form the hair fiber. It has been suggested that the GE comprises transit-amplifying cells and that the duration of anagen is determined by their limited proliferative potential. However, we show here that keratinocytes derived from the GE of ovine follicles grow extensively in vitro, arguing against this hypothesis.Materials and Methods:Primary cultures of keratinocytes were initiated from microdissected GE tissue from ovine vibrissae and wool follicles. Clonal lines of keratinocytes were derived by limiting dilution. Their growth potential was determined by exhaustive serial passaging. Expression of differentiation markers was evaluated by real-time polymerase chain reaction.Results:Initiation of these cultures required that interaction between the GE and dermal papilla was maintained. However, the keratinocytes could subsequently be cloned and were grown as pure cell populations for 26-52 cell doublings. This proliferative potential is several orders of magnitude greater than required to maintain a single anagen phase. The keratinocytes were indistinguishable from ORS keratinocytes from the same follicles, expressing K14 while undifferentiated, and upregulating the epidermal and inner root sheath markers, loricrin and KRT27 on differentiation. Thus, these cells initially depend on papilla-derived signals to grow, but can revert to an ORS-like phenotype in vitro. Their extensive proliferative capacity shows that the GE is not an exclusively transit-amplifying cell population.

Neurorestoration Induced by Mesenchymal Stem Cells: Potential Therapeutic Mechanisms for Clinical Trials

Stem cells are emerging as therapeutic candidates in a variety of diseases because of their multipotent capacities. Among these, mesenchymal stem cells (MSCs) derived from bone marrow, umbilical cord blood or adipose tissue, comprise a population of cells that exhibit extensive proliferative potential and retain the ability to differentiate into multiple tissue-specific lineage cells including osteoblasts, chondrocytes, and adipocytes. MSCs have also been shown to enhance neurological recovery, although the therapeutic effects seem to be derived from an indirect paracrine effect rather than direct cell replacement. MSCs secrete neurotrophic factors, promote endogenous neurogenesis and angiogenesis, encourage synaptic connection and remyelination of damaged axons, decrease apoptosis, and regulate inflammation primarily through paracrine actions. Accordingly, MSCs may prevail as a promising cell source for cell-based therapy in neurological diseases.

Human adipose‐derived stem cells (hASCs) proliferate and differentiate in osteoblast‐like cells on trabecular titanium scaffolds

The use of stem cells in regenerative medicine is an appealing area of research that has received a great deal of interest in recent years. The population called human adipose tissue-derived stem cells (hASCs) share many of the characteristic of its counterpart of marrow including extensive proliferative potential and the ability to undergo multilineage differentiation along classical mesenchymal lineages: adipogenesis, chondrogenesis, osteogenesis, and myogenesis. The aim of this study was to evaluate with biochemical and morphological methods the adhesion and differentiation of hASCs grown on trabecular titanium scaffolds. The hASCs isolated from subcutaneous adipose tissue after digestion with collagenase were seeded on monolayer and on trabecular titanium scaffolds and incubated at 37 degrees C in 5% CO(2) with osteogenic medium or control medium.The results showed that hASCs were able to adhere to titanium scaffolds, to proliferate, to acquire an osteoblastic-like phenotype, and to produce a calcified extracellular matrix with protein, such as, decorin, fibronectin, osteocalcin, osteonectin, osteopontin, and type I collagen. These data suggest that this kind of scaffold/cells construct is effective to regenerate damaged tissue and to restore the function of bone tissue.

Highly efficient lentiviral transduction of phenotypically and genotypically characterized endothelial progenitor cells from adult peripheral blood

Postnatal vasculogenesis has been implicated as an important mechanism for neovascularization via bone marrow-derived endothelial progenitor cells (EPCs) circulating in peripheral blood. In preparation of the utilization of EPCs in clinical protocols, we have generated blood-derived EPCs according to two established protocols by culturing either nonadherent mononuclear cells on fibronectin or adherent mononuclear cells on collagen. To explore the feasibility of these EPCs for their potential clinical use as target cells for genetic transduction to enhance their thromboresistance, newly designed retroviral and lentiviral gene ontology expression vectors were tested. Whereas cell clusters derived from the nonadherent cells demonstrated an only limited proliferative potential, cell colonies derived from collagen-adherent cells expanded more than a million-fold. Characterization of the exponentially growing cells by surface antigen and gene expression profiling revealed a consistently strong expression of characteristic endothelial markers, whereas expression of leukocyte markers was gradually lost. Using a single-step transduction protocol, we were able to achieve gene transfer efficiency of up to 99%. Our results suggest that the generated blood-derived EPC population might be attractive target cells for tissue engineering and gene therapy protocols due to their well defined phenotype, extensive proliferative potential, and efficient genetic transducibility, three important qualities that need to be defined prior to any clinical use.

Abstract 5314: Immunoregulatory properties of CD44+ cancer stem-like cells in squamous cell carcinoma of the head and neck

Abstract There is increasing evidence in certain malignancies that tumor cells are organized into a hierarchy originating from a small population of cancer stem cells (CSCs) that possesses extensive proliferative and self-renewal potential, and that these CSCs are responsible for maintaining the tumors. In squamous cell carcinoma of the head and neck (SCCHN), CD44+ cells with significant tumorigenic potential have been identified as CSCs; however, the immunological properties of such CSCs have not yet been elucidated. In this study, we investigated the immunological functions of CD44+ cells from a SCCHN cell line. Analysis of flow cytometry demonstrated that the expression of HLA-A2 molecules was deficient in the parental cell line, whereas the CD44+ cell population restored HLA-A2 expression, although at very low levels. Moreover, CD44+ cells exhibited weak HLA class II expression on the cell surface. Interestingly, down-regulation of TAP2 was found in CD44+ cells. The CD44+ cell population produced significantly higher levels of IL-8, G-CSF, and TGF-β than the CD44- cell population. Moreover, CD44+ cells have been shown to not only more strongly inhibit the proliferation of T cells activated with anti-CD3/CD28 mAb, but also to more efficiently induce CD4+CD25+FOXP3+ Treg cells and myeloid-derived suppressor cells as compared with CD44- cells. Additionally, CD44+ cells also suppressed Th1 responses (IFN-γ production by PBMCs) and enhanced regulatory T cell responses (IL-10 production by PBMCs). Thus, CSCs may have higher immunosuppressive ability promoting evasion from immunosurveillance; therefore, the development of novel immunotherapeutic strategies to efficiently overcome CSCs-driven immune suppression is urgently needed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5314.

Adipose Tissue–derived Mesenchymal Stem Cells Expressing Prodrug-converting Enzyme Inhibit Human Prostate Tumor Growth

The ability of human adipose tissue-derived mesenchymal stem cells (AT-MSCs), engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT), to convert the relatively nontoxic 5-fluorocytosine (5-FC) into the highly toxic antitumor 5-fluorouracil (5-FU) together with their ability to track and engraft into tumors and micrometastases makes these cells an attractive tool to activate prodrugs directly within the tumor mass. In this study, we tested the feasibility and efficacy of these therapeutic cells to function as cellular vehicles of prodrug-activating enzymes in prostate cancer (PC) therapy. In in vitro migration experiments we have shown that therapeutic AT-MSCs migrated to all the prostate cell lines tested. In a pilot preclinical study, we observed that coinjections of human bone metastatic PC cells along with the transduced AT-MSCs into nude mice treated with 5-FC induced a complete tumor regression in a dose dependent manner or did not even allow the establishment of the tumor. More importantly, we also demonstrated that the therapeutic cells were effective in significantly inhibiting PC tumor growth after intravenous administration that is a key requisite for any clinical application of gene-directed enzyme prodrug therapies.