Mesenchymal stem cell secretome and regenerative therapy after cancer

Cancer treatment generally relies on tumor ablative techniques that can lead to major functional or disfiguring defects. These post-therapy impairments require the development of safe regenerative therapy strategies during cancer remission. Many current tissue repair approaches exploit paracrine (immunomodulatory, pro-angiogenic, anti-apoptotic and pro-survival effects) or restoring (functional or structural tissue repair) properties of mesenchymal stem/stromal cells (MSC). Yet, a major concern in the application of regenerative therapies during cancer remission remains the possible triggering of cancer recurrence. Tumor relapse implies the persistence of rare subsets of tumor-initiating cancer cells which can escape anti-cancer therapies and lie dormant in specific niches awaiting reactivation via unknown stimuli. Many of the components required for successful regenerative therapy (revascularization, immunosuppression, cellular homing, tissue growth promotion) are also critical for tumor progression and metastasis. While bi-directional crosstalk between tumorigenic cells (especially aggressive cancer cell lines) and MSC (including tumor stroma-resident populations) has been demonstrated in a variety of cancers, the effects of local or systemic MSC delivery for regenerative purposes on persisting cancer cells during remission remain controversial. Both pro- and anti-tumorigenic effects of MSC have been reported in the literature. Our own data using breast cancer clinical isolates have suggested that dormant-like tumor-initiating cells do not respond to MSC signals, unlike actively dividing cancer cells which benefited from the presence of supportive MSC. The secretome of MSC isolated from various tissues may partially diverge, but it includes a core of cytokines (i.e. CCL2, CCL5, IL-6, TGFβ, VEGF), which have been implicated in tumor growth and/or metastasis. This article reviews published models for studying interactions between MSC and cancer cells with a focus on the impact of MSC secretome on cancer cell activity, and discusses the implications for regenerative therapy after cancer.     

Strategies to enhance immunomodulatory properties and reduce heterogeneity in mesenchymal stromal cells during ex vivo expansion

Therapies using mesenchymal stromal cells (MSCs) to treat immune and inflammatory conditions are now at an exciting stage of development, with many MSC-based products progressing to phase II and III clinical trials. However, a major bottleneck in the clinical translation of allogeneic MSC therapies is the variable immunomodulatory properties of MSC products due to differences in their tissue source, donor heterogeneity and processes involved in manufacturing and banking. This variable functionality of MSC products likely contributes to the substantial inconsistency observed in the clinical outcomes of phase III trials of MSC therapies; several trials have failed to reach the primary efficacy endpoint. In this review, we discuss various strategies to consistently maintain or enhance the immunomodulatory potency of MSCs during ex vivo expansion, which will enable the manufacture of allogeneic MSC banks that have high potency and low variability. Biophysical and biochemical priming strategies, the use of culture additives such as heparan sulfates, and genetic modification can substantially enhance the immunomodulatory properties of MSCs during in vitro expansion. Furthermore, robust donor screening, the use of biomarkers to select for potent MSC subpopulations, and rigorous quality testing to improve the release criteria for MSC banks have the potential to reduce batch-to-batch heterogeneity and enhance the clinical efficacy of the final MSC product. Machine learning approaches to develop predictive models of individual patient response can enable personalized therapies and potentially establish correlations between in vitro potency measurements and clinical outcomes in human trials.     

Enhancing survival,engraftment, and osteogenic potential of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are promising candidates for bone regeneration therapies due to their plasticity and easiness of sourcing. MSC-based treatments are generally considered a safe procedure, however, the long-term results obtained up to now are far from satisfactory. The main causes of these therapeutic limitations are inefficient homing, engraftment, and osteogenic differentiation. Many studies have proposed modifications to improve MSC engraftment and osteogenic differentiation of the transplanted cells. Several strategies are aimed to improve cell resistance to the hostile microenvironment found in the recipient tissue and increase cell survival after transplantation. These strategies could range from a simple modification of the culture conditions, known as cell-preconditioning, to the genetic modification of the cells to avoid cellular senescence. Many efforts have also been done in order to enhance the osteogenic potential of the transplanted cells and induce bone formation, mainly by the use of bioactive or biomimetic scaffolds, although alternative approaches will also be discussed. This review aims to summarize several of the most recent approaches, providing an up-to-date view of the main developments in MSC-based regenerative techniques.     

IL-12 enhances efficacy and shortens enrichment time in cytokine-induced killer cell immunotherapy

Cytokine-induced killer (CIK) cells are T cell derived ex vivo expanded cells with both NK and T cell properties. They exhibit potent anti-tumor efficacy against various malignancies in preclinical models and have proven safe and effective in clinical studies. We combined CIK cell adoptive immunotherapy with IL-12 cytokine immunotherapy in an immunocompetent preclinical breast cancer model. Combining CIK cells with IL-12 increased anti-tumor efficacy in vivo compared to either therapy alone. Combination led to full tumor remission and long-term protection in 75% of animals. IL-12 treatment sharply increased the anti-tumor efficacy of short-term cultured CIK cells that exhibited no therapeutic effect alone. Bioluminescence imaging based in vitro cytotoxicity and in vivo homing assays revealed that short-term cultured CIK cells exhibit full cytotoxicity in vitro, but display different tumor homing properties than fully expanded CIK cells in vivo. Our data suggest that short-term cultured CIK cells can be “educated” in vivo, producing fully expanded CIK cells upon IL-12 administration with anti-tumor efficacy in a mouse model. Our findings demonstrate the potential to improve current CIK cell-based immunotherapy by increasing efficacy and shortening ex vivo expansion time. This holds promise for a highly efficacious cancer therapy utilizing synergistic effects of cytokine and cellular immunotherapy.     

Can the outcomes of mesenchymal stem cell‐based therapy for myocardial infarction be improved? Providing weapons and armour to cells

Use of mesenchymal stem cell (MSC) transplantation after myocardial infarction (MI) has been found to have infarct‐limiting effects in numerous experimental and clinical studies. However, recent meta‐analyses of randomized clinical trials on MSC‐based MI therapy have highlighted the need for improving its efficacy. There are two principal approaches for increasing therapeutic effect of MSCs: (i) preventing massive MSC death in ischaemic tissue and (ii) increasing production of cardioreparative growth factors and cytokines with transplanted MSCs. In this review, we aim to integrate our current understanding of genetic approaches that are used for modification of MSCs to enable their improved survival, engraftment, integration, proliferation and differentiation in the ischaemic heart. Genetic modification of MSCs resulting in increased secretion of paracrine factors has also been discussed. In addition, data on MSC preconditioning with physical, chemical and pharmacological factors prior to transplantation are summarized. MSC seeding on three‐dimensional polymeric scaffolds facilitates formation of both intercellular connections and contacts between cells and the extracellular matrix, thereby enhancing cell viability and function. Use of genetic and non‐genetic approaches to modify MSC function holds great promise for regenerative therapy of myocardial ischaemic injury.     

The (in) auspicious role of mesenchymal stromal cells in cancer: be it friend or foe

Recent progress in the research of mesenchymal stromal cells/multipotent stromal cells (MSC) has revealed numerous beneficial innate characteristics, suggesting potential value in an array of cellular therapies. MSC are easily isolated from bone marrow (BM), fat and other tissues, and are readily propagated in vitro. Transplanted/injected MSC have been shown to migrate to a variety of organs and tissues; however, sites of inflammation and pathology elicit enhanced MSC homing for tissue remodeling and repair. Tumors utilize many of the same inflammatory mediators uncovered in wound healing and likewise provide a site for preferential MSC homing. Although incorporation into the tumor microenvironment is apparent, the role of recruited MSC in the tumor microenvironment remains unclear. Some published studies have shown enhancement of tumor growth and development, perhaps through immunomodulatory and pro-angiogenic properties, while others have shown no apparent effect or have demonstrated inhibition of tumor growth and extended survival. This controversy remains at the forefront as clinical applications of MSC commence in anti-tumor therapies as well as as adjuncts to stem cell transplantation and in ameliorating graft-versus-host disease. Careful analysis of past studies and thoughtful design of future experiments will help to resolve the discrepancies in the field and lead to clinical utility of MSC in disease treatment. This review highlights the current theories of the role of MSC in tumors and explores current controversies.     

Homing and migration of mesenchymal stromal cells: How to improve the efficacy of cell therapy?

Mesenchymal stromal cells(MSCs) are currently being investigated for use in a wide variety of clinical applications. For most of these applications, systemic delivery of the cells is preferred. However, this requires the homing and migration of MSCs to a target tissue. Although MSC hominghas been described, this process does not appear to be highly efficacious because only a few cells reach the target tissue and remain there after systemic administration. This has been ascribed to low expression levels of homing molecules, the loss of expression of such molecules during expansion, and the heterogeneity of MSCs in cultures and MSC culture protocols. To overcome these limitations, different methods to improve the homing capacity of MSCs have been examined. Here, we review the current understanding of MSC homing, with a particular focus on homing to bone marrow. In addition, we summarize the strategies that have been developed to improve this process. A better understanding of MSC biology, MSC migration and homing mechanisms will allow us to prepare MSCs with optimal homing capacities. The efficacy of therapeutic applications is dependent on efficient delivery of the cells and can, therefore, only benefit from better insights into the homing mechanisms.     

Expression and function of T cell homing molecules in Hodgkin’s lymphoma

Circulating T lymphocytes enter a tissue if they express appropriate chemokine receptors and adhesion molecules to engage ligands presented at this site. To aid rational development of T cell-based therapies for Hodgkin's lymphoma (HL), we have assessed the expression and function of homing receptors on tumour-infiltrating T cells in HL and compared them with T cells from unaffected lymph nodes and colorectal cancer tissue. Chemokine receptors CXCR3, CXCR4 and CCR7 were expressed on a large proportion of T cells within HL tissue and mediated chemotaxis to purified chemokine. The corresponding ligands (CXCL10, CXCL12, CCL21) were expressed on the malignant cells and/or vascular endothelium. Adhesion molecules including CD62L were widely expressed on HL-derived T cells and their corresponding ligands were detected on vessels within the tumour. This homing phenotype was distinct from T cells isolated from colorectal cancer, but matched closely the phenotype of T cells from unaffected lymph nodes. Thus, T cell recruitment to HL resembles entry of na?ve/central memory T cells into normal lymph nodes. This has important implications for current approaches to treat HL using T cells activated and expanded in vitro that lack CCR7 and CD62L expression.     

Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system

The need for efficient and reliable technologies for clinical‐scale expansion of mesenchymal stromal cells (MSC) has led to the use of disposable bioreactors and culture systems. Here, we evaluate the expansion of cord blood‐derived MSC in a disposable fixed bed culture system. Starting from an initial cell density of 6.0 × 10⁷ cells, after 7 days of culture, it was possible to produce of 4.2(±0.8) × 10⁸ cells, which represents a fold increase of 7.0 (±1.4). After enzymatic retrieval from Fibra‐Cell disks, the cells were able to maintain their potential for differentiation into adipocytes and osteocytes and were positive for many markers common to MSC (CD73, CD90, and CD105). The results obtained in this study demonstrate that MSC can be efficiently expanded in the culture system. This novel approach presents several advantages over the current expansion systems, based on culture flasks or microcarrier‐based spinner flasks and represents a key element for MSC cellular therapy according to GMP compliant clinical‐scale production system. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 568–572, 2013     

Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute

There is great interest in mesenchymal stromal cells (MSCs) for cell-therapy and tissue engineering approaches. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns when used in clinical grade preparations. The aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL), already shown to promote MSC growth, are endowed with biological properties appropriate for cell-therapy approaches. We confirm previously published data showing that MSCs expanded in either FCS or PL display comparable morphology, phenotype, and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. We further extended these data by investigating the immune-regulatory effect of MSCs on the alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to: (i) decrease alloantigen-induced cytotoxic activity; (ii) favor differentiation of CD4+ T-cell subsets expressing a Treg phenotype; (iii) increase early secretion of IL-10 in MLC supernatant, as well as induce a striking augmentation of IL-6 production. As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and reducing early IFNgamma-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by molecular karyotyping and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the immunological functional plasticity of MSCs and suggest that MSCs-PL can be used as an alternative to MSCs-FCS, although these latter cells might be more suitable for preventing/treating alloreactivity-related immune complications.     

GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion

Background aimsBone marrow (BM) mesenchymal stromal/stem cells (MSC) are therapeutic tools in regenerative medicine and oncology. MSC isolation is often performed starting from a separation step based on research-grade 1.077 g/mL density gradient media (DGM). However, MSC clinical application should require the introduction of good manufacturing practice (GMP) reagents. We took advantage of two novel GMP DGM with densities of 1.077 and 1.073 g/mL (Ficoll-Paque? PREMIUM and Ficoll-Paque PREMIUM 1.073, respectively) to test whether these reagents could isolate MSC efficiently while simultaneously comparing their performance.MethodsBM samples were processed using either 1.077 or 1.073 g/mL GMP DGM. BM mononucleated cell (MNC) fractions were analyzed for viability, immunophenotype, clonogenic potential, ex vivo expansion and differentiation potential.ResultsNo differences were noticed in cell recovery and viability between the groups. Fluorescence-activated cell-sorting (FACS) analyzes on freshly isolated cells indicated that the 1.073 g/mL GMP DGM more efficiently depleted the CD45⁺ fraction in comparison with 1.077 GMP DGM. Moreover, in the 1.073 group, fibroblastic colony-forming units (CFU-F) were 1.5 times higher and the final MSC yield 1.8 times increased after four passages. Both reagents isolated MSC with the expected phenotype; however, 1.073-isolated MSC showed a higher expression of CD90, CD146 and GD2. Additionally, MSC from both groups were capable of fully differentiating into bone, adipose cells and cartilage.ConclusionsBoth GMP DGM enriched MSC from BM samples, suggesting that these reagents would be suitable for clinical-grade expansions. In addition, the density of 1.073 g/mL provides a significant advantage over 1.077 g/mL GMP DGM, impacting the quantity of MSC obtained and reducing the ex vivo expansion time for optimized cell-based clinical applications.     

Cytosine deaminase expressing human mesenchymal stem cells mediated tumour regression in melanoma bearing mice

Background

Previously, we validated capability of human adipose tissue‐derived mesenchymal stem cells (AT‐MSC) to serve as cellular vehicles for gene‐directed enzyme prodrug molecular chemotherapy. Yeast fusion cytosine deaminase : uracil phosphoribosyltransferase expressing AT‐MSC (CD_y‐AT‐MSC) combined with systemic 5‐fluorocytosine (5FC) significantly inhibited growth of human colon cancer xenografts. We aimed to determine the cytotoxic efficiency to other tumour cells both in vitro and in vivo.

Methods

CD_y‐AT‐MSC/5FC‐mediated proliferation inhibition against a panel of human tumour cells lines was evaluated in direct and indirect cocultures in vitro. Antitumour effect was tested on immunodeficient mouse model in vivo.

Results

Although culture expansion of CD_y‐AT‐MSC sensitized these cells to 5FC mediated suicide effect, expanded CD_y‐AT‐MSC/5FC still exhibited strong bystander cytotoxic effect towards human melanoma, glioblastoma, colon, breast and bladder carcinoma in vitro. Most efficient inhibition (91%) was observed in melanoma A375 cell line when directly cocultured with 2% of therapeutic cells CDy‐AT‐MSC/5FC. The therapeutic paradigm of the CDy‐AT‐MSC/5FC system was further evaluated on melanoma A375 xenografts on nude mice in vivo. Complete regression in 89% of tumours was achieved when 20% CD_y‐AT‐MSC/5FC were co‐injected along with tumour cells. More importantly, systemic CD_y‐AT‐MSC administration resulted in therapeutic cell homing into subcutaneous melanoma and mediated tumour growth inhibition.

Conclusions

CD_y‐AT‐MSC capability of targeting subcutaneous melanoma offers a possibility to selectively produce cytotoxic agent in situ. Our data further demonstrate beneficial biological properties of AT‐MSC as a cellular vehicle for enzyme/prodrug therapy approach to molecular chemotherapy. Copyright © 2008 John Wiley & Sons, Ltd.     

Pancreas-derived mesenchymal stromal cells share immune response-modulating and angiogenic potential with bone marrow mesenchymal stromal cells and can be grown to therapeutic scale under Good Manufacturing Practice conditions

Background aimsMesenchymal stromal cells (MSCs) isolated from various tissues are under investigation as cellular therapeutics in a wide range of diseases. It is appreciated that the basic biological functions of MSCs vary depending on tissue source. However, in-depth comparative analyses between MSCs isolated from different tissue sources under Good Manufacturing Practice (GMP) conditions are lacking. Human clinical-grade low-purity islet (LPI) fractions are generated as a byproduct of islet isolation for transplantation. MSC isolates were derived from LPI fractions with the aim of performing a systematic, standardized comparative analysis of these cells with clinically relevant bone marrow-derived MSCs (BM MSCs).MethodsMSC isolates were derived from LPI fractions and expanded in platelet lysate-supplemented medium or in commercially available xenogeneic-free medium. Doubling rate, phenotype, differentiation potential, gene expression, protein production and immunomodulatory capacity of LPIs were compared with those of BM MSCs.ResultsMSCs can be readily derived in vitro from non-transplanted fractions resulting from islet cell processing (i.e., LPI MSCs). LPI MSCs grow stably in serum-free or platelet lysate-supplemented media and demonstrate in vitro self-renewal, as measured by colony-forming unit assay. LPI MSCs express patterns of chemokines and pro-regenerative factors similar to those of BM MSCs and, importantly, are equally able to attract immune cells in vitro and in vivo and suppress T-cell proliferation in vitro. Additionally, LPI MSCs can be expanded to therapeutically relevant doses at low passage under GMP conditions.ConclusionsLPI MSCs represent an alternative source of GMP MSCs with functions comparable to BM MSCs.     

Clinical-scale selection and viral transduction of human naïve and central memory CD8+ T cells for adoptive cell therapy of cancer patients

The adoptive transfer of lymphocytes genetically engineered to express tumor-specific antigen receptors is a potent strategy to treat cancer patients. T lymphocyte subsets, such as naïve or central memory T cells, selected in vitro prior to genetic engineering have been extensively investigated in preclinical mouse models, where they demonstrated improved therapeutic efficacy. However, so far, this is challenging to realize in the clinical setting, since good manufacturing practices (GMP) procedures for complex cell sorting and genetic manipulation are limited. To be able to directly compare the immunological attributes and therapeutic efficacy of naïve (T_N) and central memory (T_CM) CD8⁺ T cells, we investigated clinical-scale procedures for their parallel selection and in vitro manipulation. We also evaluated currently available GMP-grade reagents for stimulation of T cell subsets, including a new type of anti-CD3/anti-CD28 nanomatrix. An optimized protocol was established for the isolation of both CD8⁺ T_N cells (CD4^?CD62L⁺CD45RA⁺) and CD8⁺ T_CM (CD4^?CD62L⁺CD45RA^?) from a single patient. The highly enriched T cell subsets can be efficiently transduced and expanded to large cell numbers, sufficient for clinical applications and equivalent to or better than current cell and gene therapy approaches with unselected lymphocyte populations. The GMP protocols for selection of T_N and T_CM we reported here will be the basis for clinical trials analyzing safety, in vivo persistence and clinical efficacy in cancer patients and will help to generate a more reliable and efficacious cellular product.     

CD45<Superscript>−</Superscript>CD14<Superscript>+</Superscript>CD34<Superscript>+</Superscript> murine bone marrow low-adherent mesenchymal primitive cells preserve multilineage differentiation potential in long-term <Emphasis Type="Italic">in vitro</Emphasis> culture

Bone marrow-derived cells have been postulated as a source of multipotent mesenchymal stem cells (MSC). However, the whole fraction of MSC remains heterogeneous and the expansion of primitive subset of these cells is still not well established. Here, we optimized the protocol for propagating the low-adherent subfraction of MSC which results in long-term expansion of population characterized by CD45⁻CD14⁺CD34⁺ phenotype along with expression of common MSC markers. We established that the expanded MSC are capable of differentiating into endothelial cells highly expressing angiogenic markers and exhibiting functional properties of endothelium. Moreover, we found these cells to be multipotent and capable of giving rise into cells from neuronal lineages. Interestingly, the expanded MSC form characteristic cellular spheres in vitro indicating primitive features of these cells. In sum, we isolated the novel multipotent subpopulation of CD45⁻CD14⁺ CD34⁺ bone marrow-derived cells that could be maintained in long-term culture without losing this potential.     

Genetically Engineered Mesenchymal Stem Cells as a Proposed Therapeutic for Huntington’s Disease

There is much interest in the use of mesenchymal stem cells/marrow stromal cells (MSC) to treat neurodegenerative disorders, in particular those that are fatal and difficult to treat, such as Huntington's disease. MSC present a promising tool for cell therapy and are currently being tested in FDA-approved phase I-III clinical trials for many disorders. In preclinical studies of neurodegenerative disorders, MSC have demonstrated efficacy, when used as delivery vehicles for neural growth factors. A number of investigators have examined the potential benefits of innate MSC-secreted trophic support and augmented growth factors to support injured neurons. These include overexpression of brain-derived neurotrophic factor and glial-derived neurotrophic factor, using genetically engineered MSC as a vehicle to deliver the cytokines directly into the microenvironment. Proposed regenerative approaches to neurological diseases using MSC include cell therapies in which cells are delivered via intracerebral or intrathecal injection. Upon transplantation, MSC in the brain promote endogenous neuronal growth, encourage synaptic connection from damaged neurons, decrease apoptosis, reduce levels of free radicals, and regulate inflammation. These abilities are primarily modulated through paracrine actions. Clinical trials for MSC injection into the central nervous system to treat amyotrophic lateral sclerosis, traumatic brain injury, and stroke are currently ongoing. The current data in support of applying MSC-based cellular therapies to the treatment of Huntington's disease is discussed.     

Effective and persistent antitumor activity of HER2-directed CAR-T cells against gastric cancer cells in vitro and xenotransplanted tumors in vivo

Human epidermal growth factor receptor 2 (HER2) proteins are overexpressed in a high proportion of gastric cancer (GC) cases and affect the maintenance of cancer stem cell (CSC) subpopulations, which are used as targets for the clinical treatment of patients with HER2-positive GC. Despite improvements in survival, numerous HER2-positive patients fail treatment with trastuzumab, highlighting the need for more effective therapies. In this study, we generated a novel type of genetically modified human T cells, expressing a chimeric antigen receptor (CAR), and targeting the GC cell antigen HER2, which harbors the CD137 andCD3ζ moieties. Our findings show that the expanded CAR-T cells, expressing an increased central memory phenotype, were activated by the specific recognition of HER2 antigens in an MHC-independent manner, and effectively killed patient-derived HER2-positive GC cells. In HER2-positive xenograft tumors, CAR-T cells exhibited considerably enhanced tumor inhibition ability, long-term survival, and homing to targets, compared with those of non-transduced T cells. The sphere-forming ability and in vivo tumorigenicity of patient-derived gastric cancer stem-like cells, expressing HER2 and the CD44 protein, were also inhibited. Our results support the future development and clinical application of this adoptive immunotherapy in patients with HER2-positive advanced GC.     

Good manufacturing practice-compliant animal-free expansion of human bone marrow derived mesenchymal stroma cells in a closed hollow-fiber-based bioreactor

Mesenchymal stroma cells (MSC) are increasingly recognized for various applications of cell-based therapies such as regenerative medicine or immunomodulatory treatment strategies. Standardized large-scale expansions of MSC under good manufacturing practice (GMP)-compliant conditions avoiding animal derived components are mandatory for further evaluation of these novel therapeutic approaches in clinical trials.We applied a novel automated hollow fiber cell expansion system (CES) for in vitro expansion of human bone marrow derived MSC employing a GMP-compliant culture medium with human platelet lysate (HPL). Between 8 and 32 ml primary bone marrow aspirate were loaded into the hollow fiber CES and cultured for 15–27 days. 2–58 million MSC were harvested after primary culture. Further GMP-compliant cultivation of second passage MSC for 13 days led to further 10–20-fold enrichment. Viability, surface antigen expression, differentiation capacity and immunosuppressive function of MSC cultured in the hollow fiber CES were in line with standard criteria for MSC definition. We conclude that MSC can be enriched from primary bone marrow aspirate in a GMP-conform manner within a closed hollow fiber bioreactor and maintain their T lymphocyte inhibitory capacity. Standardized and reliable conditions for large scale MSC expansion pave the way for safe applications in humans in different therapeutic approaches.     

Vaccines for colorectal cancer.

Despite recent advances in the treatment of colorectal cancer, the overall survival rate for those patients with advanced locoregional disease remains less than 50%. Although adjuvant systemic chemotherapy has improved survival of these patients, more effective therapies are needed. Immunotherapy is an approach that could have a particular role in the adjuvant therapy of colorectal cancer. There is now convincing evidence that the immune system can specifically recognize and destroy malignant cells. Although both antibody- and T-cell-mediated anti-tumor responses have been documented, the cellular immune response with its direct cytotoxic mechanisms is felt to be the principal anti-tumor arm of the immune system. Analysis of the T cells that recognize tumors has led to the identification and characterization of many tumor-associated antigens including several colorectal antigens. Current approaches to developing a vaccine for colorectal cancer use our expanded understanding of these tumor-associated antigens and the conditions that allow development of an effective cellular immune response to them.