In vitro expansion of CD4+CD25highFOXP3+CD127low/- regulatory T cells from peripheral blood lymphocytes of healthy Mycobacterium tuberculosis-infected humans.

CD4+CD25highFOXP3+ regulatory T (Treg) cells have recently been found at elevated levels in the peripheral blood of tuberculosis patients, compared to Mycobacterium tuberculosis latently infected (LTBI) healthy individuals and non-infected controls. Here, we show that CD4+CD25highFOXP3+ T lymphocytes can be expanded in vitro from peripheral blood mononuclear cells (PBMC) of LTBI individuals, but not of uninfected controls by incubating them with BCG in the presence of TGF-beta. These expanded cells from the PBMC of LTBI subjects expressed CTLA-4, GITR and OX-40, but were CD127low/- and have therefore the phenotype of Treg cells. In addition, they inhibited in a dose-dependant manner the proliferation of freshly isolated mononuclear cells in response to polyclonal stimulation, indicating that they are functional Treg lymphocytes. In contrast, incubation of the PBMC with BCG alone preferentially induced activated CD4+ T cells, expressing CD25 and/or CD69 and secreting IFN-gamma. These results show that CD4+CD25highFOXP3+ Treg cells can be expanded or induced in the peripheral blood of LTBI individuals in conditions known to predispose to progression towards active tuberculosis and may therefore play an important role in the pathogenesis of the disease.

Isolation of monocytes from human peripheral blood using immuno-affinity expanded-bed adsorption

A novel technique for the separation of monocytes from human peripheral blood preparations has been developed. The technique is based on the use of expanded-bed adsorption and a solid perfluorocarbon derivatized with avidin or streptavidin for the indirect positive or negative capture of cells labeled with biotinylated monoclonal antibodies. The perfluorocarbon support was prepared and characterized and the contactor design and operating conditions, that enable cells to be selectively isolated, were investigated. Experiments consisted of applying an immunolabeled pulse of 1 x 10(8) peripheral blood mononuclear cells (PBMCs), isolated by density gradient centrifugation, directly onto a refrigerated expanded bed. The major cell types remaining were T-lymphocytes, B-lymphocytes, and monocytes. Monocytes could be positively adsorbed, following labeling with anti-CD14 mAb, with a clearance of up to 89% and a depletion factor of 7.6. They could also be

Isolation of a bone marrow-derived stem cell line with high proliferation potential and its application for preventing acute fatal liver failure

Transplantation of hepatocytes or hepatocyte-like cells of extrahepatic origin is a promising strategy for treatment of acute and chronic liver failure. We examined possible utility of hepatocyte-like cells induced from bone marrow cells for such a purpose. Clonal cell lines were established from the bone marrow of two different rat strains. One of these cell lines, rBM25/S3 cells, grew rapidly (doubling time, approximately 24 hours) without any appreciable changes in cell properties for at least 300 population doubling levels over a period of 300 days, keeping normal diploid karyotype. The cells expressed CD29, CD44, CD49b, CD90, vimentin, and fibronectin but not CD45, indicating that they are of mesenchymal cell origin. When plated on Matrigel with hepatocyte growth factor and fibroblast growth factor-4, the cells efficiently differentiated into hepatocyte-like cells that expressed albumin, cytochrome P450 (CYP) 1A1, CYP1A2, glucose 6-phosphatase, tryptophane-2,3-dioxygenase, tyrosine aminotransferase, hepatocyte nuclear factor (HNF)1 alpha, and HNF4alpha. Intrasplenic transplantation of the differentiated cells prevented fatal liver failure in 90%-hepatectomized rats. In conclusion, a clonal stem cell line derived from adult rat bone marrow could differentiate into hepatocyte-like cells, and transplantation of the differentiated cells could prevent fatal liver failure in 90%-hepatectomized rats. The present results indicate a promising strategy for treating human fatal liver diseases.

HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells

Reendothelialization involves endothelial progenitor cell (EPC) homing, proliferation, and differentiation, which may be influenced by fluid shear stress and local flow pattern. This study aims to elucidate the role of laminar flow on embryonic stem (ES) cell differentiation and the underlying mechanism. We demonstrated that laminar flow enhanced ES cell-derived progenitor cell proliferation and differentiation into endothelial cells (ECs). Laminar flow stabilized and activated histone deacetylase 3 (HDAC3) through the Flk-1-PI3K-Akt pathway, which in turn deacetylated p53, leading to p21 activation. A similar signal pathway was detected in vascular endothelial growth factor-induced EC differentiation. HDAC3 and p21 were detected in blood vessels during embryogenesis. Local transfer of ES cell-derived EPC incorporated into injured femoral artery and reduced neointima formation in a mouse model. These data suggest that shear stress is a key regulator for stem cell differentiation into EC, especially in EPC differentiation, which can be used for vascular repair, and that the Flk-1-PI3K-Akt-HDAC3-p53-p21 pathway is crucial in such a process.

Immune haemolytic anaemia following T cell-depleted allogeneic bone marrow transplantation for chronic myeloid leukaemia:association with leukaemic relapse and treatment with donor lymphocyte infusions

Immune haemolytic anaemia (IHA) is a recognised complication after allogeneic stem cell transplantation (SCT) and occurs more frequently if marrow cells have been subjected to T cell depletion (TCD). Among 58 consecutive patients who underwent TCD-allogeneic SCT from volunteer unrelated donors for the treatment of CML at the Hammersmith Hospital during a 3-year period (1 March 1996 to 28 February 1999) we identified nine cases of IHA. All patients had a strongly positive direct and indirect antiglobulin test and in eight patients the serological findings were typical of warm-type haemolysis often with antibody specificities within the Rh system. All nine cases had clinically significant haemolysis and were treated initially with prednisolone and immunoglobulin. The onset of IHA coincided with the occurrence of leukaemic relapse in six cases, and the presence of host haemopoiesis confirmed by lineage-specific chimerism in all four cases studied. Five patients received donor lymphocyte infusions (DLI); in three molecular remission and the restoration of full donor chimerism coincided with resolution of haemolysis. We conclude that in the context of leukaemic relapse, DLI is an effective therapy for IHA following allografts involving TCD.

Short Report:Engraftment of T-cell-depleted allogeneic haematopoietic stem cells using a reduced intensity conditioning regimen

Graft-versus-host disease (GVHD) remains a significant complication in patients undergoing allogeneic stem cell transplantation (SCT) using a reduced intensity conditioning regimen. Although T-cell depletion (TCD) reduces the risk of GVHD after a myeloablative conditioning regimen, it is associated with an increased risk of graft failure. We have therefore examined whether TCD compromises engraftment using a fludarabine-based conditioning regimen. Fifteen patients have been transplanted using such a regimen of whom 13 underwent ex vivo TCD. All but one patient demonstrated durable engraftment and no patient receiving a TCD product developed severe GVHD. Thus, TCD may play a role in GvHD prophylaxis using such regimens.

Common cancer stem cell gene variants predict colon cancer recurrence

PURPOSE: Recent evidence suggests that cancer stem cells (CSC) are responsible for key elements of colon cancer progression and recurrence. Germline variants in CSC genes may result in altered gene function and/or activity, thereby causing interindividual differences in a patient's tumor recurrence capacity and chemoresistance. We investigated germline polymorphisms in a comprehensive panel of CSC genes to predict time to tumor recurrence (TTR) in patients with stage III and high-risk stage II colon cancer.

EXPERIMENTAL DESIGN: A total of 234 patients treated with 5-fluorouracil-based chemotherapy at the University of Southern California were included in this study. Whole blood samples were analyzed for germline polymorphisms in genes that have been previously associated with colon CSC (CD44, Prominin-1, DPP4, EpCAM, ALCAM, Msi-1, ITGB1, CD24, LGR5, and ALDH1A1) by PCR-RFLP or direct DNA-sequencing.

RESULTS: The minor alleles of CD44 rs8193 C>T, ALCAM rs1157 G>A, and LGR5 rs17109924 T>C were significantly associated with increased TTR (9.4 vs. 5.4 years; HR, 0.51; 95% CI: 0.35-0.93; P = 0.022; 11.3 vs. 5.7 years; HR, 0.56; 95% CI: 0.33-0.94; P = 0.024, and 10.7 vs. 5.7 years; HR, 0.33; 95% CI: 0.12-0.90; P = 0.023, respectively) and remained significant in the multivariate analysis stratified by ethnicity. In recursive partitioning, a specific gene variant profile including LGR5 rs17109924, CD44 rs8193, and ALDH1A1 rs1342024 represented a high-risk subgroup with a median TTR of 1.7 years (HR, 6.71, 95% CI: 2.71-16.63, P < 0.001).

CONCLUSION: This is the first study identifying common germline variants in colon CSC genes as independent prognostic markers for stage III and high-risk stage II colon cancer patients.

Selective accumulation of differentiated FOXP3(+) CD4 (+) T cells in metastatic tumor lesions from melanoma patients compared to peripheral blood

Precise identification of regulatory T cells is crucial in the understanding of their role in human cancers. Here, we analyzed the frequency and phenotype of regulatory T cells (Tregs), in both healthy donors and melanoma patients, based on the expression of the transcription factor FOXP3, which, to date, is the most reliable marker for Tregs, at least in mice. We observed that FOXP3 expression is not confined to human CD25(+/high) CD4(+) T cells, and that these cells are not homogenously FOXP3(+). The circulating relative levels of FOXP3(+) CD4(+) T cells may fluctuate close to 2-fold over a short period of observation and are significantly higher in women than in men. Further, we showed that FOXP3(+) CD4(+) T cells are over-represented in peripheral blood of melanoma patients, as compared to healthy donors, and that they are even more enriched in tumor-infiltrated lymph nodes and at tumor sites, but not in normal lymph nodes. Interestingly, in melanoma patients, a significantly higher proportion of functional, antigen-experienced FOXP3(+) CD4(+) T was observed at tumor sites, compared to peripheral blood. Together, our data suggest that local accumulation and differentiation of Tregs is, at least in part, tumor-driven, and illustrate a reliable combination of markers for their monitoring in various clinical settings.

Umbilical Cord Blood Banking

The umbilical cord, previously considered as waste and discarded at birth, is a source of haematopoietic stem cells. Current therapeutic uses of umbilical cord blood stem cells and the promise of these cells for the treatment of degenerative diseases in the future have led to the establishment of cord blood banks in many parts of the world. Although umbilical cord blood banking raises many ethical and legal issues, this article focuses on the controversy created by the coexistence of public and private cord blood banks in many countries. Policy statements adopted by professional associations and advisory groups indicate that, based on the current state of medical evidence, childbearing women with no current or potential familial need of stem cell transplantation should be encouraged to donate cord blood to public banks.

Immunothérapie cellulaire de la leucémie aiguë lymphoblastique de l'enfant à partir de sang de cordon dans un modèle murin xénogénique

La leucémie aigüe lymphoblastique de précurseurs des cellules B (pré-B LAL) est le cancer le plus fréquent chez l’enfant. La transplantation de cellules souches hématopoïétiques (TCSH) est nécessaire dans environ 20 à 30 % des enfants ayant une pré-B LAL. Les rechutes après TCSH sont habituellement réfractaires aux thérapies actuelles, et par conséquent, il est important de développer et d’optimiser de nouvelles stratégies thérapeutiques. Dans cette étude, nous nous sommes intéressés aux cellules « cytokine-induced killer » (CIK). En effet, ces cellules ont été montrées comme hautement cytotoxique contre beaucoup de types de cancers. Cependant, leur activité cytotoxique contre les pré-B LAL n’est pas vraiment efficace. Par conséquent, nous avons étudié la possibilité de combiner l’immunothérapie des cellules CIK avec l’interféron alpha (IFN-α) afin d’optimiser l’activité lytique de ces cellules contre les cellules pré-B LAL. De plus, vu qu’il a été démontré que l’activité cytotoxique des cellules CIK provient de la fraction CD56+, plus particulièrement les cellules CD3+CD56+, nous avons décidé d’utiliser la fraction CD56+ (cellules CD56+) dans l’ensemble de nos expériences. Nous avons observé in vitro que les cellules CD56+ lysent mieux les lignées cellulaires pré-B LAL comparativement aux cellules CIK non purifiées. Aussi, leur activité cytotoxique peut être augmentée par le traitement avec l’IFN-α. Par ailleurs, nous avons démontré l’efficacité des cellules CD56+ traitées par l’IFN-α contre les lignées cellulaires pré- B LAL in vivo, dans le modèle de souris NOD/SCID/gamma c- (NSG). La survie des souris est significativement prolongée lorsqu’elles reçoivent les cellules pré-B LAL avec les cellules CD56+ traitées par l’IFN-α. Nous avons par la suite étudié le mécanisme d’action des cellules CD56+ contre les lignées cellulaires pré-B LAL. Nous avons observé que les cellules CD56+ provenant de sang de cordon sont plus efficaces que les cellules CD56+ provenant de sang I périphérique pour tuer les lignées cellulaires pré-B LAL. Nous avons également montré que les cellules CD56+ utilisent seulement la voie NKG2D ou bien les voies NKG2D et TRAIL selon la lignée cellulaire pré-B LAL cible et selon la provenance de la source des cellules CD56+. Par ailleurs, nous avons remarqué que les cellules CIK sont sensibles à l’apoptose par Fas, et que cette sensibilité influence leur activité cytotoxique contre les cellules tumorales. En conclusion, les cellules CD56+ sont cytotoxiques contre les lignées cellulaires pré-B LAL, et leur effet lytique est augmenté par l’IFN-α aussi bien in vitro qu’in vivo dans le modèle de souris NSG. Ces données précliniques sont encourageantes pour tester cette nouvelle approche d’immunothérapie dans le traitement contre la pré-B LAL.

Efficient animal-serum free 3D cultivation method for adult human neural crest-derived stem cell therapeutics

Due to their broad differentiation potential and their persistence into adulthood, human neural crest-derived stem cells (NCSCs) harbour great potential for autologous cellular therapies, which include the treatment of neurodegenerative diseases and replacement of complex tissues containing various cell types, as in the case of musculoskeletal injuries. The use of serum-free approaches often results in insufficient proliferation of stem cells and foetal calf serum implicates the use of xenogenic medium components. Thus, there is much need for alternative cultivation strategies. In this study we describe for the first time a novel, human blood plasma based semi-solid medium for cultivation of human NCSCs. We cultivated human neural crest-derived inferior turbinate stem cells (ITSCs) within a blood plasma matrix, where they revealed higher proliferation rates compared to a standard serum-free approach. Three-dimensionality of the matrix was investigated using helium ion microscopy. ITSCs grew within the matrix as revealed by laser scanning microscopy. Genetic stability and maintenance of stemness characteristics were assured in 3D cultivated ITSCs, as demonstrated by unchanged expression profile and the capability for self-renewal. ITSCs pre-cultivated in the 3D matrix differentiated efficiently into ectodermal and mesodermal cell types, particularly including osteogenic cell types. Furthermore, ITSCs cultivated as described here could be easily infected with lentiviruses directly in substrate for potential tracing or gene therapeutic approaches. Taken together, the use of human blood plasma as an additive for a completely defined medium points towards a personalisable and autologous cultivation of human neural crest-derived stem cells under clinical grade conditions.

Improved method for ex ovo-cultivation of developing chicken embryos for human stem cell xenografts

The characterization of human stem cells for the usability in regenerative medicine is particularly based on investigations regarding their differentiation potential in vivo. In this regard, the chicken embryo model represents an ideal model organism. However, the access to the chicken embryo is only achievable by windowing the eggshell resulting in limited visibility and accessibility in subsequent experiments. On the contrary, ex ovo-culture systems avoid such negative side effects. Here, we present an improved ex ovo-cultivation method enabling the embryos to survive 13 days in vitro. Optimized cultivation of chicken embryos resulted in a normal development regarding their size and weight. Our ex ovo-approach closely resembles the development of chicken embryos in ovo, as demonstrated by properly developed nervous system, bones, and cartilage at expected time points. Finally, we investigated the usability of our method for trans-species transplantation of adult stem cells by injecting human neural crest-derived stem cells into late Hamburger and Hamilton stages (HH26-HH28/E5-E6) of ex ovo-incubated embryos. We demonstrated the integration of human cells allowing experimentally easy investigation of the differentiation potential in the proper developmental context. Taken together, this ex ovo-method supports the prolonged cultivation of properly developing chicken embryos enabling integration studies of xenografted mammalian stem cells at late developmental stages.

Culture bag systems for clinical applications of adult human neural crest-derived stem cells

Introduction Facing the challenging treatment of neurodegenerative diseases as well as complex craniofacial injuries such as those common after cancer therapy, the field of regenerative medicine increasingly relies on stem cell transplantation strategies. Here, neural crest-derived stem cells (NCSCs) offer many promising applications, although scale up of clinical-grade processes prior to potential transplantations is currently limiting. In this study, we aimed to establish a clinical-grade, cost-reducing cultivation system for NCSCs isolated from the adult human nose using cGMP-grade Afc-FEP bags. Methods We cultivated human neural crest-derived stem cells from inferior turbinate (ITSCs) in a cell culture bag system using Afc-FEP bags in human blood plasma-supplemented medium. Investigations of viability, proliferation and expression profile of bag-cultured ITSCs were followed by DNA-content and telomerase activity determination. Cultivated ITSCs were introduced to directed in vitro differentiation assays to assess their potential for mesodermal and ectodermal differentiation. Mesodermal differentiation was determined using an enzyme activity assay (alkaline phosphatase, ALP), respective stainings (Alizarin Red S, Von Kossa and Oil Red O), and RT-PCR, while immunocytochemistry and synaptic vesicle recycling were applied to assay neuroectodermal differentiation of ITSCs. Results When cultivated within Afc-FEP bags, ITSCs grew three-dimensionally in a human blood plasma-derived matrix, thereby showing unchanged morphology, proliferation capability, viability and expression profile in comparison to three dimensionally-cultured ITSCs growing in standard cell culture plastics. Genetic stability of bag-cultured ITSCs was further accompanied by unchanged telomerase activity. Importantly, ITSCs retained their potential to differentiate into mesodermal cell types, particularly including ALP-active, Alizarin Red S-, and Von Kossa-positive osteogenic cell types, as well as adipocytes positive in Oil Red O assays. Bag culture further did not affect the potential of ITSCs to undergo differentiation into neuroectodermal cell types coexpressing β-III-tubulin and MAP2 and exhibiting the capability for synaptic vesicle recycling. Conclusions Here, we report for the first time the successful cultivation of human NCSCs within cGMP-grade Afc-FEP bags using a human blood plasma-supplemented medium. Our findings particularly demonstrate the unchanged differentiation capability and genetic stability of the cultivated NCSCs, suggesting the great potential of this culture system for future medical applications in the field of regenerative medicine.

Effects of lipid-lowering drugs on reverse cholesterol transport gene expressions in peripheral blood mononuclear and HepG2 cells

Aims: The ATP-binding cassette transporters, ABCA1 and ABCG1, are LXR-target genes that play an important role in reverse cholesterol transport. We examined the effects of inhibitors of the cholesterol absorption (ezetimibe) and synthesis (statins) on expression of these transporters in HepG2 cells and peripheral blood mononuclear cells (PBMCs) of individuals with primary (and nonfamilial) hypercholesterolemia (HC). Materials & methods: A total of 48 HC individuals were treated with atorvastatin (10 mg/day/4 weeks) and 23 were treated with ezetimibe (10 mg/day/4 weeks), followed by simvastatin (10 mg/day/8 weeks) and simvastatin plus ezetimibe (10 mg of each/day/4 weeks). Gene expression was examined in statin- or ezetimibe-treated and control HepG2 cells as well as PBMCs using real-time PCR. Results: In PBMCs, statins and ezetimibe downregulated ABCA1 and ABCG1 mRNA expression but did not modulate NR1H2 (LxR-beta) and NR1H3 (LXR-alpha) levels. Positive correlations of ABCA1 with ABCG1 and of NR1H2 with NR1H3 expressions were found in all phases of the treatments. In HepG2 cells, ABCA1 mRNA levels remained unaltered while ABCG1 expression was increased by statin (1.0-10.0 mu M) or ezetimibe (5.0 mu M) treatments. Atorvastatin upregulated NR1H2 and NR1H3 only at 10.0 mu M, meanwhile ezetimibe (1.0-5.0 mu M) downregulated NR1H2 but did not change NR1H3 expression. Conclusion: Our findings reveal that lipid-lowering drugs downregulate ABCA1 and ABCG1 mRNA expression in PBMCs of HC individuals and exhibit differential effects on HepG2 cells. Moreover, they indicate that the ABCA1 and ABCG1 transcript levels were not correlated directly to LXR mRNA expression in both cell models treated with lipid-lowering drugs.