A new standardized clinical-grade protocol for banking human umbilical cord tissue cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) isolated from human umbilical cord tissue (UCT) can be considered the perfect candidates for cell-based therapies and regenerative medicine. UCT-derived MSCs can be cryogenically stored in cell banks and expanded as needed for therapeutic uses. STUDY DESIGN AND METHODS: We developed a new method for UCT-MSC isolation, cryopreservation, and expansion, following all criteria required by a stem cell bank. UCT-MSCs were isolated either by manual dissociation (MM) or by a semiautomatic dissociation system (SAM). In both protocols UCTs were treated enzymatically using Type IV collagenase good manufacturing practices (GMP) graded and hyaluronidase (medicinal product). Isolated UCT-MSCs were cryopreserved and analyzed after thawing for phenotype; for proliferation rate; and for their osteogenic, adipogenic, and chondrogenic differentiation capabilities. RESULTS: We found that SAM reduced the time of tissue enzyme exposure and enabled us to obtain a homogeneous single-cell suspension deprived of tissue fragments. The isolated cells in both groups showed high expression of MSC markers CD105, CD73, and CD90 and similar differentiation capabilities, phenotype, and proliferation potential. Moreover, the final yield of MSCs was comparable between the two techniques. CONCLUSION: In this study, we have established a reliable and standardized protocol to isolate UCT-MSCs from UCT for cell banking purposes. Processing the whole umbilical tissue with GMP-graded enzymes using a semiautomatic dissociator allowed us to obtain a single-cell suspension product with a known number of isolated cells that can be cryopreserved right after isolation and thawed as needed for expansion and clinical use.

Cord blood-derived neuronal cells by ectopic expression of Sox2 and c-Myc.

The finding that certain somatic cells can be directly converted into cells of other lineages by the delivery of specific sets of transcription factors paves the way to novel therapeutic applications. Here we show that human cord blood (CB) CD133(+) cells lose their hematopoietic signature and are converted into CB-induced neuronal-like cells (CB-iNCs) by the ectopic expression of the transcription factor Sox2, a process that is further augmented by the combination of Sox2 and c-Myc. Gene-expression analysis, immunophenotyping, and electrophysiological analysis show that CB-iNCs acquire a distinct neuronal phenotype characterized by the expression of multiple neuronal markers. CB-iNCs show the ability to fire action potentials after in vitro maturation as well as after in vivo transplantation into the mouse hippocampus. This system highlights the potential of CB cells and offers an alternative means to the study of cellular plasticity, possibly in the context of drug screening research and of future cell-replacement therapies.

Methylation and silencing of miRNA-124 by EVI1 and self-renewal exhaustion of hematopoietic stem cells in murine myelodysplastic syndrome.

By expressing EVI1 in murine bone marrow (BM), we previously described a myelodysplastic syndrome (MDS) model characterized by pancytopenia, dysmegakaryopoiesis, dyserythropoiesis, and BM failure. The mice invariably died 11-14 months after BM transplantation (BMT). Here, we show that a double point mutant EVI1-(1+6Mut), unable to bind Gata1, abrogates the onset of MDS in the mouse and re-establishes normal megakaryopoiesis, erythropoiesis, BM function, and peripheral blood profiles. These normal features were maintained in the reconstituted mice until the study was ended at 21 months after BMT. We also report that EVI1 deregulates several genes that control cell division and cell self-renewal. In striking contrast, these genes are normalized in the presence of the EVI1 mutant. Moreover, EVI1, but not the EVI1 mutant, seemingly deregulates these cellular processes by altering miRNA expression. In particular, the silencing of miRNA-124 by DNA methylation is associated with EVI1 expression, but not that of the EVI1 mutant, and appears to play a key role in the up-regulation of cell division in murine BM cells and in the hematopoietic cell line 32Dcl3. The results presented here demonstrate that EVI1 induces MDS in the mouse through two major pathways, both of which require the interaction of EVI1 with other factors: one, results from EVI1-Gata1 interaction, which deregulates erythropoiesis and leads to fatal anemia, whereas the other occurs by interaction of EVI1 with unidentified factors causing perturbation of the cell cycle and self-renewal, as a consequence of silencing miRNA-124 by EVI1 and, ultimately, ensues in BM failure.

Point mutations in two EVI1 Zn fingers abolish EVI1-GATA1 interaction and allow erythroid differentiation of murine bone marrow cells.

EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in myelodysplastic syndrome (MDS). The expression of the corresponding gene is a very poor prognostic marker for MDS patients and is associated with severe defects of the erythroid lineage. We have recently shown that the constitutive expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including anemia, dyserythropoiesis, and dysmegakaryopoiesis. These lineages are regulated by the DNA-binding transcription factor GATA1. EVI1 has two zinc finger domains containing seven motifs at the N terminus and three motifs at the C terminus. Supported by results of assays utilizing synthetic DNA promoters, it was earlier proposed that erythroid-lineage repression by EVI1 is based on the ability of this protein to compete with GATA1 for DNA-binding sites, resulting in repression of gene activation by GATA1. Here, however, we show that EVI1 is unable to bind to classic GATA1 sites. To understand the mechanism utilized by EVI1 to repress erythropoiesis, we used a combination of biochemical assays, mutation analyses, and in vitro bone marrow differentiation. The results indicate that EVI1 interacts directly with the GATA1 protein rather than the DNA sequence. We further show that this protein-protein interaction blocks efficient recognition or binding to DNA by GATA1. Point mutations that disrupt the geometry of two zinc fingers of EVI1 abolish the protein-protein interaction, leading to normal erythroid differentiation of normal murine bone marrow in vitro.

Down-regulation of MLL-AF9, MLL and MYC expression is not obligatory for monocyte-macrophage maturation in AML-M5 cell lines carrying t(9;11)(p22;q23).

The MLL-AF9 oncogene originates from the translocation t(9;11)(p22;q23), which is mainly associated with monocytic acute myeloid leukaemia (AML-M5; FAB-classification). In AML-M5 THP-1 cells carrying t(9;11) (p22;q23) and expressing MLL-AF9, we previously showed that MLL-AF9 expression is down-regulated during monocyte-macrophage maturation. We have subsequently observed that in a 'rapid-growing' variant of the THP-1 cell line (THP-1-R) MLL-AF9 down-regulation does not occur. MLL fusion proteins (including MLL-AF9) deregulate MYC transactivation activity, and both presence and absence of MYC down-regulation have been reported during monocyte-macrophage maturation in THP-1 cells. In the present study, we analyze the expression patterns of MLL-AF9, MLL wild-type and MYC after induction of monocyte-macrophage terminal differentiation in the AML-M5 cell lines, THP-1, THP-1-R, Mono-Mac-6 (MM6) and MOLM-13, all of which carry t(9;11)(p22;q23) and express MLL-AF9. RT-PCR analysis indicated that down-regulation of MLL-AF9, MLL or MYC is not necessary to abolish malignant phenotypes by induction of terminal monocyte-macrophage differentiation in leukaemic cells carrying t(9;11)(p22;q23).

Interleukin-15 enhances cytokine induced killer (CIK) cytotoxic potential against epithelial cancer cell lines via an innate pathway.

CIK cells are a subset of effector lymphocytes endowed with a non-MHC restricted anti-tumor activity making them an appealing and promising cell population for adoptive immunotherapy. CIK are usually generated ex-vivo by initial priming with Interferon-γ (IFN-γ) and monoclonal antibody against CD3 (anti-CD3), followed by culture in medium containing Interleukin-2 (IL-2). Interleukin-15 (IL-15) shares with IL-2 similar biological functions and recently it has been reported to induce CIK with increased anti-leukemic potential. The aim of the study was to compare the killing efficacy of CIK generated by IL-2 alone or IL-2 and IL-15 toward tumor targets of different origins, leukemic cells and malignant cells from epithelial solid tumors. CIK bulk cultures were examined for cell proliferation, surface phenotype and cytotoxic potential against tumor cell lines K562, HL60, HeLa and MCF-7. The results showed that IL-15 is able to induce a selective expansion of CIK cells, but it is less effective in sustaining CIK cell proliferation compared to IL-2. Conversely, our data confirm and reinforce the feature of IL-15 to induce CIK cells with a potent cytotoxic activity mostly against tumor cells from epithelial solid malignancies via NKG2D-mediated mechanism.

Generation and characterization of bioluminescent xenograft mouse models of MLL-related acute leukemias and in vivo evaluation of luciferase-targeting siRNA nanoparticles.

Chromosomal translocations involving the MLL gene on 11q23 present frequent abnormalities in pediatric, adult and therapy-related acute leukemias, and are generally associated with aggressive disease and poor prognosis. Here, we report bioluminescent acute leukemia xenograft mouse models of the most frequent and aggressive MLL-related acute leukemias (infant and adult MLL-AF9, MLL-ENL, MLL-AF4). Four acute leukemia cell lines carrying MLL-related translocations were stably transduced with a firefly luciferase transgene and injected intravenously into NOD/SCID mice. Leukemia progression was monitored by in vivo bioluminescence imaging (BLI). All mice developed MLL-related acute leukemia. The four MLL-related acute leukemia models showed a different course of infant and adult MLL-AF9 acute myeloid leukemia, and a rapid aggressiveness of MLL-ENL acute lymphoblastic leukemia and MLL-AF4 acute biphenotypic leukemia. Tissue analysis and RT-PCR of bone marrow, spleen and liver from the mice confirmed the BL results. To validate BLI for the detection of a therapeutic response, systemic treatment with an anti-luciferase-targeting siRNA (siLuc) complexed with cationic nanoparticles was administered to mice with MLL-AF4 acute lymphoblastic leukemia. The BLI signal showed a reduction following treatment with siLuc compared to the control mice. These mouse models present MLL-related acute leukemia evolution similar to the human counterparts. Moreover, they are non-invasive, rapid and sensitive models, suitable for the in vivo study of MLL-related acute leukemias. Finally, BLI showed in vivo luminescence down modulation obtained by systemic treatment with luciferase-targeting siRNA nanoparticle complexes, confirming that these MLL-related leukemia mouse models are optimal for the evaluation and selection of delivery systems for siRNA and other new biotechnological pharmaceuticals.

In vivo bioluminescence imaging of murine xenograft cancer models with a red-shifted thermostable luciferase.

PURPOSE: Conventional in vivo bioluminescence imaging using wild-type green-emitting luciferase is limited by absorption and scattering of the bioluminescent signal through tissues. Imaging methods using a red-shifted thermostable luciferase from Photinus pyralis were optimized to improve the sensitivity and image resolution. In vivo bioluminescence imaging performance of red- and green-emitting luciferases were compared in two different xenograft mouse models for cancer. METHODS: Human hepatoblastoma cell line (HepG2) and human acute monocytic leukemia cell line (Thp1) cells were genetically engineered using retroviral vector technology to stably express the red-shifted or the wild-type green luciferase. A xenograft model of liver cancer was established by subcutaneous injection of the HepG2-engineered cells in the flank regions of mice, and a leukemia model was generated by intravenous injection of the engineered Thp1 cells. The cancer progression was monitored with an ultrasensitive charge-coupled device camera. The relative intensities of the green- and red-emitting luciferases were measured, and the resulting spatial resolutions of the images were compared. Imaging was performed with both intact and scarified live animals to quantify the absorption effects of the skin and deep tissue. RESULTS: The red-emitting luciferase was found to emit a bioluminescence signal with improved transmission properties compared to the green-emitting luciferase. By imaging the HepG2 models, which contained tumors just beneath the skin, before and after scarification, the percentage of light absorbed by the skin was calculated. The green bioluminescent signal was 75 +/- 8% absorbed by the skin, whereas the red signal was only 20 +/- 6% absorbed. The Thp1 model, which contains cancer cells within the bones, was likewise imaged before and after scarification to calculate the percentage of light absorbed by all tissue under the skin. This tissue was responsible for 90 +/- 5% absorption of the green signal, but only 65 +/- 6% absorption of the red signal. CONCLUSION: Two different bioluminescent mouse cancer models demonstrate the utility of a new red-shifted thermostable luciferase, Ppy RE-TS, that improved the in vivo imaging performance when compared with wild-type P. Pyralis luciferase. While wild-type luciferase is currently a popular reporter for in vivo imaging methods, this study demonstrates the potential of red-emitting firefly luciferase mutants to enhance the performance of bioluminescence imaging experiments.

Down-regulation of HOXA4, HOXA7, HOXA10, HOXA11 and MEIS1 during monocyte-macrophage differentiation in THP-1 cells.

The translocation t(9;11)(p22;q23) generates the MLL-AF9 oncogene and is commonly associated with monocytic acute myeloid leukemia (AML-M5; FAB-classification). For the oncogenicity of MLL-AF9, the (over)expression of several other genes, including selected HOXA cluster genes as well as MEIS1 (a HOX cofactor), is required. We previously showed that the down-regulation of MLL-AF9 expression is not obligatory for monocyte-macrophage maturation in AML-M5 cells carrying t(9;11)(p22;q23). In this study, we analyzed the expression patterns of HOXA4, 5, 6, 7, 9, 10 and 11 (defined as 'HOXA-code' genes) and MEIS1 by semiquantitative RT-PCR during the monocyte-macrophage differentiation induced by phorbol 12-myristate 13-acetate (PMA) in THP-1 cells carrying t(9;11)(p22;q23) and expressing MLL-AF9. The analyses were performed in THP-1 cells expressing MLL-AF9 even after PMA treatment. The results showed that all the analyzed genes were expressed in untreated THP-1 cells. After the induction of differentiation, we observed a down-regulation of HOXA4, 7, 10, 11 and MEIS1, an up-regulation of HOXA6, and no significant variation in the expression of HOXA5 and 9. These data indicate that the expression of most HOXA-code genes, as well as MEIS1, could be implicated in the differentiation blockage observed in MLL-AF9-related leukemias.