Intravenous administration of bone marrow-derived multipotent mesenchymal stromal cells enhances the recruitment of CD11b(+) myeloid cells to the lungs and facilitates B16-F10 melanoma colonization.

The discovery that the regenerative properties of bone marrow multipotent mesenchymal stromal cells (BM-MSCs) could collaterally favor neoplastic progression has led to a great interest in the function of these cells in tumors. However, the effect of BM-MSCs on colonization, a rate-limiting step of the metastatic cascade, is unknown. In this study, we investigated the effect of BM-MSCs on metastatic outgrowth of B16-F10 melanoma cells. In in vitro experiments, direct co-culture assays demonstrated that BM-MSCs stimulated the proliferation of B16-F10 cells in a dose-dependent manner. For in vivo experiments, luciferase-expressing B16-F10 cells were injected through tail vein and mice were subsequently treated with four systemic injections of BM-MSCs. In vivo bioluminescent imaging during 16 days demonstrated that BM-MSCs enhanced the colonization of lungs by B16-F10 cells, which correlated with a 2-fold increase in the number of metastatic foci. Flow cytometry analysis of lungs demonstrated that although mice harboring B16-F10 metastases displayed more endothelial cells, CD4 T and CD8 T lymphocytes in the lungs in comparison to metastases-free mice, BM-MSCs did not alter the number of these cells. Interestingly, BM-MSCs inoculation resulted in a 2-fold increase in the number of CD11b(+) myeloid cells in the lungs of melanoma-bearing animals, a cell population previously described to organize "premetastatic niches" in experimental models. These findings indicate that BM-MSCs provide support to B16-F10 cells to overcome the constraints that limit metastatic outgrowth and that these effects might involve the interplay between BM-MSCs, CD11b(+) myeloid cells and tumor cells.

Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds.

The seed-based production of recombinant proteins is an efficient strategy to achieve the accumulation, correct folding, and increased stability of these recombinant proteins. Among potential plant molecular farming systems, soybean [Glycine max (L.) Merrill] is a viable option for the production of recombinant proteins due to its high protein content, known regulatory sequences, efficient gene transfer protocols, and a scalable production system under greenhouse conditions. We report here the expression and stable accumulation of human coagulation factor IX (hFIX) in transgenic soybean seeds. A biolistic process was utilised to co-introduce a plasmid carrying the hFIX gene under the transcriptional control of the α' subunit of a ß-conglycinin seed-specific promoter and an α-Coixin signal peptide in soybean embryonic axes from mature seeds. The 56-kDa hFIX protein was expressed in the transgenic seeds at levels of up to 0.23% (0.8 g kg(-1) seed) of the total soluble seed protein as determined by an enzyme-linked immunosorbent assay (ELISA) and western blot. Ultrastructural immunocytochemistry assays indicated that the recombinant hFIX in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Mass spectrometry characterisation confirmed the presence of the hFIX recombinant protein sequence. Protein extracts from transgenic seeds showed a blood-clotting activity of up to 1.4% of normal plasma. Our results demonstrate the correct processing and stable accumulation of functional hFIX in soybean seeds stored for 6 years under room temperature conditions (22 ± 2°C).

Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146+ perivascular cells and fibroblasts.

OBJECTIVE: The relationship of multipotent mesenchymal stromal cells (MSC) with pericytes and fibroblasts has not been established thus far, although they share many markers of primitive marrow stromal cells and the osteogenic, adipogenic, and chondrogenic differentiation potentials. MATERIALS AND METHODS: We compared MSCs from adult or fetal tissues, MSC differentiated in vitro, fibroblasts and cultures of retinal pericytes obtained either by separation with anti-CD146 or adhesion. The characterizations included morphological, immunophenotypic, gene-expression profile, and differentiation potential. RESULTS: Osteogenic, adipocytic, and chondrocytic differentiation was demonstrated for MSC, retinal perivascular cells, and fibroblasts. Cell morphology and the phenotypes defined by 22 markers were very similar. Analysis of the global gene expression obtained by serial analysis of gene expression for 17 libraries and by reverse transcription polymerase chain reaction of 39 selected genes from 31 different cell cultures, revealed similarities among MSC, retinal perivascular cells, and hepatic stellate cells. Despite this overall similarity, there was a heterogeneous expression of genes related to angiogenesis, in MSC derived from veins, artery, perivascular cells, and fibroblasts. Evaluation of typical pericyte and MSC transcripts, such as NG2, CD146, CD271, and CD140B on CD146 selected perivascular cells and MSC by real-time polymerase chain reaction confirm the relationship between these two cell types. Furthermore, the inverse correlation between fibroblast-specific protein-1 and CD146 transcripts observed on pericytes, MSC, and fibroblasts highlight their potential use as markers of this differentiation pathway. CONCLUSION: Our results indicate that human MSC and pericytes are similar cells located in the wall of the vasculature, where they function as cell sources for repair and tissue maintenance, whereas fibroblasts are more differentiated cells with more restricted differentiation potential.

Evaluation of theranostic nanocarriers for near-infrared imaging and photodynamic therapy on human prostate cancer cells.

This paper evaluates how effectively chloroaluminum phthalocyanine (ClAlPc) entrapped in colloidal nanocarriers, such as nanocapsule (NC) and nanoemulsion (NE), induces photodamage in human prostate cancer cells (LNCaP) during photodynamic therapy (PDT). The MTT cell viability assay showed that both ClAlPc-NC and ClAlPc-NE induced phototoxicity and efficiently killed LNCaP cells at low ClAlPc-NC and ClAlPc-NE concentrations (0.3µgmL-1) as well as under low light doses of 4Jcm-2 and 7Jcm-2, respectively, upon PDT with a 670-nm diode laser line. Confocal imaging studies indicated that ClAlPc-NC and ClAlPc-NE were preferentially localized in the perinuclear region of LNCaP cells both in the dark and upon irradiation with laser light. After PDT treatment, ClAlPc-NC-treated LNCaP cells exhibited a higher green fluorescence signal, possibly due to the larger shrinkage of the actin cytoskeleton, compared to ClAlPc-NE-treated LNCaP cells. Additionally, ClAlPc-NC or ClAlPc-NE and mitochondria showed a relatively high co-localization level. The cellular morphology did not change in the dark, but confocal micrographs recorded after PDT revealed that LNCaP cells treated with ClAlPc-NC or ClAlPc-NE underwent morphological alterations. Our preliminary in vitro studies reinforced the hypothesis that biocompatible theranostic ClAlPc-loaded nanocarriers could act as an attractive photosensitizer system in PDT and could serve as an interesting molecular probe for the early diagnosis of prostate cancer and other carcinomas.

Growth and functional harvesting of human mesenchymal stromal cells cultured on a microcarrier-based system.

Human mesenchymal stromal cells (hMSCs) cells are attractive for applications in tissue engineering and cell therapy. Because of the low availability of hMSCs in tissues and the high doses of hMSCs necessary for infusion, scalable and cost-effective technologies for in vitro cell expansion are needed to produce MSCs while maintaining their functional, immunophenotypic and cytogenetic characteristics. Microcarrier-based culture systems are a good alternative to traditional systems for hMSC expansion. The aim of the present study was to develop a scalable bioprocess for the expansion of human bone marrow mesenchymal stromal cells (hBM-MSCs) on microcarriers to optimize growth and functional harvesting. In general, the results obtained demonstrated the feasibility of expanding hBM-MSCs using microcarrier technology. The maximum cell concentration (n = 5) was ~4.82 ± 1.18 × 10(5) cell mL(-1) at day 7, representing a 3.9-fold increase relative to the amount of inoculated cells. At the end of culture, 87.2% of the cells could be harvested (viability = 95%). Cell metabolism analysis revealed that there was no depletion of important nutrients such as glucose and glutamine during culture, and neither lactate nor ammonia byproducts were formed at inhibitory concentrations. The cells that were recovered after the expansion retained their immunophenotypic and functional characteristics. These results represent an important step toward the implementation of a GMP-compliant large-scale production system for hMSCs for cellular therapy.

Maturation of dendritic cells following exposure to different maturational stimuli / Maturação das células dendríticas após exposição a diversos estímulos

Dendritic cells (DCs) are professional antigen-presenting cells that are highly effective to immunize against pathogens and tumor antigens. In order to obtain mature DCs several in vitro methods have been reported. Selecting the most efficient and effective method of generating morphologic and phenotypic DCs within 7 days of culture is an essential prerequisite for success in immunotherapy strategies. Herein, we report a method of obtaining an enriched monocyte population from blood donors and performed a comparison of DC maturation in response to four agents. Monocyte populations with 91 percent ± 5 of purity were obtained from 15 healthy donors. The resulting monocyte populations were cultured in the presence of GM-CSF and IL-4 during 5 days. At day 5 different maturation conditions were performed and morphological and phenotypical changes were analyzed. Our study demonstrates that TNF-alpha or PGE1 by themselves can induce the expression of CD1a 2.4 and 2.7 times respectively more than DC cultures in the absence of maturing agents. On the other hand, for other costimulatory or accessory molecules (CD80, CD86, CD83 and CD40) TNF-alpha was more potent in the induction of expression than PGE1, although in the presence of TNF-alpha plus PGE1 this effect is more pronounced compared to TNF-alpha alone. Under TNF-alpha plus PGE1 treatment the phenotypical maturation of immature DCs are comparable to LPS and therefore TNF-alpha+ PGE1 might be useful for generating ex-vivo DCs to use in protocols of cell vaccination. Further functional evaluation of these mature DCs is warranted.

Células dendríticas (CDs) são células apresentadoras de antígenos altamente eficientes para a imunização contra patógenos e antígenos tumorais. A obtenção de CDs maturis tem sido descrita por diferentes métodos. Portanto, a escolha do método mais apropriado para gerar CDs em cultura de sete dias é pré-requisito essencial para as estratégias imunoterápicas. Aqui relatamos um método de obtenção de uma população enriquecida em monócitos de doadores de sangue e comparamos a maturação das CDs sob o estímulo de quatro agentes. Uma população de monócitos, com pureza de 91 por cento ± 5, foi obtida de 15 doadores. A população monocitária foi mantida em cultura por cinco dias com GM- CSF e IL - 4. No 5° dia, após diferentes condições de maturação, foram analisadas as modificações morfológicas e fenotípicas. Nossos estudos demonstram que o TNF-alfa ou o PGE1 por si só podem induzir a expressão de CD1a de 2.4 a 2.7 vezes, respectivamente, mais do que culturas de CDs com ausência dos agentes de maturação. Alternativamente, para com outras moléculas coestimuladoras ou acessórias (CD80, CD86, CD83 e CD 40) o TNF-alfa foi mais potente na expressão do que o PGE1, embora na presença de ambos o efeito seja mais pronunciado. A maturação fenotípica sob TNF-alfa + PGE1 pode ser comparável ao LPS, concluindo que o TNF-alfa + PGE1 pode ser útil para geração ex-vivo de CDs e útil para protocolos de vacinação celular. Avaliação funcional das CDs é recomendável.