Experimental and economic evaluation of different culture systems for mesenchymal stromal/stem cell expansion for clinical applications

The translation of cell therapies into clinical practice requires a scalable, efficient and cost-effective manufacturing process. This study presents an integrated experimental and cost analysis of different cell culture technologies for commercial manufacture of a novel umbilical cord-derived cell therapy, currently in early phase clinical trials for the treatment of acute graft-versus-host disease (aGvHD). The experimental analysis assessed the expansion and harvest potential of mesenchymal stromal cells (MSCs), derived from umbilical cord matrix (UCM-MSCs), in different scalable cell culture technologies: a multi-layer vessel (ML), a stirred tank bioreactor with microcarrriers (STR), a hollow fiber bioreactor (HF) and a packed-bed bioreactor (PB). The presentation will highlight differences in cell proliferation rate, expansion fold and harvesting efficiency across the technologies. The cells retained their functional properties post culture in all the technologies evaluated. The experimental results were incorporated into a bioprocess economics tool comprising a stochastic cost of goods (COG) and sizing model to evaluate the commercial economic feasibility and robustness of the technologies. The financial and risk rank orders predicted by the tool will be presented, as well as their sensitivity to the reimbursement scenario selected. The model determined industrially relevant scenarios for which no technology will yield a satisfactory gross margin, indicating that many studies are still needed to establish an optimized manufacturing process

Epidemiological factors related to slow progression of the acquired immune deficiency syndrome (AIDS)

Com o objetivo de identificação de fatores envolvidos na progressão lenta para aids, realizou-se estudo transversal para avaliação de dados epidemiológicos de indivíduos infectados pelo Vírus da Imunodeficiência Humana tipo 1 (HIV-1), atendidos no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto-USP. Foram selecionados pacientes, conforme critérios definidos, constituindo duas populações: população 1, composta por lentos progressores (P1), que possuía anticorpos anti-HIV há mais de oito anos e com ocorrência de menos de duas doenças oportunistas no último ano, e a população 2 (P2), pacientes rápidos progressores, com diagnóstico de infecção pelo HIV e doença manifesta a menos de dois anos e com mais de duas doenças oportunistas, diagnosticadas no último ano. Todos os indivíduos foram submetidos a questionário, contendo dados demográficos, profissão, ocorrência de outras doenças sexualmente transmissíveis, forma de contágio, data de diagnóstico e hábitos. O período do estudo foi de março de 1998 a outubro de 1999. Obtivemos na P1: doze homens e quatro mulheres, idade média 30,7 anos, forma de contágio predominantemente sangüínea, tempo de progressão da doença 10,5 anos; P2: 12 homens e 4 mulheres; idade média 34,8 anos, forma de contágio predominantemente sexual, tempo de progressão da doença de 1,5 anos. Tabagismo foi o principal vício em ambas as populações. Quando interrogados sobre a causa do bom estado de saúde, os indivíduos da P1 responderam estar ela relacionada à fé e ao uso adequado das medicações. Os pacientes da P2 não foram interrogados sobre a causa de seu estado de saúde. Quanto à prática sexual, nove pacientes da P1 mantinham relações, sendo cinco sem uso regular do preservativo. Na P2, apenas um paciente utilizava preservativo. Dois pacientes da P1 e um da P2 revelaram ter apresentado DST, Sífilis e Papiloma Vírus Humano. Em vista do reduzido número de pacientes, não foi possível estabelecer associação entre as variáveis estudadas e os padrões de progressão da doença. Os dados sobre hábitos não parecem contribuir para o padrão de desenvolvimento da doença. O estudo oferece um perfil epidemiológico dessas populações de pacientes.To determine the factors involved in slow progression to aids, a transverse study was conducted for the evaluation of the epidemiological data of individuals infected with type 1 human immunodeficiency virus seen at the University Hospital of the Faculty of Medicine of Ribeirão Preto-USP. Patients were choosed in conformity some judgment, establish two populations: population 1, consisting of slow progressors (P1), had been carrying HIV antibodies for more than 8 years, with the occurrence of less than 2 opportunistic diseases in the last year, and population 2 (P2), consisting of rapid progressors, had a diagnosis of HIV infection and overt disease detected less than 2 years before and more than 2 opportunistic diseases diagnosed during the last year. All patients responded to a questionnaire concerning demographic data, profession, occurrence of other sexually transmissible diseases, form of contagion, date of diagnosis, and habits. The study was conducted from March 1998 to October 1999. We obtain in the P1: 12 men and 4 women, mean age 30.7 years, predominant form of contagion: blood route, time of disease progression 10.5 years; P2 12 men and 4 women; mean age 34,8 years, predominant form of contagion: sexual, time of disease progression 1.5 years. Tabagisme was the principal vice in about populations. When asked about motivation yours good health, the subjects of P1 answered to be relationed the faith and medications use. The patients of the P2 did not answer about yours health state. Whatever the sexual custom, 9 patients of the P1 had sexual relations, five without regular use of condom. In the P2 only um patient used condom. Two patients of the P1 and one of P2 declared to have STD, syphilis and Human Papiloma Virus (HPV). Because there are reduces number of patients it’s impossible to make asociation between the variables studies and mesures of the disease progression. The dates about habits don’t contribute for the disease development. The study offers an epidemiological profile of these patient populations

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% ± 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.<br>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% ± 5, foi obtida de 15 doadores. A população monocitária foi mantida em cultura por cinco dias com GM- CSF e IL - 4. No 5º 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

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. (C) 2008 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc

What Do the Transcriptome and Proteome of Menstrual Blood-Derived Mesenchymal Stem Cells Tell Us about Endometriosis?

Given the importance of menstrual blood in the pathogenesis of endometriosis and the multifunctional roles of menstrual mesenchymal stem cells (MenSCs) in regenerative medicine, this issue has gained prominence in the scientific community. Moreover, recent reviews highlight how robust the integrated assessment of omics data are for endometriosis. To our knowledge, no study has applied the multi-omics approaches to endometriosis MenSCs. This is a case-control study at a university-affiliated hospital. MenSCs transcriptome and proteome data were obtained by RNA-seq and UHPLC-MS/MS detection. Among the differentially expressed proteins and genes, we emphasize ATF3, ID1, ID3, FOSB, SNAI1, NR4A1, EGR1, LAMC3, and ZFP36 genes and MT2A, TYMP, COL1A1, COL6A2, and NID2 proteins that were already reported in the endometriosis. Our functional enrichment analysis reveals integrated modulating signaling pathways such as epithelial&ndash;mesenchymal transition (&uarr;) and PI3K signaling via AKT to mTORC1 (&darr; in proteome), mTORC1 signaling, TGF beta signaling, TNFA signaling via NFkB, IL6 STAT3 signaling, and response to hypoxia via HIF1A targets (&uarr; in transcriptome). Our findings highlight primary changes in the endometriosis MenSCs, suggesting that the chronic inflammatory endometrial microenvironment can modulate these cells, providing opportunities for endometriosis etiopathogenesis. Moreover, they identify challenges for future research leveraging knowledge for regenerative and precision medicine in endometriosis