1,720,968 research outputs found
Culture of Human Limbal Epithelial Stem Cells on Tenon's Fibroblast Feeder-Layers: A Translational Approach.
No full textThe coculture technique is the standard method to expand ex vivo limbal stem cells (LSCs) by using inactivated embryonic murine feeder layers (3T3). Although alternative techniques such as amniotic membranes or scaffolds have been proposed, feeder layers are still considered to be the best method, due to their ability to preserve some critical properties of LSCs such as cell growth and viability, stemness phenotype, and clonogenic potential.Furthermore, clinical applications of LSCs cultured on 3T3 have taken place. Nevertheless, for an improved Good Manufacturing Practice (GMP) compliance, the use of human feeder-layers as well as a fine standardization of the process is strictly encouraged.Here, we describe a translational approach in accordance with GMP regulations to culture LSCs onto human Tenon's fibroblasts (TFs). In this chapter, based on our experience we identify and analyze issues that often are encountered by researchers and discuss solutions to common problems
Deletion of NF-kappa/p50 influences the outcome of Notch3-dependent T cell leukemia
No full textThe deregulation of Notch3 signaling inside T-cell compartment of transgenic (N3-tg) mice, induces an aggressive form of T-cell acute lymphoblastic lymphoma (T-ALL), sustained by a constitutive activation of the NF-B canonical pathway, mainly represented by the p50/p65 heterodimer. To clarify the Notch/NF-B relationships in the development of T-ALL, we generated double mutant mice, deleted of the NF-B/p50 subunit in a Notch3 transgenic background (N3-tg/p50-/-). The follow-up of N3-tg/p50-/- versus N3-tg mice revealed that p50 deletion strongly inhibits the development of Notch3-dependent T-ALL. Surprisingly, double mutant succumb earlier than N3-tg mice, displaying the trait of a myeloproliferative disease, with an aberrant accumulation of Mac1+Gr1+ myeloid cells in both spleen and peripheral blood, as well as of granulocyte/monocyte progenitors in the bone marrow. Our preliminary results suggest that Notch3 overexpression in T-cell compartment is able to influence, possibly in trans, the equilibrium of the myeloid compartment and that the ablation of NF-B canonical pathway may impact on the outcomes of a T-cell specific deregulation of Notch signaling. We provide a useful experimental model to extend our understanding of Notch/NF-B interplay in hemopoietic system and to unravel novel relationships between lymphoid and myeloid differentiation
A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon's fibroblasts
No full textGood manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon's fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2-3 cm(2) undamaged Tenon's biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon's fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon's and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes
T-cell specific deregulation of Notch3 receptor induces alterations in the development of myeloid compartment unveiled by the deletion of NF-kappaB/p50 expression
No full textPurpose/Objective: T-cell specific deregulation of Notch3 in transgenicmice (N3-tg), induces the development of a T-cell acute lymphoblastic leukemia (T-ALL), sustained by the constitutive activation of NF-kB canonical pathway. Besides, Notch signalling modulation in bone-marrow stromal cells or in hematopoietic stem cells, has been related to alterations in differentiation/proliferation processes of myeloid cells. To clarify the Notch/NF-kB relationships in the progression of T-ALL and the effects of a T-cell specific deregulation of Notch on myeloid compartment, we decided to delete NF-kB canonical pathway in N3-tg mice. Materials and methods: We generated N3-tg/p50-/- mice, deleted of the NF-kB/p50 subunit in a Notch3 transgenic background. The follow-up of double mutant versus N3-tg mice versus relative controls was conducted and immunophenotyping of hematopoietic cell subsets was performed at different age and in multiple tissues from the indicated animals by flow-cytometry tecniques. Total RNA and protein extract samples, derived from sorted T- or myeloid-cells of our mice models, were processed for RT-qPCR and Western blotting analysis, respectively, to test the expression of Notch-related molecules. Results: The progression of T-ALL, as defined by the peripheral expansion of immature CD4+ CD8+ T cells, was strongly inhibited in N3-tg/p50-/- versus N3-tg mice. However, the double mutant mice succumb earlier than N3-tg counterparts displaying a dramatic increase of Mac1+ Gr1+ myeloid cells in both spleen and blood, as well as of granulocyte/monocyte progenitors in the bone marrow. The expansion of myeloid subsets was detectable at a lower extent also in N3-tg versus wild-type mice. Preliminary data indicate that Mac1+ Gr1+ cells do not express Notch3, suggesting that this receptor may influence the equilibrium of the myeloid compartment mainly in trans, possibly through its interaction with the Jagged-1 ligand. Conclusions: Our results suggest that the NF-kB canonical pathway deletion inhibits the T-ALL progression, thus unveiling the influence of Notch signalling modulation on the behaviour of myeloid cells. We provide a useful model to extend our understanding of Notch/NF-kB interplay in driving the relationships between lymphoid and myeloid compartments in the context of hematological malignancy
The Outcome of Notch3-dependent T Cell Leukemia Is Modified By NF-kappaB Deletion
No full textBackground: The Notch3 deregulation inside T-cell compartment of transgenic (N3-tg) mice, induces an aggressive form of T-cell acute lymphoblastic leukemia (T-ALL), strongly sustained by an NF-κB constitutive activation, mainly represented by the p50/p65-dependent canonical pathway. To clarify the Notch/NF-κB relationships in the onset/progression of T-ALL, we decided to inhibit NF-κB canonical pathway in N3-tg mice. Methods: We generated N3-tg/p50-/- mice, deleted of the NF-κB/p50 subunit in a Notch3 transgenic background. The follow-up of N3-tg/p50-/- versus N3- tg mice was conducted and hematopoietic cell analysis was performed at different ages and in multiple tissues from the indicated animals by flow-cytometry techniques. Results: The p50 deletion inhibited the progression of T-ALL in N3-tg/p50-/- mice, as defined primarily by the peripheral expansion of immature CD4+CD8+ T cells. Surprisingly, the double mutant mice succumb earlier than N3-tg counterparts. Moribund N3-tg/p50-/- mice display the trait of a myeloproliferative disease, with the dramatic expansion of Mac1+Gr1+ myeloid cells in both spleen and blood, as well as of granulocyte/monocyte progenitors in the bone marrow. Preliminary data indicate that these cells do not express Notch3, suggesting that in the absence of p50 expression, Notch3 is able to mainly influence the equilibrium of the myeloid compartment in trans. Conclusions: The results presented suggest that the ablation of NF-κB canonical pathway may strongly impact on the outcomes of a T cell specific deregulation of Notch signaling. Thus, providing a useful experimental model to extend our understanding of Notch/NF-κB interplay and to unravel novel strategies for the therapy of different hematological malignancies
Optimization of the isolation and expansion method of human mediastinal–adipose tissue derived mesenchymal stem cells with virally inactivated GMP-grade platelet lysate
No full textMesenchymal stem cells (MSCs) are adult multipotent cells currently employed in several clinical trials due to their immunomodulating, angiogenic and repairing features. The adipose tissue is certainly considered an eligible source of MSCs. Recently, putative adipose tissue derived MSCs (ADMSCs) have been isolated from the mediastinal depots. However, very little is known about the properties, the function and the potential of human mediastinal ADMSCs (hmADMSCs). However, the lack of standardized methodologies to culture ADMSCs prevents comparison across. Herein for the first time, we report a detailed step by step description to optimize the isolation and the expansion methodology of hmADMSCs using a virally inactivated good manufacturing practice (GMP)-grade platelet lysate, highlighting the critical aspects of the procedure and providing useful troubleshooting suggestions. Our approach offers a reproducible system which could provide standardization across laboratories. Moreover, our system is time and cost effective, and it can provide a reproducible source of adipose stem cells to enable future studies to unravel new insights regard this promising stem cell population
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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