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| 2. |
- Begum, Setara, 1974, et al.
(författare)
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Characterization and engraftment of long-term serum-free human fetal liver cell cultures.
- 2010
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Ingår i: Cytotherapy. - : Elsevier BV. - 1477-2566 .- 1465-3249. ; 12:2, s. 201-11
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND AIMS: Cultured human hepatocytes have extensive diagnostic and clinical applications. However, the setting-up of new in vitro culture techniques allowing the long-term survival and functional maintenance of adult human hepatocytes represents a formidable challenge. Fetal liver cells (FLC) are attractive candidate donor cells because of their high proliferative capacity. METHODS: Using cell culture and molecular techniques, we studied the in vitro and in vivo characteristics of FLC grown long-term in serum-free conditions. RESULTS: Serum-free FLC obtained from 6-10-week-old human fetal livers grew as multiple clusters in suspension and could be subcultured for at least six passages. These cells maintained stable hepatocyte phenotypes and gene expression patterns in culture for up to 6 months. When a cluster of these cells in various passages was placed on collagen-coated plates, they formed a monolayer and morphologically resembled hepatocytes. The cells expressed alpha -fetoprotein, cytokeratin (CK) 8, CK18 and CK19 and albumin (ALB). Hepatocyte nuclear factor 4alpha and 1beta and cytochrome P450 (CYP) 3A4 and CYP3A7 mRNA expression was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). Cells at different passages, when transplanted into nude mice with liver injury, engrafted successfully, as detected by in situ hybridization using a human-specific DNA probe. Colonies of human-specific CK8, CK18, c-Met nuclear antigen (Ag), mitochondrial Ag, hepatocyte-specific Ag and ALB-expressing cells were present in the livers of recipient animals. CONCLUSIONS: Primary human FLC can be kept in culture consistently over a long time period and are potential candidates for cell therapy and in vitro diagnostics.
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| 5. |
- Grisendi, Giulia, et al.
(författare)
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GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion
- 2010
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Ingår i: Cytotherapy. - : Elsevier BV. - 1465-3249 .- 1477-2566. ; 12:4, s. 466-477
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND AIMS: Bone marrow (BM) mesenchymal stromal/stem cells (MSC) are therapeutic tools in regenerative medicine and oncology. MSC isolation is often performed starting from a separation step based on research-grade 1.077 g/mL density gradient media (DGM). However, MSC clinical application should require the introduction of good manufacturing practice (GMP) reagents. We took advantage of two novel GMP DGM with densities of 1.077 and 1.073 g/mL (Ficoll-Paque PREMIUM and Ficoll-Paque PREMIUM 1.073, respectively) to test whether these reagents could isolate MSC efficiently while simultaneously comparing their performance. METHODS: BM samples were processed using either 1.077 or 1.073 g/mL GMP DGM. BM mononucleated cell (MNC) fractions were analyzed for viability, immunophenotype, clonogenic potential, ex vivo expansion and differentiation potential. RESULTS: No differences were noticed in cell recovery and viability between the groups. Fluorescence-activated cell-sorting (FACS) analyzes on freshly isolated cells indicated that the 1.073 g/mL GMP DGM more efficiently depleted the CD45(+) fraction in comparison with 1.077 GMP DGM. Moreover, in the 1.073 group, fibroblastic colony-forming units (CFU-F) were 1.5 times higher and the final MSC yield 1.8 times increased after four passages. Both reagents isolated MSC with the expected phenotype; however, 1.073-isolated MSC showed a higher expression of CD90, CD146 and GD2. Additionally, MSC from both groups were capable of fully differentiating into bone, adipose cells and cartilage. CONCLUSIONS: Both GMP DGM enriched MSC from BM samples, suggesting that these reagents would be suitable for clinical-grade expansions. In addition, the density of 1.073 g/mL provides a significant advantage over 1.077 g/mL GMP DGM, impacting the quantity of MSC obtained and reducing the ex vivo expansion time for optimized cell-based clinical applications.
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| 6. |
- Götherström, Cecilia, et al.
(författare)
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Fetal and adult multipotent mesenchymal stromal cells are killed by different pathways
- 2011
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Ingår i: Cytotherapy. - : Elsevier BV. - 1465-3249 .- 1477-2566. ; 13:3, s. 269-278
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Tidskriftsartikel (refereegranskat)abstract
- Background aims. Multipotent mesenchymal stromal cells, also known as mesenchymal stem cells (MSC), can be isolated from adult and fetal tissues. Recently, there has been considerable interest in MSC because they have features favorable for transplantation, namely their multipotency and non-immunogenic properties. Methods. We analyzed how human MSC derived from first-trimester fetal liver and adult bone marrow interact with naive and activated innate natural killer (NK) cells. NK cell function was studied by measuring killing of MSC, as well as degranulation (CD107a) induced by MSC. To assess the importance of NK cell killing, expression of surface epitopes was analyzed by flow cytometry on MSC before and after stimulation with interferon (IFN)gamma gamma. Results. Fetal and adult MSC express several ligands to activating NK cell receptors as well as low levels of HLA class I, with large inter-individual variation. Naive peripheral blood NK cells did not lyse fetal or adult MSC, whereas interleukin (IL)2 activated allogeneic as well as autologous NK cells did. Pre-incubation of MSC with IFN-gamma gamma increased their levels of HLA class I, protecting them from NK cell recognition. Fetal and adult MSC were preferably killed via the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) pathways, respectively. Blocking NKG2D reduced NK cell degranulation in both fetal and adult MSC. Conclusions. Fetal and adult MSC differ in their interactions with NK cells. Both fetal and adult MSC are susceptible to lysis by activated NK cells, which may have implications for the use of MSC in cell therapy.
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- Hulspas, Ruud, et al.
(författare)
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Purification of regulatory T cells with the use of a fully enclosed high-speed microfluidic system
- 2014
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Ingår i: Cytotherapy. - : Elsevier BV. - 1465-3249 .- 1477-2566. ; 16:10, s. 1384-1389
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Tidskriftsartikel (refereegranskat)abstract
- Background aims. Despite promising advances in cellular therapies, it will be difficult to fully test or implement new therapies until advances are made in the processes for cell preparation. This study describes the use of an advanced prototype of a flow-cytometry cell purification system constructed for operation in a clinical environment to prepare regulatory T cells defined as CD4(+)/CD25(bright)/CD127(neg/low). Methods. The sort performance of the Gigasort system was directly compared with available droplet sorters using mixtures of highly fluorescent and non-fluorescent 5-mu m polystyrene particles. CD4(+)-enriched cell preparations were processed with the use of a sterile, disposable fluid handling unit with a chip containing parallel microfluidic-based sorters. Results. Similar purity and sort efficiency as found with droplet sorters were obtained with the 24-channel chip sorter system. Starting with 450 million fresh peripheral blood mononuclear cells, 150,000 to 1.7 million cells that were, on average, 85% FoxP3-positive and 97% viable, were obtained in <4 h. Conclusions. This study presents a technology adapted to regulatory requirements for clinical cell purification and that achieves high throughput and cell-friendly conditions by use of a microfluidic chip with 24 parallel microsorters, providing a rapid, sterile method of purifying regulatory T cells accurately and with excellent viability.
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| 9. |
- Joshi, Meghnad, 1977, et al.
(författare)
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Fetal liver-derived mesenchymal stromal cells augment engraftment of transplanted hepatocytes.
- 2012
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Ingår i: Cytotherapy. - : Elsevier BV. - 1477-2566 .- 1465-3249. ; 14:6, s. 657-69
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Tidskriftsartikel (refereegranskat)abstract
- One important problem commonly encountered after hepatocyte transplantation is the low numbers of transplanted cells found in the graft. If hepatocyte transplantation is to be a viable therapeutic approach, significant liver parenchyma repopulation is required. Mesenchymal stromal cells (MSC) produce high levels of various growth factors, cytokines and metalloproteinases, and have immunomodulatory effects. We therefore hypothesized that co-transplantation of MSC with human fetal hepatocytes (hFH) could augment in vivo expansion after transplantation. We investigated the ability of human fetal liver MSC (hFLMSC) to augment expansion of phenotypically and functionally well-characterized hFH.
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| 10. |
- Novikova, Liudmila N, et al.
(författare)
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Neuroprotective and growth-promoting effects of bone marrow stromal cells after cervical spinal cord injury in adult rats
- 2011
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Ingår i: Cytotherapy. - : Elsevier BV. - 1465-3249 .- 1477-2566. ; 13:7, s. 873-887
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Tidskriftsartikel (refereegranskat)abstract
- Background aims. Bone marrow stromal cells (BMSC) have been shown to provide neuroprotection after transplantation into the injured central nervous system. The present study investigated whether adult rat BMSC differentiated along a Schwann cell lineage could increase production of trophic factors and support neuronal survival and axonal regeneration after transplantation into the injured spinal cord. Methods. After cervical C4 hemi-section, 5-bromo-2-deoxyuridine (BrdU)/green fluorescent protein (GFP)-labeled BMSC were injected into the lateral funiculus at 1 mm rostral and caudal to the lesion site. Spinal cords were analyzed 2-13 weeks after transplantation. Results and Conclusions. Treatment of native BMSC with Schwann cell-differentiating factors significantly increased production of brain-derived neurotrophic factor in vitro. Transplanted undifferentiated and differentiated BMSC remained at the injection sites, and in the trauma zone were often associated with neurofilament-positive fibers and increased levels of vascular endothelial growth factor. BMSC promoted extensive in-growth of serotonin-positive raphaespinal axons and calcitonin gene-related peptide (CGRP)-positive dorsal root sensory axons into the trauma zone, and significantly attenuated astroglial and microglial cell reactions, but induced aberrant sprouting of CGRP-immunoreactive axons in Rexed's lamina III. Differentiated BMSC provided neuroprotection for axotomized rubrospinal neurons and increased the density of rubrospinal axons in the dorsolateral funiculus rostral to the injury site. The present results suggest that BMSC induced along the Schwann cell lineage increase expression of trophic factors and have neuroprotective and growth-promoting effects after spinal cord injury.
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| 11. |
- Patil, Pradeep B, 1982, et al.
(författare)
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CD271 identifies functional human hepatic stellate cells, which localize in pen-sinusoidal and portal areas in liver after partial hepatectomy
- 2014
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Ingår i: Cytotherapy. - : Elsevier BV. - 1465-3249 .- 1477-2566. ; 16:7, s. 990-999
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Tidskriftsartikel (refereegranskat)abstract
- Background aims. Hepatic stellate cells (HSCs) are liver-resident mesenchymal cells involved in essential processes in the liver. However, knowledge concerning these cells in human livers is limited because of the lack of a simple isolation method. Methods. We isolated fetal and adult human liver cells by immunomagnetic beads coated with antibodies to a mesenchymal stromal cell marker (CD271) to enrich a population of HSCs. The cells were characterized by cell cultivation, immunocytochemistry, flow cytometry, reverse-transcription polymerase chain reaction and immunohistochemistry. Cells were injected into nude mice after partial hepatectomy to study in vivo localization of the cells. Results. In vitro, CD271(+) cells were lipid-containing cells expressing several HSC markers: the glial fibrillary acidic protein, desmin, vimentin and alpha-smooth muscle actin but negative for CK8, albumin and hepatocyte antigen. The cells produced several inflammatory cytokines such as interleukin (IL)-6, IL-1A, IL-1B and IL-8 and matrix metalloproteinases MMP-1 and MMP-3 and inhibitors TIMP-1 and TIMP-2. In vivo, fetal CD271(+) cells were found in the pen-sinusoidal space and around portal vessels, whereas adult CD271(+) cells were found mainly in the portal connective tissue and in the walls of the portal vessels, which co-localized with alpha-smooth muscle actin or desmin. CD271(-) cells did not show this pattern of distribution in the liver parenchyma. Conclusions. The described protocol establishes a method for isolation of mesenchymal cell precursors for hepatic stellate cells, portal fibroblasts and vascular smooth muscle cells. These cells provide a novel culture system to study human hepatic fibrogenesis, gene expression and transcription factors controlling HSC regulation.
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| 12. |
- Pearce, Kim F., et al.
(författare)
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Regulation of advanced therapy medicinal products in Europe and the role of academia
- 2014
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Ingår i: Cytotherapy. - : Elsevier BV. - 1477-2566 .- 1465-3249. ; 16:3, s. 289-297
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Forskningsöversikt (refereegranskat)abstract
- Background aims. Advanced therapy medicinal products (ATMP) are gene therapy, somatic cell therapy or tissue-engineered products regulated under (EC) No. 1394/2007 to ensure their free movement within the European Union while guaranteeing the highest level of health protection for patients. Academic good manufacturing practice (GMP) centers are major contributors in the development of ATMPs and this study assessed the impact of regulations on them. Methods. European academic and non-industrial facilities (n = 747) were contacted, and a representative sample of 50 replied to a detailed questionnaire. Experienced centres were further selected in every Member State (MS) for semi-structured interviews. Indicators of ATMP production and development success were statistically assessed, and opinions about directive implementation were documented. Results. Facilities experienced in manufacturing cell therapy transplant products are the most successful in developing ATMPs. New centres lacking this background struggle to enter the field, and there remains a shortage of facilities in academia participating in translational research. This is compounded by heterogeneous implementation of the regulations across MS. Conclusions. GMP facilities successfully developing ATMPs are present in all MS. However, the implementation of regulations is heterogeneous between MS, with substantial differences in the definition of ATMPs and in the approved manufacturing environment. The cost of GMP compliance is underestimated by research funding bodies. This is detrimental to development of new ATMPs and commercialization of any that are successful in early clinical trials. Academic GMP practitioners should strengthen their political visibility and contribute to the development of functional and effective European Union legislation in this field.
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