| 1. |
- Lenshof, Andreas, et al.
(författare)
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Efficient Purification of CD4+ Lymphocytes from Peripheral Blood Progenitor Cell Products Using Affinity Bead Acoustophoresis
- 2014
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4930 .- 1552-4922. ; 85A:11, s. 933-941
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Tidskriftsartikel (refereegranskat)abstract
- Processing of peripheral blood progenitor cells (PBPC) for clinical transplantation or research applications aims to effectively isolate or deplete specific cell populations, utilizing primarily magnetic or fluorescence activated sorting methods. Here, we investigated the performance of microfluidic acoustophoresis for the separation of lymphocyte subsets from PBPC, and present a novel method for affinity-bead-mediated acoustic separation of cells which can otherwise not be acoustically discriminated. As the acoustic force on a particle depends on particle size, density and compressibility, targeting of cells by affinity specific beads will generate cell-bead complexes that exhibit distinct acoustic properties relative to nontargeted cells and are, thus, possible to isolate. To demonstrate this, PBPC samples (n = 22) were obtained from patients and healthy donors. Following density gradient centrifugation, cells were labeled with anti-CD4-coated magnetic beads (Dynal) and isolated by acoustophoresis and, for comparison, standard magnetic cell sorting technique in parallel. Targeted CD4+ lymphocytes were acoustically isolated with a mean (±SD) purity of 87 ± 12%, compared with 96 ± 3% for control magnetic sorting. Viability of sorted cells was 95 ± 4% (acoustic) and 97 ± 3% (magnetic), respectively. The mean acoustic separation efficiency of CD4+ lymphocytes to the target fraction was 65 ± 22%, compared with a mean CD4+ lymphocyte recovery of 56 ± 15% for magnetic sorting. Functional testing of targeted CD4+ lymphocytes demonstrated unimpaired mitogen-mediated proliferation capacity and cytokine production. Hematopoietic progenitor cell assays revealed a preserved colony forming ability of nontarget cells post sorting. We conclude that the acoustophoresis platform can be utilized to efficiently isolate bead-labeled CD4+ lymphocytes from PBPC samples in a continuous flow format, with preserved functional capacity of both target and nontarget cells. These results open up for simultaneous affinity-bead-mediated separation of multiple cell populations, something which is not possible with current standard magnetic cell separation technology
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| 2. |
- Li, Hongzhe, et al.
(författare)
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Low/Negative Expression of PDGFR-α Identifies the Candidate Primary Mesenchymal Stromal Cells in Adult Human Bone Marrow.
- 2014
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Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 3:6, s. 965-974
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Tidskriftsartikel (refereegranskat)abstract
- Human bone marrow (BM) contains a rare population of nonhematopoietic mesenchymal stromal cells (MSCs), which are of central importance for the hematopoietic microenvironment. However, the precise phenotypic definition of these cells in adult BM has not yet been reported. In this study, we show that low/negative expression of CD140a (PDGFR-α) on lin(-)/CD45(-)/CD271(+) BM cells identified a cell population with very high MSC activity, measured as fibroblastic colony-forming unit frequency and typical in vitro and in vivo stroma formation and differentiation capacities. Furthermore, these cells exhibited high levels of genes associated with mesenchymal lineages and HSC supportive function. Moreover, lin(-)/CD45(-)/CD271(+)/CD140a(low/-) cells effectively mediated the ex vivo expansion of transplantable CD34(+) hematopoietic stem cells. Taken together, these data indicate that CD140a is a key negative selection marker for adult human BM-MSCs, which enables to prospectively isolate a close to pure population of candidate human adult stroma stem/progenitor cells with potent hematopoiesis-supporting capacity.
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| 3. |
- 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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| 4. |
- Rolandsson Enes, Sara, et al.
(författare)
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Primary mesenchymal stem cells in human transplanted lungs are CD90/CD105 perivascularly located tissue-resident cells
- 2014
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Ingår i: BMJ Open Respiratory Research. - : BMJ Publishing Group. - 2052-4439. ; 1:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Mesenchymal stem cells (MSC) have not only been implicated in the development of lung diseases, but they have also been proposed as a future cell-based therapy for lung diseases. However, the cellular identity of the primary MSC in human lung tissues has not yet been reported. This study therefore aimed to identify and characterise the ‘bona fide’ MSC in human lungs and to investigate if the MSC numbers correlate with the development of bronchiolitis obliterans syndrome in lung-transplanted patients. METHODS: Primary lung MSC were directly isolated or culture-derived from central and peripheral transbronchial biopsies of lung-transplanted patients and evaluated using a comprehensive panel of in vitro and in vivo assays. RESULTS: Primary MSC were enriched in the CD90/CD105 mononuclear cell fraction with mesenchymal progenitor frequencies of up to four colony-forming units, fibroblast/100 cells. In situ staining of lung tissues revealed that CD90/CD105 MSCs were located perivascularly. MSC were tissue-resident and exclusively donor lung-derived even in biopsies obtained from patients as long as 16 years after transplantation. Culture-derived mesenchymal stromal cells showed typical in vitro MSC properties; however, xenotransplantation into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice showed that lung MSC readily differentiated into adipocytes and stromal tissues, but lacked significant in vivo bone formation. CONCLUSIONS: These data clearly demonstrate that primary MSC in human lung tissues are not only tissue resident but also tissue-specific. The identification and phenotypic characterisation of primary lung MSC is an important first step in identifying the role of MSC in normal lung physiology and pulmonary diseases.
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| 5. |
- Rolandsson Enes, Sara, et al.
(författare)
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Specific subsets of mesenchymal stroma cells to treat lung disorders - Finding the Holy Grail.
- 2014
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Ingår i: Pulmonary Pharmacology & Therapeutics. - : Elsevier BV. - 1522-9629 .- 1094-5539. ; 29:2, s. 93-95
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Forskningsöversikt (refereegranskat)abstract
- Accumulating studies, both in animals and human clinical trials with mesenchymal stroma cells (MSC) support the hypothesis of therapeutic effects of these cells in various disorders. However, despite success in immune-mediated disorders such as Crohns' disease, lung disorders such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary disease (IPF) treated with MSC have so far not yielded a revolutionary effect on clinical symptoms. Promising data on immunomodulatory effects in COPD have kept nourishing the research into finding specific traits of MSC beneficial in disease. A heterogeneous population of injected cells might drown a potential therapeutic role of a specific group of MSC. Thus careful analysis of MSC regarding their molecular capabilities such as delivering specific therapeutic vesicles to the environment, or plain cytokine/chemokine fingerprinting might prove useful in augmenting therapies against lung diseases.
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