| 1. |
- Li, Hongzhe, et al.
(författare)
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Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing
- 2023
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Ingår i: eLife. - : eLife Sciences Publications, Ltd. - 2050-084X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Hematopoiesis is regulated by the bone marrow (BM) stroma. However, cellular identities and functions of the different BM stromal elements in humans remain poorly defined. Based on single-cell RNA sequencing (scRNAseq), we systematically characterized the human non-hematopoietic BM stromal compartment and we investigated stromal cell regulation principles based on the RNA velocity analysis using scVelo and studied the interactions between the human BM stromal cells and hematopoietic cells based on ligand-receptor (LR) expression using CellPhoneDB. scRNAseq led to the identification of six transcriptionally and functionally distinct stromal cell populations. Stromal cell differentiation hierarchy was recapitulated based on RNA velocity analysis and in vitro proliferation capacities and differentiation potentials. Potential key factors that might govern the transition from stem and progenitor cells to fate-committed cells were identified. In situ localization analysis demonstrated that different stromal cells were localized in different niches in the bone marrow. In silico cell-cell communication analysis further predicted that different stromal cell types might regulate hematopoiesis through distinct mechanisms. These findings provide the basis for a comprehensive understanding of the cellular complexity of the human BM microenvironment and the intricate stroma-hematopoiesis crosstalk mechanisms, thus refining our current view on human hematopoietic niche organization.
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| 2. |
- Rosa, Fábio F, et al.
(författare)
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Single-cell transcriptional profiling informs efficient reprogramming of human somatic cells to cross-presenting dendritic cells
- 2022
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Ingår i: Science Immunology. - : American Association for the Advancement of Science (AAAS). - 2470-9468. ; 7:69, s. 1-18
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Tidskriftsartikel (refereegranskat)abstract
- Type 1 conventional dendritic cells (cDC1s) are rare immune cells critical for the induction of antigen-specific cytotoxic CD8+ T cells, although the genetic program driving human cDC1 specification remains largely unexplored. We previously identified PU.1, IRF8, and BATF3 transcription factors as sufficient to induce cDC1 fate in mouse fibroblasts, but reprogramming of human somatic cells was limited by low efficiency. Here, we investigated single-cell transcriptional dynamics during human cDC1 reprogramming. Human induced cDC1s (hiDC1s) generated from embryonic fibroblasts gradually acquired a global cDC1 transcriptional profile and expressed antigen presentation signatures, whereas other DC subsets were not induced at the single-cell level during the reprogramming process. We extracted gene modules associated with successful reprogramming and identified inflammatory signaling and the cDC1-inducing transcription factor network as key drivers of the process. Combining IFN-γ, IFN-β, and TNF-α with constitutive expression of cDC1-inducing transcription factors led to improvement of reprogramming efficiency by 190-fold. hiDC1s engulfed dead cells, secreted inflammatory cytokines, and performed antigen cross-presentation, key cDC1 functions. This approach allowed efficient hiDC1 generation from adult fibroblasts and mesenchymal stromal cells. Mechanistically, PU.1 showed dominant and independent chromatin targeting at early phases of reprogramming, recruiting IRF8 and BATF3 to shared binding sites. The cooperative binding at open enhancers and promoters led to silencing of fibroblast genes and activation of a cDC1 program. These findings provide mechanistic insights into human cDC1 specification and reprogramming and represent a platform for generating patient-tailored cDC1s, a long-sought DC subset for vaccination strategies in cancer immunotherapy.
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| 3. |
- Ahlstrand, Erik, 1974-, et al.
(författare)
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Highly Reduced Survival in Essential Thrombocythemia and Polycythemia Vera Patients with Vascular Complications during Follow-up
- 2020
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Ingår i: European Journal of Haematology. - : Munksgaard Forlag. - 0902-4441 .- 1600-0609. ; 104:3, s. 271-278
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: To explore the relative importance of risk factors, treatments and blood counts for the occurrence of vascular complications and their impact on life expectancy in Essential Thrombocythemia (ET) and Polycythemia Vera (PV).METHODS: Nested case-control study within the Swedish MPN registry. From a cohort of 922 ET patients and 763 PV patients, 71 ET and 81 PV cases with vascular complications were compared to matched controls.RESULTS: Incidence of vascular complications were 2.0 and 3.4 events per 100 patient-years in ET and PV, respectively. At diagnosis, no significant risk factor differences were observed between cases and controls in neither of the diseases. At the time of vascular event, ET complication cases did not differ significantly from controls but in PV, cases had significantly higher WBCs and were to a lesser extent treated with antithrombotic and cytoreductive therapy. Life expectancy was significantly decreased in both ET and PV cases compared to controls.CONCLUSIONS: The risk of vascular complications is high in both ET and PV and these complications have a considerable impact on life expectancy. The protective effect of antithrombotic and cytoreductive therapy for vascular complications in PV underscores the importance of avoiding undertreatment.
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| 4. |
- Ahrens, Norbert, et al.
(författare)
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Mesenchymal stem cell content of human vertebral bone marrow
- 2004
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Ingår i: Transplantation. - 1534-6080. ; 78:6, s. 925-929
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Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal stem cells (MSCs) are capable of down-regulating alloimmune responses and promoting the engraftment of hematopoietic stem cells. MSCs may therefore be suitable for improving donor-specific tolerance induction in solid-organ transplantation. Cells from cadaveric vertebral bone marrow (V-BM), aspirated iliac crest-BM, and peripheral blood progenitor cells were compared. Cells were characterized by flow cytometry and colony assays. MSCs generated from V-BM were assayed for differentiation capacity and immunomodulatory function. A median 5.7 x 10(8) nucleated cells (NCs) were recovered per vertebral body. The mesenchymal progenitor, colony-forming unit-fibroblast, frequency in V-BM (11.6/10(5) NC, range: 6.0-20.0) was considerably higher than in iliac crest-BM (1.4/10(5) NC, range: 0.4-2.6) and peripheral blood progenitor cells (not detectable). MSC generated from V-BM had the typical MSC phenotype (CD105(pos)CD73(pos)CD45(neg)CD34(neg)), displayed multilineage differentiation potential, and suppressed alloreactivity in mixed lymphocyte reactions. V-BM may be an excellent source for MSC cotransplantation approaches.
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| 5. |
- Bexell, Daniel, et al.
(författare)
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Bone Marrow Multipotent Mesenchymal Stroma Cells Act as Pericyte-like Migratory Vehicles in Experimental Gliomas.
- 2009
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Ingår i: Molecular Therapy. - : Elsevier BV. - 1525-0024 .- 1525-0016. ; 2008:Nov 4., s. 183-190
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Tidskriftsartikel (refereegranskat)abstract
- Bone marrow-derived multipotent mesenchymal stroma cells (MSCs) have emerged as cellular vectors for gene therapy of solid cancers. We implanted enhanced green fluorescent protein-expressing rat MSCs directly into rat malignant gliomas to address their migratory capacity, phenotype, and effects on tumor neovascularization and animal survival. A single intratumoral injection of MSCs infiltrated the majority of invasive glioma extensions (72 +/- 14%) and a substantial fraction of distant tumor microsatellites (32 +/- 6%). MSC migration was highly specific for tumor tissue. Grafted MSCs integrated into tumor vessel walls and expressed pericyte markers alpha-smooth muscle actin, neuron-glia 2, and platelet-derived growth factor receptor-beta but not endothelial cell markers. The pericyte marker expression profile and perivascular location of grafted MSCs indicate that these cells act as pericytes within tumors. MSC grafting did not influence tumor microvessel density or survival of tumor-bearing animals. The antiangiogenic drug Sunitinib markedly reduced the numbers of grafted MSCs migrating within tumors. We found no MSCs within gliomas following intravenous (i.v.) injections. Thus, MSCs should be administered by intratumoral implantations rather than by i.v. injections. Intratumorally grafted pericyte-like MSCs might represent a particularly well-suited vector system for delivering molecules to affect tumor angiogenesis and for targeting cancer stem cells within the perivascular niche.Molecular Therapy (2008); doi:10.1038/mt.2008.229.
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| 6. |
- Bexell, Daniel, et al.
(författare)
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Rat Multipotent Mesenchymal Stromal Cells Lack Long-Distance Tropism to 3 Different Rat Glioma Models
- 2012
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Ingår i: Neurosurgery. - 0148-396X. ; 70:3, s. 731-739
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Viral gene therapy of malignant brain tumors has been restricted by the limited vector distribution within the tumors. Multipotent mesenchymal stromal cells (MSCs) and other precursor cells have shown tropism for gliomas, and these cells are currently being explored as potential vehicles for gene delivery in glioma gene therapy. OBJECTIVE: To investigate MSC migration in detail after intratumoral and extratumoral implantation through syngeneic and orthotopic glioma models. METHODS: Adult rat bone marrow-derived MSCs were transduced to express enhanced green fluorescent protein and implanted either directly into or at a distance from rat gliomas. RESULTS: We found no evidence of long-distance MSC migration through the intact striatum toward syngeneic D74(RG2), N32, and N29 gliomas in the ipsilateral hemisphere or across the corpus callosum to gliomas located in the contralateral hemisphere. After intratumoral injection, MSCs migrated extensively, specifically within N32 gliomas. The MSCs did not proliferate within tumors, suggesting a low risk of malignant transformation of in vivo grafted cell vectors. Using a model for surgical glioma resection, we found that intratumorally grafted MSCs migrate efficiently within glioma remnants after partial surgical resection. CONCLUSION: The findings point to limitations for the use of MSCs as vectors in glioma gene therapy, although intratumoral MSC implantation provides a dense and tumor-specific vector distribution.
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| 7. |
- Bexell, Daniel, et al.
(författare)
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Toward Brain Tumor Gene Therapy Using Multipotent Mesenchymal Stromal Cell Vectors.
- 2010
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Ingår i: Molecular Therapy. - : Elsevier BV. - 1525-0024 .- 1525-0016. ; May 4, s. 1067-1075
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Tidskriftsartikel (refereegranskat)abstract
- Gene therapy of solid cancers has been severely restricted by the limited distribution of vectors within tumors. However, cellular vectors have emerged as an effective migratory system for gene delivery to invasive cancers. Implanted and injected multipotent mesenchymal stromal cells (MSCs) have shown tropism for several types of primary tumors and metastases. This capacity of MSCs forms the basis for their use as a gene vector system in neoplasms. Here, we review the tumor-directed migratory potential of MSCs, mechanisms of the migration, and the choice of therapeutic transgenes, with a focus on malignant gliomas as a model system for invasive and highly vascularized tumors. We examine recent findings demonstrating that MSCs share many characteristics with pericytes and that implanted MSCs localize primarily to perivascular niches within tumors, which might have therapeutic implications. The use of MSC vectors in cancer gene therapy raises concerns, however, including a possible MSC contribution to tumor stroma and vasculature, MSC-mediated antitumor immune suppression, and the potential malignant transformation of cultured MSCs. Nonetheless, we highlight the novel prospects of MSC-based tumor therapy, which appears to be a promising approach.
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| 8. |
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| 9. |
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| 10. |
- Brune, Jan Claas, et al.
(författare)
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Mesenchymal stromal cells from primary osteosarcoma are non-malignant and strikingly similar to their bone marrow counterparts.
- 2011
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Ingår i: International Journal of Cancer. - : Wiley. - 0020-7136 .- 1097-0215. ; 129, s. 319-330
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Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal stromal cells (MSC) are multipotent cells that can be isolated from a number of human tissues. In cancer, MSC have been implicated with tumor growth, invasion, metastasis, drug resistance and were even suggested as possible tumor-initiating cells in osteosarcoma (OS). However, MSC from OS and their possible tumor origin have not yet been thoroughly investigated. Therefore, primary OS mesenchymal progenitors and OS-derived MSC were studied. OS samples contained very high frequencies of mesenchymal progenitor cells as measured by the CFU-F assay (median: 1,117 colonies per 10(5) cells, range: 133 - 3,000, n=6). This is considerably higher compared to other human tissues such as normal bone marrow (1.3 ± 0.2 colonies per 10(5) cells, n=8). OS-derived MSC (OS-MSC) showed normal MSC morphology and expressed the typical MSC surface marker profile (CD105/CD73/CD90/CD44/HLA-classI/CD166 positive, CD45/CD34/CD14/CD19/HLA-DR/CD31 negative). Furthermore, all OS-MSC samples could be differentiated into the osteogenic lineage, and all but one sample into adipocytes and chondrocytes. Genetic analysis of OS-MSC as well as OS-derived spheres showed no tumor-related chromosomal aberrations. OS-MSC expression of markers related to tumor-associated fibroblasts (fibroblast surface protein, alpha-smooth muscle actin, vimentin) was comparable to bone marrow MSC and OS-MSC growth was considerably affected by tyrosine kinase inhibitors. Taken together, our results demonstrate that normal, non-malignant mesenchymal stroma cells are isolated from OS when MSC culture techniques are applied. OS-MSC represent a major constituent of the tumor microenvironment, and they share many properties with bone marrow-derived MSC. © 2010 UICC.
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| 11. |
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| 12. |
- Bräunig, Sandro, et al.
(författare)
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Three-dimensional spatial mapping of the human hematopoietic microenvironment in healthy and diseased bone marrow
- 2023
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Ingår i: Cytometry Part A. - 1552-4922. ; 103:10, s. 763-776
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Tidskriftsartikel (refereegranskat)abstract
- The bone marrow hematopoietic microenvironment (HME) plays a pivotal role in regulating normal and diseased hematopoiesis. However, the spatial organization of the human HME has not been thoroughly investigated yet. Therefore, we developed a three-dimensional (3D) immunofluorescence model to analyze changes in the cellular architecture in control and diseased bone marrows (BMs). BM biopsies from patients with myeloproliferative neoplasms (MPNs) were stained sequentially for CD31, CD34, CD45, and CD271 with repetitive bleaching steps to realize five color images with DAPI as a nuclear stain. Hematopoietically normal age-matched BM biopsies served as controls. Twelve subsequent slides per sample were stacked to create three-dimensional bone marrow reconstructions with the imaging program Arivis Visions 4D. Iso-surfaces for niche cells and structures were created and exported as mesh objects for spatial distribution analysis in the 3D creation suite Blender. We recapitulated the bone marrow architecture using this approach and produced comprehensive 3D models of endosteal and perivascular BM niches. MPN bone marrows displayed apparent differences compared to the controls, especially concerning CD271 staining density, megakaryocyte (MK) morphology, and distribution. Furthermore, measurements of the spatial relationships of MKs and hematopoietic stem and progenitor cells with vessels and bone structures in their corresponding niche environments revealed the most pronounced differences in the vascular nice in polycythemia vera. Taken together, using a repetitive staining and bleaching approach allowed us to establish a 5-color analysis of human BM biopsies, which is difficult to achieve with conventional staining approaches. Based on this, we generated 3D BM models which recapitulated key pathological features and, importantly, allowed us to define the spatial relationships between different bone marrow cell types. We, therefore, believe that our method can provide new and valuable insights into bone marrow cellular interaction research.
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| 13. |
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| 14. |
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| 15. |
- Dykes, Josefina, et al.
(författare)
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Efficient removal of platelets from peripheral blood progenitor cell products using a novel micro-chip based acoustophoretic platform.
- 2011
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:8
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Tidskriftsartikel (refereegranskat)abstract
- Excessive collection of platelets is an unwanted side effect in current centrifugation-based peripheral blood progenitor cell (PBPC) apheresis. We investigated a novel microchip-based acoustophoresis technique, utilizing ultrasonic standing wave forces for the removal of platelets from PBPC products. By applying an acoustic standing wave field onto a continuously flowing cell suspension in a micro channel, cells can be separated from the surrounding media depending on their physical properties.
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| 16. |
- Dykes, Josefina, et al.
(författare)
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Rapid and effective CD3 T-cell depletion with a magnetic cell sorting program to produce peripheral blood progenitor cell products for haploidentical transplantation in children and adults.
- 2007
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Ingår i: Transfusion. - : Wiley. - 1537-2995 .- 0041-1132. ; 47:11, s. 2134-2142
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Effective T-cell depletion is a prerequisite for haploidentical peripheral blood progenitor cell (PBPC) transplantation. This study was performed to investigate the performance of magnetic cell sorting–based direct large-scale T-cell depletion, which is an attractive alternative to standard PBPC enrichment procedures. STUDY DESIGN AND METHODS: PBPCs were harvested from 11 human leukocyte antigen (HLA)-haploidentical donors. T cells labeled with anti-CD3–coated beads were depleted with a commercially available magnetic separation unit (CliniMACS, Miltenyi Biotec) with either the Depletion 2.1 (D2.1, n = 11) or the novel Depletion 3.1 (D3.1, n = 12) program. If indicated, additional CD34+ selections were performed (n = 6). Eleven patients received T-cell-depleted grafts after reduced-intensity conditioning. RESULTS: The median log T-cell depletion was better with the D2.1 compared to the D3.1 (log 3.6 vs. log 2.3, p < 0.05) and was further improved by introducing an immunoglobulin G (IgG)-blocking step (log 4.5 and log 3.4, respectively). The D3.1 was superior to the D2.1 (p < 0.05) in median recovery of CD34+ cells (90% vs. 78%) and in median recovery of CD3– cells (87% vs. 76%). The median processing times per 1010 total cells were 0.90 hours (D2.1) and 0.35 hours (D3.1). The transplanted grafts (directly T-cell–depleted products with or without positively selected CD34+ cells) contained a median of 10.5 × 106 per kg CD34+, 0.93 × 105 per kg CD3+, and 11.6 × 106 per kg CD56+. Rapid engraftment was achieved in 10 patients. The incidences of acute graft-versus-host disease were less than 10 percent (Grade I/II) and 0 percent (Grade III/IV). CONCLUSION: The novel D3.1 program with IgG blocking enables highly effective, time-saving large-scale T-cell depletion. Combining direct depletion techniques with standard CD34+ selection enables the composition of grafts optimized to the specific requirements of the patients.
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| 17. |
- Enes, Sara Rolandsson, et al.
(författare)
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MSC from fetal and adult lungs possess lung-specific properties compared to bone marrow-derived MSC
- 2016
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6, s. 29160-
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Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal stromal cells (MSC) are multipotent cells with regenerative and immune-modulatory properties. Therefore, MSC have been proposed as a potential cell-therapy for bronchiolitis obliterans syndrome (BOS). On the other hand, there are publications demonstrating that MSC might be involved in the development of BOS. Despite limited knowledge regarding the functional role of tissue-resident lung-MSC, several clinical trials have been performed using MSC, particularly bone marrow (BM)-derived MSC, for various lung diseases. We aimed to compare lung-MSC with the well-characterized BM-MSC. Furthermore, MSC isolated from lung-transplanted patients with BOS were compared to patients without BOS. Our study show that lung-MSCs are smaller, possess a higher colony-forming capacity and have a different cytokine profile compared to BM-MSC. Utilizing gene expression profiling, 89 genes including lung-specific FOXF1 and HOXB5 were found to be significantly different between BM-MSC and lung-MSC. No significant differences in cytokine secretion or gene expression were found between MSC isolated from BOS patients compared recipients without BOS. These data demonstrate that lung-resident MSC possess lung-specific properties. Furthermore, these results show that MSC isolated from lung-transplanted patients with BOS do not have an altered phenotype compared to MSC isolated from good outcome recipients.
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| 18. |
- Garofalo, Fabio, et al.
(författare)
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Statistic estimation of cell compressibility based on acoustophoretic separation data
- 2020
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Ingår i: Microfluidics and Nanofluidics. - : Springer Science and Business Media LLC. - 1613-4982 .- 1613-4990. ; 24:8
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Tidskriftsartikel (refereegranskat)abstract
- We present a new experimental method that measures the compressibility of phenotype-specific cell populations. This is done by performing statistical analysis of the cell counts from the outlets of an acoustophoresis chip as a function of the increasing actuator voltage (i.e. acoustic energy density) during acoustophoretic separation. The theoretical separation performance curve, henceforth, Side-Stream Recovery (SSR), vs the piezo-actuator voltage (V) is derived by moment analysis of a one-dimensional model of acoustophoresis separation, accounting for distributions of the cell or microparticle properties and the system parameters (hydrodynamics, radiation force, drag enhancement, and acoustic streaming). The acoustophoretic device is calibrated with polymer microbeads of known properties by fitting the experimental SSR with the theoretical SSR , in which the acoustic energy density is considered proportional to the squared voltage, i.e. Eac=αV2. The fitting parameter α for the calibration procedure is the device effectivity, reflecting the efficiency in performing acoustophoretic microparticle displacement. Once calibrated, the compressibility of unknown cells is estimated by fitting experimental SSR cell data points with the theoretical SSR curve. In this procedure, the microparticle compressibility is the fitting parameter. The method is applied to estimate the compressibility of a variety of cell populations showing its utility in terms of rapid analysis and need for minute sample amounts.
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| 19. |
- Ghazanfari, Roshanak, et al.
(författare)
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Human Non-Hematopoietic CD271pos/CD140alow/neg Bone Marrow Stroma Cells Fulfill Stringent Stem Cell Criteria in Serial Transplantations
- 2016
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Ingår i: Stem Cells and Development. - : Mary Ann Liebert Inc. - 1547-3287 .- 1557-8534. ; 25:21, s. 1652-1658
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Tidskriftsartikel (refereegranskat)abstract
- Human bone marrow contains a population of non-hematopoietic stromal stem/progenitor cells (BMSCs), which play a central role for bone marrow stroma and the hematopoietic microenvironment. However, the precise characteristics and potential stem cell properties of defined BMSC populations have not yet been thoroughly investigated. Using standard adherent colony-forming unit fibroblast (CFU-F) assays, we have previously shown that BMSCs were highly enriched in the nonhematopoietic CD271pos/CD140alow/neg fraction of normal adult human bone marrow. In this study, we demonstrate that prospectively isolated CD271pos/CD140alow/neg BMSCs expressed high levels of hematopoiesis supporting genes and signature mesenchymal and multipotency genes on a single cell basis. Furthermore, CD271pos/CD140alow/neg BMSCs gave rise to non-adherent sphere colonies (mesenspheres) with typical surface marker profile and trilineage in vitro differentiation potential. Importantly, serial transplantations of CD271pos/CD140alow/neg BMSC-derived mesenspheres (single cell and bulk) into immunodeficient NOD scid gamma (NSG) mice showed increased mesensphere numbers and full differentiation potential after both primary and secondary transplantations. In contrast, BMSC self-renewal potential decreased under standard adherent culture conditions. These data therefore indicate that CD271pos/CD140alow/neg BMSCs represent a population of primary stem cells with MSC phenotype and sphere-forming capacity that fulfill stringent functional stem cell criteria in vivo in a serial transplantation setting.
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| 20. |
- Ghazanfari, Roshanak, et al.
(författare)
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Human Primary Bone Marrow Mesenchymal Stromal Cells and Their in vitro Progenies Display Distinct Transcriptional Profile Signatures
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Bone marrow mesenchymal stromal cells (BM-MSCs) are a rare population of cells that gives rise to skeletal tissues and the hematopoietic stroma in vivo. Recently, we have demonstrated that BM-MSCs fulfill stringent in vivo stem cell criteria when propagated as non-adherent mesenspheres but not as adherent-cultured cells. Motivated by these profound functional differences, the current study aimed to identify potential important MSC regulators by investigating global gene expression profiles of adherent and non-adherent culture-derived BM-MSCs in comparison with primary BM-MSCs. A substantial number of genes were differentially expressed between primary and culture-expanded cells already early upon culture, and numerous genes were found to be different when comparing adherent and non-adherent BM-MSCs. Cluster analysis identified 16 sets of genes of which two displayed comparable gene expression levels in primary and non-adherent cultured cells, but not in adherent cultured cells. This pattern suggested that these clusters contained candidate regulators of BM-MSCs. Gene expression differences were confirmed for selected genes and BM-MSC transcription factors by protein analysis and RT-PCR, respectively. Taken together, these data demonstrated profound gene expression changes upon culture of primary BM-MSCs. Moreover, gene cluster differences provide the basis to uncover the regulatory mechanisms that control primary and cultured BM-MSCs.
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| 21. |
- Hyrenius-Wittsten, Axel, et al.
(författare)
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Genomic profiling and directed ex vivo drug analysis of an unclassifiable myelodysplastic/myeloproliferative neoplasm progressing into acute myeloid leukemia
- 2016
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Ingår i: Genes Chromosomes and Cancer. - : Wiley. - 1045-2257. ; 55:11, s. 847-854
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Tidskriftsartikel (refereegranskat)abstract
- Myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN-U) are rare genetically heterogeneous hematologic diseases associated with older age and a poor prognosis. If the disease progresses into acute myeloid leukemia (AML), it is often refractory to treatment. To gain insight into genetic alterations associated with disease progression, whole exome sequencing and single nucleotide polymorphism arrays were used to characterize the bone marrow and blood samples from a 39-year-old woman at MDS/MPN-U diagnosis and at AML progression, in which routine genetic diagnostics had not identified any genetic alterations. The data revealed the presence of a partial tandem duplication of the MLL gene as the only detectable copy number change and 11 non-silent somatic mutations, including DNMT3A R882H and NRAS G13D. All somatic lesions were present both at initial MDS/MPN-U diagnosis and at AML presentation at similar mutant allele frequencies. The patient has since had two extramedullary relapses and is at high risk of a future bone marrow relapse. A directed ex vivo drug sensitivity analysis showed that the patient's AML cells are sensitive to, for example, the MEK inhibitor trametinib and the proteasome inhibitor bortezomib, indicating that she may benefit from treatment with these drugs.
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| 22. |
- Isern, Joan, et al.
(författare)
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Self-Renewing Human Bone Marrow Mesenspheres Promote Hematopoietic Stem Cell Expansion
- 2013
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 3:5, s. 1714-1724
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Tidskriftsartikel (refereegranskat)abstract
- Strategies for expanding hematopoietic stem cells (HSCs) include coculture with cells that recapitulate their natural microenvironment, such as bone marrow stromal stem/progenitor cells (BMSCs). Plastic-adherent BMSCs may be insufficient to preserve primitive HSCs. Here, we describe a method of isolating and culturing human BMSCs as nonadherent mesenchymal spheres. Human mesenspheres were derived from CD45(-) CD31(-) CD71(-) CD146(+) CD105(+) nestin(+) cells but could also be simply grown from fetal and adult BM CD45(-)-enriched cells. Human mesenspheres robustly differentiated into mesenchymal lineages. In culture conditions where they displayed a relatively undifferentiated phenotype, with decreased adherence to plastic and increased self-renewal, they promoted enhanced expansion of cord blood CD34(+) cells through secreted soluble factors. Expanded HSCs were serially transplantable in immunodeficient mice and significantly increased long-term human hematopoietic engraftment. These results pave the way for culture techniques that preserve the self-renewal of human BMSCs and their ability to support functional HSCs.
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| 23. |
- Kadefors, Måns, et al.
(författare)
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CD105+CD90+CD13+ identifies a clonogenic subset of adventitial lung fibroblasts
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal cells are important components of specified niches in the lung, and can mediate a wide range of processes including tissue regeneration and repair. Dysregulation of these processes can lead to improper remodeling of tissue as observed in several lung diseases. The mesenchymal cells responsible remain poorly described, partially due to the heterogenic nature of the mesenchymal compartment and the absence of appropriate markers. Here, we describe that CD105+CD90+ mesenchymal cells can be divided into two populations based on their expression of CD13/aminopeptidase N (CD105+CD90+CD13− and CD105+CD90+CD13+). By prospective isolation using FACS, we show that both these populations give rise to clonogenic fibroblast-like cells, but with an increased clonogenic and proliferative capacity of CD105+CD90+CD13+ cells. Transcriptomic and spatial analysis pinpoints an adventitial fibroblast subset as the origin of CD105+CD90+CD13+ clonogenic mesenchymal cells in human lung.
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| 24. |
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| 25. |
- Ku, Anson, et al.
(författare)
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Acoustic Enrichment of Extracellular Vesicles from Biological Fluids
- 2018
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 90:13, s. 8011-8019
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) have emerged as a rich source of biomarkers providing diagnostic and prognostic information in diseases such as cancer. Large-scale investigations into the contents of EVs in clinical cohorts are warranted, but a major obstacle is the lack of a rapid, reproducible, efficient, and low-cost methodology to enrich EVs. Here, we demonstrate the applicability of an automated acoustic-based technique to enrich EVs, termed acoustic trapping. Using this technology, we have successfully enriched EVs from cell culture conditioned media and urine and blood plasma from healthy volunteers. The acoustically trapped samples contained EVs ranging from exosomes to microvesicles in size and contained detectable levels of intravesicular microRNAs. Importantly, this method showed high reproducibility and yielded sufficient quantities of vesicles for downstream analysis. The enrichment could be obtained from a sample volume of 300 μL or less, an equivalent to 30 min of enrichment time, depending on the sensitivity of downstream analysis. Taken together, acoustic trapping provides a rapid, automated, low-volume compatible, and robust method to enrich EVs from biofluids. Thus, it may serve as a novel tool for EV enrichment from large number of samples in a clinical setting with minimum sample preparation.
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| 26. |
- 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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| 27. |
- Lenshof, Andreas, et al.
(författare)
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High performance multiplex acoustophoresis for WBC subpopulation isolation
- 2017
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Ingår i: MicroTAS 2017 : Savannah, Georgia, USA - Savannah, Georgia, USA. - 1556-5904. - 9780692941836 ; 2017, s. 1385-1386
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Konferensbidrag (refereegranskat)abstract
- Recently, acoustophoresis has been used to fractionate white blood cells (WBC) into subpopulations, Grenvall et al. [1]. However, at a sample throughput of 8-10 μl/min the separation has limited bioanalytical application. In order to substantially increase throughput, we have redesigned and developed a new separation system that enables unmatched WBC separation performance at a volume throughput of 200μl/min and a cell concentration of 106 cells/ml.
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| 28. |
- Li, Hongzhe, et al.
(författare)
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Early growth response 1 regulates hematopoietic support and proliferation in human primary bone marrow stromal cells
- 2020
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Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 1592-8721 .- 0390-6078. ; 105:5, s. 1206-1215
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Tidskriftsartikel (refereegranskat)abstract
- Human bone marrow stromal cells are key elements of the hematopoietic environment and they play a central role in bone and bone marrow physiology. However, how key stromal cell functions are regulated is largely unknown. We analyzed the role of the immediate early response transcription factor EGR1 as key stromal cell regulator and found that EGR1 was highly expressed in prospectively-isolated primary bone marrow stromal cells, downregulated upon culture, and low in non-colony-forming CD45neg stromal cells. Furthermore, EGR1 expression was lower in proliferative regenerating adult and fetal primary cells compared to adult steady-state bone marrow stromal cells. Overexpression of EGR1 in stromal cells induced potent hematopoietic stroma support as indicated by an increased production of transplantable CD34+CD90+ hematopoietic stem cells in expansion co-cultures. The improvement of bone marrow stroma support function was mediated by increased expression of hematopoietic supporting genes, such as VCAM1 and CCL28. Furthermore, EGR1 overexpression markedly decreased stromal cell proliferation whereas EGR1 knockdown caused the opposite effects. These findings thus show that EGR1 is a key stromal transcription factor with a dual role in regulating proliferation and hematopoietic stroma support function that is controlling a genetic program to coordinate the specific functions of bone marrow stromal cells in their different biological contexts.
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| 29. |
- Li, Hongzhe, et al.
(författare)
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Isolation and characterization of primary bone marrow mesenchymal stromal cells
- 2016
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Ingår i: Annals of the New York Academy of Sciences. - : Wiley. - 0077-8923 .- 1749-6632. ; 1370:1, s. 109-118
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Tidskriftsartikel (refereegranskat)abstract
- Bone marrow (BM) contains a rare population of mesenchymal stromal cells (MSCs), which have been characterized as nonhematopoietic skeletal progenitor cells with central importance for the hematopoietic microenvironment. Classically, MSCs are isolated by plastic adherence and subsequent culture. However, as cultured stromal cells differ from their in vivo progenitors, it is important to identify the phenotype of the primary MSCs to study these cells in more detail. In the past years, several surface markers have been reported to be suitable for effective enrichment of BM-MSCs, and recent data indicate that the putative MSC stem/progenitor cell population in human adult BM is highly enriched in Lin− CD45− CD271+ CD140a (PDGFRα)low/− cells. Moreover, surface marker combinations have been described for the isolation of MSCs from murine BM. On the basis of these findings, the role of primary MSCs can now be studied in normal and, importantly, diseased BM. Furthermore, genetically engineered mouse models have been developed as powerful tools to investigate well-defined BM stromal cell populations in vivo. Our discussion aims to provide a concise overview of the current state of the art in BM-MSC isolation in humans and briefly present murine MSC isolation approaches and genetic models.
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| 30. |
- 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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| 31. |
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| 32. |
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| 33. |
- Li, Ou, et al.
(författare)
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Human Embryonic Stem Cell-Derived Mesenchymal Stroma Cells (hES-MSCs) Engraft In Vivo and Support Hematopoiesis without Suppressing Immune Function : Implications for Off-The Shelf ES-MSC Therapies
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal stroma cells (MSCs) have a high potential for novel cell therapy approaches in clinical transplantation. Commonly used bone marrow-derived MSCs (BM-MSCs), however, have a restricted proliferative capacity and cultures are difficult to standardize. Recently developed human embryonic stem cell-derived mesenchymal stroma cells (hES-MSCs) might represent an alternative and unlimited source of hMSCs. We therefore compared human ES-cell-derived MSCs (hES-MP002.5 cells) to normal human bone marrow-derived MSCs (BM-MSCs). hES-MP002.5 cells had lower yet reasonable CFU-F capacity compared with BM-MSC (8±3 versus 29±13 CFU-F per 100 cells). Both cell types showed similar immunophenotypic properties, i.e. cells were positive for CD105, CD73, CD166, HLA-ABC, CD44, CD146, CD90, and negative for CD45, CD34, CD14, CD31, CD117, CD19, CD 271, SSEA-4 and HLA-DR. hES-MP002.5 cells, like BM-MSCs, could be differentiated into adipocytes, osteoblasts and chondrocytes in vitro. Neither hES-MP002.5 cells nor BM-MSCs homed to the bone marrow of immune-deficient NSG mice following intravenous transplantation, whereas intra-femoral transplantation into NSG mice resulted in engraftment for both cell types. In vitro long-term culture-initiating cell assays and in vivo co-transplantation experiments with cord blood CD34+ hematopoietic cells demonstrated furthermore that hES-MP002.5 cells, like BM-MSCs, possess potent stroma support function. In contrast to BM-MSCs, however, hES-MP002.5 cells showed no or only little activity in mixed lymphocyte cultures and phytohemagglutinin (PHA) lymphocyte stimulation assays. In summary, ES-cell derived MSCs might be an attractive unlimited source for stroma transplantation approaches without suppressing immune function.
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| 34. |
- Lim, Hooi Ching, et al.
(författare)
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Development of acoustically isolated extracellular plasma vesicles for biomarker discovery in allogeneic hematopoietic stem cell transplantation
- 2021
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Ingår i: Biomarker research. - : Springer Science and Business Media LLC. - 2050-7771. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Infection and graft-versus-host disease (GvHD) are the major causes for mortality and morbidity of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Plasma-derived extracellular vesicles (EVs) contain disease-related proteins, DNAs and RNAs, and have recently been suggested as potential biomarker candidates for transplantation complications. However, EV isolation from small plasma volumes in clinical biomarker studies using conventional methods is challenging. We therefore investigated if EVs isolated by novel automated acoustic trapping could be developed as potential biomarkers for allo-HSCT complications by performing a clinical proof-of-principle study. Results: Plasma samples were collected from twenty consecutive patients with high-risk/relapsed hematologic malignancies undergoing allo-HSCT before transplantation and post-transplant up to 12 weeks. EVs were isolated from small plasma sample volumes (150 μl) by an automated, acoustofluidic-based particle trapping device, which utilizes a local λ/2 ultrasonic standing wave in a borosilicate glass capillary to capture plasma EVs among pre-seeded polystyrene microbeads through sound scatter interactions. We found that EVs could be reliably isolated from all plasma samples (n = 173) and that EV numbers increased more than 2-fold in the majority of patients after transplantation. Also, sufficient quantities of RNA for downstream microRNA (miRNA) analysis were obtained from all samples and EV miRNA profiles were found to differ from whole plasma profiles. As a proof of principle, expression of platelet-specific miR-142-3p in EVs was shown to correlate with platelet count kinetics after transplantation as expected. Importantly, we identified plasma EV miRNAs that were consistently positively correlated with infection and GvHD, respectively, as well as miRNAs that were consistently negatively correlated with these complications. Conclusions: This study demonstrates that acoustic enrichment of EVs in a clinical biomarker study setting is feasible and that downstream analysis of acoustically-enriched EVs presents a promising tool for biomarker development in allo-HSCT. Certainly, these findings warrant further exploration in larger studies, which will have significant implications not only for biomarker studies in transplantation but also for the broad field of EV-based biomarker discovery.
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| 35. |
- Lindgren, Marie, 1971, et al.
(författare)
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Survival and risk of vascular complications in myelofibrosis—A population-based study from the Swedish MPN group
- 2022
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Ingår i: European Journal of Haematology. - : Wiley. - 0902-4441 .- 1600-0609. ; 109:4, s. 336-342
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Tidskriftsartikel (refereegranskat)abstract
- Objective: To gain knowledge of underlying risk factors for vascular complications and their impact on life expectancy in myelofibrosis. Methods: From a cohort of 392 myelofibrosis patients registered in the Swedish MPN registry 58 patients with vascular complications during follow-up were identified. Patients with vascular complications were compared with both 1:1 matched controls and the entire myelofibrosis cohort to explore potential risk factors for vascular complications and their impact on survival. Results: Incidence of vascular complications was 2.8 events per 100 patient-years and the majority of complications were thrombotic. Patients with complications were significantly older and had lower hemoglobin when compared to the entire cohort. In the case–control analysis, no significant risk factor differences were observed. The major cause of death was vascular complications and median survival was significantly impaired in patients with vascular complications (48 months) compared to controls (92 months). Inferior survival in patients with vascular complications was found to be dependent on IPSS risk category in a Cox regression model. Conclusion: Vascular complications have a considerable impact on survival in MF. At diagnosis, risk assessment by IPSS does not only predict survival but is also associated with the risk of vascular complications.
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| 36. |
- Madelung, Ann, et al.
(författare)
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A novel immunohistochemical sequential multi-labelling and erasing technique enables epitope characterization of bone marrow pericytes in primary myelofibrosis.
- 2012
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Ingår i: Histopathology. - : Wiley. - 0309-0167. ; 60:4, s. 554-560
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Tidskriftsartikel (refereegranskat)abstract
- Madelung A, Bzorek M, Bondo H, Zetterberg E, Bjerrum O W, Hasselbalch H C, Scheding S & Ralfkiaer E (2012) Histopathology A novel immunohistochemical sequential multi-labelling and erasing technique enables epitope characterization of bone marrow pericytes in primary myelofibrosis Aim: In Philadelphia (Ph)-negative chronic myeloproliferative neoplasms, increased microvascular density, bizarre vessel architecture and increased number of pericytes are among the distinct histopathological features. The aim of this study was to characterize bone marrow pericytes in primary myelofibrosis (PMF) using a novel multi-labelling immunohistochemical technique. Methods and results: Bone marrow biopsies from a normal donor (n = 1) and patients with PMF (n = 3) were subjected to an immunohistochemical sequential multi-labelling and erasing technique (SE-technique). Antigens of interest in the first and/or second layer were detected with an immunoperoxidase system and visualized with aminoethylcarbazole. After imaging, erasing and blocking of immunoreagents, the slides were stained with a traditional double immunolabelling procedure. In addition, we applied a Photoshop(®) colour palette, creating a single composite image of the sequential staining procedures. We successfully applied four layers of antibodies on one slide using CD146, smooth muscle actin, CD34, CD271 and Ki67 in different combinations. The SE-technique significantly improves morphological and phenotypical studies in bone marrow specimens. Conclusions: To our knowledge, the SE-technique is the first to multi-label antigens, identifying vessel and pericyte architecture in bone marrow by light microscopy. This technique may unravel novel aspects of the composition of the microvessel structures in patients with PMF and related neoplasms.
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| 37. |
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| 38. |
- Ohlsson, Pelle, et al.
(författare)
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Integrated Acoustic Separation, Enrichment, and Microchip Polymerase Chain Reaction Detection of Bacteria from Blood for Rapid Sepsis Diagnostics
- 2016
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 88:19, s. 9403-9411
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Tidskriftsartikel (refereegranskat)abstract
- This paper describes an integrated microsystem for rapid separation, enrichment, and detection of bacteria from blood, addressing the unmet clinical need for rapid sepsis diagnostics. The blood sample is first processed in an acoustophoresis chip, where red blood cells are focused to the center of the channel by an acoustic standing wave and sequentially removed. The bacteria-containing plasma proceeds to a glass capillary with a localized acoustic standing wave field where the bacteria are trapped onto suspended polystyrene particles. The trapped bacteria are subsequently washed while held in the acoustic trap and released into a polymer microchip containing dried polymerase chain reaction (PCR) reagents, followed by thermocycling for target sequence amplification. The entire process is completed in less than 2 h. Testing with Pseudomonas putida spiked into whole blood revealed a detection limit of 1000 bacteria/mL for this first-generation analysis system. In samples from septic patients, the system was able to detect Escherichia coli in half of the cases identified by blood culture. This indicates that the current system detects bacteria in patient samples in the upper part of the of clinically relevant bacteria concentration range and that a further developed acoustic sample preparation system may open the door for a new and faster automated method to diagnose sepsis.
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| 39. |
- Olm, Franziska, et al.
(författare)
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Acoustophoresis enables the label-free separation of functionally different subsets of cultured bone marrow stromal cells
- 2021
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Ingår i: Cytometry. Part A : the journal of the International Society for Analytical Cytology. - : Wiley. - 1552-4930. ; 99:5, s. 476-487
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Tidskriftsartikel (refereegranskat)abstract
- Culture-expanded mesenchymal stromal cells (MSCs) are promising candidates for clinical cell-based therapies. MSC products are heterogeneous and we therefore investigated whether acoustophoresis, an ultrasound-based separation technology, could be used for the label-free enrichment of functionally different MSC populations. Acoustophoresis uses an ultrasonic standing wave in a microchannel which differentially affects the movement of cells depending on their acoustophysical properties, such as size, density, and compressibility. Human bone marrow MSCs were generated by standard adherent culture in xenofree medium and separated by microchip acoustophoresis. MSCs with up to 20% higher proliferation and 1.7-fold increased clonogenic potential were enriched in the side outlet of the chip compared to the input sample. These cells were significantly smaller (average diameter 14.5 ± 0.4 μm) compared to the center outlet fraction (average diameter 17.1 ± 0.6 μm) and expressed higher levels of genes related to proliferation and stem cell properties (i.e. Ki-67 (1.9-fold), Nanog1 (6.65-fold), Oct4 (2.9-fold), and CXCL12 (1.8-fold), n = 3) in the side outlet compared to input. Fractions of MSCs in G0 /G1 cell cycle phase were significantly enriched in the side fraction and an up to 2.8-fold increase of cells in S/G2 /M phases were observed in center fractions compared to side fractions and 1.3-fold increased compared to the input sample. Acoustophoresis did not compromise MSC phenotype, proliferation, clonogenic capacity, and viability (generally 87-98%), nor did it affect differentiation or immunomodulatory capacities. These results demonstrate that label-free acoustic separation can enrich functionally different MSC subsets which can potentially be employed to produce better-defined stromal cell products from cultured MSCs. Hence, acoustophoresis is a potentially promising separation technology to provide improved cell products for research and possible future clinical use.
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| 40. |
- Olm, Franziska, et al.
(författare)
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Label-free neuroblastoma cell separation from hematopoietic progenitor cell products using acoustophoresis - towards cell processing of complex biological samples
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9:1, s. 8777-8777
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Tidskriftsartikel (refereegranskat)abstract
- Processing of complex cell preparations such as blood and peripheral blood progenitor cell (PBPC) transplants using label-free technologies is challenging. Transplant-contaminating neuroblastoma cells (NBCs) can contribute to relapse, and we therefore aimed to provide proof-of-principle evidence that label-free acoustophoretic separation can be applied for diagnostic NBC enrichment and removal ("purging") from human blood and PBPC products. Neuroblastoma cells spiked into blood and PBPC preparations served as model systems. Acoustophoresis enabled to enrich NBCs from mononuclear peripheral blood cells and PBPC samples with recovery rates of up to 60-97%. When aiming at high purity, NBC purities of up to 90% were realized, however, compromising recovery. Acoustophoretic purging of PBPC products allowed substantial tumour cell depletion of 1.5-2.3 log. PBPC loss under these conditions was considerable (>43%) but could be decreased to less than 10% while still achieving NBC depletion rates of 60-80%. Proliferation of cells was not affected by acoustic separation. These results provide first evidence that NBCs can be acoustically separated from blood and stem cell preparations with high recovery and purity, thus indicating that acoustophoresis is a promising technology for the development of future label-free, non-contact cell processing of complex cell products.
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| 41. |
- Olm, Franziska, et al.
(författare)
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Label-free separation of neuroblastoma patient-derived xenograft (PDX) cells from hematopoietic progenitor cell products by acoustophoresis
- 2021
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Ingår i: Stem Cell Research & Therapy. - : Springer Science and Business Media LLC. - 1757-6512. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundGraft-contaminating tumor cells correlate with inferior outcome in high-risk neuroblastoma patients undergoing hematopoietic stem cell transplantation and can contribute to relapse. Motivated by the potential therapeutic benefit of tumor cell removal as well as the high prognostic and diagnostic value of isolated circulating tumor cells from stem cell grafts, we established a label-free acoustophoresis-based microfluidic technology for neuroblastoma enrichment and removal from peripheral blood progenitor cell (PBPC) products.MethodsNeuroblastoma patient-derived xenograft (PDX) cells were spiked into PBPC apheresis samples as a clinically relevant model system. Cells were separated by ultrasound in an acoustophoresis microchip and analyzed for recovery, purity and function using flow cytometry, quantitative real-time PCR and cell culture.ResultsPDX cells and PBPCs showed distinct size distributions, which is an important parameter for efficient acoustic separation. Acoustic cell separation did not affect neuroblastoma cell growth. Acoustophoresis allowed to effectively separate PDX cells from spiked PBPC products. When PBPCs were spiked with 10% neuroblastoma cells, recoveries of up to 98% were achieved for PDX cells while more than 90% of CD34+ stem and progenitor cells were retained in the graft. At clinically relevant tumor cell contamination rates (0.1 and 0.01% PDX cells in PBPCs), neuroblastoma cells were depleted by more than 2-log as indicated by RT-PCR analysis of PHOX2B, TH and DDC genes, while > 85% of CD34+ cells could be retained in the graft.ConclusionThese results demonstrate the potential use of label-free acoustophoresis for PBPC processing and its potential to develop label-free, non-contact tumor cell enrichment and purging procedures for future clinical use.
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| 42. |
- 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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| 43. |
- Petersson, Klara, et al.
(författare)
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Acoustofluidic hematocrit determination
- 2018
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Ingår i: Analytica Chimica Acta. - : Elsevier BV. - 0003-2670. ; 1000, s. 199-204
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Tidskriftsartikel (refereegranskat)abstract
- Hematocrit (HCT) measurements of blood from patients, blood donors and athletes are routinely performed on a daily basis. These measurements are often performed in centralized hospital labs by whole blood analyzers, which leads to long time-to-result. On site measurements, based on centrifugation can be done, but these assays require manual handling, are slow and can just measure HCT in contrast to the central lab whole blood analyzers. In this work, we present a microfluidic based method to measure HCT in blood samples by acoustic separation of whole blood into discrete regions of plasma and red blood cells. Comparison of the areas of the red blood cell and plasma regions gives an accurate HCT value, with a linear correlation to the centrifugation-based reference method. A readout can be performed within 2 s of acoustic actuation providing a readout accuracy of approximately 3% points (pp) HCT. Additional accuracy can be achieved by extending the acoustic actuation to 20 s, yielding an error of less than 1 pp HCT. This acoustic tool is optimal for integration into a lab-on-a-chip device with in-line measurements of different clinical parameters.
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| 44. |
- 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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| 45. |
- Rolandsson Enes, Sara, et al.
(författare)
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Quantitative proteomic characterization of lung-MSC and bone marrow-MSC using DIA-mass spectrometry
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
-
Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal stromal cells (MSC) are ideal candidates for cell therapies, due to their immune-regulatory and regenerative properties. We have previously reported that lung-derived MSC are tissue-resident cells with lung-specific properties compared to bone marrow-derived MSC. Assessing relevant molecular differences between lung-MSC and bone marrow-MSC is important, given that such differences may impact their behavior and potential therapeutic use. Here, we present an in-depth mass spectrometry (MS) based strategy to investigate the proteomes of lung-MSC and bone marrow-MSC. The MS-strategy relies on label free quantitative data-independent acquisition (DIA) analysis and targeted data analysis using a MSC specific spectral library. We identified several significantly differentially expressed proteins between lung-MSC and bone marrow-MSC within the cell layer (352 proteins) and in the conditioned medium (49 proteins). Bioinformatics analysis revealed differences in regulation of cell proliferation, which was functionally confirmed by decreasing proliferation rate through Cytochrome P450 stimulation. Our study reveals important differences within proteome and matrisome profiles between lung- and bone marrow-derived MSC that may influence their behavior and affect the clinical outcome when used for cell-therapy.
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| 46. |
- 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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| 47. |
- Sun, Jianmin, et al.
(författare)
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Mesenchymal – Development and Regeneration Potential.
- 2015. - 2nd Edition
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Ingår i: The SAGE Encyclopedia of Stem Cell Research. - 2455 Teller Road, Thousand Oaks California 91320 : SAGE Publications, Inc. - 9781483347684 - 9781483347660 ; , s. 752-755
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Bokkapitel (refereegranskat)
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| 48. |
- Sun, Jianmin, et al.
(författare)
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Mesenchymal Stem Cells
- 2015. - 2nd Edition
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Ingår i: The SAGE Encyclopedia of Stem Cell Research. - 2455 Teller Road, Thousand Oaks California 91320 : SAGE Publications, Inc. - 9781483347684 - 9781483347660 ; , s. 762-764
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 49. |
- Svensson, Andreas, et al.
(författare)
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Identification of two distinct mesenchymal stromal cell populations in human malignant glioma
- 2017
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Ingår i: Journal of Neuro-Oncology. - : Springer Science and Business Media LLC. - 0167-594X .- 1573-7373. ; 131:2, s. 245-254
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Tidskriftsartikel (refereegranskat)abstract
- Gene profiling has revealed that malignant gliomas can be divided into four distinct molecular subtypes, where tumors with a mesenchymal gene expression are correlated with short survival. The present investigation was undertaken to clarify whether human malignant gliomas contain endogenous mesenchymal stromal cells (MSC), fulfilling consensus criteria defined by The International Society for Cellular Therapy, recruited from the host. We found that MSC-like cells can be isolated from primary human malignant gliomas. Two distinct MSC-like cell populations, differing in their expression of the CD90 surface marker, were discovered after cell sorting. RNA sequencing revealed further genetic differences between these two cell populations and MSC-like cells lacking CD90 produced higher amounts of VEGF and PGE2 compared to cells with the true MSC phenotype, implying that the CD90− MSC-like cells most probably are more active in tumor vascularization and immunosuppression than their CD90+ counterpart. The results highlight the CD90− subpopulation as an important tumor component, however, its functional effects in glioma remains to be resolved. Using the protocols presented here, it will be possible to isolate, characterize and analyze brain tumor-derived MSC-like cells in more detail and to further test their functions in vitro and in in vivo xenograft models of glioma.
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- Toporski, Jacek, et al.
(författare)
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High-dose iodine-131-metaiodobenzylguanidine with haploidentical stem cell transplantation and posttransplant immunotherapy in children with relapsed/refractory neuroblastoma.
- 2009
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Ingår i: Biology of Blood and Marrow Transplantation. - : Elsevier BV. - 1083-8791. ; 15:9, s. 1077-1085
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Tidskriftsartikel (refereegranskat)abstract
- We evaluated the feasibility and efficacy of using high-dose iodine-131-metaiodobenzylguanidine ((131)I-MIBG) followed by reduced-intensity conditioning (RIC) and transplantation of T cell-depleted haploidentical peripheral blood stem cells (designated haplo-SCT) to treat relapsing/refractory neuroblastoma (RRNB). Five RRNB patients were enrolled: 4 with relapse (3 after autologous SCT) and 1 with induction therapy failure. The preparative regimen included high-dose (131)I-MIBG on day -20, followed by fludarabine (Flu), thiotepa, and melphalan (Mel) from day -8 to -1. Granulocyte-colony stimulating factor (G-CSF)-mobilized, T cell-depleted haploidentical paternal stem cells were infused on day 0 together with cultured donor mesenchymal stem cells. A single dose of rituximab was given on day +1. After cessation of short immunosuppression (mycophenolate, OKT3), 4 children received donor lymphocyte infusion (DLI). (131)I-MIBG infusion and RIC were well tolerated. All patients engrafted. No primary acute graft-versus-host disease (aGVHD) was observed. Four children developed aGVHD after DLI and were successfully treated. Analysis of immunologic recovery showed fast reappearance of potentially immunocompetent natural killer (NK) and T cells, which might have acted as effector cells responsible for the graft-versus-tumor (GVT) effect. Two children are alive and well, with no evidence of disease 40 and 42 months after transplantation. One patient experienced late progression with new bone lesions (sternum) 38 months after haplo-SCT, and is being treated with local irradiation and reinstituted DLI. One patient rejected the graft, was rescued with autologous backup, and died of progressive disease 5 months after transplantation. Another child relapsed 7 months after transplantation and died 5 months later. High-dose (131)I-MIBG followed by RIC and haplo-SCT for RRNB is feasible and promising, because 2 of 5 children on that regimen achieved long-lasting remission. Further studies are needed to evaluate targeted therapy and immune-mediated tumor control in high-risk neuroblastoma.
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