| 1. |
- 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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| 2. |
- Berg, Malin, 1976, et al.
(författare)
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Replacement of a Tracheal Stenosis with a Tissue-Engineered Human Trachea Using Autologous Stem Cells: A Case Report
- 2014
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Ingår i: Tissue Engineering. Part A. - 1937-3341 .- 1937-335X. ; 20:1-2, s. 389-397
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Tidskriftsartikel (refereegranskat)abstract
- Cell-based therapies, involving tissue engineering represent interesting and potentially important strategies for treatment of patients with various disorders. Here, using a detergent-enzymatic method we prepared an intact 3-dimensional scaffold of an extracellular matrix (ECM) derived from a human cadaver donor trachea, which we repopulated with autologous stem cells and implanted into a 76-year old patient with tracheal stenosis including lower part of the larynx. Although the graft provided the patient with an open airway, a week after surgery, the mucous membrane of the graft was covered by a 1-2mm thick fungal infection, which was treated with local and systemic anti-fungal therapy. The airway lumen was postoperatively controlled by fiberbrandoscopy and found stable and sufficient. However, twenty-three days later the patient died due to cardiac arrest but with a patent, open, stable tracheal transplant and intact anastomoses. Histopathological results of the transplanted tracheal graft at autopsy showed a squamous but not ciliated epithelium, neovascularization, bundles of -sma positive muscle cells, serous glands and nerve fibres with S-100 positive nerve cells in the submucosa and intact chondrocytes in the cartilage. Our findings suggest that although autologous stem cells- engineered tracheal matrices may represent a tool for clinical tracheal replacement. Further preclinical studies are required for generating functional airway grafts and long term effects of such grafts.
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| 3. |
- Breimer, Michael, 1951, et al.
(författare)
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Multicenter evaluation of a novel endothelial cell crossmatch test in kidney transplantation.
- 2009
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Ingår i: Transplantation. - 1534-6080. ; 87:4, s. 549-56
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Despite their clinical importance, clinical routine tests to detect anti-endothelial cell antibodies (AECA) in organ transplantation have not been readily available. This multicenter prospective kidney transplantation trial evaluates the efficacy of a novel endothelial cell crossmatch (ECXM) test to detect donor-reactive AECA associated with kidney allograft rejection. METHODS: Pretransplant serum samples from 147 patients were tested for AECA by a novel flow cytometric crossmatch technique (XM-ONE) using peripheral blood endothelial progenitor cells as targets. Patient enrolment was based on acceptance for transplantation determined by donor lymphocyte crossmatch results. RESULTS: Donor-reactive AECA were found in 35 of 147 (24%) patients. A significantly higher proportion of patients with a positive ECXM had rejections (16 of 35, 46%) during the follow-up of at least 3 months compared with those without AECA (13 of 112, 12%; P<0.00005). Both IgG and IgM AECAs were associated with graft rejections. Mean serum creatinine levels were significantly higher in patients with a positive ECXM test at 3 and 6 months posttransplant. CONCLUSIONS: XM-ONE is quick, easy to perform on whole blood samples and identifies patients at risk for rejection and reduced graft function not identified by conventional lymphocyte crossmatches.
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| 4. |
- Chougule, Priti, et al.
(författare)
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Isolation and characterization of human primary enterocytes from small intestine using a novel method.
- 2012
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Ingår i: Scandinavian journal of gastroenterology. - : Informa UK Limited. - 1502-7708 .- 0036-5521. ; 47:11, s. 1334-43
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Tidskriftsartikel (refereegranskat)abstract
- Abstract Cell culture studies of enterocytes are important in many fields. However, there are difficulties in obtaining cell lines from adult human intestine, such as microbial contamination of cultures from the tissue samples, short life span of enterocytes, overgrowth of mesenchymal cells, etc. Various model used to obtain adult intestinal cell lines are very complex requiring use of feeder layer or gel matrices. The aim of this study was to establish a novel method for the simple and reproducible isolation of human enterocytes. Enterocytes were isolated from SI samples (n = 5) obtained from cadaveric donors using a mechanical procedure, and separation with immunomagnetic beads coated with anti-EpCAM antibodies. Light and electron microscopy, flow cytometry and immunocytochemistry techniques were used to characterize the isolated cells. Immunohistochemical staining of normal SB biopsies confirmed that the cell cultures maintained an in vivo phenotype as reflected in cytokeratin expression CK18, CK20 and expression of intestine-specific markers such as sucrase isomaltase and maltase glucoamylase. Furthermore, the cells strongly expressed TLR-5, 6, 7, 8 and 10 and several molecules such as CD40, CD86, CD44, ICAM-1 and HLA-DR which are important in triggering cell-mediated immune responses. This novel technique provides a unique in vitro system to study the biology of enterocytes in normal conditions as well as to study inflammatory processes in various small bowel disorders.
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| 5. |
- Holgersson, Jan, et al.
(författare)
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A case of acute vascular rejection caused by endothelial-reactive non-HLA antibodies.
- 2007
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Ingår i: Clinical transplants. - 0890-9016. ; , s. 535-8
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- We describe a female patient who, despite negative conventional cross-matches, lost her first kidney graft in an acute humoral rejection. Prior to the second, AB0-incompatible (A1B to A1) living-donor kidney transplant, the patient had negative T- and B-cell cross-matches but had a positive donor-reactive endothelial cell cross-match. Following pre-transplant protein A and GlycoSorb-ABO immunoadsorptions to remove blood group B and anti-endothelial cell antibodies, Mabthera, and IVIG administrations, she was successfully transplanted. By the second post-operative day, creatinine levels were down to 96 micromol/L from 611 micromol/L pre-operatively. On day 9 creatinine rose again, and on the same day the endothelial cell crossmatch became positive for IgG, whereas the T-cell cross-match remained negative and the anti-A1B titers remained low. A kidney biopsy taken on day 10 post-transplant showed a picture of an acute vascular, antibody-mediated rejection. Following rejection treatment and repeated protein A and Glyco-Sorb-ABO immunoadsorptions, the patient's kidney function was again normalized. The use of a recently developed kit (XM-ONE) for the detection of anti-endothelial cell antibodies allowed us to identify a patient at risk for developing acute antibody-mediated rejection as well as to monitor treatment efficacy and post-transplant complications.
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| 6. |
- Joshi, Meghnad, 1977, et al.
(författare)
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Chemokine-Mediated Robust Augmentation of Liver Engraftment: A Novel Approach.
- 2015
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Ingår i: Stem cells translational medicine. - : Oxford University Press (OUP). - 2157-6564 .- 2157-6580. ; 4:1, s. 21-30
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Tidskriftsartikel (refereegranskat)abstract
- Effective repopulation of the liver is essential for successful clinical hepatocyte transplantation. The objective was to improve repopulation of the liver with human hepatocytes using chemokines. We used flow cytometry and immunohistochemistry assays to identify commonly expressed chemokine receptors on human fetal and adult hepatocytes. The migratory capacity of the cells to various chemokines was tested. For in vivo studies, we used a nude mouse model of partial hepatectomy followed by intraparenchymal injections of chemokine ligands at various concentrations. Human fetal liver cells transformed with human telomerase reverse transcriptase were used for intrasplenic cell transplantation. Repopulation and functionality were assessed 4 weeks after transplantation. The receptor CXCR3 was commonly expressed on both fetal and adult hepatocytes. Both cell types migrated efficiently toward corresponding CXC chemokine ligands 9, 10, and 11. In vivo, animals injected with recombinant chemokines showed the highest cell engraftment compared with controls (p < .05). The engrafted cells expressed several human hepatic markers such as cytokeratin 8 and 18 and albumin as well as transferrin, UGT1A1, hepatocyte nuclear factor (1α, 1β, and 4α), cytochrome CYP3A1, CCAAT/enhancer binding protein (α and β), and human albumin compared with controls. No inflammatory cells were detected in the livers at 4 weeks after transplantation. The improved repopulation of transplanted cells is likely a function of the chemokines to mediate cell homing and retention in the injured liver and might be an attractive strategy to augment repopulation of transplanted hepatocytes in vivo.
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| 7. |
- 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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| 8. |
- Patil, Pradeep B, 1982, et al.
(författare)
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Recellularization of acellular human small intestine using bone marrow stem cells.
- 2013
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Ingår i: Stem cells translational medicine. - : Oxford University Press (OUP). - 2157-6564 .- 2157-6580. ; 2:4, s. 307-15
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Tidskriftsartikel (refereegranskat)abstract
- We aimed to produce an acellular human tissue scaffold with a view to test the possibility of recellularization with bone marrow stem cells to produce a tissue-engineered small intestine (TESI). Human small-bowel specimens (n = 5) were obtained from cadaveric organ donors and treated sequentially with 6% dimethyl sulfoxide in hypotonic buffer, 1% Triton X-100, and DNase. Each small intestine (SI) piece (6 cm) was recellularized with EPCAM+ and CD133+ allogeneic bone marrow stem cells. Histological and molecular analysis demonstrated that after decellularization, all cellular components and nuclear material were removed. Our analysis also showed that the decellularized human SI tissue retained its histoarchitecture with intact villi and major structural proteins. Protein films of common extracellular matrix constituents (collagen I, laminin, and fibronectin) were found in abundance. Furthermore, several residual angiogenic factors were found in the decellularized SI. Following recellularization, we found viable mucin-positive goblet cells, CK18+ epithelial cells in villi adjacent to a muscularis mucosa with α-actin+ smooth muscle cells, and a high repopulation of blood vessels with CD31+ endothelial cells. Our results show that in the future, such a TESI would be ideal for clinical purposes, because it can be derived from the recipient's own immunocompatible bone marrow cells, thus avoiding the use of immunosuppression.
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| 9. |
- Aydin, Y., et al.
(författare)
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Activation of PERK-Nrf2 oncogenic signaling promotes Mdm2-mediated Rb degradation in persistently infected HCV culture
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- The mechanism of how chronic hepatitis C virus (HCV) infection leads to such a high rate of hepatocellular carcinoma (HCC) is unknown. We found that the PERK axis of endoplasmic reticulum (ER) stress elicited prominent nuclear translocation of Nrf2 in 100% of HCV infected hepatocytes. The sustained nuclear translocation of Nrf2 in chronically infected culture induces Mdm2-mediated retinoblastoma protein (Rb) degradation. Silencing PERK and Nrf2 restored Mdm2-mediated Rb degradation, suggesting that sustained activation of PERK/Nrf2 axis creates oncogenic stress in chronically infected HCV culture model. The activation of Nrf2 and its nuclear translocation were prevented by ER-stress and PERK inhibitors, suggesting that PERK axis is involved in the sustained activation of Nrf2 signaling during chronic HCV infection. Furthermore, we show that HCV clearance induced by interferon-alpha based antiviral normalized the ER-stress response and prevented nuclear translocation of Nrf2, whereas HCV clearance by DAAs combination does neither. In conclusion, we report here a novel mechanism for how sustained activation of PERK axis of ER-stress during chronic HCV infection activates oncogenic Nrf2 signaling that promotes hepatocyte survival and oncogenesis by inducing Mdm2-mediated Rb degradation.
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| 10. |
- Elebring, Erik, 1990, et al.
(författare)
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Cold-perfusion decellularization of whole-organ porcine pancreas supports human fetal pancreatic cell attachment and expression of endocrine and exocrine markers
- 2017
-
Ingår i: Journal of Tissue Engineering. - : SAGE Publications. - 2041-7314. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Despite progress in the field of decellularization and recellularization, the outcome for pancreas has not been adequate. This might be due to the challenging dual nature of pancreas with both endocrine and exocrine tissues. We aimed to develop a novel and efficient cold-perfusion method for decellularization of porcine pancreas and recellularize acellular scaffolds with human fetal pancreatic stem cells. Decellularization of whole porcine pancreas at 4 degrees C with sodium deoxycholate, Triton X-100 and DNase efficiently removed cellular material, while preserving the extracellular matrix structure. Furthermore, recellularization of acellular pieces with human fetal pancreatic stem cells for 14 days showed attached and proliferating cells. Both endocrine (C-peptide and PDX1) and exocrine (glucagon and -amylase) markers were expressed in recellularized tissues. Thus, cold-perfusion can successfully decellularize porcine pancreas, which when recellularized with human fetal pancreatic stem cells shows relevant endocrine and exocrine phenotypes. Decellularized pancreas is a promising biomaterial and might translate to clinical relevance for treatment of diabetes.
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| 11. |
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| 12. |
- Hernandez, Nidia Maritza, 1979, et al.
(författare)
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Antibodies to kidney endothelial cells contribute to a "leaky" glomerular barrier in patients with chronic kidney diseases.
- 2012
-
Ingår i: American journal of physiology. Renal physiology. - : American Physiological Society. - 1522-1466 .- 1931-857X. ; 302:7
-
Tidskriftsartikel (refereegranskat)abstract
- Anti-endothelial cell antibodies (AECA) are reported to cause endothelial dysfunction but their clinical importance with tissue specific endothelial cells is not clear. We hypothesized that AECA reactive with human kidney endothelial cells (HKEC) may cause renal endothelial dysfunction in patients with chronic kidney diseases. We found that a (p < 0.001) higher fraction of end-stage renal disease patients (ESRD) (56%) have AECA reactive with glomerular endothelial cells as compared to healthy controls (5%). Presence of antibodies was associated with female sex (p < 0.001), systolic hypertension (p < 0.01) and elevated tumour necrosis factor-alpha (TNF-α, p < 0.05). These antibodies markedly decrease expression of adherens and tight junction proteins VE-cadherin, Claudin-1 and ZO-1 and provoked a rapid increase in cytosolic free Ca(2+) and rearrangement of actin filaments in HKEC as compared to controls, followed by an enhancement in protein flux and phosphorylation of VE-cadherin, events associated with augmented endothelial cell permeability. Staining of kidney biopsies from ESRD patients with AECA but not controls demonstrated a marked decrease in adherens and tight junctions in glomerular endothelium, confirming our in vitro data. In summary, our data demonstrate a causal link between AECA and their capacity to induce alterations in glomerular vascular permeability.
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| 13. |
- Joshi, M. G., et al.
(författare)
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Xenobiotic-Induced Fetal Hepatocyte Maturation
- 2019
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Ingår i: Applied In Vitro Toxicology. - : Mary Ann Liebert Inc. - 2332-1512 .- 2332-1539. ; 5:4, s. 180-186
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Human fetal liver progenitor cells (hFLPCs) offer an emerging limitless source for generating mature hepatocytes that can be useful in cell therapies, as well as in pharmacological and toxicological studies. However, hFLPCs have very limited use over adult hepatocytes because of lower expression of drug-metabolizing enzymes. Here, we studied a novel method to achieve physiological maturity of fetal hepatocytes by exposing them to PXR and HNF4α agonist. Materials and Methods: We investigated the expression of cytochrome P450s (CYP3A4 and CYP2B10), glutathione S-transferase Mu 1 (GSTM1), UDP-glucuronosyltransferase 1-1 (UGT1A1), and drug transporters ATP-binding cassette subfamily C member (ABCC2 and ABCC3) by exposing them to rifampicin (Rif, 10 and 30μM), linoleic acid (50 and 100μM), and 0.1% dimethyl sulfoxide (DMSO) for 4, 8, and 12 days. The real-time polymerase chain reaction and western blotting were used to determine mRNA and protein expression of CYPs. Results: Increased CYP expression on the mRNA and protein level was detected in hFLPCs, which are exposed to DMSO- and Rif-treated cells that were much higher than in untreated. There was increase in levels of CYP2B10 protein with respect to time when hFLPCs exposed to DMSO. Discussion and Conclusions: Thus, this is the first report on expression of few phase I, II, and III enzymes in hFLPCs challenged with PXR and HNF4α agonists and to generate hFLPCs expressing drug-metabolizing enzymes similar to that of primary human adult hepatocytes.
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| 14. |
- Kuna, Vijay Kumar, 1987, et al.
(författare)
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Decellularization and Recellularization Methodology for Human Saphenous Veins
- 2018
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Ingår i: Jove-Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; :137
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Tidskriftsartikel (refereegranskat)abstract
- Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and poor patency. Tissue engineering offers a novel solution to generate personalized grafts with a natural extracellular matrix containing the recipients cells using the method of decellularization and recellularization. We show a detailed method for performing decellularization of the human saphenous vein and recellularization by perfusion of peripheral blood. The vein was decellularized by perfusing 1% Triton X-100, 1% tri-n-butyl-phosphate (TnBP) and 2,000 Kunitz units of deoxyribonuclease (DNase). Triton X-100 and TnBP were perfused at 35 mL/min for 4 h while DNase was perfused at 10 mL/min at 37 degrees C for 4 h. The vein was washed in ultrapure water and PBS and then sterilized in 0.1% peracetic acid. It was washed again in PBS and preconditioned in endothelial medium. The vein was connected to a bioreactor and perfused with endothelial medium containing 50 IU/mL heparin for 1 h. Recellularization was performed by filling the bioreactor with fresh blood, diluted 1:1 in Steen solution, and adding endocrine gland-derived vascular endothelial growth factors (80 ng/mL), basic fibroblast growth factors (4 mu L/mL), and acetyl salicylic acid (5 mu g/mL). The bioreactor was then moved into an incubator and perfused for 48 h at 2 mL/min while maintaining glucose between 3 - 9 mmol/L. Later, the vein was washed with PBS, filled with endothelial medium and perfused for 96 h in the incubator. Treatment with Triton X-100, TnBP and DNase decellularized the saphenous vein in 5 cycles. The decellularized vein looked white in contrast to normal and recellularized veins (light red). The hematoxylin & eosin (H&E) staining showed the presence of nuclei only in normal but not in decellularized veins. In the recellularized vein, H&E-staining showed the presence of cells on the lumina! surface of the vein.
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| 15. |
- Kuna, Vijay Kumar, 1987, et al.
(författare)
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Isolation and Decellularization of a Whole Porcine Pancreas
- 2018
-
Ingår i: Jove-Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; :140
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Tidskriftsartikel (refereegranskat)abstract
- Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an allogeneic or xenogeneic source with human cells. A demonstration of methods for the efficient dissection, decellularization, and recellularization of porcine pancreas might benefit the field. Akin to human pancreases, porcine pancreases have a special anatomical arrangement with three lobes (splenic, duodenal, and connection) rounded by the duodenum and small intestine. The duodenal lobe of the pancreas connects to the duodenum by several small blood vessels. Tissue engineering of the pancreas is complicated because of its exocrine and endocrine nature. In this paper, we show a detailed protocol to dissect the whole porcine pancreas and decellularize it with detergents while saving its structure and some extracellular matrix components. To achieve complete perfusion, the aorta is chosen as inlet and the portal vein as outlet. The other blood vessels (hepatic artery, splenic vein, splenic artery, mesenteric artery and vein tree) and bile duct are ligated. To prevent the formation of thrombus, the pig is heparinized and, immediately after dissection, the organ is flushed with cold heparin. To inhibit the action of exocrine enzymes, the pancreas decellularization is set at 4 degrees C. The decellularization is performed by perfusion of Triton X-100, sodium deoxycholate, and deoxyribonuclease, with an intermittent and final extensive washing. With a successful decellularization, the pancreas appears white, and a histological evaluation with hematoxylin and eosin shows an absence of nuclei with a preserved extracellular matrix structure. Thus, the proposed method can be used to successfully dissect and decellularize whole porcine pancreas.
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| 16. |
- Kuna, Vijay Kumar, 1987, et al.
(författare)
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Successful tissue engineering of competent allogeneic venous valves
- 2015
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Ingår i: Journal of Vascular Surgery. - : Elsevier Inc.. - 0741-5214. ; 3:4, s. 421-430
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Tidskriftsartikel (refereegranskat)abstract
- Objective The purpose of this study was to evaluate whether tissue-engineered human allogeneic vein valves have a normal closure time (competency) and tolerate reflux pressure in vitro. Methods Fifteen human allogeneic femoral vein segments containing valves were harvested from cadavers. Valve closure time and resistance to reflux pressure (100 mm Hg) were assessed in an in vitro model to verify competency of the vein valves. The segments were tissue engineered using the technology of decellularization (DC) and recellularization (RC). The decellularized and recellularized vein segments were characterized biochemically, immunohistochemically, and biomechanically. Results Four of 15 veins with valves were found to be incompetent immediately after harvest. In total, 2 of 4 segments with incompetent valves and 10 of 11 segments with competent valves were further decellularized using detergents and DNAse. DC resulted in significant decrease in host DNA compared with controls. DC scaffolds, however, retained major extracellular matrix proteins and mechanical integrity. RC resulted in successful repopulation of the lumen and valves of the scaffold with endothelial and smooth muscle cells. Valve mechanical parameters were similar to the native tissue even after DC. Eight of 10 veins with competent valves remained competent even after DC and RC, whereas the two incompetent valves remained incompetent even after DC and RC. The valve closure time to reflux pressure of the tissue-engineered veins was <0.5 second. Conclusions Tissue-engineered veins with valves provide a valid template for future preclinical studies and eventual clinical applications. This technique may enable replacement of diseased incompetent or damaged deep veins to treat axial reflux and thus reduce ambulatory venous hypertension. Copyright © 2015 by the Society for Vascular Surgery. Published by Elsevier Inc.
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| 17. |
- Liu, Zhiwen, et al.
(författare)
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Wnt ligands 3a and 5a regulate proliferation and migration in human fetal liver progenitor cells
- 2021
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Ingår i: Translational Gastroenterology and Hepatology. - : AME Publishing Company. - 2415-1289. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Background: Since human fetal liver progenitor cells (hFLPC) can differentiate into multiple liver cell types in vitro and in vivo, hFLPC may be a suitable source for cell therapy and regeneration strategies. Imperative for effective clinical applications of hFLPC is the enhanced knowledge of growth factors that mediate and improve migration and proliferation. The canonical wingless/int-1 (Wnt) signal transduction pathway is known to play a key role in proliferation and migration of stem cells. So, we investigated a role for Wnt3a and Wnt5a ligands in regulating the proliferation and migration of hFLPC. Methods: We used alamarBlue assay and transwell migration assay and examined proliferation and migration of hFLPC to Wnt3a and Wnt5a. In addition, the target genes of Wnt signal transduction pathway was identified using microarray analysis and validated by quantitative real-time polymerase chain reaction (qPCR). Results: We found that Wnt3a or Wnt5a independently significantly increased migration and proliferation in a dose-dependent manner which was significantly inhibited by Wnt inhibitors Wnt-C59 or KN-62. Addition of Wnt3a to hFLPC resulted in increased mRNA expression of the known Wnt target genes Axin-2, DKK2, while Wnt5a increased CXCR7, all of which are closely associated with an enhanced proliferation capacity of stem cells. Conclusions: Thus, we report that Wnt3a and Wnt5a may play an important role in the proliferation and migration of hFLPC by possibly regulating key target genes-involved in these processes. Incorporating recombinant human Wnt3a and Wnt5a in regenerative strategies using liver stem/progenitor cells might improve the process of liver regeneration.
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| 18. |
- Methe, Ketaki, et al.
(författare)
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An alternative approach to decellularize whole porcine heart
- 2014
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Ingår i: BioResearch Open Access. - : Mary Ann Liebert Inc. - 2164-7844 .- 2164-7860. ; 3:6, s. 327-338
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Tidskriftsartikel (refereegranskat)abstract
- Scaffold characteristics are decisive for repopulating the acellular tissue with cells. A method to produce such a scaffold from intact organ requires a customized decellularization protocol. Here, we have decellularized whole, intact porcine hearts by serial perfusion and agitation of hypotonic solution, an ionic detergent (4% sodium deoxycholate), and a nonionic detergent (1% Triton X-100). The resultant matrix was characterized for its degree of decellularization, morphological and functional integrity. The protocol used resulted in extensive decellularization of the cardiac tissue, but the cytoskeletal elements (contractile apparatus) of cardiomyocytes remained largely unaffected by the procedure although their membranous organelles were completely absent. Further, several residual angiogenic growth factors were found to be present in the decellularized tissue.
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| 19. |
- Olausson, Michael, 1956, et al.
(författare)
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In vivo application of tissue-engineered veins using autologous peripheral whole blood: A proof of concept study
- 2014
-
Ingår i: EBioMedicine. - : Elsevier BV. - 2352-3964. ; 1:1, s. 72-79
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Tidskriftsartikel (refereegranskat)abstract
- Vascular diseases are increasing health problems affecting >25 million individuals in westernized societies. Such patients could benefit fromtransplantation of tissue-engineered vascular grafts using autologous cells. One challenge that has limited this development is the need for cell isolation, and risks associated with ex vivo expanded stem cells. Herewe demonstrate a novel approach to generate transplantable vascular grafts using decellularized allogeneic vascular scaffolds, repopulatedwith peripheralwhole blood (PWB) in vitro in a bioreactor. Circulating, VEGFR-2+/CD45+ and a smaller fraction of VEGFR-2+/CD14+ cells contributed to repopulation of the graft. SEMmicrographs showed flat cells on the luminal surface of the grafts consistentwith endothelial cells. For clinical validation, two autologous PWBtissue-engineered vein conduits were prepared and successfully used for bypass procedures in two pediatric patients. These results provide a proof of principle for the generation of transplantable vascular grafts using a simple autologous blood sample, making it clinically feasible globally.
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| 20. |
- Olausson, Michael, 1956, et al.
(författare)
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Transplantation of an allogeneic vein bioengineered with autologous stem cells: a proof-of-concept study.
- 2012
-
Ingår i: Lancet. - 1474-547X. ; 380:9838, s. 230-237
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Extrahepatic portal vein obstruction can have severe health consequences. Variceal bleeding associated with this disorder causes upper gastrointestinal bleeding, leading to substantial morbidity and mortality. We report the clinical transplantation of a deceased donor iliac vein graft repopulated with recipient autologous stem cells in a patient with extrahepatic portal vein obstruction. METHODS: A 10 year old girl with extrahepatic portal vein obstruction was admitted to the Sahlgrenska University Hospital in Gothenburg, Sweden, for a bypass procedure between the superior mesenteric vein and the intrahepatic left portal vein (meso Rex bypass). A 9 cm segment of allogeneic donor iliac vein was decellularised and subsequently recellularised with endothelial and smooth muscle cells differentiated from stem cells obtained from the bone marrow of the recipient. This graft was used because the patient's umbilical vein was not suitable and other strategies (eg, liver transplantation) require lifelong immunosuppression. FINDINGS: The graft immediately provided the recipient with a functional blood supply (25-30 cm/s in the portal vein and 40 mL/s in the artery was measured intraoperatively and confirmed with ultrasound). The patient had normal laboratory values for 9 months. However, at 1 year the blood flow was low and, on exploration, the shunt was patent but too narrow due to mechanical obstruction of tissue in the mesocolon. Once the tissue causing the compression was removed the graft dilated. We therefore used a second stem-cell populated vein graft to lengthen the previous graft. After this second operation, the portal pressure was reduced from 20 mm Hg to 13 mm Hg and blood flow was 25-40 cm/s in the portal vein. With restored portal circulation the patient has substantially improved physical and mental function and growth. The patient has no anti-endothelial cell antibodies and is receiving no immunosuppressive drugs. INTERPRETATION: An acellularised deceased donor vein graft recellularised with autologous stem cells can be considered for patients in need of vascular vein shunts without the need for immunosuppression. FUNDING: Swedish Government.
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| 21. |
- 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
-
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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| 22. |
- Patil, Pradeep B, 1982, et al.
(författare)
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Phenotypic and in vivo functional characterization of immortalized human fetal liver cells.
- 2014
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Ingår i: Scandinavian journal of gastroenterology. - : Informa UK Limited. - 1502-7708 .- 0036-5521. ; 49:6, s. 705-714
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Tidskriftsartikel (refereegranskat)abstract
- Abstract We report the establishment and characterization of immortalized human fetal liver progenitor cells by expression of the Simian virus 40 large T (SV40 LT) antigen. Well-characterized cells at various passages were transplanted into nude mice with acute liver injury and tested for functional capacity. The SV40LT antigen-immortalized fetal liver cells showed a morphology similar to primary cells. Cultured cells demonstrated stable phenotypic expression in various passages, of hepatic markers such as albumin, CK 8, CK18, transcription factors HNF-4α and HNF-1α and CYP3A/7. The cells did not stain for any of the tested cancer-associated markers. Albumin, HNF-4α and CYP3A7 expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry showed expression of some progenitor cell markers. In vivo study showed that the cells expressed both fetal and differentiated hepatocytes markers. Our study suggests new approaches to expand hepatic progenitor cells, analyze their fate in animal models aiming at cell therapy of hepatic diseases.
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| 23. |
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| 24. |
- Sumitran-Holgersson, Suchitra, 1961, et al.
(författare)
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Fetal liver cell transplantation
- 2013
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Ingår i: Human Fetal Tissue Transplantation. - London : Springer. - 9781447141716 ; 9781447141716, s. 219-235
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Fetal liver cell transplantation is a promising technique and is entering its clinical application phase. Temporary support of liver function is clearly obtained, and short-term benefit can be achieved for patients. The challenge is to obtain long-term efficacy and demonstrate engraftment and repopulation of the recipient liver. Numerous animal studies have been performed; however, there are many challenging issues that remain to be solved, including increasing engraftment and repopulation and in vitro cell expansion. This knowledge can be used to improve the function of hepatocyte-like cells for drug testing, bioartificial livers, and transplantation. © 2013 Springer-Verlag London. All rights are reserved.
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| 25. |
- Tait, Brian D, et al.
(författare)
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Consensus Guidelines on the Testing and Clinical Management Issues Associated With HLA and Non-HLA Antibodies in Transplantation.
- 2013
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Ingår i: Transplantation. - 1534-6080. ; 95:1, s. 19-47
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The introduction of solid-phase immunoassay (SPI) technology for the detection and characterization of human leukocyte antigen (HLA) antibodies in transplantation while providing greater sensitivity than was obtainable by complement-dependent lymphocytotoxicity (CDC) assays has resulted in a new paradigm with respect to the interpretation of donor-specific antibodies (DSA). Although the SPI assay performed on the Luminex instrument (hereafter referred to as the Luminex assay), in particular, has permitted the detection of antibodies not detectable by CDC, the clinical significance of these antibodies is incompletely understood. Nevertheless, the detection of these antibodies has led to changes in the clinical management of sensitized patients. In addition, SPI testing raises technical issues that require resolution and careful consideration when interpreting antibody results. METHODS: With this background, The Transplantation Society convened a group of laboratory and clinical experts in the field of transplantation to prepare a consensus report and make recommendations on the use of this new technology based on both published evidence and expert opinion. Three working groups were formed to address (a) the technical issues with respect to the use of this technology, (b) the interpretation of pretransplantation antibody testing in the context of various clinical settings and organ transplant types (kidney, heart, lung, liver, pancreas, intestinal, and islet cells), and (c) the application of antibody testing in the posttransplantation setting. The three groups were established in November 2011 and convened for a "Consensus Conference on Antibodies in Transplantation" in Rome, Italy, in May 2012. The deliberations of the three groups meeting independently and then together are the bases for this report. RESULTS: A comprehensive list of recommendations was prepared by each group. A summary of the key recommendations follows. Technical Group: (a) SPI must be used for the detection of pretransplantation HLA antibodies in solid organ transplant recipients and, in particular, the use of the single-antigen bead assay to detect antibodies to HLA loci, such as Cw, DQA, DPA, and DPB, which are not readily detected by other methods. (b) The use of SPI for antibody detection should be supplemented with cell-based assays to examine the correlations between the two types of assays and to establish the likelihood of a positive crossmatch (XM). (c) There must be an awareness of the technical factors that can influence the results and their clinical interpretation when using the Luminex bead technology, such as variation in antigen density and the presence of denatured antigen on the beads. Pretransplantation Group: (a) Risk categories should be established based on the antibody and the XM results obtained. (b) DSA detected by CDC and a positive XM should be avoided due to their strong association with antibody-mediated rejection and graft loss. (c) A renal transplantation can be performed in the absence of a prospective XM if single-antigen bead screening for antibodies to all class I and II HLA loci is negative. This decision, however, needs to be taken in agreement with local clinical programs and the relevant regulatory bodies. (d) The presence of DSA HLA antibodies should be avoided in heart and lung transplantation and considered a risk factor for liver, intestinal, and islet cell transplantation. Posttransplantation Group: (a) High-risk patients (i.e., desensitized or DSA positive/XM negative) should be monitored by measurement of DSA and protocol biopsies in the first 3 months after transplantation. (b) Intermediate-risk patients (history of DSA but currently negative) should be monitored for DSA within the first month. If DSA is present, a biopsy should be performed. (c) Low-risk patients (nonsensitized first transplantation) should be screened for DSA at least once 3 to 12 months after transplantation. If DSA is detected, a biopsy should be performed. In all three categories, the recommendations for subsequent treatment are based on the biopsy results. CONCLUSIONS: A comprehensive list of recommendations is provided covering the technical and pretransplantation and posttransplantation monitoring of HLA antibodies in solid organ transplantation. The recommendations are intended to provide state-of-the-art guidance in the use and clinical application of recently developed methods for HLA antibody detection when used in conjunction with traditional methods.
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