Publications by authors named "Meghnad Joshi"

17 Publications

  • Page 1 of 1

Tissue engineered human ear pinna derived from decellularized goat ear cartilage: clinically useful and biocompatible auricle construct.

Cell Tissue Bank 2021 Mar 3. Epub 2021 Mar 3.

Department of Stem Cells and Regenerative Medicine, D Y Patil Education Society (Deemed University), E 869, D. Y. Patil Vidyanagar, Kasba Bawda, Kolhapur, MS, 416006, India.

Surgery of the entire ear pinna even today presents a challenge to reconstructive surgeons, in the absence of a universally acceptable, quality construct for clinical use. In this article, the authors present a technique to generate a flexible, human size ear with the aim to meet this limitation for ear reconstructive surgeries. The construct was engineered by using a decellularized goat ear cartilage. This was characterized by hematoxylin-eosin (H/E), diamidino-2-phenylindole (DAPI), Masson's trichrome (MT), Alcian Blue (AB) staining and Scanning Electron Microscopy (SEM) for extracellular matrix (ECM) analysis. The decellularization protocol followed yielded complete removal of all cellular components without changing the properties of the ECM. In vivo biocompatibility of the ear pinna showed demonstrable recellularization. Recellularization was tracked using HE, DAPI, MT, AB staining, toluidine staining, SEM, vascular-associated protein (VAP) and CD90 expressing cells. VAP expression revealed specific vasculogenic pattern (angiogenesis). CD90 expression reflected the presence of the stromal cell. The graft maintained the properties of ECM and displayed chondrocyte recruitment. In summary, the decellularized goat ear pinna (cartilage) exhibited xenograft biocompatibility, stable mechanical properties and in vivo chondrocyte recruitment. Subsequently developed tissue-engineered ear pinna offer potential for cartilage flexibility and individualization of ear shape and size for clinical application.
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http://dx.doi.org/10.1007/s10561-021-09911-1DOI Listing
March 2021

Antiviral properties of placental growth factors: A novel therapeutic approach for COVID-19 treatment.

Placenta 2020 09 6;99:117-130. Epub 2020 Aug 6.

Department of Medicine, D Y Patil Medical College, D Y Patil Education Society (Deemed University), E 869 D. Y. Patil Vidyanagar, KasbaBawda, Kolhapur, 416006, MS, India.

The current challenge of the COVID-19 pandemic is complicated by the limited therapeutic options against the virus, with many being anecdotal or still undergoing confirmatory trials, underlining the urgent need for novel strategies targeting the virus. The pulmotropic virus causes loss of oxygenation in severe cases with acute respiratory distress syndrome (ARDS) and need for mechanical ventilation. This work seeks to introduce placental extract-derived biologically active components as a therapeutic option and highlights their mechanism of action relevant to COVID-19 virus. Human placenta has been used in clinical practice for over a century and there is substantial experience in clinical applications of placental extract for different indications. Aqueous extract of human placentacontains growth factors, cytokines/chemokines, natural metabolic and other compounds, anti-oxidants, amino acids, vitamins, trace elements and biomolecules, which individually or in combination show accelerated cellular metabolism, immunomodulatory and anti-inflammatory effects, cellular proliferation and stimulation of tissue regeneration processes. Placental extract treatment is proposed as a suitable therapeutic approach consideringthe above properties which could protect against initial viral entry and acute inflammation of alveolar epithelial cells, reconstitute pulmonary microenvironment and regenerate the lung. We reviewed useful therapeutic information of placental biomolecules in relation to COVID-19 treatment. We propose the new approach of using placental growth factors, chemokines and cytokine which will execute antiviral activity in coordination with innate and humoral immunity and improve patient's immunological responses to COVID-19. Executing a clinical trial using placental extract as preventive, protective and/or therapeutic approach for COVID-19treatment could advance the development of a most promising therapeutic candidate that can join the armamentaria against the COVID-19 virus.
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http://dx.doi.org/10.1016/j.placenta.2020.07.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406421PMC
September 2020

Decellularized amnion scaffold with activated PRP: a new paradigm dressing material for burn wound healing.

Cell Tissue Bank 2018 Sep 5;19(3):423-436. Epub 2018 Mar 5.

Department of Stem Cells and Regenerative Medicine, D.Y.Patil University, D. Y. Patil Vidyanagar, Kasba Bawda, Kolhapur, MS, 416006, India.

Direct application of amnion has greater risk of immunological rejection and infection. Decellularization is an effective method to lower the risk of immune complications and infections. The bioreactor assembly with multiple cassettes was designed for decellurization of multiple amnions with different cell types simultaneously in single run. A detergent-based protocol was modified to remove all cellular components from amnion and diminish the DNA content to render it non-immunogenic. Amnion (n = 10) were treated with 2% sodium dodecyl sulphate (SDS), 5% dimethyl sulfoxide (DMSO) and 2% sodium deoxycholeate (SD). Decellularized amnion samples were analyzed by haematoxylin-eosin staining (HE), Alcian blue pH 1 (AB-pH-1), 4,6-diamnionidino-2-phenylindol (DAPI), Massion's trichrome stain, DNA quantification, mechanical testing and scanning electron microscopy (SEM). Histological analysis showed complete removal of cellular components and the histoarchitecture of scaffold remained intact. Amnion scaffold activated with platelet rich plasma (PRP) and calcium chloride composition supported better adherence to the wound than amnion alone. Only single application showed good healing. In vivo assessment of activated amnion revealed stable dressing. It has good promising outcome. At day 7, histologically the wounds treated with activated amnion were almost closed without scarring and showed well differentiated epidermis, proliferation of keratinocytes, hair follicles and basement membrane as compared to controls and silver nitrate gel dressings in a mouse (Mus musculus). Cryopreservation had no adverse effect on the mechanical properties of the amnion scaffold. Cryopreservation of decellularized amnion by Dulbecco's modified eagle medium (DMEM) was expected to prepare off-the-shelf skin substitutes and preserve them to be immediately available upon request of patients' needs.
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http://dx.doi.org/10.1007/s10561-018-9688-zDOI Listing
September 2018

Comprehensive cytotoxicity studies of superparamagnetic iron oxide nanoparticles.

Biochem Biophys Rep 2018 Mar 8;13:63-72. Epub 2018 Jan 8.

Centre for Interdisciplinary Research, D.Y.Patil University, Kolhapur, India.

Recently lots of efforts have been taken to develop superparamagnetic iron oxide nanoparticles (SPIONs) for biomedical applications. So it is utmost necessary to have in depth knowledge of the toxicity occurred by this material. This article is designed in such way that it covers all the associated toxicity issues of SPIONs. It mainly emphasis on toxicity occurred at different levels including cellular alterations in the form of damage to nucleic acids due to oxidative stress and altered cellular response. In addition focus is been devoted for in vitro and in vivo toxicity of SPIONs, so that a better therapeutics can be designed. At the end the time dependent nature of toxicity and its ultimate faith inside the body is being discussed.
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http://dx.doi.org/10.1016/j.bbrep.2017.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766481PMC
March 2018

Novel Approach Toward the Generation of Tissue Engineered Heart Valve by Using Combination of Antioxidant and Detergent: A Potential Therapy in Cardiovascular Tissue Engineering.

Tissue Eng Regen Med 2017 Dec 18;14(6):755-762. Epub 2017 Sep 18.

1Department of Stem Cells and Regenerative Medicine, D. Y. Patil University, D. Y. Patil Vidyanagar, Kasba Bawda, Kolhapur, Maharashtra 416006 India.

To develop decellularized heart valve scaffold from porcine for heart valve regeneration. Porcine heart valves were decellularized with unique optimized approach by using 1% sodium dodecyl sulfate solution and 5% dimethyl sulfoxide for the first time. Effect of decellularization process on scaffold were characterized by hematoxylin-eosin, 4',6-diamidino-2-phenylindole, Masson's trichrome, alcian blue staining and scanning electron microscopy for extracellular matrix (ECM) analysis in scaffold. The results showed that developed protocol for decellularization of heart valve scaffold shown complete removal of all cellular components, without changing the properties of ECM. The developed protocol was successfully used for heart valve ECM scaffolds development from porcine. The developed protocol seems to be promising solution for the heart valve tissue engineering application.
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http://dx.doi.org/10.1007/s13770-017-0070-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171666PMC
December 2017

Chemokine-mediated robust augmentation of liver engraftment: a novel approach.

Stem Cells Transl Med 2015 Jan 3;4(1):21-30. Epub 2014 Dec 3.

Laboratory for Transplantation Biology and Regenerative Medicine, Department of Surgery, and Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden; The Transplant Institute, Sahlgrenska University Hospital, Gothenburg, Sweden; NovaHep AB, Stockholm, Sweden

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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http://dx.doi.org/10.5966/sctm.2014-0053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4275005PMC
January 2015

TEMPORARY REMOVAL: CD271 identifies functional human hepatic stellate cells, which localize in peri-sinusoidal and portal areas in liver after partial hepatectomy.

Cytotherapy 2014 07 13;16(7):990-9. Epub 2014 May 13.

Sahlgrenska University Hospital, Laboratory for Transplantation and Regenerative Medicine, University of Gothenburg, Gothenburg, Sweden. Electronic address:

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 α-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 peri-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 α-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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http://dx.doi.org/10.1016/j.jcyt.2014.03.001DOI Listing
July 2014

Phenotypic and in vivo functional characterization of immortalized human fetal liver cells.

Scand J Gastroenterol 2014 Jun 15;49(6):705-14. Epub 2014 Apr 15.

Laboratory of Transplantation and Regenerative Medicine, Sahlgrenska Academy at University of Gothenburg , Gothenburg , Sweden.

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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http://dx.doi.org/10.3109/00365521.2013.830328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4059185PMC
June 2014

Expression of major histocompatibility complex class I-related chain A/B (MICA/B) in pancreatic carcinoma.

Int J Oncol 2014 Jan 31;44(1):99-104. Epub 2013 Oct 31.

Department of Surgery and Institute for Research of Digestive System, Lithuanian University of Health Sciences, Kaunas, Lithuania.

Major histocompatibility complex class I-related chain A and B (MICA/B) are two stress-inducible ligands that bind to the immunoreceptor NKG2D and play an important role in mediating cytotoxicity of NK and T cells. Release of MIC molecules from the cell surface is thought to constitute an immune escape mechanism of tumor cells and thus could be associated with more aggressive course of tumor growth. In this study, we investigated the expression of MICA/B in ductal pancreatic carcinoma and serum in relation to tumor stage, differentiation and survival. MICA/B expression in tumor tissues and sera from patients with pancreatic cancer were analyzed by immunohistochemical staining (IHC), western blotting and ELISA, respectively. MICA/B expression was present in 17 of 22 (77%) of the tumors but not in normal pancreatic ductal epithelial cells. Poorly differentiated tumors showed more pronounced MICA/B expression compared to differentiated tumors, but did not correlate significantly to other tumor characteristics. MICA/B-negative tumors displayed significantly lower incidence of lymph node metastases (p<0.01), and less mortality within 3 years following resection (p<0.02). In conclusion, tissue levels of MICA/B expression were elevated in pancreatic cancer cells without elevated levels in serum, despite well-recognized acute phase reactants in serum. Poorly differentiated tumors showed high MICA/B expression, which was related to extended tumor lymph node metastases and less frequent long-term survival.
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http://dx.doi.org/10.3892/ijo.2013.2156DOI Listing
January 2014

Replacement of a tracheal stenosis with a tissue-engineered human trachea using autologous stem cells: a case report.

Tissue Eng Part A 2014 Jan 12;20(1-2):389-97. Epub 2013 Oct 12.

1 Department of Otolaryngology, Head and Neck Surgery, Sahlgrenska University Hospital , University of Gothenburg, Gothenburg, Sweden .

Cell-based therapies involving tissue engineering represent interesting and potentially important strategies for the treatment of patients with various disorders. In this study, using a detergent-enzymatic method, we prepared an intact three-dimensional scaffold of an extracellular matrix 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 the lower part of the larynx. Although the graft provided the patient with an open airway, a week after the surgery, the mucous membrane of the graft was covered by a 1-2 mm thick fungal infection, which was treated with local and systemic antifungal therapy. The airway lumen was postoperatively controlled by fiber endoscopy and found stable and sufficient. However, after 23 days, the patient died due to cardiac arrest but with a patent, open, and stable tracheal transplant and intact anastomoses. Histopathological results of the transplanted tracheal graft during autopsy showed a squamous but not ciliated epithelium, neovascularization, bundles of α-sma-positive muscle cells, serous glands, and nerve fibers 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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http://dx.doi.org/10.1089/ten.TEA.2012.0514DOI Listing
January 2014

Therapeutic potential of autologous stem cell transplantation for cerebral palsy.

Case Rep Transplant 2012 4;2012:825289. Epub 2012 Oct 4.

StemOne Biologicals Pvt. Ltd., Gat No. 801, Urwade, Gadewadi, Tal-Mulshi, Pune 412108, India.

Background. Cerebral palsy (CP) is a severe disabling disease with worldwide incidence being 2 to 3 per 1000 live births. CP was considered as a noncurable, nonreparative disorder, but stem cell therapy offers a potential treatment for CP. Objective. The present study evaluates the safety and efficacy of autologous bone-marrow-derived mononuclear cell (BMMNCs) transplantation in CP patient. Material and Methods. In the present study, five infusions of autologous stem cells were injected intrathecally. Changes in neurological deficits and improvements in function were assessed using Gross Motor Function Classification System (GMFCS-E&R) scale. Results. Significant motor, sensory, cognitive, and speech improvements were observed. Bowel and bladder control has been achieved. On the GMFCS-E&R level, the patient was promoted from grade III to I. Conclusion. In this study, we report that intrathecal infusion of autologous BMMNCs seems to be feasible, effective, and safe with encouraging functional outcome improvements in CP patient.
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http://dx.doi.org/10.1155/2012/825289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3505957PMC
December 2012

Fetal liver-derived mesenchymal stromal cells augment engraftment of transplanted hepatocytes.

Cytotherapy 2012 Jul 16;14(6):657-69. Epub 2012 Mar 16.

Department of Surgery, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden.

Background Aims: 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.

Methods: Two million hFH (passage 6) were either transplanted alone or together (1:1 ratio) with green fluorescence protein-expressing hFLMSC into the spleen of C57BL/6 nude mice with retrorsine-induced liver injury.

Results: After 4 weeks, engraftment of cells was detected by fluorescence in situ hybridization using a human-specific DNA probe. Significantly higher numbers of cells expressing human cytokeratin (CK)8, CK18, CK19, Cysteine-rich MNNG HOS Transforming gene (c-Met), alpha-fetoprotein (AFP), human nuclear antigen, mitochondrial antigen, hepatocyte-specific antigen and albumin (ALB) were present in the livers of recipient animals co-transplanted with hFLMSC compared with those without. Furthermore, expression of human hepatocyte nuclear factor (HNF)-4α and HNF-1β, and cytochrome P450 (CYP) 3A7 mRNA was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) in these animals. In addition, significantly increased amounts of human ALB were detected. Importantly, hFLMSC did not transdifferentiate into hepatocytes.

Conclusions: Our study reports the use of a novel strategy for enhanced liver repopulation and thereby advances this experimental procedure closer to clinical liver cell therapy.
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http://dx.doi.org/10.3109/14653249.2012.663526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3411318PMC
July 2012

Antibodies to kidney endothelial cells contribute to a "leaky" glomerular barrier in patients with chronic kidney diseases.

Am J Physiol Renal Physiol 2012 Apr 21;302(7):F884-94. Epub 2011 Dec 21.

Dept. of Transplantation Surgery, Laboratory for Transplantation and Regenerative Medicine, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska Science Park, Medicinaregatan 8A, Gothenburg, Sweden.

Anti-endothelial cell antibodies (AECA) have been reported to cause endothelial dysfunction, but their clinical importance for 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 report that a higher fraction (56%) of end-stage renal disease (ESRD) patients than healthy controls (5%) have AECA reactive against kidney endothelial cells (P <0.001). The presence of antibodies was associated with female gender (P < 0.001), systolic hypertension (P < 0.01), and elevated TNF-α (P < 0.05). These antibodies markedly decrease expression of both adherens and tight junction proteins VE-cadherin, claudin-1, and zonula occludens-1 and provoked a rapid increase in cytosolic free Ca(2+) and rearrangement of actin filaments in HKEC compared with controls. This was followed by an enhancement in protein flux and phosphorylation of VE-cadherin, events associated with augmented endothelial cell permeability. Additionally, 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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http://dx.doi.org/10.1152/ajprenal.00250.2011DOI Listing
April 2012

Improved intestinal preservation using an intraluminal macromolecular solution: evidence from a rat model.

Transplantation 2010 Feb;89(3):285-90

Department of Surgery, University of Gothenburg, The Institute for Clinical Sciences, Gothenburg, Sweden.

Background: Intestinal preservation injury consists of progressive submucosal edema, with fluid originating both from the lumen and the interstitium. Although vascular flushing aims to control electrolyte shifts in the tissue, the lumen is not addressed, and luminal water and electrolytes enter the tissue during ischemia. Because macromolecular solutions may retain water and electrolytes intraluminally, we investigated whether these solutions administered intraluminally may alleviate preservation injury.

Methods: Sprague-Dawley rat intestines were perfused with University of Wisconsin solution. After excision of the intestines, we intraluminally introduced solutions containing polyethylene glycol 3350 with high (125 mEq) or low (65 mEq) sodium before cold preservation. Controls underwent only vascular flush. After 8, 14, or 20 hr of cold storage, the intestines were analyzed for extent of tissue injury, water retention, brush-border maltase, and tight junction proteins zonula occludens-1 and claudin-3.

Results: Intraluminal composition changed over time, indicating sodium absorption and potassium secretion. After 8 and 14 hr of cold storage, intestines from the low-sodium group had the best morphology and least edema, followed by the controls. Maltase activity slightly decreased in all groups over time and was not affected by the intraluminal polyethylene glycol solutions. Various degrees of delocalization and degradation of zonula occludens-1 and claudin-3 were recorded within the tight junctions, with the most significant effects in intestines from the high-sodium group.

Conclusions: Intraluminal macromolecular solutions may modulate the preservation injury in University of Wisconsin- perfused intestines. Low-sodium solutions administered immediately before preservation may improve preservation injury, but high-sodium solutions may be detrimental.
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http://dx.doi.org/10.1097/TP.0b013e3181c9905aDOI Listing
February 2010

Characterization and engraftment of long-term serum-free human fetal liver cell cultures.

Cytotherapy 2010 Apr;12(2):201-11

Department of Transplantation Surgery, Sahlgrenska University Hospital, University of Gothenburg, Gothenburg, Sweden.

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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http://dx.doi.org/10.3109/14653240903398053DOI Listing
April 2010

Generation of hepatocyte-like cells from in vitro transdifferentiated human fetal pancreas.

Cell Transplant 2009 ;18(2):183-93

Division of Transplantation Surgery, Karolinska University Hospital-Huddinge, Karolinska Institutet, Stockholm, Sweden.

Although the appearance of hepatic foci in the pancreas has been described in animal experiments and in human pathology, evidence for the conversion of human pancreatic cells to liver cells is still lacking. We therefore investigated the developmental plasticity between human embryonic pancreatic cells and liver cells. Cells were isolated and expanded from 7-8-week-old human fetal pancreata (HFP) and were characterized for the absence and presence of pancreatic and hepatic markers. In vitro expanded HFP were treated with fibroblast growth factor 2 (FGF2) and dexamethasone (DX) to induce a liver phenotye in the cells. These treated cells in various passages were further studied for their capacity to be functional in hepatic parenchyma following retrorsine-induced injury in nude C57 black mice. Amylase- and EPCAM-positive-enriched cells isolated from HFP and treated with FGF2 and DX lost expression of pancreatic markers and gained a liver phenotype. Hepatic differentiation was based on the expression (both at the mRNA and protein level) of liver markers albumin and cytokeratin 19. When transplanted in vivo into nude mice treated with retrorsine, both cell types successfully engrafted and functionally differentiated into hepatic cells expressing human albumin, glycogen, dipeptidyl peptidase, and gamma-glutamyltranspeptidase. These data indicate that human fetal pancreatic cells have a capacity to alter their gene expression profile in response to exogenous treatment with FGF2 and DX. It may be possible to generate an unlimited supply of hepatocytes in vitro for cell therapy.
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http://dx.doi.org/10.3727/096368909788341333DOI Listing
August 2009

Evidence for no relevance of anti-major histocompatibility complex class I-related chain a antibodies in liver transplantation.

Liver Transpl 2008 Dec;14(12):1793-802

Division of Clinical Immunology, Huddinge University Hospital, Karolinska Institutet, Stockholm, Sweden.

The polymorphic major histocompatibility complex class I-related chain A (MICA) antigen is being increasingly recognized as a potential target molecule for immune cells during allograft rejection. Here we studied whether MICA is a target antigen for antibodies in liver transplant patients. Eighty-four patients were investigated for the presence of MICA antibodies before and after liver transplantation with MICA-transfected cells and flow cytometry. MICA typing was performed by polymerase chain reaction. Expression of MICA in liver cells was determined by reverse-transcription polymerase chain reaction, Western blotting, and flow cytometry. Liver biopsy specimens from liver transplant patients were examined for MICA expression. A total of 22 of 84 (26%) patients had MICA antibodies either pre-transplant (8/84, 9.5%) or post-transplant (14/84, 17%). No correlation between rejection frequencies (14/22, 63%) or other clinical parameters was observed in patients with MICA antibody versus those without MICA antibody (29/62, 47% P = not significant). We found weak messenger RNA expression for MICA in liver cells but no protein or cell surface expression. In addition, no MICA expression in liver biopsy sections from liver transplant patients was observed at any time point, including rejections. Thus, our preliminary results demonstrate no causal relationship between the presence of MICA antibodies and liver allograft rejections. Therefore, it is likely that MICA may not be an important target antigen during liver allograft rejections.
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http://dx.doi.org/10.1002/lt.21620DOI Listing
December 2008