Bruna Maria Manzini1, Adriana da Silva Santos Duarte1, Sundararaj Sankaramanivel2, Aline Lisie Ramos2, Paulo Latuf-Filho3, Cecilia Escanhoela4, Paulo Kharmandayan5, Sara Teresinha Olalla Saad6, Ilka Boin7, Ângela Cristina Malheiros Luzo8. 1. Umbilical Cord Blood Bank, Hematology Hemotherapy Centre/INCT do Sangue, University of Campinas, São Paulo, Brazil. 2. Hematology Hemotherapy Centre/INCT do Sangue, University of Campinas, São Paulo, Brazil. 3. Research Center in Pediatrics, Faculty of Medical Sciences, University of Campinas, São Paulo, Brazil. 4. Pathology Department, Faculty of Medical Sciences, University of Campinas, São Paulo, Brazil. 5. Plastic Surgery Department, Faculty of Medical Sciences, University of Campinas, São Paulo, Brazil. 6. Internal Medicine Department, Faculty of Medical Sciences, Haematology Hemotherapy Centre/INCT do Sangue, University of Campinas, São Paulo, Brazil. 7. Liver Transplantation Unit-Gastroenterology Department, Faculty of Medical Sciences, University of Campinas São Paulo, Brazil. 8. Umbilical Cord Blood Bank, Hematology Hemotherapy Centre/INCT do Sangue, University of Campinas, São Paulo, Brazil; Liver Transplantation Unit-Gastroenterology Department, Faculty of Medical Sciences, University of Campinas São Paulo, Brazil. Electronic address: luzo@unicamp.br.
Abstract
BACKGROUND AIMS: End-stage liver diseases frequently require liver transplantation. Cell therapy could be an alternative. This study aimed to analyze whether undifferentiated mesenchymal stromal cells (U-MSCs) or MSC-derived hepatocyte-like cells (DHLCs) from adipose tissue (AT), umbilical cord blood (UCB) and bone marrow (BM) would better restore damaged liver. METHODS: AT was obtained from lipo-aspiration, UCB from an Umbilical Cord Blood Bank and BM from a BM Transplantation Unit. AT (collagenase digestion), UCB and BM (Ficoll gradient) were cultured (Dulbecco's modified Eagle's medium, low glucose, FBS) for 3 days. Detached adherent cells, at passage 4, were characterized as MSCs. Genetic stability was investigated by means of telomerase enzyme activity and karyotype. Hepatocyte differentiation protocol was performed with the use of Dulbecco's modified Eagle's medium, hepatocyte growth factor, basic fibroblast growth factor and nicotinamide (7 days); maturation medium (oncostatin, dexamethasone, insulin, transferrin and selenium) was added at 36 days. Hepatogenesis analyses were performed by use of morphology and albumin, AF, tyrosine-aminotransferase and glutamine synthetase gene expression and quantitative reverse transcription-polymerase chain reaction on days 9, 18, 25 and 36. Functionality was assessed through glycogen storage detection, indocyanine green absorption and transplantation procedure. U-MSCs and DHLCs were injected 48 h after induced fulminant hepatitis (intraperitoneal injection of carbon tetrachloride) in SCID/BALB-c mice. Histopathologic analyses were performed on days 7 and 15. Human origin included albumin and CK19 human markers. RESULTS: All MSCs differentiated into functional hepatocyte-like cells, stored glycogen and absorbed indocyanine green. AT-MSC DHLC gene expression was more consistent with a normal hepatogenic-differentiation profile. UCB-MSCs expanded weakly, impairing their use for the transplantation procedure. AT and BM U-MSCs and DHLCs regenerated liver injury equally. Regenerated hepatocytes exhibited human origin. CONCLUSIONS: AT might be the source and U-MSCS the stem cells useful for liver-regenerative therapy.
BACKGROUND AIMS: End-stage liver diseases frequently require liver transplantation. Cell therapy could be an alternative. This study aimed to analyze whether undifferentiated mesenchymal stromal cells (U-MSCs) or MSC-derived hepatocyte-like cells (DHLCs) from adipose tissue (AT), umbilical cord blood (UCB) and bone marrow (BM) would better restore damaged liver. METHODS: AT was obtained from lipo-aspiration, UCB from an Umbilical Cord Blood Bank and BM from a BM Transplantation Unit. AT (collagenase digestion), UCB and BM (Ficoll gradient) were cultured (Dulbecco's modified Eagle's medium, low glucose, FBS) for 3 days. Detached adherent cells, at passage 4, were characterized as MSCs. Genetic stability was investigated by means of telomerase enzyme activity and karyotype. Hepatocyte differentiation protocol was performed with the use of Dulbecco's modified Eagle's medium, hepatocyte growth factor, basic fibroblast growth factor and nicotinamide (7 days); maturation medium (oncostatin, dexamethasone, insulin, transferrin and selenium) was added at 36 days. Hepatogenesis analyses were performed by use of morphology and albumin, AF, tyrosine-aminotransferase and glutamine synthetase gene expression and quantitative reverse transcription-polymerase chain reaction on days 9, 18, 25 and 36. Functionality was assessed through glycogen storage detection, indocyanine green absorption and transplantation procedure. U-MSCs and DHLCs were injected 48 h after induced fulminant hepatitis (intraperitoneal injection of carbon tetrachloride) in SCID/BALB-c mice. Histopathologic analyses were performed on days 7 and 15. Human origin included albumin and CK19 human markers. RESULTS: All MSCs differentiated into functional hepatocyte-like cells, stored glycogen and absorbed indocyanine green. AT-MSC DHLC gene expression was more consistent with a normal hepatogenic-differentiation profile. UCB-MSCs expanded weakly, impairing their use for the transplantation procedure. AT and BM U-MSCs and DHLCs regenerated liver injury equally. Regenerated hepatocytes exhibited human origin. CONCLUSIONS: AT might be the source and U-MSCS the stem cells useful for liver-regenerative therapy.
Authors: Ioannis G Papanikolaou; Charalambos Katselis; Konstantinos Apostolou; Themistoklis Feretis; Maria Lymperi; Manousos M Konstadoulakis; Apostolos E Papalois; George C Zografos Journal: Stem Cells Int Date: 2017-03-13 Impact factor: 5.443