Enhanced Hepatic Differentiation Of Human Cord Blood -Books Pdf

Enhanced hepatic differentiation of human cord blood
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Enhanced hepatic differentiation of,human cord blood derived. mesenchymal stem cells,using activin A based protocol. Directed by Professor Hyun Ok Kim,The Doctoral Dissertation. submitted to the Department of Medicine,the Graduate School of Yonsei University. in partial fulfillment of the requirements for the degree. of Doctor of Philosophy,Sinyoung Kim,This certifies that the Doctoral.
Dissertation of Sinyoung Kim is,Thesis Supervisor Hyun Ok Kim. Kwang Hyub Han Thesis Committee Member 1,Yoo Hong Min Thesis Committee Member 2. Dong Wook Kim Thesis Committee Member 3,Kyung Sik Kim Thesis Committee Member 4. The Graduate School,Yonsei University,ACKNOWLEDGEMENTS. While pursuing my doctoral degree with various research projects a. number of generous individuals guided and supported me Firstly I owe. much gratitude to my dissertation advisor Professor Hyun Ok Kim who. has not only advised me through the scientific endeavors of my projects. but also encouraged me with enthusiasm and inspired me in every situation. Let me take this opportunity to also express my sincere gratitude to other. members of the dissertation committee Professor Kwang Hyub Han. Professor Yoo Hong Min Professor Dong Wook Kim and Professor. Kyung Sik Kim for all of their professional advice in leading me through. my thesis completion And I want to thank Professor Han Soo Kim for the. direction he has provided and the focus he allowed me to achieve. And most of all I would like to thank my parents for their patience my. wife Yunsook Oh for her unrelenting support and inspiration at times. when I needed it most and my lovely daughter for making everyday a. 9 May 2009,Sinyoung Kim,TABLE OF CONTENTS,ABSTRACT 1.
I INTRODUCTION 4,II MATERIALS AND METHODS 7, 1 Isolation and primary culture of cord blood derived mesenchymal stem. cells CBMSCs 7,2 Immunophenotyping of CBMSCs 8,3 In vitro differentiation assays 8. A Osteogenic differentiation 8,B Chondrogenic differentiation 9. C Adipogenic differentiation 10,4 Hepatic differentiation protocol 10. 5 RNA isolation and reverse transcriptase polymerase chain reaction. 6 Treatment of animals 14,7 Biochemical analysis 15.
8 Immunohistochemical analysis 15,9 Statistical analysis 16. III RESULTS 17,1 Isolation and expansion of CBMSCs 17. 2 Immunophenotypes of CBMSCs 17,3 In vitro differentiation assays 19. 4 In vitro hepatic differentiation potential 19, 5 In vivo hepatic differentiation of CBMSCs in cirrhotic rat model 23. 6 Changes of liver function after CBMSC infusion 26. IV DISCUSSION 30,V CONCLUSION 34,REFERENCES 35,ABSTRACT IN KOREAN 42.
LIST OF FIGURES,Figure 1 Photomicrographs of isolated CBMSCs 17. Figure 2 Immunophenotypes of CBMSCs 18, Figure 3 Multi lineage in vitro differentiation capacity of. Figure 4 Hepatic differentiation of CBMSCs in vitro 22. Figure 5 Immunohistochemical detection of human,hepatocytes 24. Figure 6 Immunohistochemical detection of human cells 25. Figure 7 Representative figures showing fibrotic area in. placebo and CBMSC infused group 28, Figure 8 Comparison of fibrotic area between the placebo. and CBMSC infused group 29,LIST OF TABLES, Table 1 Primer pairs used for polymerase chain reactions to.
detect hepatocyte specific gene expression 13, Table 2 Comparison of serum markers related to liver function. between placebo and CBMSC infused group 26, Table 3 Quantitative measurement of picro sirius red stained. fibrotic area 28, Enhanced hepatic differentiation of human cord blood derived. mesenchymal stem cells using activin A based protocol. Sinyoung Kim,Department of Medicine,The Graduate School Yonsei University. Directed by Professor Hyun Ok Kim, Recent studies have shown that mesenchymal stem cells MSCs which are.
isolated from various sources such as bone marrow cord blood adipose tissue. amniotic fluid and the placenta could differentiate into hepatocyte like cells in. vitro However these in vitro differentiation protocols are not practical for. clinical usage as more than 28 days is required to differentiate MSCs into. hepatic lineages and the differentiation efficiency is low In addition the in vivo. hepatic differentiation of MSCs and therapeutic efficacy of MSC administration. in chronic liver injury models remains controversial The goal of the present. study was to improve and modify the hepatic differentiation protocol of cord. blood derived MSC CBMSC based on the current knowledge of liver. ontogeny Also we investigated the therapeutic effects of systemic CBMSC. infusion in cirrhotic rat model, MSCs were isolated from human cord blood using the standard plastic. adherence method and its surface phenotype and multi lineage differentiation. potential were examined Isolated CBMSCs were differentiated into hepatic. lineages using the activin A based protocol or previously published two step. protocol The activin A based protocol was modified from the two step protocol. to reflect cytokine expression during embryonic hepatogenesis activin A plus. fibroblast growth factor FGF 4 followed by a combination of hepatocyte. growth factor FGF 4 oncostatin M and dexamethasone Differentiated. hepatocyte like cells were examined for hepatocyte specific markers such as. alpha fetoprotein AFP and albumin Ex vivo expanded CBMSCs were infused. in Wistar rats with thioacetamide induced chronic liver injury Biochemical. markers liver fibrosis and engraftment of CBMSCs were assessed 1 or 4 weeks. after cell or placebo infusion, After induction under each protocol cuboidal morphology which is. characteristic of hepatocytes was observed These hepatocyte like cells which. were induced by the two step protocol did not express mRNA of hepatocyte. specific AFP and albumin at 4 weeks of induction However using the activin. A based protocol CBMSCs five among 12 cell lines from different donors. revealed expression of AFP albumin and cytokeratin 19 as early as at 2 weeks. of induction Four weeks after CBMSC infusion into rats with chronic liver. injury there was no difference in serological markers of liver injury and area of. liver fibrosis between the cell and placebo infusion groups Infused CBMSCs. were detected in the perivascular and fibrous region of the liver and did not. acquire mature hepatic phenotypes, CBMSCs have the potential to differentiate into hepatic lineages in vitro and. activin A is hereby essential to promote differentiation of CBMSCs towards. functional hepatocyte like cells However improvement of liver function and. liver fibrosis after infusion of CBMSCs could not be found in our experimental. cirrhotic rat model, Key words cord blood mesenchymal stem cell hepatocyte differentiation. Enhanced hepatic differentiation of human cord blood derived. mesenchymal stem cells using activin A based protocol. Sinyoung Kim,Department of Medicine,The Graduate School Yonsei University.
Directed by Professor Hyun Ok Kim,I INTRODUCTION, Liver transplantation is currently the only successful treatment for acute. hepatic failure or ends stage liver disease Several problems are associated with. liver transplantation which include lack of donors surgical complications. rejection and high cost Hepatocyte transplantation has been the alternative. option for bridging patients to liver transplantation However primary. hepatocytes are only available from a restricted number of donor organs which. are usually not allocated for organ transplantation1 Also primary hepatocytes. have a poor proliferative potential and probably are not sufficient to effectively. repopulate the host liver and it is difficult to maintain in vitro cultures without. loss of functions2 Additionally their usage does not solve the problem of. immune rejection, Therefore novel sources of transplantable cells such as adult stem cells will. have to be established Stem cells have been identified in a variety of tissues. where they play critical roles in the tissues maintenance and repair But. identification and characterization of hepatic stem cells has been technically. difficult because sensitive and stable single cell based assays have not yet been. developed Several studies have shown that bone marrow derived. hematopoietic stem cells HSCs have the capacity to differentiate into a. variety of non hematopoietic cells such as hepatocytes3 4 myocardium5 and. neurons6 However HSCs contribute little to hepatocyte formation under either. physiological or pathological conditions although they provide cytokines and. growth factors that promote hepatocyte functions by the paracrine mechanism7. The other potential candidate stem cells are mesenchymal stem cells MSCs. which could be obtained from various sources such as bone marrow8 cord. blood9 amniotic fluid10 placenta11 or adipose tissue12 Cord blood has a. number of advantages over the other sources of MSCs in terms of cell. procurement which include abundance lack of donor attrition and a low risk. of viral transmission for disease such as the herpes family viruses Furthermore. MSCs have a reduced risk of allogenic transplant rejection because they are. immuno privileged with a low major histocompatibility complex MHC I and. no MHC II class expression13 14 For these reasons cord blood derived MSCs. CBMSCs could be a prominent source of cells for transplantation for various. The hepatic differentiation potential of MSCs has been recently described15. Multipotent adult progenitor cells which were discovered by Schwartz et al. were the first plastic cells found within bone marrow that gained the ability to. undergo hepatic differentiation under the fibroblast growth factor FGF 4 and. hepatocyte growth factor HGF 18 Thus far several studies have revealed the. hepatic differentiation potential of MSCs using different protocols Proposed. protocols included stimulation of MSCs with hepatogenic factors added either. as a mixture FGF HGF19 FGF HGF oncostatin M OSM 20 or. separately HGF OSM21 FGF HGF OSM15 FGF HGF OSM22, However the resultant populations were far from homogeneous and the. hepatocyte like cells differentiated by the abovementioned protocols showed. hepatocyte specific mRNA expression at over 30 days of induction Such a. long period of induction time did require large quantities of cytokines and may. have problems related to long term in vitro cultivation. Activin A a member of the TGF superfamily has been identified as a. potential regulator for the induction of mesoderm and endoderm in embryonic. stem cells23 24 Activin A has also demonstrated to provide an efficient signal. into human embryonic stem cells for definitive endodermal differentiation via. activin nodal signaling pathway25 Cai et al differentiated human embryonic. stem cells into functional hepatic cells after sequential treatment with activin A. and FGF4 bone morphogenetic protein 226 This process generally mimicked. natural embryonic liver development observed in vivo In the present study we. investigated to modify and improve the hepatic differentiation protocol of. CBMSC using activin A based on the current knowledge of liver ontogeny. The administration of MSCs has been shown to improve liver function in. animal model of chronic liver injury27 28 However evidence supporting the in. vivo ability of transplanted human MSCs to enter liver parenchyma and to. acquire markers of hepatocyte like differentiation is still lacking29 Also we. investigated the therapeutic effect of systemic CBMSC infusion in the cirrhotic. II MATERIALS AND METHODS,1 Isolation and primary culture of CBMSCs. Umbilical cord blood samples from normal full term deliveries were. collected after obtaining written informed consent and were stored at 22. before processing Mononuclear cells MNCs in cord blood were separated. within 6 hours of collection by Ficoll Hypaque density 1 077 Pharmacia. Biotech Uppsala Sweden using density gradient centrifugation at 435g. for 25 minutes MNCs were removed from the interface and washed three. times with phosphate buffered saline PBS MNCs were set in culture at a. density of 3 106 cells per cm2 into fibronectin Sigma Aldrich St Louis. MO USA coated 6 well plates Nunc Rochester NY USA in Endothelial. Growth Medium 2 EGM 2 Gibco Grand Island NY USA or MSCGM. medium MSCGM BulletKit CellSystems St Katharinen Germany The. media was changed every 3 days After colonies formed between days 14. through 20 cells were harvested using 0 1 trypsin EDTA solution Gibco. and cultured thereafter in Dulbecco s modified Eagle medium DMEM. Gibco containing 10 fetal bovine serum FBS and Penicillin. Streptomycin solution Gibco,2 Immunophenotyping of CBMSCs.
For flow cytometric analysis the cells were harvested using 0 1 trypsin. EDTA treatment and detached 5 105 cells were resuspended in 200 L PBS. and incubated for 20 min at room temperature with the following antibodies. CD14 CD29 CD31 CD34 CD44 CD45 CD73 CD90 and CD105 all of. which were conjugated with either fluorescein isothiocyanate FITC or. phycoerythrin PE all from BD Sciences San Jose CA USA An isotype. control that was FITC or PE labeled was included in each experiment and. specific staining was measured from the cross point of the isotype with. specific antibody graphs The fluorescence intensity of the cells was. evaluated by flow cytometry using a flow cytometer Cytomics FC500. Beckman Coulter Hialeah FL USA,3 In vitro differentiation assays. A Osteogenic differentiation, To induce osteogenic differentiation CBMSCs were seeded at a density.

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