Wright State University CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2011 Differentiation of Megakaryocytes/Platelets and Neurons from Human Endometrial Stromal Progenitor Cells Jinju Wang Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Pharmacology, Toxicology and Environmental Health Commons Repository Citation Wang, Jinju, "Differentiation of Megakaryocytes/Platelets and Neurons from Human Endometrial Stromal Progenitor Cells" (2011). Browse all Theses and Dissertations. 481. https://corescholar.libraries.wright.edu/etd_all/481 This Thesis is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. Differentiation of Megakaryocytes/Platelets and Neurons from Human Endometrial Stromal Progenitor Cells A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By Jinju Wang B.S., NanHua University, Hunan, China, 2007 2011 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL August 5, 2011 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Jinju Wang ENTITLED Differentiation of Megakaryocytes/Platelets and Neurons from Human Endometrial Stromal Progenitor Cells BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science. Yanfang Chen, M.D., Ph.D. Thesis Director Mariana Morris, Ph.D., Chair Department of Pharmacology & Toxicology Committee on Final Examination Mariana Morris, Ph.D. Lawrence S. Amesse, Ph.D. James E. Olson, Ph.D. Andrew Hsu, Ph.D. Dean, Graduate School ABSTRACT Wang, Jinju. M.S., Department of Pharmacology and Toxicology, Wright State University, 2011. Differentiation of Megakaryocytes/Platelets and Neurons from Human Endometrial Stromal Progenitor Cells.1 1 human endometrium is a high dynamic tissue that contains stem/progenitor cells. These endometrial stromal progenitor cells (hESCs) have been differentiated into a number of mesodermal lineages. There is limited information on differentiating hESCs into neurons, and no information on differentiating hESCs into megakaryocytes (MKs) and platelets (PLTs). The aim of this work was to investigate the possibility of differentiating hESCs into two distinct lineages: MKs, with subsequent PLT formation, and neurons. We isolated hESCs from human endometrial tissue and cultured the cells for 4-6 passages. Before each differentiation experiment, the purity of hESCs was confirmed by flow cytometry analysis which revealed that the cells were highly positive (>95%) for CD90 and CD29 (stromal cell markers), and were negative (<1.3%) for CD45 and CD34 (hematopoietic cell markers). For MKs differentiation, hESCs (1.8×105 cells/well) were cultured in serum-free medium either with (differentiation) or without (control) thrombopoietin (TPO, 50 ng/ml) for 18 days. MKs differentiation was analyzed by flow cytometry and confocal microscopy. PLTs were collected from culture medium supernatant from day10-18, and were defined by flow cytometry and functional study. Our results iii show: 1) MKs were successfully generated as evidenced by expression of MK membrane markers (CD41a: 39 ± 3.0% and 1 ± 0.09%; CD42b: 28 ± 2.0% and 1.2 ± 0.06%, differentiation vs. control, n=3). 2) Immunocytochemistry analysis showed the differentiation rate of CD41a (38 ± 3.0%) and CD42b (27 ± 2.5%). 3) Generated PLTs were positively labeled with CD41a (90 ± 2%). 4) Functional study of generated PLTs revealed that thrombin (5 U/ml) stimulation up-regulated CD62P expression (26.0 ± 4% and 2.5 ± 1%, thrombin vs. control, n=3), and fibrinogen binding (32 ± 3.0% and 1 ± 0.4%, thrombin vs. control, n=3). 5) Electronic microscopic examination showed that generated PLTs had similar ultrastructure (storage granules) as normal human peripheral blood PLTs. For neuron differentiation, hESCs (1.6×105 cells/well) were cultured in medium either with (differentiation) or without (control) cytokines (FGF, EGF, NGF and BDNF) for 14 days. Differentiated neurons were analyzed by confocal microscopy and western blot analysis. Our results show: 1) neurons were successfully generated as evidenced by phenotypic expression of neuron specific markers nestin (40 ± 4%), β-tublin III (37 ± 3%), NeuN (15 ± 2%), TH (14 ± 1%), and astrocyte marker GFAP (10 ± 3%). 2) Western blot analysis showed that the expression of transcription genes (Oct4: 0.11 ± 0.01and 0.32 ± 0.01; Sox2: 0.25 ± 0.02 and 0.52 ± 0.03, differentiated vs. control, n=4) was down- regulated in differentiated neurons. Taken together, these data indicate that it’s feasible to generate MKs, functional PLTs and neurons from hESCs. The hESCs could be a potential source for cell- based therapy in regenerative medicine. iv TABLE OF CONTENTS CHAPTER I. INTRODUCTION AND PURPOSE Structure of human endometrium………………………………...…….……..1 Presence of stromal progenitor cells in human endometrium……………………………………………………………………..2 Menorrhagia: a common bleeding disorder in reproductive-age females........................................................................................................4 MKs and PLTs………………….…………………………….………………….6 Generation of MKs and PLTs from stem cells in vitro……………………….8 Ischemic stroke and neuron stem cells…………….…………………………9 Generation of neurons from stem cells in vitro ……………………………..10 II. HYPOTHESIS AND SPECIFIC AIMS…………………………………….12 III. EXPERIMENT DESIGN Materials and methods…..…………………………………………………… 14 Harvesting human endometrium tissue sample………………………..…..16 Isolation and culture of hESCs….……………………………………….…..17 Identification study of hESCs ……….……………………………………… 18 v Differentiation of MKs and PLTs from hESCs ……….…………………….19 Characterization study of MKs differentiated from hESCs ……………….20 Characterization study of PLTs generated from hESCs…………………..21 Functional study of generated PLTs with thrombin stimulation…………..22 Morphological study of PLTs by TEM……………………………………….23 Differentiation of neurons from hESCs……………………………………...24 Immunofluoresence staining of generated neurons ……………………….25 Western blot analysis of transcription gene expression in Differentiated neuronal cells ………………………………………………….26 Statistic analysis………………………………………………………………..27 IV. RESULTS Morphology of hESCs ……………………….………………………………..28 Characterization of hESCs………….………………………………..…….…28 Characterization of hESCs-derived MKs by immunocytochemistry analysis…..……………………………………………………….…………….30 Expression of surface protein markers for hESCs-derived MKs by flow cytometric analysis……………………………………………………32 Expression of surface protein markers for PLTs by flow vi cytometric analysis…………………….………………………………………34 Expression of activation-dependent antigens on generated PLTs….……35 Ultrastructural similarities between generated PLTs and peripheral blood derived PLTs…………………………………………………………….38 Expression of surface protein markers for hESCs-derived neurons by immunocytochemistry analysis……………………………………………39 Expression of transcription genes Oct4 and Sox2 in the cells after neuronal differentiation…………………………………………..….................40 V. DISCUSSION………………………………………………………………...42 VI. CONCLUSION………………………………………………………………49 VII. REFERENCES……………………………………………………………..50 vii LIST OF FIGURES 1. Diagram demonstrating the histologic structure of human endometrium…………………………………………………………….….2 2. Diagram illustrating putative location of endometrial stem/progenitor cells……………………………………………………....4 3. The flow chart shows our experiment protocol…...……………………14 4. Morphological study of hESCs under light microscope……………....28 5. hESCs identification……………………………………………………...29 6. Characterization of hESCs-derived MKs by immunocytochemistry……………………………………………………31 7. hESCs-derived MKs surface protein expression analysis by flow cytometry…………………………………………………………33 8. Generated PLTs surface protein expression analysis by flow cytometry ………………………………………………………..35 9. Functional assay of the generated PLTs ………………….………….37 10. Electron micrograph of PLTs…………...……………………………...38 11. Characterization of generated neurons………………………….…...40 12. Comparison of the transcription genes expression in neuron differentiation and control cells……......................................41 viii LIST OF ABBREVIATIONS hESCs Human endometrial stromal progenitor cells MK Megakaryocyte PLT Platelet DMEM/F12 Dulbecco’s modified eagle medium nutrient mixture F-12 (ham) IMDM Iscove’s modified dulbecco’s medium NPBM Neural progenitor basal medium TPO Recombinant human thrombopoietin BSA Bovine albumin serum FBS Fetal bovine serum BDNF Brain derived neurotrophic factor EGF Epidermal growth factor FGF Fibroblast growth factor NGF Nerve growth factor GFAP Glial fibrillary acidic protein TH Tyrosine hydroxylase PI Propidium iodide DAPI 4, 6-diamidino-2-phenylindole dihydrochloride TEM Transmission electron microscopy ix ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Yanfang Chen, for his intellectual guidance, support and encouragement during the past two years. Dr. Chen is an excellent mentor. In spite of his busy schedule, he would always find time for his students. I feel very fortunate to get the opportunity to work in his laboratory under his guidance over the years. Without his keen insights and continues support, I could not have completed this project. I would like to thank Dr. Lawrence Amesse and Dr. James Olson for serving on my thesis committee