Phân lập và Đặc tính hóa Tế bào gốc Ống Thận Người và Tác động Cadmium

University of North Dakota UND Scholarly Commons Theses and Dissertations Theses, Dissertations, and Senior Projects January 2018 Isolation And Characterization Of Stem Cell Population In Human Proximal Tubule Cells And The Effect Of Cadmium In The Isolated Proximal Tubule Stem/progenitor Cell Line. Swojani Shrestha Follow this and additional works at: https://commons.edu/theses Recommended Citation Shrestha, Swojani, "Isolation And Characterization Of Stem Cell Population In Human Proximal Tubule Cells And The Effect Of Cadmium In The Isolated Proximal Tubule Stem/progenitor Cell Line. Theses and Dissertations.edu/theses/2347 This Dissertation is brought to you for free and open access by the Theses, Dissertations, and Senior Projects at UND Scholarly Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UND Scholarly Commons.

For more information, please contact zeineb. ISOLATION AND CHARACTERIZATION OF STEM CELL POPULATION IN HUMAN PROXIMAL TUBULE CELLS AND THE EFFECT OF CADMIUM IN THE ISOLATED PROXIMAL TUBULE STEM/PROGENITOR CELL LINE. by Swojani Shrestha Bachelor of Science, Minnesota State University Moorhead, 2013 A Dissertation Submitted to the Graduate Faculty of the University of North Dakota in partial fulfillment of the requirements for the degree of Doctor of Philosophy Grand Forks, North Dakota August 2018 i c 2018 Swojani Shrestha ii PERMISSION Title Isolation and characterization of stem cell population from human proximal tubule cells and the effect of cadmium in isolated proximal tubule stem/progenitor cell lines Department Biochemistry and Molecular Biology Degree Doctor of Philosophy In presenting this dissertation in partial fulfillment of the requirements for a graduate degree from the University of North Dakota, I agree that the library of this University shall make it freely available for inspection. I further agree that permission for extensive copying for scholarly purposes may be granted by the professor who supervised my dissertation work or, in his absence, by the Chairperson of the department or the dean of the School of Graduate Studies.

It is understood that any copying or publication or other use of this dissertation or part thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of North Dakota in any scholarly use which may be made of any material in my dissertation. Swojani Shrestha Jun, 2018 iv TABLE OF CONTENTS LIST OF FIGURES…………………………………………………………….…iv LIST OF TABLES………………………………………………………………….1 Proximal Tubule Cells of the Kidney, Their Functions and Significance…………….4 Kidney Tubule Regeneration Theories.6 Stem/Progenitor Cells in Kidney………….8 Markers of Stem/Progenitor Cells in Human Proximal Tubule Cells.…8 Studies on Isolation of Stem/Progenitor Cells of Human Proximal Tubule Cells.……9 Role of Stem/Progenitor Cells in Tubular Regeneration…….11 Significance of In-vitro Cell Culture Models.12 Cell Culture Models…….20 Real Time PCR.21 Microarray Analysis of Global Gene Expression……………………22 Determination of Cell Viability by DAPI Staining………………….22 Western Blot…………………………………………………………23 Determination of LDH release……………………………………….25 Florescence Activated Cell Sorting………………………………….26 Transepithelial Resistance Measurement……………………………27 Spheroids Culture……………………………………………………28 Thick and Thin Layer Matrigel coated Cell Growth……………….29 MTT growth assay………………………………………………….30 Exposure of Cell-lines to Cd2+ and Determination of Cell Population………………………………………………………….30 Subcutaneous Injection of Cell-Lines……………………………….31 Immunohistochemistry………………………………………………31 Statistical Analysis…………………………………………………. RESULTS……………………………………………………………34 vi Global Gene Expression Pattern of HK-2, HPT and RPTEC/TERT1 Cells………………………………………………………………….34 Expression of CD24 and CD133 in the HK-2, HPT and RPTEC/TERT1 Cells……………………………………………….42 Expression of Cadherins, Claudins and Occludin in the HK-2, HPT and RPTEC/TERT1 Cell Isolates……………………………………45 Mode of Cell Death in RPTEC/TERT1 Cells in Response to Cd+2 - Induced Toxicity…………………………………………………….48 Isolation of CD133+CD24+ and CD24+ Cell Population from RPTEC/TERT1 Cell Cultures…………………………………….…50 Expression of CD133 and CD24 Markers in RPTEC/TERT1, CD133+CD24+ and CD24+ Cells Cultures…………………….54 Morphology, Transepithelial Resistance and Growth Rate Determination of RPTEC/TERT, CD133+CD24+ and CD24+ Cells………………………………………………….56 Ki67 Expression in RPTEC/TERT1, CD133+CD24+ and CD24+ cells………………………………………………….58 Expression of AQP-1 and CAL Markers in RPTEC/TERT1, CD133+CD24+ and CD24+ Cells………………………….60 Ability of RPTEC/TERT1, CD133+CD24+ and CD24+ Cells to Form Spheroids…………………………………………………….61 Change in CD133+CD24+ and CD24+ Cell Population after Exposure to Cd2+ for 34 Days………………………………………………….63 Growth of RPTEC/TERT1, CD133+CD24+, CD24+ and HPT Cells with Matrigel in Culture Plates………………………………………68 Histology and Immunohistochemical staining of tubular structures formed by various populations of RPTEC/TERT1 cells…………….77 vii Characteristic Features of In-Vitro Human Proximal Epithelial Cell Culture……………………………………………………………….77 Immortalized RPTEC/TERT1 Cells as a Model System to Study Human Proximal Tubule Cells…………………………………….79 Mode of Cell Death after Cadmium Insult in RPTEC/TERT1 Cells.82 Expression of CD133 and CD24 Markers in Primary Human Proximal Tubule Cultures…………………………………………………….83 Characterization of CD133+CD24+ and CD24+ Cells isolated from RPTEC/TERT1 Cells……………………………………………….83 Exposure to Cadmium decreases CD24+ Cell Population while increases CD133+CD24+ Cell Population……………………….…85 Ki67 Expression in Cultured RPTEC/TERT1 after Cadmium Insult.87 Demonstration of the ability of various populations of cells to form tubular structures in-vitro and in-vivo……………………………….92 viii LIST OF FIGURES Figure Page I-1.

Three regions of the kidney……………………………………………………. Parts of the nephron………………………………………………………………. Regeneration mechanism in epithelial tubular cells after injury…………………. Schematic of dome formation………………………………………………….

Hierarchical clustering of various proximal tubule cell transcriptomes………. Principal component analysis using all 67,528 non-control transcript clusters……………………………………………………………………. Venn diagram showing differentially expressed genes in HK-2 cells and RPTEC/TERT1 cells in comparison to mortal human proximal tubule cell (HPT) isolates………………………………………………………………………………. Transposed principal component analysis after performing discriminant analysis of pre-defined groups……………………………………………………………….

Hierarchical clustering of transcript cluster expression after statistical filtering…. Flow cytometry analysis of CD24 and CD133 populations in proximal tubule cells…. Expression of stem cell markers in cultures of human proximal tubule cells………………………………………………………………………. Expression of cadherins in cultures of human proximal tubule cells………………………………………………………………………………….

Expression of tight junction proteins in cultures of human proximal tubule cells…………………………………………………………………………. Effect of Cd+2 on RPTEC/TERT1 cells………………………………………48 ix III-11. Effect of Cd+2 on HK-2 cells………………………………………………. Flow cytometry analysis of CD133+CD24+ and CD24+ populations in RPTEC/TERT1 cells ran in triplicate……………………………………………….

Flow cytometry analysis of CD133+CD24+ cells to test purity at each passage number………………………………………………………………………………. Flow cytometry analysis of CD24+ cells to test purity at each passage number………………………………………………………………………………. Expression of CD133 and CD24 in various populations of renal tubular cells……………………………………………………………………. Light level microscopy of different proximal tubular cell cultures after sorting…………………………………………………………………56 III-17.

Transepithelial resistance measurement of RPTEC/TERT1, CD133+CD24+ and CD24+ cells…………………………………………………………………………. Determination of growth rates of various populations of renal tubular cells. Flow cytometric analysis of number of cells stained positive for Ki67 in fully confluent cultures RPTEC/TERT1, CD133+CD24+ and CD24+ cells……………. Expression of aquaporin-1 (AQP-1) and calbindin (CAL) in various populations of renal tubular cells……………………………………………………………….

Light level microscopy of spheroids generated from RPTEC/TERT1, CD133+CD24+, CD24+ cells………………………………………………………. Effect of Cd+2 on the viability RPTEC/TERT1 cells exposed to 0, 4.5, 18, 27 and 45 μM Cd+2 for 16 days…………………………………………………63 III-23. Light level microscopy of CD133+CD24+ cells treated with 4.5 and 9µM Cd2+ for 34 days…………………………………………………………………. Light level microscopy of CD24+ cells treated with 4.5 and 9µM Cd2+ for 34 days………………………………………………………………………………….

Effect of cadmium on the viability RPTEC/TERT1 cells……………………67 x III-26. Effect of cadmium on the viability of various populations of renal tubular cells…………………………………………………………………………………. Light level microscopy of RPTEC/TERT1 cells plated with matrigel in a 48- well plate……………………………………………………………………………. Light level microscopy of CD133+CD24+ cells plated with matrigel in a 48- well plate…………………………………………………………………………….

Light level microscopy of CD24+ cells plated with matrigel in a 48-well plate…………………………………………………………………………. Light level microscopy of HPT cells plated with matrigel in a 48-well plate. Light level microscopy of RPTEC/TERT1, CD133+CD24+ and CD24+ cells plated on the surface of thin matrigel coated 48-well plate…………………………. Immunohistochemical analysis of proximal tubule stem cell markers in subcutaneous nodules formed in immuno-compromised mice………………………72 III-33.

Immunohistochemical analysis of tubular markers in subcutaneous nodules formed in immuno-compromised mice………………………………………………73 III-34. Immunohistochemical analysis of keratin proteins, filament protein and nephron development marker in subcutaneous nodules formed in immuno-compromised mice……………………………………….74 xi LIST OF TABLES Table Page II-1. List of primary antibodies used for western blot. List of primary antibodies used for immunofluorescence.

List of primary antibodies used for immunohistochemistry.32 xii ACKNOWLEDGEMENTS I would first like to thank Dr. Don Sens who accepted me as a graduate student in his lab and provided me with an opportunity to gain extensive knowledge and training on science and research. Joe Provost, my undergraduate advisor, I would have never imagined going to graduate school, so much thanks to him. I would like to thank my advisors Dr.

Seema Somji as well as Dr. Scott Garrett for providing insightful guidance on my research, training on different cellular and molecular biology techniques and being available anytime to answer my questions. My special thanks to Dr. Seema Somji for believing in my potentials and providing me with tremendous encouragement throughout my graduate degree career.

I am overwhelmed and fortunate to be a part of this department who recognized my abilities and allowed me to thrive as a researcher. I would like to thank Dr. Jane Dunlevy for training me with her expertise in confocal microscopy and Dr. Zhou and Ms.

Wang for helping me with animal handling techniques and taking immunohistological images. I would like to thank Dr. Min Wu and Dr. Kathy Sukalski for giving their time to talk about how to succeed in graduate school when I first started the program.

I would like to extend many thanks to Dr. Tristan Darland for his willingness to become my faculty at large. Thank you to Steve for providing me extensive training on flow cytometry. xiii I am extremely grateful and thankful to Dr.

Mary Ann Sens and Dr. Don Sens for the support they offered me during my difficult times. I will never be able to repay their kindness. I considered myself extremely lucky for having one of the most amazing lab mates and colleagues, Menglan, Bethany, Andrea, Emily, Zach as well as other lab members.

I don’t miss being far away from home and family because of them. Lastly, I thank my parents for all their sacrifices and my brother and sister-in-law for always supporting me. I love them all and they will forever be in my heart. xiv ABSTRACT The proximal tubules of the kidney are target sites of injury by various toxicants.

Cadmium (Cd2+), an environmental nephrotoxicant can cause adverse effects and overt renal damage. To decipher the mechanisms involved in nephrotoxicity, an in-vitro model system is required. Mortal cultures of human proximal tubule (HPT) cells isolated from the renal cortex are used as models, but are difficult to acquire and have limited passage number. The immortalized HK-2 cell line, has served as a model but it lacks vectorial active transport and shows signs of lost epithelial features.

Recently a new proximal tubule cell line, the RPTEC/TERT1, was developed. For this study we performed global gene expression analysis of this cell line in comparison to the HK-2 and HPT cells showed that the RPTEC/TERT1 cells had gene expression patterns similar to HPT cells when compared to the HK-2 cells. The HPT and the RPTEC/TERT1 cell lines showed higher expression of renal stem/progenitor like cell population, CD133+CD24+ when compared to the HK-2 cells. The level of expression of tight junctional molecules was also similar between the RPTEC/TERT1 and the HPT cells.

Acute exposure to Cd2+ resulted in necrosis of the RPTEC/TERT1 cells whereas the HK-2 cells died by apoptosis. Thus, we verified that the RPTEC/TERT1 are similar to HPT cells and can serve as a good model system to study mechanisms involved in Cd2+ induced renal damage. Recent studies suggest that tubular regeneration after toxic insult may involve progenitor/ stem like cells expressing CD133 as well as CD24 markers that resides among the renal tubular cells. Our previous study shows that the RPTEC/TERT1 cells comprise of 25-30% of CD24+ cells whereas 70-75% of the CD133+CD24+ cells.

To determine the response of these populations xv of cells to renal insult, the two populations of cells were sorted from the RPTEC/TERT1 cells, following which they were cultured and treated with 4.5μM and 9μM Cd2+ for approximately 34 days. The results demonstrate that the CD133+CD24+ cells are more resistant to cadmium exposure as there was no change in the number of double positive cells in response to cadmium treatment whereas the number of CD24+ cells significantly decrease. Both populations of cells form domes in culture indicative of vectorial active transport. The CD133+CD24+ cells show faster growth rate when compared to the CD24+ cells.