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Ezhil Final. Ppt

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Ezhil Final. Ppt

  1. 1. Isolation of Human hepatic stem cells and characterization by using α - feto protein, Cytokeratin-19 & Albumin Presented by S. Ezhil Mangai INTERNAL GUIDE : L.G. MOHANA DIVYA Lecturer, Department of biotechnology, KSR college of Technology. EXTERNAL GUIDE : DR. ALEEM KHAN, Scientist, Department of stem cell therapy, CLRD.
  2. 2. Introduction: <ul><li>Stem cells : </li></ul><ul><li>Progenitor cells. </li></ul><ul><li>undifferentiated cells. </li></ul><ul><li>self-renewal . </li></ul><ul><li>Differentiate under influence of suitable tropic factors. </li></ul><ul><li>Types of stem cells : </li></ul><ul><li>Pluripotent </li></ul><ul><li> Embryonic stem cells. </li></ul><ul><li> Embryonic Germ cells. </li></ul><ul><li>Multipotent </li></ul><ul><li>Adult stem cells. </li></ul><ul><li>Sources : </li></ul><ul><li>Blastocyst – embryonic stem cells. </li></ul><ul><li>Fetus – fetal stem cells. </li></ul><ul><li>Adult stem cells – neuronal, bone marrow, pancreatic , hepatic etc… </li></ul>
  3. 3. <ul><li>Advancement in stem cell research – new sources of adult stem cells. </li></ul><ul><li>New sources – hair follicles , Dental pulp , Retinal stem cells , Menstrual blood etc. </li></ul><ul><li>Stem cells of blood – hematopoietic stem cells. </li></ul><ul><li>Applications: </li></ul><ul><li>Regenerative medicine. </li></ul><ul><li>Toxicology studies. </li></ul><ul><li>Cosmetic therapy. </li></ul><ul><li>Dental therapies </li></ul><ul><li>Demand for organ transplants - Cell based therapies alternatives for transplants </li></ul><ul><li>Liver is a metabolizing organ of the body where most of the detoxification reactions take place. </li></ul><ul><li>Hepatocyte transplants – conceptual alternative for patients suffering from chronic liver diseases – lack of proliferation rate – unsuccessful in transplants. </li></ul>
  4. 4. <ul><li>So far the source of stem cells from human fetal liver is taken from first trimester which contains highly proliferative stem cells whose lineage specificity is not yet been fully accomplished. </li></ul><ul><li>In my present study, I have taken up the source of stem cells from human fetal hepatic tissue during the second trimester. </li></ul><ul><li>Hence we are checking for expression of liver specific markers during 14 – 20 weeks of gestation. </li></ul><ul><li>Hepatic progenitors – highly expandable due to high proliferation and can be used in liver regeneration </li></ul><ul><li>Hepatic progenitors are isolated by using specific stem cell markers. </li></ul>
  5. 5. LIVER SPECIFIC MARKERS <ul><li>Alpha fetoprotein :  </li></ul><ul><li>Alpha-fetoprotein (AFP) is a serum glycoprotein produced at high levels during fetal life by the liver and the visceral endoderm of the yolk sac and at lower levels by the developing gastrointestinal tract </li></ul><ul><li>Albumin:                         </li></ul><ul><li>Albumin (Latin: albus, white) refers generally to any protein with water solubility, which is moderately soluble in concentrated salt solutions, and experiences heat coagulation (protein denaturation). </li></ul>
  6. 6. <ul><li>Cytokeratins 18 and 19:  </li></ul><ul><li>  Cytokeratins are intermediate filament keratins found in the intracytoplasmic cytoskeleton of epithelial tissue. There are two types of cyto keratins: the low weight, acidic type I cytokeratins and the high weight, basic or neutral type II cytokeratins. Ck-18 & 19 belongs to type-1 cytokeratins.            </li></ul>
  7. 7. Objectives: <ul><li>To isolate the stem cells from human fetal liver. </li></ul><ul><li>To characterize the isolated human fetal liver stem cells. </li></ul><ul><li>Gene expression analysis of liver Specific markers (AFP , ALB , CK18 & 19 ). </li></ul>
  8. 8. Materials and Methods: Materials: Fetal liver cells from Aborted Fetus. Methods: <ul><li>Fetal liver is regained by dissection of fetus. </li></ul><ul><li>CD 34 labeling and magnetic sorting (MACS). </li></ul><ul><li>Cell viability (Trypan blue Assay) </li></ul><ul><li>Hemocytometer (Cell count ) </li></ul><ul><li>Immunocytochemistry by FITC labeled CD34 antibodies. </li></ul><ul><li>MTT assay. </li></ul><ul><li>Gene expression analysis using liver specific markers. </li></ul>
  9. 9. LIVER CELL EXTRACTION <ul><li>Homogenization is done with collagenase 4 for complete extraction of liver cells. </li></ul><ul><li>Cell washing is done with Hanks buffer 3-4 times . </li></ul>
  10. 10. MAGNETIC ASSOCIATED CELL SORTER <ul><li>MACS with LS max column is used for separation of stem </li></ul><ul><li>cells containing CD 34+ and CD 34- cells. </li></ul><ul><li>The separation of +ve and –ve cells is due to the magnetic </li></ul><ul><li>field applied and the binding of specific antibodies with the cells. </li></ul><ul><li>The cells which do not bind with the specific antibody elute first </li></ul><ul><li>and are referred as CD 34- cells. </li></ul><ul><li>The cells are washed with Hanks Buffer 2-3 times. </li></ul><ul><li>The cells are preserved in 4 °c with the addition of Hanks buffer. </li></ul>
  11. 11. CELL COUNTING <ul><li>Cell counting is the process of measuring total number of cells per ml. </li></ul><ul><li>It is done with the help of Hemocytometer </li></ul><ul><li>The cells are mixed with trypan blue dye in the ratio 1:10 such as dye and cells respectively. </li></ul><ul><li>The cells are made into exact concentration and placed on the square slide covered with cover slip. </li></ul><ul><li>The slide is placed on the microscope and observed for total number of cells. </li></ul><ul><li>The calculation is done with the formula </li></ul><ul><li>number of cells = number of * dilution </li></ul><ul><li> per cubic mm cells counted factor </li></ul>
  12. 12. VIABILITY ASSAY <ul><li>The cell viability test was performed in total cells and in MACS sorted cells by dye exclusion method. </li></ul><ul><li>Trypan blue is a vital stain used to selectively colour dead tissues or cells blue. It is a diazo dye. </li></ul><ul><li>The dead cells intake dye which results in blue colour. Whereas the viable cells remain colorless . </li></ul>
  13. 13. IMMUNOCYTOCHEMISTRY <ul><li>To characterize the isolated stem cells. </li></ul><ul><li>It visualizes the sub cellular localization of antigen in isolated stem cell populations. </li></ul><ul><li>Immunocytochemistry refers to a process of localizing proteins in the cells exploiting the binding of antibodies to antigen. </li></ul><ul><li>The visual marker may be a fluorophore (like FITC, Rhodamine), colloidal metal, hapten, radioactive marker or more commonly for light microscopy an enzyme. Ideally, maximal signal strength along with minimal background or non-specific staining is required to give optimal antigen demonstration </li></ul>
  14. 14. MTT ASSAY <ul><li>MTT assay is a laboratory test and a standard colorimetric assay (an assay which measures changes in color) for measuring cellular proliferation (cell growth). </li></ul><ul><li>It can also be used to determine cytotoxicity of potential medicinal agents and other toxic materials. </li></ul><ul><li>Yellow MTT (3-(4,5- Dimethylthiazol -2-yl)-2,5- diphenyltetrazolium bromide , a tetrazole) is reduced to purple formazan in the mitochondria of living cells. </li></ul><ul><li>A solubilization solution (usually either dimethyl sulfoxide, an acidified ethanol solution, or a solution of the detergent sodium dodecyl sulfate in dilute hydrochloric acid) is added to dissolve the insoluble purple formazan product into a colored solution. </li></ul><ul><li> </li></ul>
  15. 15. GENE EXPRESSION STUDIES <ul><li>The RNA was isolated from the total cells, CD34 positive cells and CD34 negative cells . The RNA bands are viewed in agarose  gel electrophoresis </li></ul><ul><li>Reverse transcription is a process for the conversion of mRNA into cDNA. The cDNA bands were confirmed and characterization study was carried out in PCR.  </li></ul><ul><li>PCR was performed using the cDNA isolated from the total cells and the MACS sorted cells (positive & negative) as a template to detect the expression of AFP, ALB, CK18 & CK19 genes in a human fetal liver. </li></ul>
  16. 16. Results and Discussion: 1. Fetal Dissection Ventral view of human fetal liver before perfusion. 1 , 2 , 3 , 4 – Liver lobes. 5 – Hepatic and portal vein. 6- Hepatic lymph nodes. 7- gall bladder.
  17. 17. 2. MACS Magnetic assorted cell sorter Pre-running column with saline Collection of CD34-ve cells Column is separated from magnetic field and Eluted with PBS. CD34+ve cells separated.
  18. 18. 3 . Viability Test ( Trypan Blue Staining ). Total number of cells = 2368cells/50 μ l Viable cells = 1600cells/50 μ l Dead cells = 768cells/50 μ l Total number of cells in area = 23.68X10 9 %viability = 67.56% Trypan blue staining of CD34+ve cells from human fetal liver cells.
  19. 19. 4 . Cell Counting Cells Cell concentration Before MACS After MACS Total cells Positive cells Negative cells 5 million/ml 1 million/ml 4 million/ml
  20. 20. 5. Immunocytochemistry FITC conjugated CD34+ve cells under Fluorescent microscope CD34+ve cells
  21. 21. 6 . MTT Assay Percentage of activity ( μ g/ml) OD at 580nm 35 39 0.1349 0.1790
  22. 22. 7. Gene Expression Analysis (AFP , ALB , CK -18 , CK -19)
  23. 24. Conclusion: <ul><li>The main purpose of our study is to isolate the CD34 +ve cells which are progenitors in the total liver cell population. </li></ul><ul><li>Characterization studies have been done for the isolated CD34+ve cells to know the morphology and study the properties of the progenitors </li></ul><ul><li>Gene expression analysis for AFP , ALB , CK18 & 19 is being done to check for the liver specific makers in the CD34+ve cells. </li></ul><ul><li>AFP , ALB , CK18 & 19 – illustrates the normal morphogenesis of liver </li></ul><ul><li>The Cells which are being isolated and characterized are being used for clinical trials on human patients in our laboratory. </li></ul>

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