Isolation of Human hepatic stem cells and

characterization by using α- feto protein, Cytokeratin-19

& Albumin Presented by S. Ezhil Mangai

INTERNAL GUIDEINTERNAL GUIDE : L.G. MOHANA DIVYA : L.G. MOHANA DIVYA Lecturer, Department of biotechnology, Lecturer, Department of biotechnology, KSR college of Technology. KSR college of Technology.

EXTERNAL GUIDEEXTERNAL GUIDE : DR. ALEEM KHAN, : DR. ALEEM KHAN, Scientist, Department of stem cell therapy,Scientist, Department of stem cell therapy, CLRD.CLRD.

Introduction: Stem cells :

Progenitor cells.

undifferentiated cells.

self-renewal .

Differentiate under influence of suitable tropic factors.

Types of stem cells : Pluripotent

Embryonic stem cells.

Embryonic Germ cells.

Multipotent

Adult stem cells. Sources:

Blastocyst – embryonic stem cells.

Fetus – fetal stem cells.

Adult stem cells – neuronal, bone marrow, pancreatic , hepatic etc…

• Advancement in stem cell research – new sources of adult stem cells.

• New sources – hair follicles , Dental pulp , Retinal stem cells , Menstrual blood etc.

• Stem cells of blood – hematopoietic stem cells.

Applications: Regenerative medicine. Toxicology studies. Cosmetic therapy. Dental therapies

• Demand for organ transplants - Cell based therapies alternatives for transplants• Liver is a metabolizing organ of the body where most of the detoxification reactions take place.

• Hepatocyte transplants – conceptual alternative for patients suffering from chronic liver diseases – lack of proliferation rate – unsuccessful in transplants.

• 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.

• In my present study, I have taken up the source of stem cells from human fetal hepatic tissue during the second trimester.

• Hence we are checking for expression of liver specific markers during 14 – 20 weeks of gestation.

• Hepatic progenitors – highly expandable due to high proliferation and can be used in liver regeneration

• Hepatic progenitors are isolated by using specific stem cell markers.

LIVER SPECIFIC MARKERSAlpha fetoprotein: 

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

Albumin:                     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).

Cytokeratins 18 and 19: 

 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.           

Objectives:

To isolate the stem cells from human fetal liver.

To characterize the isolated human fetal liver stem cells.

Gene expression analysis of liver Specific markers (AFP , ALB , CK18 & 19 ).

Materials and Methods:Materials:

Fetal liver cells from Aborted Fetus.

Methods:Fetal liver is regained by dissection of fetus.

CD 34 labeling and magnetic sorting (MACS).

Cell viability (Trypan blue Assay)

Hemocytometer (Cell count )

Immunocytochemistry by FITC labeled CD34 antibodies.

MTT assay.

Gene expression analysis using liver specific markers.

LIVER CELL EXTRACTION Homogenization is done with collagenase 4 for complete

extraction of liver cells.

Cell washing is done with Hanks buffer 3-4 times.

MAGNETIC ASSOCIATED CELL SORTER

MACS with LS max column is used for separation of stem cells containing CD 34+ and CD 34- cells.

The separation of +ve and –ve cells is due to the magnetic field applied and the binding of specific antibodies with the cells.

The cells which do not bind with the specific antibody elute first and are referred as CD 34- cells.

The cells are washed with Hanks Buffer 2-3 times.

The cells are preserved in 4°c with the addition of Hanks buffer.

CELL COUNTING Cell counting is the process of measuring total number of

cells per ml. It is done with the help of Hemocytometer The cells are mixed with trypan blue dye in the ratio 1:10

such as dye and cells respectively. The cells are made into exact concentration and placed on

the square slide covered with cover slip. The slide is placed on the microscope and observed for total

number of cells. The calculation is done with the formula

number of cells = number of * dilution per cubic mm cells counted factor

VIABILITY ASSAY

The cell viability test was performed in total cells and in MACS sorted cells by dye exclusion method.

Trypan blue is a vital stain used to selectively colour dead tissues or cells blue. It is a diazo dye.

The dead cells intake dye which results in blue colour. Whereas the viable cells remain colorless.

IMMUNOCYTOCHEMISTRY

To characterize the isolated stem cells.

It visualizes the sub cellular localization of antigen in isolated stem cell populations.

Immunocytochemistry refers to a process of localizing proteins in the cells exploiting the binding of antibodies to antigen.

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

MTT ASSAY MTT assay is a laboratory test and a standard colorimetric assay

(an assay which measures changes in color) for measuring cellular proliferation (cell growth).

It can also be used to determine cytotoxicity of potential medicinal agents and other toxic materials.

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.

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.

GENE EXPRESSION STUDIES The RNA was isolated from the total cells, CD34 positive

cells and CD34 negative cells. The RNA bands are viewed in agarose gel electrophoresis

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. 

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.

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.

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.

3 . Viability Test (Trypan Blue Staining ).

Total number of cells = 2368cells/50μl

Viable cells = 1600cells/50μl

Dead cells = 768cells/50μl

Total number of cells in area = 23.68X109

%viability = 67.56%

Trypan blue staining of CD34+ve cells from human fetal liver cells.

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

5. Immunocytochemistry

FITC conjugated CD34+ve cells under Fluorescent microscope

CD34+ve cells

6 . MTT Assay

Percentage of activity (μg/ml)

OD at 580nm

35

39

0.1349

0.1790

7. Gene Expression Analysis(AFP , ALB , CK -18 , CK -19)

Conclusion:The main purpose of our study is to isolate the CD34 +ve cells which are progenitors in the total liver cell population.

Characterization studies have been done for the isolated CD34+ve cells to know the morphology and study the properties of the progenitors

Gene expression analysis for AFP , ALB , CK18 & 19 is being done to check for the liver specific makers in the CD34+ve cells.

AFP , ALB , CK18 & 19 – illustrates the normal morphogenesis of liver

The Cells which are being isolated and characterized are being used for clinical trials on human patients in our laboratory.