Evaluate the chondrogenic differentiation of DDR1 depleted human adipose derived stem cells.
Dissertation submitted to Vellore Institute of Technology
in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
DEPARTMENT OF INTEGRATIVE BIOLOGY
SCHOOL OF BIO SCIENCES AND TECHNOLOGY
VELLORE INSTITUTE OF TECHNOLOGY
Certificate of the External Guide
This is to certify that the dissertation entitled “Evaluate the chondrogenic differentiation of DDR1 depleted in human adipose derived stem cells” submitted by Swathi Nedunchezian (13MSI0025) to the Vellore Institute of Technology, Vellore-632014, for the degree of Master of Science in Biotechnology is her original work, based on the results of the experiments and investigations carried out independently by her during the period December 2017- May 2018 of study under my guidance.
This is also to certify that the above said work has not previously submitted for the award of any degree, diploma, fellowship in any Indian or foreign University.
Place: Signature of the Guide
Date: Name and Designation
This is to certify that the dissertation entitled “Evaluate the chondrogenic differentiation of DDR1 depleted adipose derived stem cells” submitted by Swathi Nedunchezian (13MSI0025) to the Vellore Institute of Technology, Vellore-632014, for the degree of Master of Science in Biotechnology is her original work, based on the results of the experiments and investigations carried out independently by her during the period December 2017- May 2018 of study under my supervision.
This is also to certify that the above said work has not previously submitted for the award of any degree, diploma, fellowship in any Indian or foreign University.
Date Signature of the Internal Guide
HOD / Director
I hereby declare that the thesis entitled “Evaluate the suppression of the DDR1 depleted adipose derived stem cells” submitted by me, for the award of the degree of Master of Science to Vellore Institute of Technology, is a record of bonafide research work carried out by me under the supervision of Dr Rajesh Kumar.
I further declare that the work reported in this thesis has not been submitted and will not be submitted, either in part or full, for the award of any other degree or diploma in this Institute or of any other Institute or University.
Date: Swathi N
I prompt my sincere appreciation to honorable chancellor, Dr.G. Viswanathan for giving me the opportunity to be a part of VIT UNIVERSITY, Vellore.
I express my profound gratitude to my External Research Guide and mentor, Dr. chih kuang wang Department of Medicinal and Applied Chemistry, Director, Innovation Incubation Center, Office for Operation of Industry-University Cooperation, Research Fellow and Deputy Director, Orthopedic Research Center, Kaohsiung Medical University for providing all the facility required for the project and for his guidance and support for enabling me to complete the thesis within the stipulated time.
I also thank the Ministry of education M.O.E in Taiwan for giving me a wonderful opportunity for doing a project in Kaohsiung Medical University, Taiwan.
I express my deep gratitude to Dr. George Priya Doss, Associate Professor, Head of the Department, Department of Integrative Biology, School of Bio Sciences and Technology, VIT University, Vellore for his support in my project work.
I express my sincere thanks to Internal Research Guide, Dr. S. Rajesh Kumar, Research Scientist, School of Bio Sciences and Technology, VIT University, Vellore.
I am grateful to my parents and my friends for the mental support provided throughout the five years of my MSc Biotechnology (Integrated) and their prayers and wishes for all my future endeavors.
LIST OF ABBREVIATION
RA Rheumatoid arthritis
ECM Extracellular matrix
COL-? Collagenase type ?
ACT Autologous chondrocyte implantation
MSC Mesenchymal stem cells
BMSC Bone marrow derived mesenchymal stem cells
ADSC Adipose derived stem cells
hADSCHuman adipose derived stem cells
PEG Polyethylene glycol
RNA Ribonucleic acid
DNA Deoxyribonucleic acid
RT-PCR Real-time polymerase chain reaction
PVDF Polyvinylidene difluoride
DDRs Discoidin Domain receptors
sGAG Sulphated glycosaminoglycan
DMMB Dimethyl methylene blue assay
shRNA Short hairpin ribonucleic acid
DDR1 Discoidin Domain Receptor 1
FBS Fetal bovine serum
LIST OF SYMBOLS
µl Micro liter
°c Degree Celsius
µg Micro gram
ng Nano gram
AIM AND OBJECTIVES
REVIEW OF LITERATURE…………….
MATERIALS AND METHODS………….
Chapter 1: Introduction
1.1 Cartilage disease
Articular cartilage is also called as articular ligament is the one of the tissue which are often get affected by different factors, for example, maturing and injury. Among the many disease of cartilage, arthritis is the normal form and it involves the wide range of conditions caused by such factors as ageing and infection. Ageing causes about the degeneration of the articular cartilage similarly as it does to other organs and the tissues in the body. Septic arthritis is the disease which is caused due to infections. The septic arthritis is defined as the microorganism such as bacteria are invading in the joint of the bone. Rheumatoid arthritis is one of the major categories in the arthritis family. In RA the pain in the joint will start to migrate to other organs such as lungs and pleura. Sometimes the situation become complicated and results in acute or chronic illness which cannot be cured easily. Degeneration of the cartilage due to the RA is the most common form in the worldwide population. Another type of arthritis is osteoarthritis. The major function of osteoarthritis is degeneration of the articular cartilage and damage to it. It also decreases the function of the joints. Risk factors of osteoarthritis include pain and no physical activity in the body. These conditions can be improved by surgery or replacement of the bone. The individuals affected by arthritis will experience a pain in the joint and difficulties in moving the legs. This disease has been affecting peoples lives and therefore it has been a challenge to the country to develop a method to treat the disease with low cost-efficiency.
1.2 Biology of cartilage
Cartilage is a smooth elastic tissue which covers the ends of the bones at its joints. It is a stiffer tissue and also less stretchable than muscle. For example, in the knee joints, articular cartilage is the connective tissue which links the tibia and femur. Many Other tissues such as Nose, Ears, Rib cage also contains cartilage. It has the good rigidity property. And it has also the good shape restoration of the tissue. The cartilage tissue contains the specialized type of single cells called as chondrocytes, which produces the enormous number of extra cellular matrix contains rich amount of proteoglycan and elastin fibers. Lacunae is a cavity, where the single chondrocytes or mass number of chondrocytes are trapped. Due to avascular characteristic of the cartilage, the chondrocyte can remain undamaged in the low oxygen level and the nutrition level. The cells, nutrition and the waste migrate through diffusion method in the cartilage because of lack of blood vessels. The chondrocytes are originated and differentiated from the mesenchymal stem cells and later it is divided into articular cartilage, where the cells growth and division stop and start to secret the cartilage ECM.
There are three types of cartilage namely fibrous cartilage, hyaline cartilage and elastic cartilage. The composition of the components of cartilage ECM such as collagen fibers, glycosaminoglycan and elastin fibers will differ in the amount of the above three types of the cartilage.
The fibrous cartilage is found in the intervertebral discs and meniscus. The ECM of fibrocartilage is mainly composed of 70% of fibrous components and 1% of proteoglycans. The fibrocartilage is the combination of the fibrous tissue and the cartilaginous tissue, which will give the fibrous cartilage stiffness and the elasticity. The fibrocartilage has the enormous amount of col ? present in the ECM. As the collagen fibers in the fibrous cartilage arranged in the circumference dimension.
The cartilage present in the ears and epiglottis are elastic cartilage. The elastic fibers in the elastic cartilage gives the flexibility to the tissue which can enable the cartilage to change to its original shape or positions after a heavy external force. The elastic cartilage is less likely to regenerate.
Many organs and tissue contain hyaline cartilage. For example, nose, bronchi and joints. As a connection between the bones and the joint, the hyaline articular cartilage cover the closures of the bone. The essential capacity of hyaline articular ligament is weight stacking for joint movement. The low force between the nearby articular cartilage tissues in the joint, due to the oil impact of the synovial fluid, enables helpful relative development of the cartilage. One important function is that articular cartilage does not contain blood vessels in the tissue. Therefore, the proliferation of the cell rate is lower than that of other tissues. It has brought huge issues when the cartilage has experienced injury or degeneration, during this condition the cartilage self-repairs is very slowly and it is limited. It provides the sources for the regeneration of the cartilage. And it became the preliminary idea to think why the cartilage therapeutic invention is needed when the cartilage is damaged.
Hyaline cartilage is composed of 60- 85% of wet weight of water which is highly dehydrated. And 10-30% of total weight is collagen which is the rich components in the cartilage. Twenty-seven types of collagen have been recognized all of which shares a typical unit of Gly-X-Y and frame triple helix structures. In this collagen types, col ? is present in 90-95% of total collagen. Other types of collagens are present in small amount in the cartilage. The capacity of these segments is to hold the state of ligament and give elasticity. It additionally goes about as a molecular binding, framing a total network of ECM.
1.3 Articular cartilage in tissue engineering
Regenerative medicine is defined as the reproduction of the damaged organs or tissues with treatment. The main aim of the regenerative medicine is to restore the function and structure of any organs and tissue. Regenerative drugs are considered as a less painful treatment, where the drugs are biologically functioned and attack the damaged site in the tissue and rescue it and replace the function and structure of the damaged tissue. Intra articular injection or stem cells is the popular in the regenerative medicine. In vivo environment is needed for the intra articular injection or stem cells to differentiated into the particular cell lineage. The second method in regenerative medicine is transplantation of the any organs or tissues. This includes the field of tissue engineering which creates the artificial tissues or organs by involving the combination of biology, material science and engineering. The cells are taken from the patient or donors and extended to achieve the adequacy. These cells would be stacked to the scaffold which go about as a help or direction for cell growth, proliferation, differentiation and most importantly, tissue implantation to oblige cell motility and keep away from cell misfortune.
So far, now by using the principle of tissue engineering only a few artificial organs and tissues are produced. for example, pancreas, liver, heart valves and cartilage. Autologous chondrocyte implantation is the successful method for the regeneration of the cartilage. It has been observed that the over 10000 patients with cartilage disease undergone an ACT treatment and the success rate is high. In an ACT treatment the tissues are extracted from the patient or donor and the chondrocytes cells are separated and then amplified under in vitro condition. Later, the chondrocytes cells are passed through periosteum flap to the damaged site to attack and attach to the cells. This is the new method for the cartilage regeneration approach.
Mesenchymal stem cells
Mesenchymal stem cells are defined as the group of adult stem cell which is derived from the mesenchyme. The MSCs is used mostly in the cartilage regeneration. As a sort of stem cells, mesenchymal stem cells are undifferentiated cells which have possibilities to separate into particular cell types for tissue arrangement and regeneration after repairs to the tissues. they ordinarily dwell quietly in inches of tissues and when necessary, are selected to the harmed site, where they get signals from the in-situ condition for reinventing. MSCs have been a cell origin for chondrogenesis because of their relatives ease in determination and reconstructing into chondrocyte phenotype.
Mesenchymal stem cells have two particular features, which make them better than terminally differentiated cells. Initially the cells will differentiate into other cell lineages. Secondly, they will replicate themselves for multiple times and results in obtaining large number of cells. This overcomes the bottleneck in tissue engineering.
MSCs are mainly extracted and isolated from the adult patients. MSCs are chiefly found in many parts. Early it is found in bone marrow later found in the umbilical cord, adipose tissue and peripheral blood.
Bone marrow derived stem cells occurs in abundance in the bone marrow and it can be easily isolated. After the isolation of the bone marrow, it is separated using the centrifugation method and then the pellets are resuspended. The cells are plated for cultures. The morphology of the bone marrow stem cells is spindle like structure. So that it is easy to distinguish the other type of cells and bone marrow stem cells. The more number of BMSCs can be isolated or obtained by changing the media and cell passage spontaneously. Fluorescent activated cell sorting (FACS) method is used to purify the BMSCs. BMSCs can be passaged while holding their potential for differentiation, and they have been substantiated in bunches of trials to have the capacity to separate into numerous cell types, including osteocytes, chondrocytes, adipocytes, cardiomyocytes, etc.
Stem cells derived from the umbilical cord has been studied. This cell can be distinguished or differentiate into smooth muscle cells, endothelial cells and neurons as layer on osteocytes, chondrocytes and adipocytes. This area of researched have started a business in market that the babies umbilical blood is stored under frozen temperature. Later, it can be used for any demand of emergencies.
Another class of stem cells are adipose derived stem cells. The ADSCs is easily isolated in a huge number of cells. Age is not a barricade for the ADSCs. It maintains an active regeneration capability. AMSCs maintains the potentiality of osteogenesis, chondrogenesis, myogenesis and adipogenesis. It has been noted that the AMSCs shows poor chondrogenesis with other types of MSCs.
Gene delivery method
3.1 Non-viral vector method
The non-viral vector method uses any physical or chemical agents to deliver the target gene, in the form of plasmid to the cells. Physical methods are gene gun shooting and electroporation. Chemical methods include using of liposomes and PEG Polymers. The chemical agent used should be cationic therefore it will react with the anionic DNA and the target gene delivers via the cell membrane by endocytosis method. Disadvantage of using non-viral vector method is the destruction to the cells structure and also loss of functions. Liposome method causes the changes in the cell membrane due to this activity the cell permeability affects and sometimes results in cell death. Electroporation method results in low transfection efficiency and cell destruction. The low transfection efficiency affects the phenotype of the cells. It has been proved in the previous research study that the viral vector increases the transfection efficiency.
3.2 Viral vector method
The rate of transduction efficiency has been increased to 99% by using the adenoviral vector to deliver the target gene to the host. Viral vector is developed with safety and increasing efficiency. Example of viral vectors are adenoviral vector, lentiviral vector, retroviral vector, adeno-associated viral vector. The brief study has been done for the adenoviral vector and the lentiviral vector. Because of the viral structure and its function. In our research lentiviral vector is used to deliver a target gene.
3.2.1 Lentiviral vector
Lentivirus-retrovirus family. Lentiviruses are envelope structure surrounded by capsid with a particle size ranging from 80-110nm in diameter. It has diploid RNA molecules in the genome and coupled with viral replication enzymes. The different types of encoding domains in the genome of the lentivirus for example gag, pol, env. The functions of these domain are the production of the protein for replication, integration and envelope. The genes such as tat and rev act as regulatory domain. Once the injection has inserted the genomic RNA are transferred into the nucleus. The reverse transcription process takes place and the double stranded DNA is transcribed and integrated into the host genome with the help of integrase. New copies of RNA are produced by transcription process and encapsulated using proteins into the capsids.
Advantage of lentiviral vector
High transduction efficiency
Infect both dividing and non-dividing cells.
RT- PCR (reverse transcription- polymerase chain reaction is the most sensitive technique of mRNa detection and quantitation currently available. Compared to the two other commonly used techniques for quantifying mRNA levels, northern blot analysis and RNase protection assay. RT-PCR can be used to quantify mRNA levels from much smaller samples. In fact, this technique is sensitive enough to enable quantitation of RNA from a single cell. Over the last several years, the development of novel chemistries and instrumentation platforms enabling detection of PCR products on real-time basis has led to widespread adoption of real-time RT-PCR as the method of choice for quantitating changes in gene expression. RT-PCR is a method to amplify, or increase, the amount of a specific RNA sequence. Typically, the target RNA sequence is reverse transcribed into its complementary DNA (cDNA). The cDNA is subsequently amplified using a solution containing DNA polymerase and nucleotides, and primers that are complementary to the target DNA sequence. When this solution is heated, the dsDNA denatures, separating into two separate strands. As the solution cools, the primers anneal to the target sequences in the separated DNA strands. The DNA polymerase then forms a new strand by extending the primers with nucleotides, creating a complimentary copy of the target DNA sequence. When repeated, this cycle of denaturing, annealing, and extending increases exponentially the number of target DNA sequences. Ideally, no amplification occurs if the target DNA sequence is not present.
Figure 1 : Principle behind RT PCR
Western blot technique is used for the detection & analysis of proteins based on the ability of the protein to bind to their specific antibodies. This is the common technique used for life science research. It is a simple experiment with inexpensive reagents and commonly used techniques in the lab. The antibody- antigen specificity interaction enables the target protein to be identified in the midst of a protein mixture.
An analytical method where the protein sample is electrophoresized on an SDS- PAGE and transferred to polyvinylidene difluoride (PVDF) or nitrocellulose membrane. The protein is detected using specific primary and secondary enzyme labeled antibody.
Epitope is defined as the antibodies which bind to the specific sequence of amino acids. Because amino acid sequences are different from protein to protein, antibodies can recognize specific proteins among a group of many. A single protein can be identified in the sample of cell lysate which contains many number of different proteins which can be later quantified through the western blot technique. At First, proteins are separated from each other based on their size. Secondly, antibodies are used to detect the target protein. Finally, a substrate that reacts with an enzyme is used to view the antibody complex.
Principle behind the western blot technique
Western blotting is the transfer of proteins from the SDS- PAGE gel to a solid supporting membrane. There are two types of blotting apparatus used to transfer proteins to solid supports; these facilitate either wet transfer also called as tank blotting or semidry transfer. Both of them give perfect result.
Electrophoresis is a technique separates the mixture of the proteins. The one-dimensional gel electrophoresis can separate the proteins based on the molecular size when they pass through an SDS PAGE towards the anode. Here the low molecular weight proteins migrate faster than the high molecular weight protein. The SDS-PAGE contains two gel-stacking gel & separating gel which is prepared and sealed into the apparatus. The protein samples are loaded into the gel lane. The protein starts to separate by electrophoretically. The reagent such as 2-mercaptoethanol and dithiothreitol are added because it will reduce the disulphide bonds.
A protein sample is subjected to polyacrylamide gel electrophoresis. The gel is placed over a sheet of nitrocellulose membrane and the protein in the gel is transferred to the membrane electrophoretically. The membrane is dipped into the blocking buffer which contains 3% of skimmed milk solution helps to block the non-specific binding of proteins. Later the nitrocellulose membrane is incubated with the specific antibody for the protein of interest. The nitrocellulose is then incubated with a second antibody, which is specific for the first antibody. For instance, if the principal immunizer was brought up in mouse, the second counter acting agent may be named “goat hostile to mouse immunoglobulin”. This means mouse immunoglobulin was utilized to inspire a counter acting agent reaction in goats. The second neutralizer will ordinarily have a covalently appended compound which, when furnished with a chromogenic substrate, will cause a shading response. Along these lines, the sub-atomic weight and measure of the coveted protein can be described from an intricate blend of different proteins by western blotting.
Alcian Blue Staining Assay
Alcian blue staining assay is a method used to stain polysaccharides for example glycosaminoglycans present in the cartilage. Alcian blue is derived from the group of polyvalent dye which are water soluble. The blue color indicates the presence of copper in the molecule. The 3% percent of acetic acid solution, the alcian blue will stain both the sulfated and carboxylated acid mucopolysaccharides and glycoproteins. It forms a salt linkage with the acid groups.
Chapter 2: Review of literature
Chondrogenesis is defined as the development of cartilage. Chondrification is the procedure toward which cartilage is framed from condensed mesenchyme tissue, which differentiates under chondrocytes. Furthermore, starts secreting the particles that type those extracellular grids.
Discoidin Domain receptor
DDRs belongs to the receptor tyrosine kinase family. DDR1 and DDR2 are known as collagen binding receptors. The receptors are found in several transmembrane domain. The transmembrane domain is distinguished according to the motif in the extracellular domain. These N-terminal of the domains are homologous to dictyostelium discoideum. The function of DDRs is cell migration, proliferation and maintaining homeostasis of extracellular matrix. The function of DDR1 and DDR2 are not same, it may vary different. The amino acid sequence relationship between DDR1 and DDR2 remains similar. Collagen is the key ligand for the DDRs. For example, the DDR1 is mainly activated by collagen type 1 to 5 and DDR2 is activated by fibrillar collagen type ?. The previous study shows that the DDR1 is the cause for affecting the cell homeostasis during the chondrogenesis process. Since there is no proper evidence for the detailed study of the DDR1 function during the chondrogenesis in ADSCs. In this experiment we did a study for chondrogenic differentiation in hADSCs.
Suppression of the DDR1 gene to enhance the chondrogenic gene expression
In previous study, non-viral vector method is used to deliver a DDR1 shRNA into the hADSCS. The non-viral vector method is microporation. The DDR1 shRNA or plasmid is transfected into the hADSCs. To induce the chondrogenic differentiation they used three-dimensional pellet culture model. To detect the expression of the DDRs, chondrogenic genes (SOX-9, COL-?, Aggrecan) and cartilaginous matrix deposition (sGAG &COL-?) Real-time polymerase chain reaction method was used. And to determine the protein expression of the gene western blotting technique was used. The alcian blue staining method was used to determine the amount of sGAG synthesis. It has been observed that the RT-PCR results shows that the increased expression of the chondrogenic genes (SOX-9, COL-?, Aggrecan) and cartilaginous matrix deposition (sGAG, COL-?). It has been observed that the decreased expression of the terminal differential genes in the DDR1 depleted hADSCs. From the results, it is noted that the DDR1 expression can enhance the chondrogenesis in the adipose derived stem cells by increasing the expression of the chondrogenic genes and the cartilaginous matrix deposition.
Chapter 3: Aim & Objectives
To evaluate the expression of the chondrogenic genes (SOX-9, COL-? and aggrecan) and the cartilaginous matrix deposition (COL-? and sGAG) in the DDR1 depleted human adipose derived stem cells.
Isolation and culturing of the hADSCs.
DDR1 Gene knockdown using lentiviral vector.
Detecting the expression of the chondrogenic genes in the DDR1 Knock down hADSCs using RT-PCR technique.
The protein expression is detected by using Western blotting.
The amount of sulphated glycosaminoglycan (sGAG)deposition is detected by alcian blue staining assay method.
The total amount of DNA present in the sGAG is quantified by dimethyl methylene blue assay (DMMB).
Materials and Methods:
Isolation and culture of the hADSCs
The hADSCs isolated from the adipose tissue. The adipose tissue is extracted from the patient with the approval from the ethical committee at the Kaohsiung medical university.
5g of adipose tissue taken out from the patient and digest with the type ? Collagenase enzyme at 37? under CO? condition.
Centrifuge the cells at 1000 rpm for 5-10 minutes.
Wash twice with the PBS reagent.
The cell is cultured in the K-NAC medium in the 100mm of culture dish.
Preparation of the Medium – The medium is made up of keratinocyte-SFM basal Medium (GIBCO, Rockville) which is combined with bovine pituitary extract (GIBCO), human recombinant epidermal growth factor, N-acetyl-L-cysteine, ascorbic acid 2-Phosphate magnesium salt and bovine serum. The K-medium function is to amplify the cells in the Petri dishes. The medium should be refreshed once for 48hrs.
Transduction with DDR1-Specific short hairpin RNA using viral vector method
The MISSION TRC shRNA lentiviral particle is purchased and used for long term gene silencing.
Solution – Hexadimethrine bromide (2mg/ml) and puromycin (10mg/ml).
shRNA- Negative control is plKO.1 lentiviral particle (puro control), shRNA target control, Positive control is JAK1 Control lentiviral particle.
After transduction of the cells, hADSCs are cultured in a K-NAC medium without antibiotics. This need to be done till the induction of chondrogenic differentiation in a pellet culture method.
Chondrogenic differentiation induction using pellet culture method
The pellet culture method is used to induce the chondrogenic differentiation in the medium. The hADSCs that transduced with the DDR1 shRNA are typsinized and resuspended in the Dulbecco’s modified Eagle’s medium in which 3.7g of sodium bicarbonate,100ml of the FBS,10ml of penicillin or streptomycin are supplemented to the cell density of 4×10? cells/ml. The aliquots of hADSCs in the basal medium is added to the 15 ml of polypropylene conical tubes and centrifuged it for 5 minutes to get a cell pellet. Later the cell pellets in the conical tubes is placed inside the incubator for 24 hrs. to get a spherical shape. The basal medium is removed after taking out from the incubator. The cell pellets are cultured with chondrogenic medium which consist of basal medium supplemented with antibiotics, growth factors and L-ascorbic acid. The chondrogenic medium is changed for 2 days once.
RNA (Ribonucleic Acid), it is polymeric substance seen in living cells and virus. It has long single stranded chain of phosphate and ribose units with nitrogen bases adenine, guanine, cytosine and Uracil. RNA carries a genetic information for many viruses and it also play an important role in synthesis of protein. RNA isolation is a difficult step. The reagent used for RNA isolation from the cells and the tissues is Trizol reagent. Trizol maintains integrity of RNA and breaks cells and cell components. Chloroform separates the solution into aqueous and organic phases. RNA is present in the aqueous phase.
The cell pellets were collected from the pellet culture at each indicated time points.
The media is pipetted and wash twice with the 1-2ml of cold PBS.
Aspirate the PBS and add 0.5ml of TRIzol to the vial.
Scarpe the plate gently, remove the TRIzol reagent and lysate it into 1.5ml of Eppendorf tube.
Leave the Eppendorf tube at room temperature.
Add 100?l of the chloroform to the vial and shake the vial vigorously for 10-15 seconds.
Leave at room temperature for 2-5 minutes.
Centrifuge the tubes at 12,000 rpm for 10 minutes. After centrifugation three layers will appear in each tube.
The top layer which is clear and aqueous, the middle layer contains the white precipitated DNA and the Bottom layer is pink organic phase. The top layer is removed and transfer it to 1.5ml of Eppendorf tube.300?l of the isopropanol is added to the tube and vortex it gently. Leave it for room temperature for 10 minutes.
Centrifuge it again for 12,000 rpm for 5-10 minutes. At the bottom of each tube there will be a pellet barely visible. Throw the isopropanol and add 0.5 ml of the 100% of ethanol. Recentrifuge at 12000 rpm for 5 minutes.
Pour off the ethanol and make the pellets to air-dry. Add 20-25?l of DEPC Water to the RNA pellet.
RNA was quantified using nanodrop. RNAse free water was used as blank.
The concentration of RNA was found.
Quantitative Real time PCR
1µg of RNA was taken and water was added making it to 5µl which was then mixed with the Master Mix that was prepared by addition of RNAse free water (1.5µl), Random 6 miR primer (0.5µl), buffer (2µl) oligo dt (0.5µl) and enzyme (0.5µl) making up to (5µl). The total RNA was reverse transcribed into the cDNA using the superscript first-strand synthesis system(Invitrogen). Real time PCR reactions were performed and it is monitored using the SYBR green super mix and also by the quantitative real-time PCR detection system. The cDNA samples were checked for the gene of interest. The primers used for detecting SOX-9, collagen type ?(COL-?), aggrecan, collagen type ?(COL-?) and glyceraldehyde-3-phosphate-dehydrogenase(GADPH). And also, the primers for detecting the DDR1, Matrix metalloproteinase 13(MMP-13), and runt-related transcription factor 2(Runx2). The primers used for the above genes are listed in the Table.1. To determine the specificity of the reaction the melting curve was designed. The mRNA expression level for each target gene was calculated by threshold cycle(Ct) value and it is normalized to the expression of the house keeping gene GADPH using the comparative Ct method.
Gene PCR Primers sequence (forward and reverse) Annealing temperature?
SOX-9 Forward: 5′-CTT CCG CGA CGT GGA CAT-3′
Reverse: 5′-GTT GGG CGG CAG GTA CTG-3′ 55
Collagen type ?(COL-?) Forward:5′-CAA CAC TGC CAA CGT CCA GAT-3′
Reverse:5′-TCT TGC AGT GGT AGG TGA TGT TCT-3′ 61
Aggrecan Forward:5′-ACA GCT GGG GAC ATT AGT GG-3′
Reverse:5′-GTG GAA TGC AGA GGT GGT TT-3′ 61
Collagen type ?(COL-?) Forward:5′-GGC TCC TGC TCC TCT TAG-3′
Reverse:5′-CAG TTC TTG GTC TCG TCA C-3′ 61
Discoidin Domain Receptor -1(DDR1) Forward:5’AAC AAT TCC TCT CCG GCA CTG-3′
Reverse:5’GAT GAG GGC AAT GAT GAG CAG-3′ 60
Matrix Metalloproteinase 13(MMP-13) Forward:5’TCT TGA GCT GGA CTC ATT GT-3′
Reverse:5’AGC CTC TCA GTC ATG GAG CT-3′ 61
Runt-Related transcription factor 2(Runx2) Forward:5’AGA TGG GAC TGT GGT TAC TG-3′
Reverse:5’GTA GCT ACT TGG GGA GGA TT-3′ 55
Glyceraldehyde -3-Phosphate -dehydrogenase(GADPH) Forward:5’TCT CCT CTG ACT TCA ACA GCG AC-3′
Reverse:5’CCC TGT TGC TGT AGC CAA ATT C-3′ 61
Table 1: primers sequences and its annealing temperature.
Isolation of protein
The cell pellets were collected and the media are removed.
Wash the cells with PBS buffer for 3 times.
Add 300?l of lysis buffer to the Petri plate.
Keep the plates at 37? for 5 min.
Centrifuge the cells at 14000rpm for 15 min at 4?.
Throw the pellet which contains the cell membrane and nucleus.
The protein concentration is determined by using BCA protein assay kit.
The kit contains Solution A and the Solution B. It has to be diluted for 5X.
Prepare the BCA reagent in the ratio of 1:16(Sample: BCA solutions)
Dilute the 30?g/ml sample with the double distilled water.
Load the samples into the 96 well plate.
Keep the plates at 37? for 40 mins for reaction to occur.
Read the concentration of the protein at 540nm.
Preparation of the SDS-PAGE
Wipe the SDS-PAGE kit with the ethanol.
Preparation of the upper gel and the lower gel for 10%
After adding the lower gel materials into the gel slab leave it for 15-20mins
Pour the upper gel solution into the slab.
Place the comb on it
Loading the sample
Prepare 36?l of 6X dye and 4?l of Mercaptoethanol (gel loading dye).
Take 2?l of the dye and add it to the sample and the control.
Add 4?l of the lysis buffer to the control and 5?l to the DDR1.
Now place the tube in the temperature of 90? for protein to linearize for 5mins.
Meanwhile, setup the SDS-PAGE kit.
Add the samples and the control to the well in the gel. The marker is loaded in the one of the well which determines the molecular weight of the sample.
Run the gel, negative pole is on the well of the gel and positive pole is opposite end to the gel.
Transfer of membrane (wet transfer)
After the gel run, it is placed against a membrane, a current is passed across the membrane to transfer the protein onto the membrane.
The first step in the immunoblotting is the washing the membrane and blocking it with non-specific protein.
Next step, adding the primary antibody to the solution where in the membrane is floating.
After a wash, add secondary antibody which can recognize the primary antibody.
Finally ,another wash is done to remove the unbound secondary antibody.
The detection is done by chemiluminescence analyzer.
Reagents Lower gel Upper gel
Double Distilled water 4.8ml 2.96ml
40% Tris 2.5ml 500?l
1.5M Tris 2.5ml 500?l
20% SDS 50?l 20?l
10%APS 100?l 40?l
TEMED 6?l 12?l
Alcian blue staining method for the detection of sulphated glycosaminoglycan
Reagents used: 3% glacial acetic acid, alcian blue solution, nuclear fast red.
Control: Small intestine, Colon
Hydrate the slides with the distilled water.
Add 3% of acetic acid leave it for 3 minutes.
Microwave the slides for 30 seconds in the HI-power.
Wash in the running water for 2 minutes.
Add nuclear fast red and leave it for 5 minutes and wash it in running water.
Observe under the microscope.
Dimethyl methylene blue assay for the quantification of sGAG
The DNA content and sGAG deposition in the cell pellets were quantified spectrofluorometrically using the dye and the dimethyl methylene blue. A standard curve for the DMMB assay was made using an aqueous solution of chondroitin sulphate C.
Chapter 4: Results
To suppress the expression of the DDR1 in the hADSCs, the lentiviral vector method is used to transfer the DDR1 specific short hairpin RNA to the hADSCs. The hADSCs were transduced with high transduction efficiency. The figure 2(a&b) shows that the transduction of the virus into the hADSCs in the control group and the DDR1 depleted group.
To detect whether DDR1 is involved in the hADSCs chondrogenic differentiation, chondrogenic marker gene expression and cartilaginous matrix deposition in the control group and the DDR1-depleted group were examined after culturing in the three-dimensional pellet culture system with the chondrogenic medium. The real-time PCR method shows that the chondrogenic gene COL-? and Aggrecan have showed the increased expression in the DDR1-depleted group than the control group at day 0 to day 5. We found that the expression of COL-? is higher in the DDR1-depleted group than the control group.
The alcian blue staining method also shows that the sGAG deposition in the DDR1-depleted group is higher than that of control group. The DMMB assay quantification shows that the amount of sGAG deposition in the hADSCs is higher in the DDR1-depleted group than that of control group at day 5.
Figure 2: Isolation and culture of hADSCs
DDR1 knock down using lentiviral vector method
The fig 2 is the number of hADSCs cultured in the K-NAC medium. The total number of cells is 1.7×10?cells/ml. fig 3(a) shows the DDR1 knock out in the control group hADSCs & 3(b) shows the DDR1 knock out using lentiviral in the hADSCs. Fig 4 is the mRNA expression of the DDR1 in the control group and the DDR1-depleted hADSCs at day 1& day 5. It shows that the decreased expression at day 5 in the DDR1-depleted hADSCs than the control group.
The fig 5 shows the mRNA expression of the chondrogenic genes-COL ? & Aggrecan in the control group and the DDR1-depleted hADSCs group grown in the pellet culture model. It shows that the DDR1-depleted hADSCs group shows the significant increase in the gene expression at day 1 and day 5 than the control group.
The fig:6 shows the sGAG synthesis of hADSCs processed with the alcian blue staining for the control and the DDR1-depleted hADSCs.
The fig 7 indicates the sGAG synthesis by hADSCs in the control and the DDR1-depleted group by dimethyl methylene blue assay.
Figure:8 Concentration of the protein.
The fig 9 shows the protein expression of the DDR1 and the ?-actin in the hADSCs.
The fig.10 is the mRNA expression of the COL-? in the control group and the DDR1-depleted hADSCs group.
The fig.11 is the mRNA expression of the terminal differentiation genes RUNX2 and MMP-13 in the control group and the DDR1-depleted hADSCs.
The expression of the collagen receptors DDR1 & DDR2 affects the homeostasis of the articular cartilage. In this study we have proved that the suppression of the DDR1 increase the chondrogenesis of hADSCs by increasing the expression of the chondrogenic genes and the cartilaginous matrix deposition. In previous study they used the microporation method to deliver a shRNA into the hADSCs to knock down the DDR1 gene. In our study, we used the lentiviral vector method to deliver a DDR1 shRNA to knock down the DDR1 gene in the hADSCs. It has found that the terminal differentiation genes are present in the damaged articular cartilage tissue. So, the expression of the terminal differentiation genes in the damaged articular cartilage tissue shows the increased expression. It has been observed in our study that the terminal differentiation genes such as Runx2 and MMP-13 shows the decreased expression in the mRNA level in the DDR1-depleted Group in the hADSCs. From this it has been clear that the DDR1 suppression can increase the effect of chondrogenesis in the hADSCs. Hence proved that the suppression of the DDR1 in the hADSCs will be a strategy to optimize ADSC-based articular cartilage tissue engineering by enhancing the hADSCs chondrogenesis.