PEPTIDE APTAMER PVGL52 PEPTIDE APTAMER PVGL52 INDUCING MESENCHYMAL INDUCING MESENCHYMAL STEM CELL LOCALISATION STEM CELL LOCALISATION

INTO HEART:A CELL INTO HEART:A CELL THERAPY MODEL STUDY IN THERAPY MODEL STUDY IN

CHICKCHICK PRESENTED BYPRESENTED BY

R.PRIYADHARSHNIR.PRIYADHARSHNI ROLL NO:11706214043ROLL NO:11706214043

S.VANIDEVIS.VANIDEVI ROLL NO:11706214057ROLL NO:11706214057

INTRODUCTIONINTRODUCTION

In 1960S,The existence of stem cells in an irradiated In 1960S,The existence of stem cells in an irradiated mouse was reported by mouse was reported by Canadian scientists Ernest ACanadian scientists Ernest A. McCulloch and James E. Till.McCulloch and James E. Till.

Stem cellsStem cells have the remarkable potential to develop have the remarkable potential to develop into many different cell types in the body during early into many different cell types in the body during early life and growth.life and growth.

They are found in most, if not all, multi-They are found in most, if not all, multi-cellular organisms.They serve as a sort of cellular organisms.They serve as a sort of internal repair system.internal repair system.

eg:wound healingeg:wound healing

2 important characterstics of stem cells are-2 important characterstics of stem cells are-

They are unspecialized cells capable of renewing They are unspecialized cells capable of renewing themselves through cell division and differentiation themselves through cell division and differentiation giving rise to specialised cells.giving rise to specialised cells.

under certain physiologic or experimental conditions, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-they can be induced to become tissue- or organ-specific cells with special functions.specific cells with special functions.

eg:Addition of growth hormoneseg:Addition of growth hormones

Stem cells are divided or classified based on-Stem cells are divided or classified based on-

1.Source1.Source 2.Potency2.Potency

Based on Based on sourcesource stem cells can be classified into stem cells can be classified into following:following:

Embryonic stem cellsEmbryonic stem cells come from a five to six-day-old come from a five to six-day-old embryo. They have the ability to form virtually any embryo. They have the ability to form virtually any type of cell found in the human body. type of cell found in the human body.

Embryonic germ cellsEmbryonic germ cells are derived from the part of a are derived from the part of a human embryo or foetus that will ultimately produce human embryo or foetus that will ultimately produce gametes (eggs or sperm). gametes (eggs or sperm).

Adult stem cellsAdult stem cells are undifferentiated cells found are undifferentiated cells found among specialised (differentiated) cells in a tissue among specialised (differentiated) cells in a tissue or organ after birth. Based on current research, or organ after birth. Based on current research, adult stem cells appear to have a more restricted adult stem cells appear to have a more restricted ability to produce different cell types and to self-ability to produce different cell types and to self-renew than embryonic stem cells. renew than embryonic stem cells.

Umbilical cord blood stem cellsUmbilical cord blood stem cells are used to treat a are used to treat a range of blood disorders and immune system range of blood disorders and immune system

conditions.conditions.

Based on potency Stem cells are classified as- Based on potency Stem cells are classified as- "Potency" is a term that describes how many types of "Potency" is a term that describes how many types of

cells a stem cell can become.cells a stem cell can become.

TotipotentTotipotent stem cells are cells that have not begun stem cells are cells that have not begun differentiating at all. They are capable of developing differentiating at all. They are capable of developing into into anyany other type of body cell. other type of body cell.

PluripotentPluripotent cells are almost as potent as totipotent cells are almost as potent as totipotent

stem cells. They have barely started differentiating stem cells. They have barely started differentiating and can develop into almost any other type of and can develop into almost any other type of cell,except placenta.cell,except placenta.

Multipotent Multipotent stem cells are stem cells that have begun stem cells are stem cells that have begun differentiating into a general type of cell. For eg,a differentiating into a general type of cell. For eg,a blood cell giving rise to a blood cell only but not blood cell giving rise to a blood cell only but not brain cell.eg:MSCbrain cell.eg:MSC

Oligopotent Oligopotent stem cells can differentiate into only a stem cells can differentiate into only a few types of cell. For example, a lympoid stem cell few types of cell. For example, a lympoid stem cell can become any of the blood cells found in the can become any of the blood cells found in the lymphatic system (T cells, B cells, and plasma cells), lymphatic system (T cells, B cells, and plasma cells), but not a different kind of blood cell, such as a red but not a different kind of blood cell, such as a red blood cell or platelet.blood cell or platelet.

UnipotentUnipotent stem cells can only become one type of cell stem cells can only become one type of cell — their own. They are considered stem cells because — their own. They are considered stem cells because they can reproduce indefinitely. An example is skin they can reproduce indefinitely. An example is skin cells, which can renew themselves indefinitely, but cells, which can renew themselves indefinitely, but which cannot become any other type of cell.which cannot become any other type of cell.

WHAT ARE MSCs?WHAT ARE MSCs? Mesenchymal stem cellsMesenchymal stem cells, or , or MSCsMSCs, or Marrow , or Marrow

Stromal Cell are multipotent stem cells that can Stromal Cell are multipotent stem cells that can differentiate into a variety of cell types which include differentiate into a variety of cell types which include osteoblasts, chondrocytes and adipocytes.osteoblasts, chondrocytes and adipocytes.

SOURCES OF MSCsSOURCES OF MSCs

• Mesenchymal stem cells (MSCs) are adult stem cells traditionally found in the bone marrow.

Bone marrow is the flexible tissue found in the hollow interior of bones. In adults, marrow in large bones produces new blood cells . .

There are two types of bone marrow: 1.red marrow (consisting mainly of hematopoietic

tissue) 2.yellow marrow (consisting mainly of fat cells).

Bone marrow contains three types of stem cells:Bone marrow contains three types of stem cells:

Hematopoietic stem cellsHematopoietic stem cells give rise to the three classes give rise to the three classes of blood cells that are found in the circulation: white of blood cells that are found in the circulation: white blood cells (leukocytes), red blood cells blood cells (leukocytes), red blood cells (erythrocytes), and platelets (thrombocytes). (erythrocytes), and platelets (thrombocytes).

Mesenchymal stem cellsMesenchymal stem cells are found arrayed around the are found arrayed around the central sinus in the bone marrow. They have the central sinus in the bone marrow. They have the capability to differentiate into osteoblasts, capability to differentiate into osteoblasts, chondrocytes, myocytes, and many other types of chondrocytes, myocytes, and many other types of cells. cells.

Endothelial stem cellsEndothelial stem cells

The other sources of bone marrow include cord The other sources of bone marrow include cord blood,peripheral blood,fallopian tube,foetal liver and blood,peripheral blood,fallopian tube,foetal liver and lung adipose tissue,skeletal muscle,amniotic lung adipose tissue,skeletal muscle,amniotic fluid,synovium and circulatory system.fluid,synovium and circulatory system.

CHARACTERISTICSCHARACTERISTICS MorphologyMorphology::

They are bigger in size having a They are bigger in size having a large, round large, round nucleus with a prominent nucleolus, when compared nucleus with a prominent nucleolus, when compared with normal cells.with normal cells.

It also contains It also contains Golgi apparatus, rough Golgi apparatus, rough endoplasmic reticulum, mitochondria, and endoplasmic reticulum, mitochondria, and polyribosomes.polyribosomes.

Differentiation capacityDifferentiation capacity::

MSCs have a large capacity for self-renewal while MSCs have a large capacity for self-renewal while maintaining their multipotency. The standard test to maintaining their multipotency. The standard test to confirm multipotency is differentiation of the cells into confirm multipotency is differentiation of the cells into osteoblasts, adipocytes, and chondrocytes as well as osteoblasts, adipocytes, and chondrocytes as well as myocytes and possibly neuron-like cells.myocytes and possibly neuron-like cells.

Immunomodulatory effects:

Numerous studies have demonstrated that human MSC avoid allorecognition, interfere with dendritic cell and T-cell function and generate a local immunosuppressive microenvironment by secreting cytokines.(Ryan JM et al 2005)

The minimal requirement for a population of cells to qualify as MSCs, as suggested by International Society for Cytotherapy is threefold:

1.They must be plastic adherent under standard culture conditions.

2.They should express CD105,CD73 and CD90 and lack the expression of CD45,CD34,CD14,CD19 and HLA-DR surface molecules.

3.They should possess tripotential mesodermal capacity into osteoblasts,chondrocytes and adipocytes.

DIFFERENTIATION OF MSCDIFFERENTIATION OF MSC

Under defined conditions, MSCs can differentiate Under defined conditions, MSCs can differentiate into chondrocytes, osteoblasts, and adipocytes, and into chondrocytes, osteoblasts, and adipocytes, and they also serve as hematopoiesis-supporting stromal they also serve as hematopoiesis-supporting stromal cells. cells.

MSCs have also been reported, to differentiate into MSCs have also been reported, to differentiate into myocytes and cardiomyocytes and even into cells of myocytes and cardiomyocytes and even into cells of non mesodermal origin, including hepatocytes and non mesodermal origin, including hepatocytes and neurons.neurons.

Mesenchymal stem cells

Ectodermal cells Mesodermal stem cells endodermal cells

Nerve cells Adiposecells bone cartilage cardiac skeletal liver cells pancreatic

muscle muscle cells

REGULATION OF MSCREGULATION OF MSC

Growth factors such as members of the Growth factors such as members of the transforming growth transforming growth factor-beta (TGF-β) superfamily, the insulin-like growth factor-beta (TGF-β) superfamily, the insulin-like growth factors, the fibroblast growth factors, the platelet-derived factors, the fibroblast growth factors, the platelet-derived growth factor, and Wntsgrowth factor, and Wnts..

Several Hormonal factors like Several Hormonal factors like phosphatonins FGF 23 and phosphatonins FGF 23 and sFRP4, Receptors for PTH/PTHrP sFRP4, Receptors for PTH/PTHrP ((Franz Jakob et al, 2006)Franz Jakob et al, 2006)

Peptides derived from growth factors(Peptides derived from growth factors(Jae Sam Lee et Jae Sam Lee et al,2009)al,2009)

REGENERATION BY MSCREGENERATION BY MSC

MSCs are a promising cell type for regenerative medicine MSCs are a promising cell type for regenerative medicine because of their ease of isolation and expansion, their because of their ease of isolation and expansion, their multipotency and their low immunogenicity.multipotency and their low immunogenicity.

The MSCs has the following applications as a regenerative The MSCs has the following applications as a regenerative medice:medice:

regeneration of cardiac muscle.(regeneration of cardiac muscle.(Pittenger, Mark F et Pittenger, Mark F et al,2002al,2002))

regeneration of musculoskeletal tissues.(regeneration of musculoskeletal tissues.(Edward J. Edward J. Caterson et al,2001Caterson et al,2001).).

regeneration of bone marrow(regeneration of bone marrow(K Fukuda,2003) etc.K Fukuda,2003) etc.

MODEL STUDY IN MODEL STUDY IN CHICKCHICK

Model organismModel organism is a non-human species that is is a non-human species that is extensively studied to explore potential causes and extensively studied to explore potential causes and treatments for human diseasetreatments for human disease . .

These can be classed as:These can be classed as:

1.1. genetic models (with short generation times, such genetic models (with short generation times, such as the fruitfly and nematode worm)as the fruitfly and nematode worm)

2.2. experimental models, and experimental models, and

3.3. genomic models, with a pivotal position in the genomic models, with a pivotal position in the

evolutionary treeevolutionary tree..

The The chickenchicken ( (Gallus gallusGallus gallus domesticus domesticus) is a domesticated ) is a domesticated fowl.fowl.

It It has descended primarily from the Red Junglefowl (has descended primarily from the Red Junglefowl (Gallus Gallus gallusgallus) and is scientifically classified as the same species) and is scientifically classified as the same species . . it is it is an amniote and excellent for micromanipulation (e.g. tissue an amniote and excellent for micromanipulation (e.g. tissue grafting) and over-expression of gene products.grafting) and over-expression of gene products.

TAXONOMIC CLASSIFICATIONTAXONOMIC CLASSIFICATION:: Kingdom: AnimaliaKingdom: Animalia

Phylum: ChordataPhylum: ChordataClass: AvesClass: AvesOrder: GalliformesOrder: GalliformesFamily: PhasianidaeFamily: PhasianidaeGenus: GallusGenus: GallusSpecies: G. gallusSpecies: G. gallusBinomial name: Binomial name: Gallus gallusGallus gallus

OBJECTIVESOBJECTIVES

To culture stem cells and isolate the MSCs.To culture stem cells and isolate the MSCs.

To subculture the cells.To subculture the cells.

Induction of growth and differentiation.Induction of growth and differentiation.

Cell therapyCell therapy

Staining techniquesStaining techniques

RT-PCRRT-PCR

Agrose gel electrophoresisAgrose gel electrophoresis

METHODSMETHODS

MEDIUM USED:MEDIUM USED:

1.L-15 medium(Leibovitz) 1.L-15 medium(Leibovitz) 2.DMEM(Dulbecco's Modified Eagle's Medium 2.DMEM(Dulbecco's Modified Eagle's Medium

Modified )Modified )

PREPARATION OF L-15 and DMEM MEDIUM:PREPARATION OF L-15 and DMEM MEDIUM:

1.4 grams of L-15 (in powder form) was added to 1.4 grams of L-15 (in powder form) was added to 100ml of autoclaved 1XPBS.100ml of autoclaved 1XPBS.

10grams of DMEM powder in 1000ml of HBS(Hanks 10grams of DMEM powder in 1000ml of HBS(Hanks buffered-saline).buffered-saline).

MATRIX PREPARATIONMATRIX PREPARATION A solid matrix was prepared by adding 2.5grams agar A solid matrix was prepared by adding 2.5grams agar

agar and 1gram gelatin in 100ml 1XPBS.agar and 1gram gelatin in 100ml 1XPBS.

5 culture bottles were taken and 1ml of this matrix 5 culture bottles were taken and 1ml of this matrix was poured at the bottom of each culture bottle.was poured at the bottom of each culture bottle.

Above this 5ml of freshly prepared L-15 medium was Above this 5ml of freshly prepared L-15 medium was added which contains 1000ul of pencillin and 100ul added which contains 1000ul of pencillin and 100ul of amphotericinB.of amphotericinB.

To isolate marrow, kill chick by cervical dislocationTo isolate marrow, kill chick by cervical dislocation

Rinse the animal skeleton freely in 70% ethanolRinse the animal skeleton freely in 70% ethanol

Make an incision around the perimeter of the hind limbs Make an incision around the perimeter of the hind limbs

Dissect the hind limbs from the trunk of the body by cutting Dissect the hind limbs from the trunk of the body by cutting along the spinal cord along the spinal cord

Bisect each hind limb by cutting through the knee joint and Bisect each hind limb by cutting through the knee joint and remove the muscle and connective tissue .remove the muscle and connective tissue .

ISOLATION AND CULTURE OF MSC ISOLATION AND CULTURE OF MSC FROM CHICK BONE MARROWFROM CHICK BONE MARROW

After cleaning, store the bones in L-15 supplemented with After cleaning, store the bones in L-15 supplemented with penicillin/streptomycin.penicillin/streptomycin.

Now the stem cells are taken by Aspiration of Now the stem cells are taken by Aspiration of Bonemarrow(small amount of bone marrow fluid and cells are Bonemarrow(small amount of bone marrow fluid and cells are removed through a needle put into a boneremoved through a needle put into a bone))..

Centrifuge the BM cells taken at 6000rpm for 5 min and pellet itCentrifuge the BM cells taken at 6000rpm for 5 min and pellet it

Culture the cells in 5 culture bottles with 1ml of matrix and 5 Culture the cells in 5 culture bottles with 1ml of matrix and 5 ml of L-15 media .ml of L-15 media .

Incubate the bottles at 37 °C for 4 days without disturbing Incubate the bottles at 37 °C for 4 days without disturbing them.them.

After 4 days incubationAfter 4 days incubation

Add collagenase into the culture tube Add collagenase into the culture tube

Once the media becomes cloudy, the cells are detached and starts Once the media becomes cloudy, the cells are detached and starts floatingfloating

Resuspend the cells in a small volume of fresh serum-containing Resuspend the cells in a small volume of fresh serum-containing medium to inactivate the collagenasemedium to inactivate the collagenase

Transfer the required number of cells to 5 new labeled bottles Transfer the required number of cells to 5 new labeled bottles containing matrix and pre-warmed mediumcontaining matrix and pre-warmed medium..

SUBCULTURINGSUBCULTURING

The same procedure for subculturing is repeated twice, in 2 days The same procedure for subculturing is repeated twice, in 2 days interval.interval.

MSC DifferentiationMSC Differentiation:: The differentiation of the Mesenchymal stem cells was induced The differentiation of the Mesenchymal stem cells was induced

by the addition of synthetic peptides.by the addition of synthetic peptides.

10ul of 5 different test peptides was added to the 5 culture bottles10ul of 5 different test peptides was added to the 5 culture bottles

To avoid contamination 100microlitre of penicillin was added To avoid contamination 100microlitre of penicillin was added

incubated overnight at 37C.incubated overnight at 37C.

After inducing differentiation the culture bottles were checked for After inducing differentiation the culture bottles were checked for more number of cells using these steps:more number of cells using these steps:

All the culture bottles were shaken wellAll the culture bottles were shaken well

1ml of supernatant was removed and transferred to fresh 1ml of supernatant was removed and transferred to fresh eppendorf tubes.eppendorf tubes.

OD taken at 450nm.OD taken at 450nm.

Now the cultured cells were checked for its metabolic activity Now the cultured cells were checked for its metabolic activity using PHOS metabolic assay .using PHOS metabolic assay .

1ml supernatant was removed from the culture after shaking well.1ml supernatant was removed from the culture after shaking well.

Centrifuged at 6000rpm for 10 min, supernatant discarded.Centrifuged at 6000rpm for 10 min, supernatant discarded.

1ml of PBS was added to the pellet.1ml of PBS was added to the pellet.

100ul of PHOS solution was added.100ul of PHOS solution was added.

Incubated at 37ºC for 15 min .Incubated at 37ºC for 15 min .

OD measured at 450nm.OD measured at 450nm.

Following the metabolic assay, the culture with greater metabolic Following the metabolic assay, the culture with greater metabolic

activity is taken and further subcultured by dilution method. The activity is taken and further subcultured by dilution method. The

method is carried out as follows:method is carried out as follows:

Take the culture with greater activity and shake well.Take the culture with greater activity and shake well.

Centrifuge at 6000rpm for 10 min and remove the supernatant.Centrifuge at 6000rpm for 10 min and remove the supernatant.

To the pellet 1ml of DMEM medium was added and suspended.To the pellet 1ml of DMEM medium was added and suspended.

Take 5 plastic culture flasks ,add 20ml DMEM medium.Now the Take 5 plastic culture flasks ,add 20ml DMEM medium.Now the suspended cells are added as given below(dilution method):suspended cells are added as given below(dilution method):

11stst flask – 1µl of cells flask – 1µl of cells

22ndnd flask - 5µl of cells flask - 5µl of cells

33rdrd flask - 20µl of cells flask - 20µl of cells

44thth flask - 100µl of cells flask - 100µl of cells

55thth flask – remaining. flask – remaining. The flasks are then incubated overnight at 37ºC.The flasks are then incubated overnight at 37ºC. OD was measured at 450nm to find which dilution flask has OD was measured at 450nm to find which dilution flask has

better number of MSC.better number of MSC. 18ml of the medium is removed from that flask and replaced with 18ml of the medium is removed from that flask and replaced with

fresh medium, incubated fresh medium, incubated overnight at 37ºC. overnight at 37ºC.

NOTE: Further studies were carried out with the dilution flask NOTE: Further studies were carried out with the dilution flask containing maximum no of cells.containing maximum no of cells.

CELL THERAPYCELL THERAPY

The dilution having the least OD reading was taken as The dilution having the least OD reading was taken as appropriate for further invivo studies in chick.appropriate for further invivo studies in chick.

2ml of this culture was taken from the plastic flask2ml of this culture was taken from the plastic flask Centrifuged at 6000rpm for 10 min, 4ºC and supernatant Centrifuged at 6000rpm for 10 min, 4ºC and supernatant

removed.removed. To the pellet 100ul of 1XPBS solution was added and kept aside To the pellet 100ul of 1XPBS solution was added and kept aside

for injecting into chick.for injecting into chick. 4 chicks were taken.Out of which 1 was kept as control.4 chicks were taken.Out of which 1 was kept as control. 100µl of isoproterenol and 100ul cell sample was given to 3 100µl of isoproterenol and 100ul cell sample was given to 3

chicks. 1 chick taken as a control was given 100µl isoproterenol chicks. 1 chick taken as a control was given 100µl isoproterenol alone.alone.

The chicks were left for 3 days.The chicks were left for 3 days.

RESULTS AND DISCUSSIONS

OD FOR SUBCULTURED CELLS

SAMPLE OD(450nm) CONCENTRATION (mg/ml)

1 0.8176 0.08176

2 0.1223 0.01223

3 0.1760 0.01760

4 0.6230 0.06230

5 0.6530 0.06530

OD FOR METABOLIC ASSAY

SAMPLE OD(450nm)

1 0.5795

2 1.3176

3 1.0383

4 0.5745

5 0.4999

OD FOR CELLS IN PLASTIC CULTURE FLASKS

SAMPLE OD(450nm) CONCENTRATION(mg/ml)

1ul flask 0.2455 0.02455

5ul flask 0.2306 0.02306

20ul flask 0.1807 0.01807

100ul flask 0.1501 0.01501

Remaining 0.2308 0.02308

FUTURE WORKFUTURE WORK

Cell viability assayCell viability assay Cell morphology using PAP stainCell morphology using PAP stain Metabolic assaysMetabolic assays Rna isolationRna isolation RT-PCRRT-PCR Agarose gel electrophoresisAgarose gel electrophoresis

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