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

° `

 have the remarkable potential to develop

into many different cell types in the body during early
life and growth.

° They are found in most, if not all, multi-

multi-
cellular organisms.They serve as a sort of
internal repair system.
eg:wound healing
2 important characterstics of stem cells are-
are-

° They are unspecialized cells capable of renewing

themselves through cell division and differentiation
giving rise to specialised cells.

° under certain physiologic or experimental conditions,

they can be induced to become tissue-
tissue- or organ-
organ-
specific cells with special functions.
eg:Addition of growth hormones
Stem cells are divided or classified based on-
on-

1.Source
2.Potency

Based on source stem cells can be classified into

following:

° Y

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

° Y
   are derived from the part of

a human embryo or foetus that will ultimately
produce gametes (eggs or sperm).
° „


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

° O
 
 
  are used to treat a
range of blood disorders and immune system
conditions.
Based on potency Stem cells are classified as-
as-

"Potency" is a term that describes how many types of

cells a stem cell can become.

° Totipotent stem cells are cells that have not begun

differentiating at all. They are capable of developing
into any other type of body cell.

° Pluripotent cells are almost as potent as totipotent

stem cells. They have barely started differentiating
and can develop into almost any other type of
cell,except placenta.
° Multipotent stem cells are stem cells that have begun
differentiating into a general type of cell. For eg,a
blood cell giving rise to a blood cell only but not
brain cell.eg:MSC

° Oligopotent stem cells can differentiate into only a

few types of cell. For example, a lympoid stem cell
can become any of the blood cells found in the
lymphatic system (T cells, B cells, and plasma cells),
but not a different kind of blood cell, such as a red
blood cell or platelet.
° Unipotent stem cells can only become one type of cell
² their own. They are considered stem cells because
they can reproduce indefinitely. An example is skin
cells, which can renew themselves indefinitely, but
which cannot become any other type of cell.

WHAT ARE MSCs?


, or ›`
›

 


, ›`,, or Marrow
Stromal Cell are multipotent stem cells that can
differentiate into a variety of cell types which include
osteoblasts, chondrocytes and adipocytes.
SOURCES 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:

O




 (consisting mainly of hematopoietic
tissue)
2.



 (consisting mainly of fat cells).
Bone marrow contains three types of stem cells:

° Hematopoietic stem cells give rise to the three classes

of blood cells that are found in the circulation: white
blood cells (leukocytes), red blood cells
(erythrocytes), and platelets (thrombocytes).

° Mesenchymal stem cells are found arrayed around the

central sinus in the bone marrow. They have the
capability to differentiate into osteoblasts,
chondrocytes, myocytes, and many other types of
cells.

° Endothelial stem cells

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

CHARACTERISTICS
° ›

›

They are bigger in size having a large, round
nucleus with a prominent nucleolus, when compared
with normal cells.
It also contains Golgi apparatus, rough
endoplasmic reticulum, mitochondria, and
polyribosomes.
° V








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

° ?





 
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   





 for a population of cells to
qualify as MSCs, as suggested by ?




`


  


 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 MSC

° Under defined conditions, MSCs can differentiate

into chondrocytes, osteoblasts, and adipocytes, and
they also serve as hematopoiesis-
hematopoiesis-supporting stromal
cells.

° MSCs have also been reported, to differentiate into

myocytes and cardiomyocytes and even into cells of
non mesodermal origin, including hepatocytes and
neurons.
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 REGULATION OF MSC

° Growth factors such as members of the



 
 


 






 



   
  
 

 


 
 



 
 

 
 








 

 

 ..

° Several Hormonal factors like phosphatonins FGF 23 and

sFRP4, Receptors for PTH/PTHrP (Franz Jakob et al, 2006)

° Peptides derived from growth factors(

`




factors(

`





 REGENERATION BY MSC

° MSCs are a promising cell type for regenerative medicine

because of their ease of isolation and expansion, their
multipotency and their low immunogenicity.

° The MSCs has the following applications as a regenerative

medice:
regeneration of cardiac muscle.(Pittenger,
muscle.(Pittenger, Mark F et
al,2002)
al,2002)
regeneration of musculoskeletal tissues.(Edward
tissues.(Edward J.
Caterson et al,2001
al,2001
regeneration of bone marrow(K
marrow(K Fukuda,2003) etc.
MODEL STUDY IN
CHICK
° ›


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

° These can be classed as:

1. genetic models (with short generation times, such as
the fruitfly and nematode worm)
2. experimental models, and
3. genomic models, with a pivotal position in the
evolutionary tree
° The  
 (aallus gallus domesticus
domesticus)) is a domesticated
fowl.

° It has descended primarily from the Red Junglefowl ((aallus

aallus
gallus)) and is scientifically classified as the same species . it is
gallus
an amniote and excellent for micromanipulation (e.g. tissue
grafting) and over-
over-expression of gene products.

° TAXONOMIC CLASSIFICATION:
CLASSIFICATION:
Kingdom: Animalia
Phylum: Chordata
Class: Aves
Order: Galliformes
Family: Phasianidae
Genus: Gallus
Species: G. gallus
Binomial name:
Gallus gallus
OBJECTIVES
° To culture stem cells and isolate the MSCs.

° To subculture the cells.

° Induction of growth and differentiation.

° Cell therapy

° Staining techniques

° RT--PCR
RT

° Agrose gel electrophoresis

METHODS
 MEDIUM USED:

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

PREPARATION OF L-
L-15 and DMEM MEDIUM:

1.4 grams of L-
L-15 (in powder form) was added to
100ml of autoclaved 1XPBS.
10grams of DMEM powder in 1000ml of HBS(Hanks
buffered--saline).
buffered
 MATRIX PREPARATION
° A solid matrix was prepared by adding 2.5grams agar
agar and 1gram gelatin in 100ml 1XPBS.

° 5 culture bottles were taken and 1ml of this matrix

was poured at the bottom of each culture bottle.

° Above this 5ml of freshly prepared L-

L-15 medium was
added which contains 1000ul of pencillin and 100ul
of amphotericinB.
 ISOLATION AND CULTURE OF MSC
FROM CHICK BONE MARROW
To isolate marrow, kill chick by cervical dislocation

Rinse the animal skeleton freely in 70% ethanol

Make an incision around the perimeter of the hind limbs

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

Bisect each hind limb by cutting through the knee joint and
remove the muscle and connective tissue .
 After cleaning, store the bones in L-
L-15 supplemented with
penicillin/streptomycin.

Now the stem cells are taken by Aspiration of

Bonemarrow(small amount of bone marrow fluid and cells are
bone.
removed through a needle put into a bone

Centrifuge 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

ml of L-
L-15 media .

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

them.
SUBCULTURING
After 4 days incubation

Add collagenase into the culture tube

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

Resuspend the cells in a small volume of fresh serum-

serum-containing
medium to inactivate the collagenase

Transfer the required number of cells to 5 new labeled bottles

containing matrix and pre- medium
pre-warmed medium
The same procedure for subculturing is repeated twice, in 2 days
interval.

›







° The differentiation of the Mesenchymal stem cells was induced
by the addition of synthetic peptides.

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

To avoid contamination 100microlitre of penicillin was added

incubated overnight at 37C.

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

All the culture bottles were shaken well

1ml of supernatant was removed and transferred to fresh

eppendorf tubes.

OD taken at 450nm.
Now the cultured cells were checked for its metabolic activity
using PHOS metabolic assay .

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

Centrifuged at 6000rpm for 10 min, supernatant discarded.

1ml of PBS was added to the pellet.

100ul of PHOS solution was added.

Incubated at 37ºC for 15 min .

OD measured at 450nm.
Following the metabolic assay, the culture with greater metabolic
activity is taken and further subcultured by dilution method.
method. The
method is carried out as follows:
follows:

Take the culture with greater activity and shake well.

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

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

° Take 5 plastic culture flasks ,add 20ml DMEM medium.Now the
suspended cells are added as given below(dilution method):
1st flask ± 1µl of cells
2nd flask - 5µl of cells
3rd flask - 20µl of cells
4th flask - 100µl of cells
5th flask ± remaining.
° The flasks are then incubated overnight at 37ºC.
° OD was measured at 450nm to find which dilution flask has
better number of MSC.
° 18ml of the medium is removed from that flask and replaced with
fresh medium, incubated overnight at 37ºC.
NOTE: Further studies were carried out with the dilution flask
containing maximum no of cells.
CELL THERAPY

° The dilution having the least OD reading was taken as

appropriate for further invivo studies in chick.
° 2ml of this culture was taken from the plastic flask
° Centrifuged at 6000rpm for 10 min, 4ºC and supernatant
removed.
° To the pellet 100ul of 1XPBS solution was added and kept aside
for injecting into chick.
° 4 chicks were taken.Out of which 1 was kept as control.
° 100µl of isoproterenol and 100ul cell sample was given to 3
chicks. 1 chick taken as a control was given 100µl isoproterenol
alone.
° The chicks were left for 3 days.
 

 
 
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FUTURE WORK
° Cell viability assay
° Cell morphology using PAP stain
° Metabolic assays
° Rna isolation
° RT
RT--PCR
° Agarose gel electrophoresis
à  à

° Becker AJ, McCulloch EA, Till JE (1963). "Cytological
demonstration of the clonal nature of spleen colonies derived from
transplanted mouse marrow cells". uature O O:: 452±
452±4.
doi::10.1038/197452a0
doi 10.1038/197452a0.. PMID 13970094
13970094..
° â Siminovitch L, McCulloch EA, Till JE (1963). "The distribution
of colony-
colony-forming cells among spleen colonies". ournal of
Cellular and Comparative Physiology ë ë:: 327±
327±36.
doi::10.1002/jcp.1030620313.
doi 10.1002/jcp.1030620313. PMID 14086156
14086156..
° â Friedenstein AJ, Deriglasova UF, Kulagina NN, Panasuk AF,
Rudakowa SF, Luria EA, Ruadkow IA (1974). "Precursors for
fibroblasts in different populations of hematopoietic cells as
detected by the in vitro colony assay method". Y p Hematol  (2):
83±±92. PMID 4455512
83
° Friedenstein AJ, Gorskaja JF, Kulagina NN (1976). "Fibroblast
precursors in normal and irradiated mouse hematopoietic organs".
Y p Hematol  (5): 267±
267±74. PMID 976387
° Netter, Frank H. (1987), ›usculoskeletal system: anatomy,
physiology, and metabolic disorders.
disorders. Summit, New Jersey: Ciba-
Ciba-
Geigy Corporation ISBN 0914168886,
0914168886, p.134
° â Brighton, Carl T. and Robert M. Hunt (1991), "Early histologic
and ultrastructural changes in medullary fracture callus", ournal
of Bone and oint Surgery
Surgery,, 
ë :: 832-

ë 832-847
° â Engler AJ, Sen S, Sweeny HL, Discher DE (2006). "Matrix
Elasticity Directs Stem Cell Lineage Specification". Cell Oë (4):
677±±689. doi
677 doi::10.1016/j.cell.2006.06.044
10.1016/j.cell.2006.06.044.. PMID 16923388
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° â Ryan JM, Barry FP, Murphy JM, Mahon BP (2005).
"Mesenchymal stem cells avoid allogeneic rejection".  Inflamm
Lond)) : 8. doi
(Lond doi::10.1186/1476
10.1186/1476--9255-
9255-2-8. PMID 16045800
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° â Ryan JM, Barry F, Murphy JM, Mahon BP (2007). "Interferon-
"Interferon-
gamma does not break, but promotes the immunosuppressive
capacity of adult human mesenchymal stem cells". ClinClin.. Y p.
353±63
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THANK YOU