Download Adult stem cells Hessah Alshammari MSc stem cell technology

Stem cells
Hessah Alshammari
• Stem cells are distinguished from other cell
types by two important characteristics.
• They are unspecialized cells capable of renewing
themselves through cell division, sometimes after
long periods of inactivity.
• Under certain conditions, they can be induced to
become tissue- or organ-specific cells with special
functions e.g. in bone marrow, stem cells regularly
divide to repair and replace worn out or damaged
tissues.
Stem cells types
• Embryonic stem cells.
• Adult stem cells.
• Cancer stem cells.
Embryonic stem cells
• Embryonic stem cells derived
from embryo in typically four
or five days old which called
the blastocyst.
• The blastocyst includes three
structures: the trophoblast,
which is the layer of cells that
surrounds the blastocoel, a
hollow cavity inside the
blastocyst; and the inner cell
mass, which is a group of cells
at one end of the blastocoel that
develop into the embryo
proper.
Adult stem cells
• An adult stem cell is an undifferentiated cell, found among
differentiated cells in a tissue or organ that can renew itself
and can differentiate to yield some or all of the major
specialized cell types of the tissue or organ.
• The primary roles of adult stem cells in a living organism are
to maintain and repair the tissue.
• In fact, adult hematopoietic, or blood-forming, stem cells from
bone marrow have been used in transplants for 40 years
Similarities and differences between
embryonic and adult stem cells
Embryonic stem cells
adult stem cells
Differentiation
ability
Pluripotent stem cells
can become all cell types of
the body
multipotent stem cells
limited to differentiating into
different cell types of their tissue
culture
grown easily in culture
Isolation and expansion
challenging
Rejection after
transplantation
Do not cause rejection
cause rejection
Tumour
formation after
injection
tumour genesis
is not tumour genesis
Colony forming
form embryoid bodys
MSC form colony forming units
NSC form neurispheres
embryoid bodys
colony forming units
Neurispheres
Adult stem cells in human body
• Neural
stem cells.
• Heamatopeotic stem cells.
•Mesenchymal stem cells.
•Adipose stem cells.
•Skin stem cells.
•Liver stem cells.
•Multipotent adult precursor cells
MAPC.
•Mammary stem cells.
Mesenchymal stem cell
•Somatic progenitors able to self-renew and produce
mesenchymal derivatives such as osteroblast, adipocytes and
chondrocytes.
•CD29 & CD105 is MSCs marker.
Mesenchymal stem cell isolation
Isolation of bone
marrow.
Lysis blood cells using
buffer and culture
MSC.
Biomedical applications
1- Pharmacological screening.
2- Orthopaedic tissue repair (bone, cartilage, tendon).
3- Non-orthopaedic application (heart,spinal cord).
4- Gene delivery.
Pharmacological screening
Research into bone metabolism & drug discovery such as
osteoprosis (caused by decreased in bone formation and
increase fatty marrow).
Misregulation in MSC
differentiation. Therefore,
identify components
regulate MSC
differentiation is leading to
drug discovery.
Orthopaedic tissue repair (bone, cartilage, tendon)
The Effect of Implants Loaded with Autologous Mesenchymal
Stem Cells on the Healing of Canine Segmental Bone Defects*
SCOTT P. BRUDER, M.D., PH.D., BALTIMORE, KARL H. KRAUS, D.V.M., NORTH GRAFTON,
MASSACHUSETTS, VICTOR M. GOLDBERG, M.D. and SUDHA KADIYALA, PH.D., BALTIMORE,
MARYLAND
Investigation performed at Osiris Therapeutics, Baltimore, and Tufts University School of Veterinary Medicine, North
Grafton
Radiographs of a segmental
defect that had been treated
with a ceramic cylinder that
had not been loaded with
MSCs(A) and loaded with
MSC (B).
Non-orthopaedic application (heart,spinal cord)
Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells
after myocardial infarction
Luciano C. Amado,Anastasios P. Saliaris,Karl H. Schuleri, and Joshua M. Hare
MSC cardiomyoplasty augments development
of new myocardium (Left). At 8 weeks after
MI, the subendocardial rim is thicker in the
MSC group (arrows). Hematoxylin/eosin
(H&E) stain of the subendocardial rim
demonstrates cardiomyocytes in both control
and MSC (×100 magnification).
Bar graph, depicting enhanced thickness of
subendocardial viable tissue with MSCs (*, P <
0.05 vs. nontreated) after 8 weeks.
Adipose stem cells
Adipose-derived stem cells was reported first time in
2002 by Hedrick et al.
Human Adipose Tissue Is a Source of Multipotent Stem Cells
Patricia A. Zuk,* Min Zhu,* Peter Ashjian,* Daniel A. De Ugarte,* Jerry I. Huang,* Hiroshi
Mizuno,* Zeni C. Alfonso, John K. Fraser, Prosper Benhaim,* and Marc H.
Hedrick* *Departments of Surgery and Orthopedics, Regenerative Bioengineering and Repair
Laboratory, UCLA School of Medicine, Los Angeles, California 90095; and Department of
Medicine and the Jonsson Comprehensive Cancer Center, Division of Hematology and Oncology,
UCLA School of Medicine, Los Angeles, California 90095
Submitted February 25, 2002; Revised June 21, 2002; Accepted August 23, 2002
Monitoring Editor: Martin Raff
CD45, CD105, CD90 and CD29 is marker of
adipose-derived stem cells, So it is closely to MSC.
Isolation of adipose stem cells
Lipoas
pirate
wash
collagenease
digest
density gradient
centrifugation
plating
Morphology and multilineage differentiation
potential of ASCs
Primary ASCs
exhibited a
fibroblast-like
morphology as
observed under
a phase contrast
microscope
adipogenic
differentiatio
n was
confirmed by
positive Oil
Red O
staining
chondrogenic
differentiation
was verified by
immunofluoresc
ence staining for
the presence of
collagen type II
deposition
osteogenic
differentiation
was revealed
by ALP
staining
Adipose stem cells application
•Plastic & cosmetic application e.g breast
reconstruction.
• As a source of non-adipogenic cell types e.g.
Bone / cartilage repair applications.
•As a vector for gene delivery or cell therapy.
Adipose stem cells applications
Adipose-derived adult stromal cells heal critical-size mouse calvarial defects
Catherine M Cowan1,Yun-Ying Shi1, Oliver O Aalami1,Yu-Fen Chou2,
Carina Mari3, Romy Thomas1, Natalina Quarto1, Christopher H Contag4,
Benjamin Wu2 & Michael T Longaker1.
1The Department of Surgery, Stanford University School of Medicine, Stanford University, 257 Campus
Drive, Stanford, California 94305, USA
Neural stem cells in brain
Neural stem cells in the brain
-the lining of the lateral ventricles.
-the dentate gyrus of the
hippocampus.
neural stem cells markers
Nestin,Sox1 , Sox2 and Sox9
Neural stem cells
• Precursors for neuronal and glial lineages.
Isolation and culture of Neural stem cells
Brain digestion
determine the
location of NSC
in brain
Tissue
disaggregation
by using enzyme
e.g accumax
Culture NSC
In serum-free
medium with
EGF and bFGF
NSC form neurosphere in culture and
has Sox1 & Sox2 marker
Neural stem cells application
• Transplantation of NSC to repair damaged tissue (trauma,
strok), neurodegeneration (Alzheimer) and delivery of genes.
Neurosci Res. 2005 Jul;52(3):243-9.
Intravenous administration of human neural stem cells induces functional
recovery in Huntington's disease rat model.
Lee ST, Chu K, Park JE, Lee K, Kang L, Kim SU, Kim M.
Department of Neurology, Clinical Research Institute, Seoul National University
Hospital, 28, Yongon-Dong, Chongro-Gu, Seoul 110-744, South Korea.
• Fetal human NSC
•Chemically induced lesion
•Cell injection
•Analysis at 9 week
Phenotype is significantly
improved
Limitations of neural stem cells application
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Ethics of collection
Availability of healthy tissue
Viability of isolated cells
immunocompatibility
Skin stem cells
Stem cells have been found in three regions of the epidermis:
• the bulge region of the follicle
• The sebaceous gland
• The interfollicular epidermis.
Skin stem cells properties
• Have a high colony-forming ability in vitro.
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•
•
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Long-term proliferative capability.
Strong adhsion to extrcellular matrix.
Small cells with a high nuclear to cytoplasmic ratio
Divide when skin is injured.
Express keratin 19, keratin 15 and CD34.
Skin stem cells isolation
Tissue
digestion
Enzymatic
digestion
Mechanical
disaggregati
on
Culture condition:
• On irradiated feeder layer or ECM.
• In the media containing of FCS.
•Presences of FGF.
Centrifugati
on
Plating
Skin stem cells applications
• Burns and skin damage.
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Doctors from the University of Sydney and Concord Hospital have formed the
Sydney Burns Foundation in 2010.
Under laboratory conditions scientists use the patient's own skin stem cells and
grow the top layer of the skin, the 'epidermis'.
Professor Maitz treated farmer John Heffernan who suffered burns to 80 per cent of
his body in 2006. They've had to replace his skin from where ever he got burnt.
They did multiple surgeries and some areas wouldn't heal up, so they'd have to go
back and put in a bit of a graft on that. While it will close the wound, it has no
elasticity, it cannot sweat, it cannot regulate temperature.
Mr Heffernan has returned to farming but every day is a challenge as his skin is so
fragile and easily damaged.
The treatment for burns survivors has made great advances, but still has a long way
to go. The hope is that one day they'll be able to grow a full three dimensional skin
which can perform all the vital functions of healthy skin.
• Genetic conditions ( epidermolysis bullosa).
Correction of junctional epidermolysis bullosa by transplantation of genetically
modified epidermal stem cells, Michele De Luca et .al ,2006, University of
Modena and Reggio Emilia, Italy.
Mutation in laminin 5 gene (basement membrane component).
Epidermolysis Bullosa
Extensive blistering of the skin generating infected
lesions
• Genetic conditions ( epidermolysis bullosa).
Experimental strategy:
•Biopsy from palm skin
•Isolation of stem cells
•Genetic modification of mutated
LAM5
•Control for restored LAM5 production
in vitro
•Transplantation of multiple grafts (each
~55cm2)
They observed complete epidermal regeneration on
both legs at day 8, and a normal-looking epidermis was
maintained throughout the 1-year follow-up (Fig. 3a,