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Stem Cells
Embryonic stem cells
Adult (Somatic) stem cells
ES cells line
Pluripotentiality property
Immortal property
Nucleus transfer
Fusion of somatic cells and embryonic cells
Embryonic stem cell (ES)
Endoderm Mesoderm Ectoderm Germ cell line
Mesenchymal stem cell (MSC) HSC
adipocyte Gonad Death
muscle
tendon
stromal
osteoctye
neuronal
Adult (Somatic) Stem Cells
Hematopoietic stem cells (HSC)
Mesenchymal stem cells (MSC)
Neural stem cells
Hepatic stem cells
etc.
HSC Transplantation
What is stem cell?
CD 34+ ? (HSC)
CD 34- Could it be stem cell?
Is phenotype alone enough to identify stem cell?
HSC Transplantation
Autologous
Allogeneic
Syngeneic
Xenogeneic
Human Leukocyte Antigen (HLA)
HLA class I
A B C
HLA class II
DR DP DQ
HLA match = 6 antigens (A B DR)
HSC Transplantation
Allogeneic
HLA-matched related
HLA-mismatched related: Haploidentical (3 Ags mismatched)
HLA-matched unrelated
HLA-mismatched unrelated
HSC Transplantation
Sources of Stem Cell Bone Marrow
Peripheral Blood Stem Cell
Cord Blood
Fetal liver
Differences among various sources of stem cells
BM PBSC CB FL
Engraftment ++ +++ + ?
GVHD + ++ +/- -/?
HLA matching yes yes yes no
Process of HSC transplantation
Conditioning
Stem cell infusion
GVHD prophylaxis
Engraftment
Immune tolerance
Donor Recipient
Stem cell T cellNK cell
T cell NK cell Stem cell
Indications for HSC transplant
Malignant diseases: leukemia/lymphoma and hematologic malignancies and solid tumors
Non malignant diseases:
Bone marrow failure: aplastic anemia
Thalassemia
Primary immune deficiency
Neurometabolic disease
Metabolic bone disease
Other genetic diseases
Autoimmune disease
Pediatric HSC Transplant at Ramathibodi: 1989-2005
Total patient 150 patients
Autologous 30 patients
Allogeneic 120 patients
Pediatric HSC Transplant at Ramathibodi: 1989-2005
Age ranged 1 – 22 yr (median; 10 yr)
Thalassemia
α thalassemia disease
Hb bart ( _ _ / _ _ )
Hb H disease ( _ _ /_ α )
β thalassemia disease
Homozygous β thalassemia
β thalassmia / Hb E
Treatments in Thalassemia
Palliative treatment Blood transfusion Iron chelation SplenectomyCurative treatment Stem cell transplant
Pediatric Stem Cell Transplant in Thalassemia at Ramathibodi:
1989-2005Thalassemia (n= 48 pts)
Donor of stem cell Matched and mismatched related 25 pts Matched and mismatched unrelated 21 pts Haploidentical 2 ptsSource of stem cell BMT and PBSCT 44 pts Cord blood 4 pts
Figure 1 : Thalassaemia free survival for all patients N=49
Su
rviv
al p
rob
ab
ilit
y
Time (Months)
14413212010896847260483624120
-1.00.90.80.70.60.50.40.30.20.10.0
78%
Su
rviv
al p
rob
ab
ilit
y
Figure 2 : Overall survival for all patients n=49
Time (Months)
14413212010896847260483624120
1.00.90.80.70.60.50.40.30.20.10.0
- 89%
Su
rviv
al p
rob
ab
ilit
y
Figure 3 : Thalassaemia free survival in related and unrelated transplantation
groups
Time (Months)
14413212010896847260483624120
1.00.90.80.70.60.50.40.30.20.10.0
-
Related donor groupUnrelated donor group
p=0.42
82% n=28
70%
n=21
Su
rviv
al p
rob
ab
ilit
y
Figure 4 :Overall survival in related and unrelated donor transplantation groups
Time (Months)
14413212010896847260483624120
1.00.90.80.70.60.50.40.30.20.10.0
-
Related donor groupUnrelated donor group
p=0.43
92% n=28
82%
n=21
Mesengenic Process
:Caplan AI.Clin Plas Surg 1994;21(3):429-435.
Adult stem cells and plasticity
Day 2 Day 10
Osteogenic Differentiation
• MSCs were cultured for 16 days in the presence of – Dexamethazone
– L-ascorbic acid-2-phosphate
– Ascorbic acid
– Beta-glycerophosphate
• Detection of APase activity, formation of mineralized matrix
:Jaiswal N et al. J Cell Biochem 1997;64:295-312.
Induction of Osteoblast differentiation from Human Mesenchymal Stem Cells
by Dexamethasone
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Methods
hMSC passage 11-15 at confluency
Dexamethasone 10-7 M
L-ascorbate-2-phosphate 10 μM
-glycerophosphate 10 mM
Cbfa-1 Expression by RT-PCR
Cbfa-1 Coll-1 GAPDH
Day 0 5 10 15 0 5 10 15 0 5 10 15
0 5 10 15 0 5 10 15 0 5 10 15
Alkaline Phosphatase Enzyme Activity
0
4
8
12
16
20
0 5 10 15 20
Day
um
ol p
-nit
rop
he
no
l/30
m
in/u
g p
rote
in
Von Kossa Staining
Day 7 14 21 28
Stem cell transplant in Duchenne muscular dystrophy
Hematopoietic stem cell and mesenchymal stem cell transplant
Evaluation
FISH XX/XY
Dystrophin protien
Dystrophin mutation analysis
Dystrophin Staining
Pre-transplantation
Dystrophin Staining
1 year post transplantation
0
2000
4000
6000
8000
10000
12000
14000
16000
pre
Tx
1
mo
2
mo
3
mo
4
mo
5
mo
6
mo
7
mo
8
mo
9
mo
10
mo
11
mo
12
mo
13
mo
14
mo
15
mo
CK level
Genetic control of chondrocyte and osteoblast differentiation
18 srRNA
CD 34+CB
CD105 (thal)
MSC (normal)
Study of Mechanism Osteoporosis
Renal tubular acidosis
Thalassemia
Ex vivo umbilical cord blood expansion by
co-culturing with IL-1 treated mesenchymal stem cells
Day 7 Day 14 Day 21
MNC (fold) 6.0 22.4 33.9
CD34+ (fold) 1.1 52.0 89.0
MSC: Potential Precursors for Tumor Stroma
Target-Delivery Vehicles for Anticancer Agents
Interferon
IL-2
etc.
Tolerance Induction in Organ Transplantation
by Stem Cell Transplantation
Donor Recipient
Stem cell T cellNK cell
T cell NK cell
Stem cell
Pre transplant Post transplant
D R MC DLI D
MC = Mixed chimerism DLI = donor lymphocyte infusion
Candidate Effector Cells
Natural Killer Cells (NK)
Cytotoxic T Cells (CTL)
Cytokine Induced Killer Cells (CIK)
Candidate Effector Cells
NK CTL CIKPhenotype CD 3 -, 56+, 16+ CD 3+, 56-, CD 8+ CD 3+, 56+, 16-
Precursor cells NK T T MHC-restricted No Yes No
Expandable Limited Yes Yes
Require IL-2 in vivo Yes No No
Clinical Advantages of DCs
Professional antigen presenting cells
High expression of MHC I, MHC II, and co-stimulatory molecules e.g. CD86, CD80, CD40
Immature DCs have the highest capacity of antigen uptake
Mature DCs have the highest capacity of antigen presenting
APC TMHC
TCR
Adhesion
B7-1
B7-2
IL2
IL2R
Dendritic Cell CultureCulture adherent mononuclear cells in RPMI 1640 with 100 ng/ml GM-CSF and 1000 U/ml IL-4 for 5-6 days
Induce DC maturation with monocyte-conditioned media for 48 hours
Pulsed with whole tumor lysate or total RNA of tumor cells or specific chimeric RNA transcript (leukemia and solid tumors), epidermal growth factor receptor (EGFR) (glioma) and DOTAP, and opti-MEM and subsequently generate cytotoxic T cell (CTL)
Clinical Advantage of CIKs
CIKs express both CD3 And CD56
(marker of T cell and NK cell)
Non-MHC restricted cytotoxic effector cells
Fas-induced apoptosis resistance
Cytokine Induced Killer Cells Expansion
Mononuclear cell were cultured in RPMI 1640 in the presence of 1000 U/ml IFN-gamma on day 0.
50 ng/ml anti CD3 MoAb and 300 U/ml IL-2 were added on day 1.
Cultured cells were harvested on day 14 for analysis of CD3 and CD56 double expression.
LDH release assay against different pediatric cancer cell lines was performed.
Allogeneic setting of CIK/CTL and CTL
in GBM model
Autologous setting of CIK/CTL in osteosarcoma model
E:T = 12.5:1