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