Cord Blood Hematopoietic Stem Cells from biology to ex vivo - - PowerPoint PPT Presentation

Cord Blood Hematopoietic Stem Cells from biology to ex vivo expansion and plasticity

Hector Mayani, Ph.D.

Hematopoietic Stem/Progenitor Cells Laboratory Oncology Research Unit IMSS National Medical Center, Mexico City

Umbilical Cord Blood

HE BROXMEYER UCB contains hematopoietic stem and progenitor cells at significant levels E GLUCKMAN First UCB transplant

Clinical impact of UCB cells

• More than 30,000 HCT transplants have

been performed using UCB cells

• Cord blood banking (public and private)

throughout the world

• More than 700,000 UCB units stored in

public banks, and more than 1 million units in private banks worldwide

Hematopoietic Cell Transplantation HSC-based Cellular Therapy

Biological characterization and in vitro manipulation of HSC

HSC Laboratory

Funding: IMSS-CIS (Mexico) CONACYT (Mexico) Terry Fox Fund (Canada)

The Hematopoietic System

LT-HSC ST-HSC MPP CMP MkEP GMP E e M MK Mk m G n e b e p m n e b B Nk T ELP CLP B T STEM CELLS PROGENITOR CELLS PRECURSOR CELLS MATURE CELLS Nk

Mayani H. Stem Cells Dev 2010

Yolk Sac Fetal Liver Bone Marrow AGM 0.2%

Biological differences between HSC from UCB and adult BM

Hematopoietic Stem Cells

CD34 CD90 CD117 CD133 Adult Bone Marrow: 1 HSC per 60,000 nucleated cells Umbilical Cord Blood: 1 HSC per 20,000 nucleated cells CD49f

Hematopoietic progenitor cell content in UCB and adult bone marrow

Myeloid Erythroid Multipotent ABM = 164 ± 57 UCB = 160 ± 49 ABM = 137 ± 51 UCB = 179 ± 64 ABM = 4 ± 3 UCB = 21 ± 10*

• No. progenitors / 105 MNC

* p<0.05

Mayani H. et al. Stem Cells 1998

HSC/HPC from UCB possess higher proliferation* potentials than adult cells

100 200 300 400 500 600 700 800 900 1000 5 10 20 30

* Fold-increase in total cell number UCB aBM

Martínez-Jaramillo G, et al Stem Cells Dev 2004

HSC/HPC from UCB possess higher expansion* potentials than those from ABM

5 10 15 20 25 30 Myeloid Erythroid Multipotent

UCB aBM

* fold-increase in HPC number

Martínez-Jaramillo G, et al Stem Cells Dev 2004

A

20 40 60 80 100 120

6 1 2 1 8 2 4 3 3 6 4 2 4 8 5 4 6 6 6 7 2 7 8 8 4 9 9 6 1 2 1 8 1 1 4 1 2 1 2 6 1 3 2 1 3 8 1 4 4 1 5 1 5 6 1 6 2 1 6 8 Figure 2

% of cells in each phase

G0/G1 S G2/M

B

20 40 60 80 100 120

6 12 21 27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123 129 135 141 147 153 159 165 Figure 2

% of cells in each phase

G0/G1 S G2/M

UCB aBM

Cell cycle kinetics in culture

7-9 cycles 5-6 cycles

Alvarado-Moreno A, et al Stem Cells Dev 2007

CD3

500 1000 1500 2000 2500 3000 3500 4000 4500

6 1 2 1 8 2 4 3 3 6 4 2 4 8 5 4 6 6 6 7 2 7 8 8 4 9 9 6 1 2 1 8 1 1 4 1 2 1 2 6 1 3 2 1 3 8 1 4 4 1 5 1 5 6 1 6 2 1 6 8 Figure 3

Mean Fluorescence Intensity

UCB aMPB

cdk4

1000 2000 3000 4000 5000 6000 7000 8000

6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 120 126 132 138 144 150 156 162 168 Figure 4

Mean Fluorescence Intensity

UCB aMPB

UCB aBM

Expression levels of cell cycle regulators

UCB aBM Cyclin D3 cdk4

Alvarado-Moreno A, et al Stem Cells Dev 2007

2 4 6 8 10 12

CD34+CD38- CD34+CD38+ CD3+CD4+ CD3+CD8+ CD19/CD20+ CD56+ CD15/CD66b+

Mean Telomere Length (kb)

UCB MPB

HSC from UCB possess longer Telomeres than those from adult subjects

Hills et al Blood 2009

HSC: UCB vs aBM

• UCB contains a higher frequency of HSC and

multipotent HPC than aBM

• HSC/HPC from UCB possess higher

proliferation and expansion potentials in vitro than equivalent cells from aBM

• Such differences in proliferation and expansion

potentials seem to be due to differences in the expression of cell cycle regulators and telomere length

Role of hematopoietic cytokines on the in vitro biology of HSC

Doubling time in liquid cultures

B

1 2 3 4 5 6 7 8 9 12 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72

hours of culture fold-increase in total cell number

No Cytokines Cytokines

33 hours 24 hours 15 hours No cytokines Cytokines

Cell cycle dynamics in culture

A

20 40 60 80 100 120

3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78 81 84 87 90 93 96

hours of culture % of cells in each phase

G0/G1 S G2/M

B

20 40 60 80 100 120

3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78 81 84 87 90 93 96

hours of culture % of cells in each phase

G0/G1 S G2/M

No cytokines Cytokines

Cytokines = SCF, FL, TPO, IL3, IL6, GM, G, EPO

Expression levels of cell cycle stimulators

A

500 1000 1500 2000 2500 3000 3500 4000 4500

3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78 81 84 87 90 93 96

hours of culture Mean Fluorescence Intensity

No Cytokines Cytokines

B

1000 2000 3000 4000 5000 6000 7000 8000

3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78 81 84 87 90 93 96

hours of culture Mean Fluorescence Intensity

No Cytokines Cytokines

Cyclin D3 cdk4

Cytokines = SCF, FL, TPO, IL3, IL6, GM, G, EPO

No cytokines Cytokines

Expression levels of cell cycle inhibitors

A

1000 2000 3000 4000 5000 6000 7000 8000

3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78 81 84 87 90 93 96

hours of culture Mean Fluorescence Intensity

No Cytokines Cytokines

B

500 1000 1500 2000 2500

3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78 81 84 87 90 93 96

hours of culture Mean Fluorescence Intensity

No Cytokines Cytokines

No Cytokines Cytokines

Cytokines = SCF, FL, TPO, IL3, IL6, GM, G, EPO

p16 p21

10 20 30 40 50 60 70 80 90 100 d0 d14 d0 d14 Population I Population II Percentage of cells expressing Bcl-2

Expression of Bcl-2

Day 0 Day 14

Expression of c-Myc

10 20 30 40 50 60 70 80 90 d0 d14 d0 d14 Population I Population II Percentage of cells expressing c-Myc

Day 0 Day 14

Stimulatory cytokines

• Favor expression of cell cycle promoters
• Down-regulate expression of cell cycle inhibitors
• Favor expression of anti-apoptotic proteins
• Favor expression of proliferation stimulators

Strategies for ex vivo expansion of HSC

HSC expansion in culture

Liquid suspension cultures of HSC- enriched cell populations in the presence

• f recombinant stimulatory cytokines

Liquid cultures of HSC-enriched cell populations in the presence of recombinant stimulatory cytokines and stromal cells

Ex vivo expansion of HSC

Hematopoietic Progenitor Cells Hematopoietic Stem Cells

HSC differentiation

Multipotency vs Plasticity

M G E Mk E DC

Aplastic Anemia Leukemia (myeloid and lymphoid) Mielodysplasia Lymphoma Multiple Myeloma

HSC-based cell therapy for hematologic disorders

HSC CD34+ CD38- Lin-

¿Non hematopoietic cells? ¿Neural cells?

HSC

Liquid culture in 3 phases: proliferation – priming - differentiation

CD34+ CD38- Lin- 25 days

Experimental Design

Nestin NF MAP2 NeuN

Expression of neural proteins

control

• 80
• 60
• 40
• 20

20 40

• 1

1 2 3 4 5 current (nA) voltage (mV)

40 mV

• 60 mV

500 ms

TEA (1 mM)

Patch clamp on neural-like cells originated from HSC

In the presence of TEA (which blocks K+ channels)

Neural cell of hematopietic origin

In vitro manipulation of HSC

• By using particular cytokine combinations,

UCB-derived HSC can be selectively induced to differentiate into specific hematopoietic lineages

• Under the appropriate culture conditions,

UCB-derived HSC can give rise to non- hematopoietic cells that show morphological, molecular and functional features of neural cells

HSC-based cell therapy for non-hematologic disorders?

Myocardial infarction Spinal cord injuries Osteogenesis imperfecta Muscular Distrophy Osteoporosis Neurodegenerative disorders

HSC

?

? ? ? ? ? ?