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

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 Biological characterization and in vitro manipulation of HSC HSC-based Cellular Therapy

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

The Hematopoietic System STEM CELLS PROGENITOR CELLS PRECURSOR CELLS MATURE CELLS Yolk Sac T T Nk AGM Nk ELP CLP B B b b Fetal Liver e e LT-HSC ST-HSC MPP G n n GMP M m m CMP p MK Mk Bone Marrow MkEP E e e 0.2% Mayani H. Stem Cells Dev 2010

Biological differences between HSC from UCB and adult BM

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

Hematopoietic progenitor cell content in UCB and adult bone marrow No. progenitors / 10 5 MNC Myeloid Erythroid Multipotent ABM = 164 ± 57 ABM = 137 ± 51 ABM = 4 ± 3 UCB = 21 ± 10 * UCB = 160 ± 49 UCB = 179 ± 64 * p<0.05 Mayani H. et al. Stem Cells 1998

HSC/HPC from UCB possess higher proliferation* potentials than adult cells 1000 900 800 UCB 700 600 aBM 500 400 300 200 100 0 0 5 10 20 30 Martínez-Jaramillo G, et al * Fold-increase in total cell number Stem Cells Dev 2004

HSC/HPC from UCB possess higher expansion* potentials than those from ABM 30 25 UCB 20 15 aBM 10 5 0 Myeloid Erythroid Multipotent Martínez-Jaramillo G, et al * fold-increase in HPC number Stem Cells Dev 2004

UCB 120 7-9 cycles A G0/G1 S G2/M 100 % of cells in each phase 80 60 40 20 0 Cell cycle kinetics 2 8 4 0 6 2 8 4 0 6 2 8 0 6 2 8 4 0 6 2 8 4 0 6 2 8 4 0 6 1 1 2 3 3 4 4 5 6 6 7 7 8 9 9 0 0 1 2 2 3 3 4 5 5 6 6 1 1 1 1 1 1 1 1 1 1 1 1 Figure 2 aBM in culture 120 B G0/G1 5-6 cycles S 100 G2/M % of cells in each phase 80 60 40 20 0 Alvarado-Moreno A, et al 0 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 Stem Cells Dev 2007 Figure 2

Cyclin D3 4500 UCB CD3 UCB aMPB 4000 Mean Fluorescence Intensity 3500 3000 aBM 2500 2000 1500 Expression 1000 500 levels of cell 0 0 6 2 8 4 0 6 2 8 4 0 6 2 8 4 0 6 2 8 4 0 6 2 8 4 0 6 2 8 1 1 2 3 3 4 4 5 6 6 7 7 8 9 9 0 0 1 2 2 3 3 4 5 5 6 6 1 1 1 1 1 1 1 1 1 1 1 1 Figure 3 cycle cdk4 8000 UCB cdk4 regulators UCB aMPB 7000 Mean Fluorescence Intensity 6000 aBM 5000 4000 3000 2000 1000 0 Alvarado-Moreno A, et al 0 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 Stem Cells Dev 2007 Figure 4

HSC from UCB possess longer Telomeres than those from adult subjects 12 UCB MPB 10 Mean Telomere Length (kb) 8 6 4 2 0 CD34+CD38- CD34+CD38+ CD3+CD4+ CD3+CD8+ CD19/CD20+ CD56+ CD15/CD66b+ 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 9 15 hours No Cytokines B No cytokines Cytokines 8 Cytokines fold-increase in total cell number 7 6 5 24 hours 4 33 hours 3 2 1 0 0 12 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 hours of culture

Cell cycle dynamics in culture No cytokines Cytokines 120 120 G0/G1 A B G0/G1 S S G2/M G2/M 100 100 % of cells in each phase % of cells in each phase 80 80 60 60 40 40 20 20 0 0 0 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 0 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 hours of culture Cytokines = SCF, FL, TPO, IL3, IL6, GM, G, EPO

Expression levels of cell cycle stimulators Cyclin D3 cdk4 4500 8000 A No Cytokines B No Cytokines Cytokines Cytokines 4000 7000 3500 Mean Fluorescence Intensity Mean Fluorescence Intensity 6000 3000 5000 2500 4000 2000 3000 1500 2000 1000 1000 500 0 0 0 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 0 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 hours of culture No cytokines Cytokines Cytokines = SCF, FL, TPO, IL3, IL6, GM, G, EPO

Expression levels of cell cycle inhibitors p16 p21 8000 2500 No Cytokines A B No Cytokines Cytokines Cytokines 7000 2000 Mean Fluorescence Intensity Mean Fluorescence Intensity 6000 5000 1500 4000 1000 3000 2000 500 1000 0 0 0 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 0 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 hours of culture No Cytokines Cytokines Cytokines = SCF, FL, TPO, IL3, IL6, GM, G, EPO

Expression of Bcl-2 Day 0 100 90 Percentage of cells expressing Bcl-2 80 70 60 50 40 Day 14 30 20 10 0 d0 d14 d0 d14 Population I Population II

Expression of c-Myc Day 0 90 80 Percentage of cells expressing c-Myc 70 60 50 40 Day 14 30 20 10 0 d0 d14 d0 d14 Population I Population II

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 of 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

DC E M G E Mk

HSC-based cell therapy for hematologic disorders Aplastic Anemia Lymphoma Multiple Myeloma Mielodysplasia Leukemia (myeloid and lymphoid)

HSC ¿Non hematopoietic cells? ¿Neural cells? CD34 + CD38 - Lin -

Experimental Design HSC Liquid culture in 3 phases: proliferation – priming - differentiation CD34 + CD38 - Lin - 25 days

Expression of neural proteins Nestin MAP2 NeuN NF

Patch clamp on neural-like cells originated from HSC control TEA (1 mM) 40 mV -60 mV 500 ms current (nA) 5 4 Neural cell of hematopietic origin 3 2 1 In the presence of TEA (which blocks K + channels) -80 -60 -40 -20 0 20 40 -1 voltage (mV)

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