Pluripotent Stem Cells

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Pluripotent Stem Cells
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Objectives

• What is a Stem Cell
• Different Types of Stem Cells
• Clinical Use of Stem Cells
• Stem Cell Transplant
• Types of Transplants
• Mechanisms
• Procedure
• Potential Benefits and Toxicities
• Clinical Results

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Definition of a Stem Cell

• primitive cell that can
• 1) divide to reproduce itself (undergo self-
  renewal)
• has extensive self-renewal ability
• prerequisite for sustaining the population
• 2) give rise to more specialized (differentiated)
  cells
• has multilineage differentiation ability
• Can differentiate into more than one cell type
• Examples include hematopoietic stem cells (HSC)
  that give rise to all hematopoietic cells neural
  stem cells (NSC) that give rise to neurons,
  astrocytes, and oligodendrocytes
• 3) can rescue a lethally damaged tissue in vivo
  for the lifetime of the recipient.

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• Early Experiments of Stem Cells and
  Hematopoiesis
• 1) bone marrow stem cells can rescue lethally
  irradiated mice
• ONLY SMALL NUMBER OF ACTUAL STEM CELLS REQUIRED
• ENGRAFT IN NEW MARROW
• DIFFERENTIATE INTO ALL CELLS LINES
• CONTINUE TO UNDERGO SELF RENEWAL and
  DIFFERENTIATION IN NEW MARROW ENVIRONMENT

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Stem cells
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• 2) primitive hematopoietic cells form colonies
  and sustain hematopoiesis in vitro culture
  mediums
• Stem and progenitor cells can be sustained for
  prolonged periods of time in vitro in culture
  mediums
• Progenitor cells are stimulated to differentiate
  by inclusion of growth regulatory proteins
  growth factors
• Colony Forming Units
• Bust Forming Units
• With differentiation there is expression of new
  growth factor receptors
• Growth factors also important in survival of stem
  cells
• Stem Cell Factor, G-CSF, kit ligand,IL-1..

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Stem cells
red cell colonies CFU-E
red cell bursts BFU-E
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Stem cells
granulocyte colonies CFU-G
macrophage colonies CFU-M
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Stem cells
mixed colonies CFU-GEMM
granulocyte/ macrophage colonies CFU-GM
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Stem cell hierarchy
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Types of Stem Cells
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Embryonic Stem Cells(ESC)

• 4-8 cell stage of the human embryo
• blastocyst
• The Inner Cell Mass (ICM) are pluripotent stem
  cells
• Can grow indefinitely in the primitive
  uncommitted state
• Can differentiate and give rise to ALL tissues
  of the human body
• ULTIMATE PLASTICITY
• ETHICAL ISSUES

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Embryonic Stem Cells (ES) in vitro

• ES grown on feeder cell layers (mouse
  fibroblasts) leukemia inhibitory factor (LIF
  antidifferentiation cytokine)
• Maintain their pristine, undifferentiated state
• If removed-undergo spontaneous differentiation
  and initiate cell and tissue specification
• Genes can be altered in ES cells
• Ectopic transgene expression (designed gene
  deletion, substitution, mutation) used to
  manipulate genetic programs responsible for the
  development of neurons, cardiomyocytes,
  hepatocytes, blood

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Somatic Adult Stem Cells (ASC)

• Are tissue specific
• Readily found in tissues that undergo rapid
  turnover blood, skin, central nervous system
• Hematopoietic Stem Cells (HSC) most extensively
  used to characterize adult stem cells
• Thought to be committed to their given cell
  lineage
• Recent HSC studies suggest greater
  differentiation potential than previously thought
• LESS PLASTICITY
• HIGHLY AVAILABLE

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Trans-differentiation of ASCs

• Unlike ES, somatic stem cells thought to be
  restricted in differentiation capacity, and only
  able to generate cells of the derived tissue
• Suggestion that ASCs in the right milieu are
  more plastic

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Trans-differentiation of ASCs

• HSCs injected into infarcted mouse myocardium
  improved cardiac function
• Collect stem cells
• Induced coronary artery infarct-gt measure EF of
  heart
• Inject stem cells into infarct area(s)
• Over time re-measure EF of heart sif improved
  EF

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Cellular transplantation future therapeutic
options. Division of Cardiology, University of
Ottawa Heart Institute, Ottawa, Ontario,
Canada. PURPOSE OF REVIEW Cardiac
transplantation is a complex undertaking and an
imperfect solution to end-stage heart failure.
Cellular transplantation has been proposed as an
alternative solution however, clinical trials at
present are small and show variable results. The
mechanisms behind stem cell therapy have not yet
been elucidated. RECENT FINDINGS Several large
trials have been presented that address the
question of bone marrow stem cells as therapy for
acute myocardial infarction, and also the
possible benefits of therapy with granulocyte
colony-stimulating factor. Although some trials
show a modest improvement in ejection fraction or
reduction of infarct size, other trials show no
change with treatment. Fewer clinical data are
available on the treatment of chronic left
ventricular systolic function. Many questions
remain such as what cell type to use, dosing, the
ideal timing for therapy, and the technique of
cell delivery. Finally, further research
continues on the cellular milieu, enhancement of
cell engraftment, proliferation, and survival.
SUMMARY This review briefly examines the
background for stem cell therapy, as well as the
larger clinical trials of stem cell therapy for
acute myocardial infarction and chronic left
ventricular systolic dysfunction, and possible
pharmacologic enhancement options.
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Intramyocardial implantation of CD133 stem cells
improved cardiac functionPoll Voekel et al

• Cell transplantation for myocardial regeneration
  has been shown to have beneficial effects on
  cardiac function after myocardial infarction
• Ten patients with end-stage chronic ischemic
  cardiomyopathy (ejection fraction lt22)
• Autologous bone marrow CD133 cells (1.5-9.7 X
  106 cells) were injected
• Stem cell transplantation typically improved the
  heart function stage from New York Heart
  Association/Canadian Cardiovascular Society class
  III-IV to I-II.
• mean preoperative and postoperative ventricular
  ejection fractions were 15.8 /- 5 and 24.8 /-
  5, respectively

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Cord Blood Stem Cells

• Readily available
• Thought to have higher plasticity than adult stem
  cells
• No ethical issues
• Yields small number of stem cells / harvest

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ASC Stem Cell Transplant

• Most experimental and clinical data are from
  Hematopoietic Stem Cells (HSC) used to treat
  hematopoietic diseases

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Bone Marrow Harvest Transplant
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Bone Marrow Harvest Transplant
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Peripheral Blood Stem Cell Transplant

• Stem cells harvested from peripheral blood rather
  than bone marrow
• Less invasive
• Quicker engraftment
• High dose G-CSF stimulates bone marrow to release
  stem cells
• Given subcutaneously
• No severe adverse effects
• Stem cells harvested from blood by peripheral line

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Peripheral Blood Stem Cell Harvest and Transplant
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Peripheral Blood Stem Cell Harvest
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Peripheral Blood Stem Cell Transplant
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Peripheral Blood Stem Cell Transplant
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Stem Cell Transplant

• Types
• Mechanism How They Work

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Types of Transplants

• Autologous Transplant
• Patients own stem cells
• Allogeneic Transplant
• Stem cells from someone elsedonor stem cells
• Matched Sibling (Related) (MRD)
• Matched Unrelated Donor (MUD)
• Unmatched Related or Unrelated

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Autologous Transplant
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Allogeneic Transplant

• Toxicity noted in early allogeneic studies
• 2 disease of diarrhea, liver necrosis skin
• Termed Graft Versus Host Disease (GVHD)
• GVHD pts had less leukemic relapse

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Allogeneic Transplant
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• Allogeneic transplants results in
• 1) fewer leukemic relapses GRAFT VERSUS
  LEUKEMIA EFFECT (GVL)
• 2) increased toxicity GRAFT VERSUS HOST DISEASE
  (GVHD)

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Allogeneic Stem Cell Transplant Result in Donor
Immune Mediated Responses

• Donor Immune cells recognize Recipient cells as
  non-self
• Donor T-cells Attack
• 1) Normal Host Cells Graft Vs Host Disease
  (GVHD)
• 2) Neoplastic/Leukemic Host Cells Graft Vs
  Leukemia effect (GVL)
• Results in
• Greater cure rate
• Higher Toxicity / Treatment Related Mortality
  (TRM)

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MHC-HLA

• MHC molecules discriminate self non-self
• In humans HLA molecules
• function is Ag presentation to T-cells
• Via Antigen Presenting Cells (Dendritic Cells)
• T-Cells trained early in thymus to recognize this
• Required to select T-cells which are tolerant of
  self

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• Major HLA molecules (Class I II) most
  important
• Class I (HLA-A, HLA-B) molecules interact with
  CD8 Cytotoxic T-cells
• Expressed on all cells
• Class II (HLA-DR) interact with CD4 Helper
  T-cells
• Expressed on specific immune cells
• Dendritic cells, B-cells
• Minor HLA molecules

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HLA Balance most important for Allogeneic Stem
Cell Transplants

• Major Class I II HLA matched for allogeneic
  SCT
• Decreases severe GVHD
• Minor HLA antigens not matched
• Results in GVL effect
• Less severe GVHD

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Separating GVHD from GVL

• Functional differences in T-cells subsets
• IL-2 IFN ? ? Tc-1 Th-1 ? GVHD
• IL-4, IL-5 IL-10 ? Tc-2 Th-2 ? GVL
• Selection for Tc2 Th2 will preserve GVL
  prevent GVHD

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• Overall Allogeneic Stem Cell Transplant
  (compared to Autologous SCT)
• has greatest chance for CURE
• Much higher TOXICITY and TREATMENT RELATED
  MORTALITY
• 10 first 3 months

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Active Lymphoma/Leukemia
Chemotherapy
Chemotherapy
Remission
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Stem Cell Harvest

• Allogeneic Donor harvested shortly prior to
  transplant and infused into recipient
• Autologous once in remission, patients own stem
  cells harvested/collected? frozen? short time
  later thawed and infused into patient

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AUTOLOGOUS Stem Cell Transplant
Autologous Stem Cell Rescue
High Dose Chemo
Ablation
Engraftment
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ALLOGENEIC Stem Cell Transplant
Donor Stem Cells
1) Engraftment
Donor T Cells
2) Donor T Cell Graft vs Tumor
(and GVHD)
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Reducing the Toxicity of Allogeneic Transplants

• Non-myeloablative or Mini-allogeneic
  transplants
• Lower dose conditioning chemotherapy
• Less toxic than full myeloablative traditional
  BMTs
• Takes advantage of the graft vs tumor effect
• Decreased mortality of transplant
• Allogeneic transplants now possible for elderly
  medically compromised
• Recent trials showing promising results

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Nonmyeloablative Allogeneic Transplants
Mini-Transplants
Low Dose Chemo
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Increasing the Possibility of Matched Allogeneic
Transplants- the Unrelated Donor

• 25 probability of 6/6 HLA-match of any given
  sibling
• Many patients w/o matched related donor
• International Bone Marrow registry
• HLA Typing of individuals worlwide
• Now 10 million registered
• Probability of unrelated match close to 75
• Ethnic background a factor

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International BMT Registry
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Clinical Results of Stem Cell Transplants
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Autologous Stem Cell Transplants

• Relapsed Aggressive B-Cell Lymphomas and
  Hodgkins Lymphomas
• Previous poor prognosis
• 50 long term survival

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• Multiple Myeloma
• Median survival 2 ½ years with traditional chemo
• Increases median survival to 5-6 years

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Allogeneic Stem Cell Transplant

• Adult AML (intermediate risk)
• 2 year survival with high dose chemotherapy
• Long term survival 60 with allo SCT

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• Refractory Low Grade Lymphomas
• Previously no potential cure
• Allogeneic transplant only treatment shown to
  result in long term survival
• Mini-transplants 50 response rate / long term
  survival

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• Non Malignant Diseases
• Severe Autoimmune disorders (SLE)
• Severe Sickle Cell, Thalassemias
• Marrow Failure (Aplastic Anemia)
• Severe Immunodeficiency Disorders

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Umbilical Cord Blood Transplants

• Over 6000 UCB transplants worldwide
• No ethical issues
• Easy to get
• Reduced transmission of infections
• HLA mismatches much more tolerable
• Most patients will find a 4/6 match worldwide
• Major limitation limited cell dose
• ¼ of the stem cells compared to adult harvest
• Unrelated UCB 4/6 mismatch transplants similar
  success / toxicity to unrelated matched ASC
  transplants

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Hematopoietic Transplants in 2007

• Higher Success
• Less Toxicities / Lower Transplant Mortality
• Better viral and fungal prophylaxis
• Better GVHD prophylaxis and control
• Better conditioning regimens
• More Patients Eligible for Transplant
• Matched Unrelated Donor Registry
• Mini- Transplants for elderly (gt60 yo) and
  medical comorbidities

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The end
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