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Daniel Kraft, MDJuly, 2007
Stem CellsEverything You’ve Wanted to Know But Were Afraid to Ask…
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Daniel Kraft, MDStanford University School of Medicine
Daniel Kraft, MDJuly, 2007
Overview• What makes a cell a Stem Cell?
• Adult vs. Embryonic Stem Cells (ESC)?
• Current & New Applications of Adult Stem Cells?
• Stem Cell Source: Where do we get them?
• What are Cancer Stem Cells?
• Challenges of ESC & Therapeutic Cloning?
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Daniel Kraft, MDJuly, 2007
Stem Cell: Definition
Long-Term Self Renewal
•Unspecialized; pluripotent or multipotent
•Self Renewing
•Give rise to mature, specialized cells
Stem Cell
Mature Cell
Type I
Mature Cell
Type II
Progenitor
Differentiation
Daniel Kraft, MDJuly, 2007
Stem cell type Description
Embryonic Cells from human blastocysts
Fetal stem cells Cells from aborted fetuses
Umbilical cordstem cells
Cells from the umbilical cord blood of newborns
Placenta derived stem cells
Cells from the placenta and amniotic fluid of newborns
Adult stem cellsCells from adult tissues (Bone Marrow, Fat)
STEM CELL SOURCES
Daniel Kraft, MDJuly, 2007
Daniel Kraft, MDJuly, 2007
Stem cell type Description Examples
TotipotentEach cell can develop into a new individual
Cells from early (1-3 days) embryos
PluripotentCells can form any (over 200) cell types
Some cells of blastocyst (5 to 14 days)
MultipotentCells differentiated, but can form a number of other tissues
Fetal tissue, cord blood, and adult stem cells
Pluripotency—that is the ability to give rise to differentiated cell types that are derived from all three primary germ layers of the embryo, endoderm, mesoderm, and ectoderm—is
what makes ES cells unique.
STEM CELL TYPES
Daniel Kraft, MDJuly, 2007
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Adult Stem CellsEmbryonic Stem Cells
Daniel Kraft, MDJuly, 2007
Bone Marrow: A Rich Source of Adult Stem Cells
Hematopoeitic
Stem Cell
Mesenchymal
Stromal
Stem Cell
Daniel Kraft, MDJuly, 2007
Hematopoietic (Blood Forming) Stem Cells
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-First, and best characterized stem cell
-Very rare (.05-01% of marrow cells)
-Generate the blood & immune system (red blood cells, white blood cells, platelets)
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Daniel Kraft, MDJuly, 2007
Hematopoietic Stem Cell Markers
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c-kit+
Sca-1+
Lineage Negative for:B220 (B-cells)
Mac-1(Macrophage)
Gr-1 (Granulocytes)
CD3,4,8 (T-cells)
Ter119 (Erythroid)
Mouse Man
Thy1lo
Hematopoietic stem cells have distinctive surface phenotypes (shown on mouse and humans) and can be
isolated by flow cytometry.
Fluorescent Activated Cell Sorting (FACS) to identify and sort single stem cells
Negative for:CD10CD14CD15CD16CD19CD20
c-kit+
CD34+Thy1lo
Daniel Kraft, MDJuly, 2007
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Assay for Stem Cell Activity: Engraftment & Maturation
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Lethal dose of Radiation followed by Stem Cell Infusion
Hematopoietic Stem Cell
Lymphoid Progenitor
Myeloid Progentor
Stem Cells
Uchida, Weissman et al
Daniel Kraft, MDJuly, 2007
Tracking Stem Cell Engraftment by Bioluminescense
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Gene for Firefly Luciferase
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Homes to Marrow
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Time
Purified Hematopoietic Stem Cell
Cao, Wagers, Weissman, Contag et al
Daniel Kraft, MDJuly, 2007
Bone Marrow Transplantation Clinical Application of Stem Cell Therapy
• Bone Marrow from patient (Autologous) or donor (Allogeneic)
• Cells Home to Bone Marrow of the Host
• Donor Stem Cells generate new Blood & Immune System system
Autologous
Allogeneic
Daniel Kraft, MDJuly, 2007
Non-Malignant Disease
INDICATIONS FOR BLOOD AND MARROW TRANSPLANTATION
HSC03_9.ppt
Non-HodgkinLymphoma
Multiple Myeloma
TR
AN
SP
LA
NT
S
4,500
0
500
1,000
1,500
2,000
AML CMLHodgkinDisease
Neuroblastoma
AllogeneicAutologous
2,500
3,000
4,000
3,500
BreastCancer
ALL MDS / Other
Leukemia
OtherCancer
CLL
Year: 2002
Indications for hematopoietic cell transplantation in North America, 2002. Abbreviations: Non Hodgkins Lymphoma (NHL), Acute Myelogenous Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Myelodysplastic Disease (MDS), Chronic Myelogenous Leukemia (CML), Chronic Lymphocytic Leukemia (CLL). Courtesy of the Statistical Center the IBMTR & ABMTR.
Daniel Kraft, MDJuly, 2007
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Outcome Depends If / When Transplanted
Marrow transplant
Standard Rx
Chronic Myelogenous Leukemia: Transplant versus Standard Chemotherapy :
Transplanted patients have higher long term survival
Daniel Kraft, MDJuly, 2007
Growing Stem Cell Use
Cancer Therapy
Marrow/Immune Regeneration
Tissue Repair and Regeneration
400,000+ 2 million 18 million
Leukemia
Lymphoma
Multiple Myeloma
Breast Cancer
Anemias
Autoimmune diseases
Immunodeficiencies
Solid Organ Transplants
Heart and Vascular
Neurodegenerative
Liver disease
Arthritis/Orthopedics
Diabetes
Daniel Kraft, MDJuly, 2007
Bone Marrow Derived Stem Cells in Regenerative Medicine
Bone Marrow Derived Stem Bone Marrow Derived Stem Cells have shown an ability to Cells have shown an ability to repair & regenerate many tissuesrepair & regenerate many tissues
Plasticity? Secrete Factors?Plasticity? Secrete Factors?
Many clinical trials underway for Many clinical trials underway for diverse indications, including:diverse indications, including:
Heart AttacksHeart Attacks
Congestive Heart FailureCongestive Heart Failure
Peripheral Artery DiseasePeripheral Artery Disease
StrokeStroke
DiabetesDiabetes
Liver DiseaseLiver Disease
Orthopedics/Tissue Orthopedics/Tissue EngineeringEngineering
Daniel Kraft, MDJuly, 2007
Source: Business Week
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Daniel Kraft, MDJuly, 2007
Stem Cells & Treatment of Heart Attacks
Large double blinded trials: Patients own bone marrow stem cells delivered after a heart attack, improve heart function and clinical outcomes
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Intra-coronary delivery
Trans-myocardial delivery
Daniel Kraft, MDJuly, 2007
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Univ. of Pittsburgh
Daniel Kraft, MDJuly, 2007
Daniel Kraft, MDJuly, 2007
Daniel Kraft, MDJuly, 2007
Amit Patel et al.
Univ. of Pittsburgh
Marrow Stem Cell Treatment of Advanced Congestive Heart Failure
Daniel Kraft, MDJuly, 2007
Ideal:
•Contracting myocardial muscle cells from patient
•Must integrate, mechanically and electrically
Daniel Kraft, MDJuly, 2007
Neurologic Disease: replacing damaged or dysfunctional cells
Daniel Kraft, MDJuly, 2007
Neurodegenerative disease: Parkinson’s
NurOwnTM
Autologous Transplantation
Using the patients own bone marrow to generate neuronal cells
Isolate Bone Marrow Stem cells:
Daniel Kraft, MDJuly, 2007
Developing Adult Stem Cell Therapeutics:
• Liver: damaged by drugs, toxins,
infection
• Diabetes: Islet Cell Regeneration
• Kidney Repair:
• Injured Joints: Rheumatoid Arthritis
• Epidermal: Skin grafts
• Skeletal muscle: Muscular dystrophies
• Gene therapy: HIV
• Orthopedics: Enhance bone healing
• Tissue Engineering: Scaffold + cells
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Daniel Kraft, MDJuly, 2007
Sources: Harvesting Adult Stem Cells
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•Bone Marrow•Peripheral Blood•Cord Blood•Fat
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Daniel Kraft, MDJuly, 2007
Harvest of Bone Marrow in Operating
Room
Daniel Kraft, MDJuly, 2007
Need: Faster, easier, cheaper way to harvest marrow stem cells without an O.R.
Advantages: Local anesthesia, harvest large #s of marrow stem cells
Integrated: closed system cell process/delivery Use: for BMT & Regenerative Rx, Banking cells
A better way to harvest adult stem cells?“The MarrowMiner” Minimally invasive harvest device
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Daniel Kraft, MDJuly, 2007
MarrowMiner: Integrated System for Marrow Collection
Complete SystemComplete System
MarrowMiner SystemMarrowMiner System
Aspiration ShaftAspiration Shaft
Access TrocarAccess Trocar
Access GuideAccess Guide
Aspiration Aspiration CanisterCanister
MarrowMinerMarrowMiner
AspirationAspiration Chamber Chamber
Daniel Kraft, MDJuly, 2007
BioCardia Helix Catheter: an Enabling Platform for Cardiovascular Cell Therapy
BioCardia Helix Catheter: an Enabling Platform for Cardiovascular Cell Therapy
BIOCARDIA
Daniel Kraft, MDJuly, 2007
Transendocardial Delivery Localizes Therapeutic Cells to Heart
Transendocardial Delivery Localizes Therapeutic Cells to Heart
3D Video of Intramyocardial Delivery of Contrastto Provide Qualitative Understanding of Local Pharmacokinetics and Value of Helix Stability
3D Video of Intramyocardial Delivery of Contrastto Provide Qualitative Understanding of Local Pharmacokinetics and Value of Helix Stability
BIOCARDIA
Daniel Kraft, MDJuly, 2007
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Bone marrow stem cells fuse with liver cells in mice to produce a hybrid cell (blue) that contains two nuclei.Image courtesy Nature.
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This Purkinje cell glows brightly after fusing with a bone marrow cell that produces a Green fluorescent protein (GFP). Photo: Courtesy of Helen Blau
-A possible ‘Rescue’ Mechanism?
Cell Fusion: potential mechanism for explaining apparent ‘Plasticity’
Daniel Kraft, MDJuly, 2007
Cancer Stem Cells• Only a small minority of tumor cells can truly propagate and sustain the cancer
• These ‘Cancer Stem Cells’ can regenerate themselves and the rest of the tumor
• Isolating and understanding Cancer Stem Cells will lead to more effective therapies which target these cells
©2003 National Academy of Sciences, U.S.A.
Rx Cancer Cells
Target Rx of Tumor Stem Cells
Relapse
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Tumor shrinks
Targeted killing of tumor stem cells
Cure
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Daniel Kraft, MDJuly, 2007
Human Breast Cancer Stem CellsOnly selected CD24- cells contain malignant cells and can
recapitulate heterogeneous tumor
Al-Hajj, Muhammad et al. (2003) Proc. Natl. Acad. Sci. USA 100, 3983-3988
Daniel Kraft, MDJuly, 2007
Identification of brain tumor stem cells
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Identify patients with tumors
Freeze Cells
FACS Analysis and Sorting
Transplant into mouse brainFigure Courtesy: Dr. Sam Cheshire, Stanford
Daniel Kraft, MDJuly, 2007
Brain Cancer Stem Cells
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Brain Cancer:
Only the CD133+ brain tumor fraction contains cells that are capable of tumor initiation in immunodeficient mouse brains.
Injection of as few as 100 CD133+ cells produced a tumor that could be serially transplanted and was a phenocopy of the patient's original tumour
Injection of 100,000 CD133- cells
engrafted but did not cause a tumor.
Daniel Kraft, MDJuly, 2007
WRONG TARGET:
Traditional cancer therapies (top) kill rapidly dividing tumor cells (red) but may spare stem cells (blue) that can give rise to a new tumor.
RIGHT TARGET:
In theory, killing cancer stem cells (bottom) should halt a tumor's growth and perhaps even lead to its disappearance.
Cancer Stem Cells: The ultimate target
Daniel Kraft, MDJuly, 2007
Embryonic Stem Cells
Source: University of Wisconsin-Madison
And Potential Applications
A cell, derived from an embryo, that has the ability to continuously divide and differentiate (develop) into various other kind(s) of cells/tissues
Daniel Kraft, MDJuly, 2007
Daniel Kraft, MDJuly, 2007
CLONED ESC DIFFERENTIATE INTO DIFFERENT TISSUE TYPES
Neural Retinal
Bone Cartilage Epithelial
Daniel Kraft, MDJuly, 2007
Stages of Embryogenesis
Day 1Fertilized egg
Day 1Fertilized egg
Day 22-cell embryo
Day 22-cell embryo Day 3-4
Multi-cell embryoDay 3-4
Multi-cell embryo
Day 5-6BlastocystDay 5-6
BlastocystDay 11-14Tissue Differentiation
Day 11-14Tissue Differentiation
Daniel Kraft, MDJuly, 2007
Derivation and Use of Embryonic Stem Cell Lines
Isolate inner cell mass(destroys embryo)
Isolate inner cell mass(destroys embryo)
Heart muscleKidney
Liver
“Special sauce”(largely unknown)
Day 5-6BlastocystDay 5-6
Blastocyst
Inner cells(forms fetus)Inner cells
(forms fetus)
Outer cells(forms placenta)
Outer cells(forms placenta)
Heartrepaired
Culture cellsCulture cells
Daniel Kraft, MDJuly, 2007
Daniel Kraft, MDJuly, 2007
that are either genetically identical or are immune-deficient
In the host animal, the injected ES cells can develop into benign tumors called teratomas.
• These tumors contain cell types derived from all three primary germ layers of the embryo—endoderm, mesoderm, and ectoderm.
TERATOMAS
• Method for determining the pluripotency ES cells is to inject the cells into adult mice
Daniel Kraft, MDJuly, 2007
Cardiac
• Following heart damage
Nervous System• Stroke / Spinal Cord• Parkinsons• Alzheimers
• Burns
• Diabetes
• Solid Organ Generation
Potential Therapeutic ApplicationsPotential Therapeutic Applications
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Daniel Kraft, MDJuly, 2007
After spinal cord lesion Same mouse 6 mo after ESC
EMBRYONIC STEM CELL THERAPY FOR SPINAL CORD INJURY
Source: Geron / Hans Kiersted
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Daniel Kraft, MDJuly, 2007
Daniel Kraft, MDJuly, 2007
BASIC SCIENCE PROBLEMS TO BE OVERCOMEBASIC SCIENCE PROBLEMS TO BE OVERCOME
HOW TO DIRECT DIFFERENTIATION OF CELLS DOWN SPECIFIC PATHWAYS?e.g. all into muscle or all into nerve; different “cocktails” of growth factors without remaining residual ESC
EXPANSION & DIFFERENTIATION OF ESC ON CELL-FREE SYSTEM Many ESC have and continue to be grown on mouse supporting cells and non-
human serum
HOW TO OVERCOME IMMUNE REJECTION?e.g. alter histocompatibility genes; therapeutic cloning for “customized” lines
HOW TO MAKE AN ORGAN?e.g. Tissue BioEngineering to combine different cell types in three dimensional arrangements (complex organs such as Kidney & Liver)
Daniel Kraft, MDJuly, 2007
Somatic Cell Nuclear Transfer Or “Therapeutic Cloning”
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Daniel Kraft, MDJuly, 2007
Daniel Kraft, MDJuly, 2007
Enucleation: The recipient oocyte is held by a glass pipette, while a glass needle is used to remove the genetic material in the process of enucleation.
Cell Transfer: A cell containing the genetic material from the donor is placed inside the Zona Pellucida. An electrical pulse is then applied across the two cells, causing their
membranes to fuse into one complete cell.
Somatic Cell Nuclear Transfer Process
Daniel Kraft, MDJuly, 2007
Cloned Embryonic Stem Cells – Advantages/Problems
• Advantages- No rejection, “Perfect match”
• Problems- Extremely expensive (>$200,000/patient)
- Only 0.6% (1 out of 176) oocytes survived to become a cell line (required 16 egg donors to produce one cell line)
- Cloning damages DNA
- Holy Grail… Pluripotent Stem Cells from Your Own Adult Cells- ESC have same genes as cells in mature tissues…
- Can we activate ‘Stemness Genes’ and turn adult cells into pluripotent
stem cells?
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Daniel Kraft, MDJuly, 2007
Reprogramming Cells for Stem-nessInduced Pluripotent Stem Cells
• Only 4 genes can convert a skin cell (fibroboblast) to the equivalent of an ESC
• Can form all germ layers, form a complete embryo
Skin cell
Induced Pluripotent Cell
Regulatory Genes
Yamanaka et al.
Daniel Kraft, MDJuly, 2007
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Stem Cell Generation vs Intrinsic Stem Cell Stimulation
Daniel Kraft, MDJuly, 2007
Daniel Kraft, MDJuly, 2007
Religion, Ethics and Politics…
Daniel Kraft, MDJuly, 2007
CONFIDENTIAL 59
Brief Stem Cell Chronology
1963 McCulloch and Till illustrate the presence of self-renewing stem cells in mouse bone marrow
1968 Bone Marrow Transplant (BMT) between two siblings successfully treats SCID
1978 Haematopoietic stem cells (HSC) are discovered in human cord blood
1981
1992
Mouse embryonic stem cells are derived from the inner cell mass
Neural stem cells are cultured in vitro as neurospheres
1995 Dickey Amendment passed: makes it illegal for Federal money to be used for research where stem cells are derived from the destruction of the embryo.
1997 Leukemia is shown to originate from a HSC – 1st direct evidence for cancer stem cells
Dolly the sheep Cloned
1998 James Thomson derives the first human embryonic stem cell line
2000s Several reports of adult stem cell plasticity are published
2003 New source of adult stem cells discovered in children's primary teeth
2006 Embryonic Stem Cell Lines Created Without destroying the Embryo.
2006/7 President George W. Bush vetoes a bill which would have allowed Federal money to be used for research where stem cells are derived from the destruction of the embryo.
Daniel Kraft, MDJuly, 2007
• Prop-71 will fund $3 Billion over 10 years; primarily focused on research not federally supported currently, but generally all aspects of adult and embryonic stem cell research
•California Institute for Regenerative Medicine (CIRM) formed…
•Now past legal hurdles and funding research and facilities
Funding ESC Research: Prop 71
Daniel Kraft, MDJuly, 2007
Questions?
Daniel Kraft, MDStanford University
Institute for Stem Cell Biology & Regenerative Medicine
(650) 799-3744
Daniel Kraft, MDJuly, 2007
Stem Cell Links
• NIH: stemcells.nih.gov
• International Society for Stem Cell Research (ISSCR): isscr.org
• California Institute for Regenerative Medicine (CIRM) www.cirm.ca.gov
Daniel Kraft, MDJuly, 2007
•Baxter- (BAX) Isolex CD34 separation device
•Thermogenesis (KOOL)
•Cord blood processing and banking freezers
•ViaCell (VIAC)•Private cord blood banking•CB001 expanded cord blood units for transplant
•Unrestricted Somatic Stem Cells (USSCs) derived from umbilical cord blood•Soon Oocyte banking, •Regenerative Rx from cord blood derived cells
•GERON (GERN): ESC therapy
•Stem Cells Inc (STEM): Neural stem cells
Public Companies in BM and Cord Blood, ESC
Daniel Kraft, MDJuly, 2007
Aldagen :
•isolation of stem cells from marrow: initial focus on Rx of Peripheral Artery Disease
•trials BM versus Aldesorted Cells•http://www.aldagen.com
Atherysys: Human Multipotent Adults Stem Cells (MAPC)
Arteriocyte: Marrow and Cord Blood cells for ischemic disease
BioHeart: Myoblast therapy for CHF
Cytori: Fat Based MSC for Cardiovascular
Selected Private Companies in Regenerative Medicine
Daniel Kraft, MDJuly, 2007
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Daniel Kraft, MDJuly, 2007
Questions?
Daniel Kraft, MDStanford University
Center for Stem Cells & Regenerative Medicine
(650) 799-3744