Google TechTalk Re RM (Daniel Kraft 2007)

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

[email protected]


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?






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


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



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


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Adult Stem CellsEmbryonic Stem Cells


Bone Marrow: A Rich Source of Adult Stem Cells

Hematopoeitic

Stem Cell

Mesenchymal

Stromal

Stem Cell


Hematopoietic (Blood Forming) Stem Cells



-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)






Hematopoietic Stem Cell Markers



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




Assay for Stem Cell Activity: Engraftment & Maturation



Lethal dose of Radiation followed by Stem Cell Infusion

Hematopoietic Stem Cell

Lymphoid Progenitor

Myeloid Progentor

Stem Cells

Uchida, Weissman et al


Tracking Stem Cell Engraftment by Bioluminescense



Gene for Firefly Luciferase



Homes to Marrow



Time

Purified Hematopoietic Stem Cell

Cao, Wagers, Weissman, Contag et al


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


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.




Outcome Depends If / When Transplanted

Marrow transplant

Standard Rx

Chronic Myelogenous Leukemia: Transplant versus Standard Chemotherapy :

Transplanted patients have higher long term survival


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


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


Source: Business Week

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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



Intra-coronary delivery

Trans-myocardial delivery



are needed to see this picture.Amit Patel et al.

Univ. of Pittsburgh




Amit Patel et al.

Univ. of Pittsburgh

Marrow Stem Cell Treatment of Advanced Congestive Heart Failure


Ideal:

•Contracting myocardial muscle cells from patient

•Must integrate, mechanically and electrically


Neurologic Disease: replacing damaged or dysfunctional cells


Neurodegenerative disease: Parkinson’s

NurOwnTM

Autologous Transplantation

Using the patients own bone marrow to generate neuronal cells

Isolate Bone Marrow Stem cells:


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






Sources: Harvesting Adult Stem Cells



•Bone Marrow•Peripheral Blood•Cord Blood•Fat








Harvest of Bone Marrow in Operating

Room


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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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


BioCardia Helix Catheter: an Enabling Platform for Cardiovascular Cell Therapy

BioCardia Helix Catheter: an Enabling Platform for Cardiovascular Cell Therapy

BIOCARDIA


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




Bone marrow stem cells fuse with liver cells in mice to produce a hybrid cell (blue) that contains two nuclei.Image courtesy Nature.



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’


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




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


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


Brain Cancer Stem Cells



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.


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


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



CLONED ESC DIFFERENTIATE INTO DIFFERENT TISSUE TYPES

Neural Retinal

Bone Cartilage Epithelial


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


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



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


Cardiac

• Following heart damage

Nervous System• Stroke / Spinal Cord• Parkinsons• Alzheimers

• Burns

• Diabetes

• Solid Organ Generation

Potential Therapeutic ApplicationsPotential Therapeutic Applications




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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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)


Somatic Cell Nuclear Transfer Or “Therapeutic Cloning”





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


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?




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.



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Stem Cell Generation vs Intrinsic Stem Cell Stimulation



Religion, Ethics and Politics…


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.


• 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


Questions?

Daniel Kraft, MDStanford University

Institute for Stem Cell Biology & Regenerative Medicine

[email protected]

(650) 799-3744


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


•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


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





Questions?

Daniel Kraft, MDStanford University

Center for Stem Cells & Regenerative Medicine

[email protected]

(650) 799-3744

Google TechTalk Re RM (Daniel Kraft 2007)

Health & Medicine