STEM CELLS

THE HEALING POWER OF STEM CELLS

Our

tissues have regenerative powers

Tissue healing- Skin, muscles, liver

There are specific cells in the body that are immortal Stem cells
-

Bone marrow, RBC, WBC, Platelets

UNDERSTANDING STEM CELL

POTENTIAL USES

Introduce new cells into damaged tissue

Cultured Stem Cells can differentiate in culture to replace damaged tissue without risk of rejection

Most treatments are experimental Potential Treatments

Brain Damage Cancer Spinal Cord Damage Heart Damage Haematopoiesis Baldness, Teeth, Deafness, Blindness Neural Birth Defects

TYPES OF STEM CELLS

Omnipotent all types of cells Pluripotent most cell types

Derive from Omnipotent Cells

Multipotent close family of cells Oligopotent few cells: Lymphoid, Myeloid Unipotent one type of cell

FLOW CYTOMETRY
Flow Cytometry is the technological process that allows for the individual measurements of cell fluorescence and light scattering. This process is performed at rates of thousands of cells per second. This information can be used to individually sort or separate subpopulations of cells.

Flow Cytometry and sorting

Pressurized Cell Sample 488nm Formard Light Scatter Detector Ultrasonic Transducer Collimated Light Path Through Dichroic and Band Pass Filters

Pressurized 1X PBS(Sheath)

FL4 PMTs

Pulse Height (0-10Volts) Analog Data Time(useconds) SS FL1 FL2 FL3

FLOW CYTOMETRY DATA

Smaller Region, Live cells mostly

Larger Region includes all cells

EMBRYONIC STEM CELLS

Strengths Highest level of pluripotency

All somatic cell types Enhanced telomerase activity Oct-4, Nanog, SSEA-3/4

Unlimited self-renewal

Markers

Limitations Teratoma Formation Animal pathogens Immune Response Ethics

POTENTIAL SOLUTIONS

Teratoma Formation

Pre-differentiate cells in culture then insert Feeder-free culture conditions (Matrigel) Somatic cell nuclear transfer Universalize DNA

Animal pathogens

Immune Response

Ethics

EMBRYONIC STEM CELLS AND THERAPEUTIC CLONING

Embryonic stem cells were first cultured from mouse embryos in 1981. Mouse embryonic stem cells are an important experimental tool:
They can be used to introduce altered genes into mice. They provide an outstanding model system for studying the molecular and cellular events associated with cell differentiation.

EMBRYONIC STEM CELLS AND THERAPEUTIC CLONING

Human embryonic stem cell lines were first established in 1998. Clinical transplantation therapies based on embryonic stem cells may provide the best hope for treatment of diseases such as Parkinsons and Alzheimers disease, diabetes, and spinal cord injuries.

In 1997 Ian Wilmut and colleagues cloned Dolly the sheep. Dolly arose by a process called somatic cell nuclear transfer. This type of cloning in mammals is a difficult and inefficient process.

In therapeutic cloning, a nucleus from an adult human cell would be transferred to an enucleated egg. The resulting embryo could produce differentiated cells for transplantation therapy. This would bypass the problem of tissue rejection.

These technical and ethical difficulties may be overcome by using induced pluripotent stem cellsreprogramming somatic cells to resemble embryonic stem cells. The action of only four key transcription factors is sufficient to reprogram adult mouse somatic cells.

INDUCED PLURIPOTENT STEM CELLS

Strengths Patient DNA match Similar to embryonic stem cells? Limitations Same genetic pre-dispositions Viral gene delivery mechanism

POTENTIAL SOLUTIONS

Same genetic pre-dispositions

Gene

therapy in culture liposome, controlled-release

Viral gene delivery mechanism

Polymer,

Use of known onco-genes

Try

other combinations

SOLUBLE CHEMICAL FACTORS

Transduce signals
Cell type-dependent Differentiation stage-dependent

Timing

is critical

Dose-dependence

Growth Survival Motility Differentiation

ADULT STEM CELLS

Strengths Ethics, not controversial Immune-privileged

Allogenic, xenogenic transplantation Most somatic tissues

Many sources

Limitations Differentiation Capacity? Self-renewal? Rarity among somatic cells

POTENTIAL SOLUTIONS

Differentiation Capacity

Mimic stem cell niche Gene therapy

Limited Self-renewal

Limited availability
Fluorescence-activated cell sorting Adherence

Heterogenous

population works better clinically

MESENCHYMAL STEM CELLS

Easy isolation, high expansion, reproducible

WHAT ARE MESENCHYMAL STEM CELLS (MSC) ? First isolated by Friedenstein et al in 1974 Fibroblastoid cells - spindle-shaped Adherent to tissue culture glass or plastic High growth potential

WHY ARE MSC INTERESTING ?

Rat MSC genetically tagged are microinjected into a mouse blastocyte Blastocyte reimplanted into another rat Blastocyte allowed to develop into baby rat > sacrificed Gene tags are found in many of the baby rat tissues tested ->Embryonic stem cells?

IN VITRO MSC GROWTH PATTERNDOUBLING TIME 33HR

(Conget et al 1999)

HOW TO ISOLATE MSC?

Sources: BM, periosteum, synovium, fat, cord blood, peripheral blood, foetal liver and lung Adherent to culture bottle; haemopoietic stem cells do not Easily subcultured

HOW TO IDENTIFY MSC?

Morphology Adherent to glass or plastic Surface markers: SH2, SH3, SH4, STRO-1, ICAM-2, NCAM, integrins, PDGF, IL-IR Capable to be induced in vitro to differentiate into osteoblasts, chondrocytes, and adipocytes

ADIPOCYTES

TRANSDIFFERENTIATION
MSC mesodermal origin Can differentiate into cells of endodermal and ectoderml origins Neurons, hepatocytes, islet cells, skin (fusion controversy)

MSC-> Neurons (Martin et al 2002; Choong & Cheong 2003)

160 kDa neurofilament M

the nerve growth factor receptor

FIII tubulin

INSULIN FOR TYPE I DIABETES MELLITUS Insulin deficiency Can be corrected with islet cell transplant MSC ->brand new autologous islet cells MSC -> transduced with gene responsible for insulin production Allogeneic transdued MSC also feasible

Menstrual Blood Could be a Rich Source of Pluripotent Stem Cells November 25, 2007

Endometrial Regenerative Cells

HEMATOPOIETIC STEM CELLS

Best-studied, used clinically for 30+ years

HSCT - DEFINITION

Definition
any procedure where hematopoietic stem cells of any donor and any source are given to a recipient with intention of repopulating/replacing the hematopoietic system in total or in part

History

Hematopoietic stem cell transplantation in the mouse

the radiation protection phenomenon (mid-1950s) Hematopoietic stem cell tranplantation in the dog Hematopoietic stem cell tranlsplantation in human patients
19591963 : first allogeneic HSCT in humans beginning of the Modern Era of HSCT: the end of 1960

The Nobel Prize, 1990 E. Donnall Thomas

first succsessful HSCT in treatment of acute leukemias

Thomas ED, Lochte HL, Lu WC, Ferrebee JW. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N. Engl. J. Med. 1957; 257: 491.

1 / 25 000 - 100 000 of bone marrow cells

Charakteristic: CD34 CD133 Lin C-kit (CD117) BCRP

Blood, 15 Jan 2004

HSCT

Allogeneic
from

HSCT

syngeneic

sibling/related donor from unrelated donor

Autologous

HSCT

SOURCES OF STEM CELLS

Bone marrow Peripheral blood Umbilical cord blood Fetus liver

bone marrow

peripheral blood

INDICATION FOR HSCT

Neoplastic disorders

Hematological malignancies
Lymphomas (Hodgkin and non-Hodgkin) Leukemias (acute and chronic) Multiple myeloma MDS

Solid tumors Aplastic anemia Autoimmune diseases Immunodeficiency Inborn errors of metabolism

Non-neoplastic disorders

CONCEPTS OF CANCER STEM CELL



First formulated in 1875, Julius Cohnheim

proposed that stem cell misplaced during embryonal development were the source of tumors later in the life <embryonal-rest theory>

Decades ago

only a small minority of cancer cells were able to proliferate extensively

An operational & functional term

ability to self-renew dividing to another malignant stem cell and a cancer cell

CANCER STEM CELL-MODELS

(any cell)

Trends in Cell Biology 2005;15:494-501

STEM CELL PATHWAYS

 

  

WNT: APC/axin/GSK3- /Dsh; -catenin; LEF/LCF Hedgehog: sonic(Shh), Desert(Dhh), Indian(Ihh); patched, smoothened, Fused (Fu), SuFu, Gli Bmi-1: INK4a, ARF, MDM2, Cyclin D, CDK4 Notch: SHARP, HDAC, SKIP, CBF-1 PTEN: PI3K, AKT, mTOR

ORIGIN OF CANCER STEM CELL

-ENVIRONMENTAL EFFECT
mutation

Environmental factors in cancer initiation

Gastric cancer originating from BMDCs -Houghton J.

Possible origins of cancer stem cells

Convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis -Bachoo, R. M
Cancer Cell 1,269277 (2002)

Nature Review Cancer 2005;5:899-904

Science 306, 15681571 (2004) Cell 1,269277 (2002) Cancer

THERAPEUTIC IMPLICATION

Nat Rev Cancer 2003;3:895-902