Stem Cells & Neurological Disorders Said Ismail Faculty of Medicine University of Jordan

Stem Cells & Neurological Disorders

Said IsmailFaculty of Medicine University of Jordan

Outline:- Introduction

- Types & Potency of Stem Cells- Embryonic Stem Cells- Adult Stem Cells- iPSCs

- Tissue Engineering and Regenerative Medicine

- Stem Cells & Neurological Disorders:- Neural Stem Cells

- Examples of Therapeutic Applications

- Conclusion

Stem Cells and Neurological Disorders

Introduction

Benefits of stem cell research :

- Treatment of complex diseases:

Chronic Disorders: Diabetes

Neurological Disorders: Alzhimer’s

Parkinson’sSpinal Cord Injuries

Heart disorders: MI

- Regenerative medicine (Spare parts !)

Skin Cartilage Bone Cornea Heart Valves

Definition:

• stem cells:

(i) renew itself indefinitely

(ii) differentiate to multiple tissue types

A stem cell is not committed to a specific function until it receives a signal to differentiate into a specialized cell

Types & Potency

1. Embryonic:- Blastomere (4-5 day embryo) - Pluripotent

2. Adult:

- Adult tissue - multi or uni potent

Other :

- Fetal: - Aborted embryos- Pluripotent

- Umbilical: - Umbilical cord blood

- Multipotent

Potency:1.Totipotent (Fertilized egg) Generate: - all embryonic cells and tissues

- supporting tissue like placenta and umbilical cord

2. Pluripotent - Give rise to cells of all 3 germ layers (ecto-, meso-, and endoderm- Come from embryos and fetal tissue- Have active telomerase (maintain long telomers)

3. Multipotent - Give rise to multiple different cell types

4. Unipotent- Cell differentiating along only one lineage

Embryonic Stem Cell

The Embryonic Stem Cell

Source:

1. IVF embryos

2. Aborted Fetus

3. Therapeutic cloning

IVF embryos

Thousands of frozen embryos are routinely

destroyed when couples finish their

treatment.

Somatic Cell Nuclear Transfer

The nucleus of a donated egg is removed and replaced with the nucleus of a mature, "somatic cell" (a skin cell, for example).

Embryonic Stem Cell First isolated and cultured in 1998

From inner cell mass of blastocyst (4-5 day embryo).

Pluripotent with long-term self-renewal

Capable of unlimited number of divisions without differentiation

Can essentially live forever without forming tumors

Maintain normal diploid complement of chromosomes (stable karyotype)

Telomerase activity

Clonogenic: give rise to genetically identical group of cells

Expresses transcription factor Oct-4 (+ or – genes needed for proliferative state)

Spend most of their time in S phase

- In-Vitro: 300 population doublings

Human Blastocyst showing Inner Cell Mass

GROWING HESC IN VITRO:

Advantages:

- Immortal: supply endless amount of cells- Flexible: can make any body cell- Available: IVF clinics

Disadvantages:

- Hard to control their differentiation- Ethics- Immune rejection

Avoiding Immune rejection:

1. Genetically engineering stem cell to:a. Express MHC antigens of recipientb. produces stem cells with deleted MHC genes

2. Therapeutic Cloning: Clone somatic Cell nucleus of recipient into eggdevelop into blastocyst and isolate ES cellsSuch ES cells have recipient immunological profile

3. Co-transplantation with Hematopoitic Stem cells

Avoiding Immune rejection

Laboratory tests to identify ESC :

1. Immortality: Sub-culturing stem cells for many months (long-term self-renewal)

2. Morphology: Inspecting culture by microscope (for undifferentiation)

3. Surface markers & Stemnss genes: (e.g. Oct-4)

4. Karyotype stability: Examining the state of chromosomes

5. Telomerase Activity

6. Pluripotency: testing differentiation potential into diff. cells types

Here Here or Here

When is it OK….when is it NOTStem Cells and Neurological Disorders

Ethics and ESCs:

Group of cells or Human life

Adult Stem Cells

The Adult Stem Cell Undifferentiated cell found in a specialized tissue in adult. Capable of self-renewal Become specialized to cell types of the tissue from which it

originated.

Properties:- Somatic- Long-term self-renewal- give rise to mature cell types

- Generate intermediate cell (progenitors) “committed”

- Can migrate whenever needed- Uni- or Multipotent

Sources of adult stem cells : Bone marrow Blood stream Umbilical cord blood Dental pulp of the tooth Cornea and retina Skeletal muscle Liver Skin (epithelia) Gastrointestinal tract Pancreas Brain & spinal cord

Bone marrow

umbilical cord blood

Dental Pulp

Adult stem cell plasticity

- Plasticity: stem cell from one adult tissue can generate the differentiated

cell types of another tissue:“unorthodox differentiation” or “transdifferentiation”

- EX. Hematopoietic stem cell Neurons

- Possible under specific conditions

Plasticity of adult stem cells

Advantages :

1. No immune rejection2. Available: eg HSC

3. Partly specialized: easier to control differentiation

4. Flexible: under the right conditions

Disadvantages :

5. Scarce (Rare): True for many Adult SCs

6. Unavailable: Some are difficult to isolate like Neural stem cells 7. Vanishing: Don’t live in culture as long as ES cells

8. Questionable quality: more prone to DNA abnormalities

Induced Pluripotent Stem Cells (iPSCs)

Induced Pluripotent Stem Cells (iPSCs):

= Retro-differentiation = Re-programming

Producing stem cells from differentiated cells !!!

Pluripotent embryonic like stem cells are produced

Reversal of normal process

Does Not require human embryos

No donor…..No rejection

Less expensive

No Ethical issues

Main Key Genes:

- iPSCs are derived from adult somatic cells by inducing expression

of certain Stemness genes: (usually by viral vectors: risk !!!)

- eg: Master transcriptional regulators:

- other genes: c-Myc (oncogene: cancer risk !!!!)

Pluripotency:

Believed to be identical to embryonic stem (ES) cells in many respects:

- expression of certain stemness genes

- chromatin methylation patterns

- doubling time

- embryoid body formation

- teratoma formation

- viable chimera formation

- potency and differentiability

(1) Isolate and culture donor cells.

(2) Transfect stemness genes into cells by viral vectors. Red cells express those genes

(3) Harvest and culture the cells according to ES cell culture, on feeder cells (light gray)

(4) A subset of the transfected cells become iPS cells and generate ES-like colonies

Generation of induced pluripotent stem (iPS) cells

Neurogenesis of iPS Pluripotent Neuronal Stem Cells derived from Adult Leukocytes

Potential target disorders for Stem Cell Therapy:

• Leukemia• Heart damage • Anemia • Cornea damage • Retinal damage• Parkinson’s • Alzhimer’s• Diabetes • Spinal Cord Injury • Kidney Failure • Skin grafts

leukemia

Heart damage

Diabetes

Tissue Engineering &

Regenerative Medicine

Bone Repair

Skin graft grown from stem cells

Cornea

trachea from stem cells

A grown ear seeded with cartilage cells

Stem Cells &

Neurological Disorders

Which Stem Cell:

1. Neural stem cells

2. Other Adult SC (HSCs & MSCs)

3. Cord Blood SC

3. Embryonic SC

4. iPSCs

Delivery Strategy:1. Injection into brain2. Into Blood stream (Homing + immobilization by cytokines)

Graft type:3. Stem cells + Biomaterial 4. Stem Cells + Gene therapy

All have been shown to generate neural tissue

(Adult SCs are the mostlyused in clinical trials)

Comparison:

Stem Cell Embryonic Pluirpotent Ethics

Fetal Pluripotent Ethics

Cord Blood Potent RejectionAvailable

Adult Neural / Autologus Self low Numbers Same tissue Isolation

Adult (HSCs, MSCs,…) Easy isolation rejection (if allo.) Easy culture Plasticity ?!!

iPCs Pluripotent vector safetySelf

Ongoing clinical Trials in US and the world 2012

Sanberg et al.February 2012

Different strategies for stem cell delivery to repair degenerated tissue

Neural stem cells:

- Generate new neural cells throughout the lifetime

- Can migrate and replace dying neurons

- Give rise to all types of neurons, astrocytes and oligodendrocytes, …

- Capable of only Minor repairs

- Their activity is up-regulated following injury

- Found in: - Sub-ventricular zone of lateral ventricles (Most neurogenic area)

- Dentate gyrus of Hippocampus (2nd)

fewer in:- Cerebellum - Spinal Cord

Therapeutic Applications:

Main target disorders:

- Parkinson’s: localized degeneration (in substantia nigra) easier cell therapy

- Huntington’s: clear etiology, single gene disorders (Gene/Cell Therapy)

- Alzheimer : damage is less defined, widespread neuro-degeneration

- Spinal Cord injuries: very promising prospects

- Other: - Multiple Sclerosis (Siatskas and Bernard, 2009)- Ischemia / stroke- Epilepsy (Naegele et al., 2010) - Amyotrophic Lateral Sclerosis (ALS) (Wolfson et al., 2009).

Parkinson’s:

- Main Strategy:

- Replacing degenerated neurons with dopamine-producing cells

- Site:- Substantia nigra: area were most degeneration occurs in PD

- Source of SCs:

- Pieces of fetal midbrain tissue (Mendez et al., 2005)

- Autologous adult neural stem/progenitor cells (Michel et al., 2009)

- Embryonic SCs (Friling et al. 2009)

Huntington’s:

- Good Model: well characterized single gene disorder

- Main Strategy:

Blocking neuronal cell death & replacing lost neurons in striatum

- Source of SCs:

- SCs of fetal striatal primordium into striatum of HD patients (Bachoud-Lévi et al., 2006)

- Autologous adult neural stem/progenitor cells (Yu and Silva, 2008; Visnyei et al., 2006).

Alzheimer’s:

- Neuro-genesis in hippocampus deteriorates in AD patients

Example approaches: (Lunn et al., 2011)

1. Implanting Neural Stem Cells:

- Replace lost neurons

- Delay degeneration by producing Brain-Derived Neurotrophic Factor (BDNF)

2. Nerve growth factor (NGF) production:

- Genetically engineered patient fibroblasts that produce NGF …!!!

- Integration of NGF fibroblasts into a major cholinergic center of the basal forebrain provided some benefit to AD patients

Spinal cord injuries: (Salewski et al., 2010; Hu et al., 2010, Mathai et al 2008).

Stem cells can:

1. Replace neurons that died from injury

2. Generate supporting cells to re-form the myelin sheath & stimulate re- growth of damaged nerves

3. Protect cells at injury site from further damage, by releasing protective factors

Stem cells under trials:

- Embryonic SCs - Umbilical cord SCs - Adult neural SCs - Mesenchymal / bone marrow SCs - induced pluripotent Scs

Christopher Reeve

1952 - 2004

Paralyzed Patients Walking Again

http://www.youtube.com/watch?v=KGUAyKQKmmY

http://www.youtube.com/watch?v=-kygF2leZCE

http://www.youtube.com/watch?v=ZgI4tm8Tr5M

Conclusion:

- Very promising clinical trial results in the last few years

- More research needed to optimize diff. SC replacement protocols:- Cell type- Route- No. of cells- Single or multiple cell doses

- Choice between ESCs / ASCs / iPSCs: yet to be resolved

- Ethics (ESCs and Fetal tissue): Each Country has to decide

THANK YOU

Stem Cells & Neurological Disorders Said Ismail Faculty of Medicine University of Jordan

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