1. STEM CELLS BRING NEW STRATEGIES FOR DEVELOPING REPLACEMENT NEURONS2. MULTIPLE APPROACHES FOR USING STEM CELLS IN PARKINSON’S DISEASE RESEARCH3. FETAL TISSUE TRANSPLANTS IN PARKINSON’S DISEASE RESEARCH4. RAISING NEURONS FOR REPLACEMENT IN PATIENTS WITH PARKINSON’S DISEASE5. TURNING ON THE BRAIN’S OWN STEM CELLS AS A REPAIR MECHANISM6. STEM CELLS’ FUTURE ROLE IN SPINAL CORD INJURY REPAIR

7. Early Research Shows Stem Cells Can Improve Movement in Paralyzed Mice

1. STEM CELLS BRING NEW STRATEGIES FOR DEVELOPING REPLACEMENT NEURONS

• neurons in the adult human brain and spinal cord could not regenerate focused almost entirely on therapeutic approaches to limiting further damage.• In the mid-1990s: some parts of the adult human brain do, in fact, generate new neurons, at certain circumstances. - “the new neurons arise from "neural stem cells” in the fetal as well as the adult brain”

The discovery of a regenerative capacity in the adult central nervous system holds out the promise that it may eventually be possible to repair damage from terrible degenerative diseases: Parkinson’s Disease, amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease), brain and spinal cord injuries

1) grow differentiated cells in a laboratory dish by treat the cells in culture to nudge them toward the desired differentiated neuronal cell type before implantation, or implant them directly (rely on signals inside the body to direct their maturation into the right kind of brain cell. - neural precursor cells - pluripotent embryonic stem cells

2) growth hormones and other “trophic factors”—growth factors, hormones, and other signaling molecules: fire up a patient’s own stem cells and endogenous repair mechanisms

2. MULTIPLE APPROACHES FOR USING STEM CELLS IN PARKINSON’S DISEASE RESEARCH

• A progressive movement disorder that usually strikes after age 50. • Symptoms: an uncontrollable hand tremor increasing rigidity, difficulty walking, and trouble initiating voluntary movement. • result from the death of a particular set of neurons that connect the substantia nigra to the striatum, composed of the caudate nucleus and the putamen • “nigro-striatal” neurons release dopamine onto their target neurons in the striatum. • Dopamine: regulate the nerves to control body movement.• treated with a drug “levodopa”, which the brain converts into dopamine: initially helps, but side effects of the drug increase over time and its effectiveness wanes.

Parkinson’s diseaseParkinson’s disease

•Degenerative CNS disorder•Symptom : rigidity, tremor, slowness of movement, postural instability•Pathology : degeneration of dopaminergic neurons in substantia nigra decrease of dopamine in nigrostrialtal tract•Treatment (1) drugs ex) L-dopa (2) symptomatic surgery: pallidotomy, thalamectomy (3) Fetal midbrain transplantation

DA neuron

• 9 distinct group - ventral midbrain (A8, 9, 10)

- diencephalon (A11, 15)

- telecephalon (olfactory bulb A16 , retina A17)

• Midbrain DA neuron - the substantia nigra pars compacta

- the ventral tegmental area

- innervate the striatum (or the limbic

system) and neocortex

• Associated disorders

1> substantia nigral neurons - Parkinson’s disease

2> ventral tegmental DA neurons - schizophrenia, drug addiction

3. FETAL TISSUE TRANSPLANTS IN PARKINSON’S DISEASE RESEARCH

Sources of Cells for Parkinson’s disease

• Fetal Midbrain Tissue• Porcine Midbrain Tissue• Adrenal Medulla• Carotid Body• Genetically Engineered Fibroblast• Immortalized neural cell line• CNS Stem Cells

• Sonic Hedgehog (SHH), FGF-8• Nurr1• Ptx-3• Lmx1b• Neurotrophic factors : GDNF, BDNF, NT-3• cAMP• KCl (Depolarization)• BMPs (Bone Morphogenic Proteins)• Low Oxygen

Factors for Midbrain Dopaminergic Neuron

• Implant dopamine cells into the brain that can replace the lost dopamine-releasing neurons - Fully developed and differentiated dopamine neurons - Full functional recovery depends on more than cell survival and dopamine release

• The first cell transplantation in humans: in Mexico in the 1980s - the transplantation of dopamine producing cells in the adrenal glands - dopamine-producing chromaffin cells from several patients’ own adrenal glands to the nigro- striatal area of their brains only very modest and inconsistent improvement in their patients’ symptoms: the risks associated with the procedure

• in the early 1970s: transplanted developing dopamine neurons from fetal brain tissue - transplanted directly from the developing nigro-striatal pathways of embryonic mice into the anterior chamber of an adult rat’s eye (to mature into fully developed dopamine neurons)

• in the early 1980s (Anders Bjorkland and others): transplantation of fetal tissue into the damaged areas of the brains of rats and monkeys functional recovery depends on the implanted neurons growing and making functional connections at the appropriate brain locations

• human trials in the mid-1980s: tissue removed from a fetus electively aborted seven to nine weeks after conception encouraging,but inconsistent, benefit to patients a clear reduction in the severity of their symptoms increase in dopamine neuron function in the striatum: positron emission tomography (PET) the surgery revealed a robust survival of the grafted neurons

# A major weakness: all done “open label,” with no double-blind trial!!

• In the mid-1990s, NIH approved funding for two rigorous clinical trials of fetal tissue transplantation for Parkinson’s patients. - placebo control, in the form of sham surgery conducted on half the patients, and double blind test

• Curt Freed: no significant benefit in a subjective assessment of the patient’s quality of life - 5 out of 33 treated patients developed persistent dyskinesia - provide important information about the ability of dopamine neurons to survive in humans - many of the dopamine neurons survived and grew.

• Warren Olanow: use of immunosuppressive drugs to limit rejection of the implanted tissue - the tests used to assess patient response (open label test) - still on-going

Most Parkinson’s researchers are still hopeful that the cell-implantation approach will one day lead to a useful and widely used therapy for Parkinson’s Disease - a different source of cells for transplant: recovering enough developing dopamine neurons - standardize the tissue collected

• One alternative to cell implantation: fetal cells and tissues from animals - at Diacrin and Genzyme, used neural cells from the brains of fetal pigs did not improve enough to show a statistically significant difference from eight control patients who received a sham immunosuppression regimen and underwent sham surgery

4. RAISING NEURONS FOR REPLACEMENT IN PATIENTS WITH PARKINSON’S DISEASE

• What Parkinson’s researchers ultimately want is a renewable source of cells that can differentiate into functional dopamine neurons when placed in the striatum. - need the right combination of growth factors and cell culture conditions - bring undifferentiated cells along in a culture dish to a point where they are committed to becoming dopamine neurons, - put less-committed cells into a damaged brain and rely on “environmental” signals in the brain - need a reliable source: ES Cells, umbilical cord blood and human bone marrow

• A great deal of basic research remains to be done to find which of these cells provides the best way to get a workable therapy for Parkinson’s Disease

• Ron McKay and his colleagues at NIH - expand a population of neurons from embryonic mouse brain in culture in a rat model (1998) - a procedure for efficiently converting mouse pluripotent ES cells into neurons (2000)

• Privately funded researchers are following an analogous path using pluripotent human ES cells - Thomas Okarma of Geron Corporation: testing the potential of human ES cells in animal models of Parkinson’s Disease, but the results are not yet complete

Sources of CNS Stem Cells Subventricular zone from adult brain CNS tissues from embryo Embryonic stem (ES) cells Transdifferentiation

Limitation of primary stem cells for clinical Application 1. Limitation of cell supply a) source of cells b) laborious dissection c) maintaining of long-term culture 2. Limitation of differential potential : early development has been determined 3. Problems in donor cell survival & integration : inadequate gene manipulation

Efficient generation of midbrain dopaminergic Efficient generation of midbrain dopaminergic neurons from mouse ES cells neurons from mouse ES cells

(Nature Biotech, 2000)(Nature Biotech, 2000)A

Expand undifferentiated ES cells population on gelatin-coated tissue culture surface in ES cell medium in the presence of LIF

(Stage 1)

Generate EBs in suspension cultures for 4 days in ES cell medium(Stage 2)

Select nestin-positive cells for 8 days in ITSFn medium from EBs plated on tissue culture surface (Stage 3)

Expand nestin-positive cells for 7 days in N2 medium containing bFGF/laminin (Stage 4)

Induce differentiation of the expanded neuronal precursor cells

by withdrawing bFGF from N2 medium containing laminin

(Stage 5)

Bβ-actin

Nestin

Otx1Early CNS

Otx2

Pax2

Pax5

Wnt1

En1

Nurr1Midbrain DA neuronal-specific

mesencephalic

5. TURNING ON THE BRAIN’S OWN STEM CELLS AS A REPAIR MECHANISM

• looking for ways to spark the repair mechanisms already in a patient’s brain to fix damage that these mechanisms could not otherwise manage effective drug treatments that help a patient’s own stem cells and repair mechanisms work more effectively

• in the mid-1990s - when the brain is injured, stem cells in these two areas proliferate and migrate toward the site of the damage

• Recent research shows the direction that this may be heading for Parkinson’s Disease - James Fallon and colleagues :- to the rat brain: inject TGFα that activates normal repair processes in several organs :- TGF injected into healthy rat brain causes stem cells in the subventricular zone to proliferate for several days :- after damage the nigro-striatal neurons with 6-hydroxydopamine, inject TGF: a “wave of migration” of the stem cells to the damaged areas, where they differentiate into dopamine neurons :- do not show the behavioral abnormalities associated with the loss of the neurons

6. STEM CELLS’ FUTURE ROLE IN SPINAL CORD INJURY REPAIR

• Therapies for other disorders face much bigger hurdles• In many spinal injuries, the spinal cord is not actually cut and at least some of the signal- carrying neuronal axons are intact: no longer carry messages because oligodendrocytes, which make the axons’ insulating myelin sheath, are lost - stem cells can aid remyelination in rodents - injection of oligodendrocytes derived from mES cells could remyelinate axons in chemically demyelinated rat spinal cord - the treated rats regained limited use of their hind limbs compared with the controls

• Researchers at Johns Hopkins University - cells derived from embryonic germ cells can restore movement in an animal model of amyotrophic lateral sclerosis (ALS) Kerr, D.A., Llado, J., Shamblott, M., Maragakis, N., Irani, D.N., Dike, S., Sappington, A., Gearhart, J., and Rothstein, J. (2001). Human embryonic germ cell derivatives facillitate motor recovery of rats with diffuse motor neuron injury.

- ALS: progressively destroys special nerves in the spinal cord (motor neurons), that control movement :- muscle weakness over months to years paralysis and death

- a rat model of ALS: exposed to Sindbis virus, which infects the central nervous system and destroys the motor neurons in the spinal cord

- prepared cells from the embryoid bodies injected them into the fluid surrounding the spinal cord of the paralyzed rats that had their motor neurons destroyed by the Sindbis virus - Three months after the injections, many of the treated rats were able to move their hind limbs and walk, albeit clumsily, while the rats that did not receive cell injections remained paralyzed.

Early Research Shows Stem Cells Can Improve Movement in Paralyzed Mice