parkinson’s disease and transplants: with what? where? and in … · 2015-12-25 · parkinson’s...

Parkinson’s disease and transplants:

With What? Where? and in whom?

Dr. Roger Barker

The screen versions of these slides have full details of copyright and acknowledgements 1

1


Cambridge Centre

for Brain Repair

and

Department of Neurology

Cambridge CB2 2PY, [email protected]

Roger Barker


2

Tremor

= Bradykinesia

Rigidity

Parkinson’s disease - Classically:

3

Tremor

= Bradykinesia

Rigidity

Drug

therapies

DBS/

Lesion

GDNF therapy

Cell Therapies

And what

has been achieved

with this

approach to date?



Dr. Roger Barker


4

So what should any cell therapy in PD

be able to do?

Be as effective as best medical therapy (i.e. L-dopa)

Repair the brain

Replace all neuronal losses appropriately

Recreate normal circuits

Produce long-lasting benefit of all symptoms

Be safe and well-tolerated

And so far most work has been done using fetal nigral tissue

5

Neural grafts/experimental summary

Best results obtained with:allografts

embryonic tissue of right age

striatal implantation

This gives:long term survival

connectivity and synapse formation

dopamine release

behaviour al recovery

BUT IS IT A USEFUL AND

SAFE PROCEDURE FOR

PATIENTS WITH PD?

6

P at ien t 3

time

sp

ent i

n "o

ff"

(%)

0

2 0

4 0

6 0

8 0

S e l eg i l i n e 1 0 m g

P at ien t 4

- 1 2 -6 0 6 1 2 1 8 2 4 3 0 3 6

m o n t h s p re - a n d p o s t-tr a n s p l a n t a t i o n

time

spe

nt i

n "o

ff"

(%)

0

2 0

4 0

6 0

8 0

C yc l o s p o r i n e 0

L -d o p a 0

S e le g i l i n e 1 0 m g

T r a n sp l an t at i o n

su r g er y



Dr. Roger Barker


7

Transplantation of Embryonic Dopamine Neurons

for Sev ere Parkinson's Disease

RESULTS:

• Minimal benefit at 1 year

• Younger patients do better

• Significant side-effects

BUT:

• Less tissue than other groups

• Stored for up to a month prior to grafting

• Different surgical approach

• No immuno-suppr essi on

• Short follow-up

Freed et al (2001) NEJM

8

A double blind controlled trial

of bilateral fetal nigral transplantation

in Parkinson’s disease

2 year F/UP

Primary outcomeUPDRS motor score

in off

Fluorodopa PET

11 placebo

11 - 1 donor

per side

12 - 4 donor

per side

34 patients

with advanced

PD1 death

1 death1 lost to F/up

11

10

10

CyA -2 weeks to +6 months after second graft

Study Design

Olanow CW et al (2003) Ann.Neurol. 54:403-414

9

Results:• 2 post-mortems: Good graft survival with innervation

• PET scans: change of -0.02 v +0.2 v +0.4 (significant)

• UPDRS motor “off”: Change +9.4 v +3.5 v -0.7 (p=0.096, ns)

• No change in any measures

• ADVERSE EFFECTS - Off medication dyskinesia

Placebo 0 0

1 donor 0 7

4 donor 0 6

3 requiring further neurosurgical intervention



Dr. Roger Barker


10

So what hav e embryonic nigral cell

transplants in PD be able to do so far?

• Be as effective as best medical therapy (i.e. L-dopa)

In some cases, yes

• Repair the brain

Probably

• Replace all neuronal losses appropriately

No

• Recreate normal circuits

No

• Produce long-lasting benefit of all symptoms

In some cases ..

yes especially in less severe cases

• Be safe and well-tolerated

Not so in all casesFproblems with dyskinesias

11

So what are issues facing the field

of neural transplantation in PD:

What cells should be graf ted?

Where should they be placed in the CNS?

When should they be placed relativ e to disease progression

giv en dy skinesias?

Which PD patients are best suited f or this ty pe of therapy ?


12

Pathology in PD

NON-

CNS

PATHOLOGY

DA pathology:

Putamen>

Caudate>

Ventral striatum>

Prefrontal cortex

CORTICAL LEWY BODIES

NON DAPATHOLOGY

IN BRAINSTEM andOLFACTORY BULB



Dr. Roger Barker


13

Clinical features in PD

Constipation

?Autonomic

abnormalities

DA pathology:

Motor symptoms

? Tremor

Executive frontal

cortical deficits

Visuospatial deficits and dementia

HyposmiaMood disturbancesAutonomic abnormalities

Gait disorder

14

So Cell therapies in PD

Enteric neurons

?Primary cells

??Stem cells

DA pathology:

Primary nigral

DA neurons

Other primary DA

cell sources

Stem cells

Cell Lines

Viral delivery

? Which Cells

Non-dopaminergiccells including NAic

neurons:Primary cells

?Stem cells

And best results have so far been obtained with primary fetal nigral allografts/BUT OTHER CELLS TO CONSIDER..

15

Cells for transplantation

Molecular replacement Neuronal replacement

Secretory cell lines Nigral dopaminergic neurons

Peripheral neurons/ganglia

Stem cells

Encapsulated cell lines Immortalised cell lines

Genetically engineered cells Xenograf ts

Genetically modif ied cell lines

In v iv o gene transf er

Slow release poly mers/matrices

Xenografts



Dr. Roger Barker


16

Why use porcine xenografts?

Adv antages:

1. Similar size

2. Easy to breed large litters

3. Can be transgenically modified

4. Long history of use in periphery

5. ? Innervation advantage with xenografted tissue

Disadv antages:

1. Infection (PERVs)

2. Immunological rejection

3. Different to cells originally lost in disease state

17

Obstacle 1

Risk of zoonotic infection

18

Search for cross-sp ecies transmission

of porcine endogenous retrovirus

in patients treated with living pig tissue.

The XEN 111 Study Group.

Paradis K, Langford G, Long Z et al

Science 1999 Aug 20;285(5431):1236-41

160 patients treated with various living pig tissues up to 12 years earlier.

No viremia was detected in any patient.

No PERV infection was detected in any of the patients

Persistent microchimerism (presence of donor cells in the recipient)

was observed in 23 patients for up to 8.5 years.



Dr. Roger Barker


19

Infection by porcine endogenous retroviruses

after islet xenotransplantation in SCID mice

van der Laan et al. Nature (2000) 407:501- 504

PERV contF.

Pig pancreati c cells produce PERV

which can infect human cells in culture

Transplant tissue and find viral expressi on

in several tissue compartments

20

Obstacle 2

Risk of rejection

21

CD8 - Positive Cellular Infiltrate

Isograft

Day 5 Day 10 Day 21 Day 35 Day 10

Xenograft



Dr. Roger Barker


22

Obstacle 3

Functional capacity

23

Cells for transplantation

Molecular replacement Neuronal replacement

Secretory cell lines Nigral dopaminergic neurons

Peripheral neurons/ganglia

Stem cells

Encapsulated cell lines Immortalised cell lines

Genetically engineered cells Xenograf ts

Genetically modif ied cell lines

In v iv o gene transf er

Slow release poly mers/matrices

24

Self renewal Pluripotency

Why consider stem cells for grafting?

• Can be grown to

large numbers

• Can be relativ ely

easily genetically modif ied

• ? Can migrate

to sites of pathology



Dr. Roger Barker


25

ES CELLS

ADULT NPC

EMBRYONIC

NSC

BM STROMAL

CELLS

BLOOD STEM

CELLS

OTHER STEM

CELLS

EG CELLS

26

How might stem cells be involved with neurodegenerative disorders?

1. Role in pathogenesis

2. Used to repair damage by

• recruitment of endogenous stem cells

• transplant of stem cells derived in vitro

♦ to replace missing cells

♦ to deliver chemical/enzyme

♦ to target chemotherapy

27

How might stem cells be involved with neurodegenerative disorders?

1. Role in pathogenesis

2. Used to repair damage by

• recruitment of endogenous stem cells

• transplant of stem cells derived in vitro

♦ to replace missing cells

(Dopaminergic or GABAergic)



Dr. Roger Barker


28

ES CELLS

ADULT NPC

EMBRYONIC

NSC

BM STROMAL

CELLS

BLOOD STEM

CELLS

OTHER STEM

CELLS

EG CELLS

29

So what should stem cell transplants in PD be able to do?

Be as effective as best medical therapy (i.e. L-dopa)...

So, can neural stem cells be grown

and can they form useful neurons -

especially dopaminergic neurons for PD?

Repair the brain



Produce long-lasting benef it of all sy mptoms

Be saf e and well-tolerated

30

Days in vitro

0 5 10 15 20 25

Cumulative expansion

0

1

2

3

4

5

6

7

8

9

10

Hoechst

Nestin

BrdU

A

B

C

Yes - they can be grown/...



Dr. Roger Barker


31

And they do differentiate in vitro�.

Gal C GABAβ-III-tubulin

GFAP Hoechst Hoechst

Hoechst

32

But with neural stem cells there are a number of important v ariables to consider:

Species (Mouse; rat; pig; human and marmoset)

Region of harv est (Cortex; Striatum; VM; spinal cord)

Age of tissue

Extent of manipulation of cells (i.e. any genetic engineering?)

Culture conditions and passaging technique

F.. and this has important implications in PD

in terms of generating DAic neurons.

So, for examplewith human embryonic NSC...

33

There is a reduction in neurons and an increase in astrocytes over time in culture

P0 P2 P4

Region of Or igin

Ctx Str VM Cbm Cord

Pro

portio

n o

f GFA

P-p

ositive cells (%

)

0

5

10

15

20

25

30

35

40

Region of Or igin

Ctx Str VM Cbm SC

Pro

portio

n o

f ββ ββ-tubulin I

II-pos

itiv

e cells

(%

)

0

10

20

30

40

50

60

P0 P2

P4

Neurons Astrocytes



Dr. Roger Barker


34

And the only neuronal phenotype detected was GABA,

irrespective of time in culture or region of origin

•Hoechst

•Beta Tubulin III

•GABA

Region of Origin

Ct x St r VM Cbm SC

Proportio

n o

f G

AB

A-p

ositiv

e cells (%

)

0

10

20

30

40

50

60

70

80

Jain et al (2003) NeuroReport

35

BUT CAN GET differentiated mouse ES cells

to turn into DA neurons

80% Dopamine cells

Nurr1 SHHFGF8

No teratomasGood functional recovery

Striatal phenotype

Kim et al. Nature vol 418 July 2002 page 50-56

Dopamine neurons derived form embryonic stem cells function in an animal model of Parkinson’s disease

36

So what should stem cell transplants in PD be able to do?

Be as effective as best medical therapy (i.e. L-dopa)...

So, can neural stem cells be grown

and can they form useful neurons -

especially dopaminergic neurons for PD?

YES, and in some cases can get DA cells

Repair the brain



Produce long-lasting benef it of all sy mptoms

Be saf e and well-tolerated



Dr. Roger Barker


37

So what should cell transplants in PD

be able to do?

Repair the brain

Replace all neuronal losses appropriat ely


Produce long-lasting benefit of all symptoms

Be safe and well-tolerat ed

So if neural stem cells are grafted,

do they surv iv e, replace neurons,

form circuits, and repair the brain back to normal?

Be as effective as best medical therapy (i.e. L-dopa)

38

EGF / FGF-2

14 days

23-28 days

‘Stem cell’ culture

6-OHDA lesioned rat:Mesencephalon or Striatum

Porcine neural stem cell xenografts

Experimental approach

Histology Amphetamine-induced rotation

Functional

assessment63 days

Transplantation

39

The neural stem cells DO survive..

Intrastriatal Intramesencephalic

CD8 IgM



Dr. Roger Barker


40


They DO differentiate into neurons and glia...

Ne

uN

Vim

en

tin

41

And this INCLUDES Tyrosine hydroxylase-positive (i.e. Dopaminergic) neurones..


42

WITH circuit reconstruction -

Fibre projections from Intrastriatal grafts

A B

C D

lv

CPed

SNff CPu

GP



Dr. Roger Barker


43

Fibre projections from intramesencephalic grafts

A B

C D

Hyp

CPumfb

44

Weeks post-graft

0 2 4 6 8 10

Ipsi

late

ral tu

rns / 90 m

in

600

700

800

900

1000

1100

1200

But/ Met-amphetamine-induced rotation..

ControlIntramesencephalicIntrastriatal

..shows no difference so not functionally useful

45

So what can stem cell transplants in PD currently do?

• Be as effective as L-dopa therapy

•Limited success getting them

to turn into DA neurons- mainly with ES cells

• Repair the brain

•Possible but unproven

• Replace all neuronal losses appropri ately


• Recreate normal circuits


• Produce long-lasting benefit of all symptoms

•Not demonstrated

• Be safe and well-tolerated

•Probably the case for neural stem cells but

debatable for ES cells/cell lines



Dr. Roger Barker


46

So what are issues facing the field of neural transplantation in PD:


PRIMARY DOPAMINERGIC NIGRAL NEURONS

or STEM CELLS


When should they be placed relative to disease progression given dyskinesias?

Which PD patients are best suited for this type of therapy?

47


What cells should be grafted?


When should they be placed relative to disease progression given dyskinesias?


48

• Best results with striatal placement of dopaminergic cells

• Require ~100,000 dopaminergic neurons

• Putamen +/- Caudate

Dyskinesia after fetal cell transplantation for parkinsonism:

a PET study.Ma Y, Feigin A, Dhawan V, Fukuda M, Shi Q, Greene P,

Breeze R, Fahn S, Freed C, Eidelberg D.

Ann Neurol. 2002 Nov;52(5):628-34.

• Pallidal placement needed

Ann Neurol. 2003 53:206-213

Plasticity of nigrostrital pathway in Parkinson’s diseaseWhone AL, Moore RY, Piccini P, Brooks DJ

• Nigrostriatal circuit reconstruction..



Dr. Roger Barker


49

Nigrostriatal circuit reconstruction with even striatal

dopaminergic innervation

Restoration of complex sensorimotor behavior and skilled forelimb use by a modified nigral cell suspension

transplantation approach in the rat Parkinson model.

Nikkhah G, Duan WM, Knappe U, Jodicke A, Bjorklund A.

Neuroscience. 1993 Sep;56(1):33-43.

Fibre projections from intramesencephalic porcine neural precursor cell grafts

A B

C D

Hyp

CPumfb

Long distance directed axonal growth from human dopaminergic mesencephalic neuroblasts

implanted along the nigrostriatal pathwayin 6-hydroxydopamine lesioned adult rats.

Wictorin K, Brundin P, Sauer H, Lindvall O, Bjorklund A.

J Comp Neurol. 1992 Sep 22;323(4):475-94.

Nigrostriatal circuit reconstruction..

50

Site of nigrostriatal reinnervation may influence outcome

Dorsolateral DAic cell loss -

stooped posture; postural instabil ity;

bradykinesia; gait disturbance

Dorsomedial quadrant DAic cell loss -

impaired speech, hypomimia

ROSS GW et al (2004) Ann.Neur ol. 56: 532-539.

51




Ideally in Nigra with Striatal Reinnerv ation and ?PallidumF..

??? OTHER CELL LOSSES

When should they be placed

relative to disease progression given dyskinesias?




Dr. Roger Barker


52







53

Stages of Parkinson’s disease

Palliative phase

Time

[DA]Diagnostic stage

Maintenance Phase

Complex Phase

0 5 10 15

DYSKINESIAS

Dyskinesias associated with:

• Pulsatile stimulation of DA-R (?drug regime)

• Stage of disease

• Downstream changes

in striatal projection neurons inc. LTD

• Pallidal dopaminergic loss

10% of patients per treatment year develop dyskinesias

54

Neural grafting

Neurotrophic factors

Neuroprotective factors

DA agonists

L-dopa

Amantadine

COMTI

MAOI

Stages and treatment of Parkinson’s disease

Palliative phase

Time

[DA]Diagnostic stage

Maintenance Phase

Complex Phase

0 5 10 15



Dr. Roger Barker


55

At onset of Dyskinesias in mild-moderateParkinson’s disease







56







57

The heterogeneity of PD

Genes Env ironment Co-Morbidity

Pathology

Clinical Phenoty pe



Dr. Roger Barker


58

The CamPaIGN Studyan attempt to define the incidence and types of PD

at presentation and in population based cohort of patients

Foltynie et al Brain 2004

Case Definition

UKPDSBB criteria

Case AscertainmentGPs

Neurology Clinics

Geriatric Clinics

Old Age Psychiatry Clinics

PD Nurses

Hospital Discharge Records

59

and how can these patients be characterised in terms of sub-groups of patients?

• Motor phenotypes

• Cognitive phenotypes

• ? relationship to genotypes

• ? relationship to pathology

60








Dr. Roger Barker


61

PIGD Mixed TD Probability of no difference

Age at symptom onset

71.7 (48.1- 89.2)

65.2 (14.5-84.5)

63.6 (31.2-81.0)

0.0001

Duration of disease 2.4

(0.3-20)2.7

(0.2-38.2)3.2

(0.1- 20.7)0.5500

Gender 39M37F

18M13F

28M24F

0.8200

FDR c PD01

23

696

01

247

00

406

51

0.0150

12m timed walk

15.9 (7-40) 11.7 (7-23) 10.3 (6-16) >0.0001

Hand taps (combined score each

hand)

156 (58-280) 189 (73-288) 186(114-264)

>0.0001

Motor Phenotypes

Cognition also different

but NB age

TD: tremor dominantPIGD: postural instabilityand gait disturbance

62






63

146

159 PDBB patients

13

3

5

112

30

92

14

12

16

3

5

MMSE >24

MMSE <24

PR >

16

PR >

16

PR <16

PR <16

TOL >8

TOL >8

TOL <8

TOL <8

TOL <8

TOL <8

36% (14 +30 +13 = 57) of newly diagnosed PD patients have cognitive impairment

146

13

112

30

3

5

92



Dr. Roger Barker


64

Cognitive Response

Lew is et al (2003) Neuropsychologia

None Easy Hard

Manipulation

0

0.5

1

1.5

2

2.5

3

3.5

4

Tim

e (

s)

Controls

PDEU

PDEI

65• PD unimpaired •PD impaired

fMRI changes with manipulation on VWMT

Lewis et al (2003) J.Neurosci.

66

But what causes these differences?

Is it due to pathological changes

in the frontal cortex and/or basal ganglia?

Or

Is it due to different functional genetic polymorphisms

in genes acting on this network?



Dr. Roger Barker


67

BDNF: ? genotype determines pattern

of dopamine cell loss in midbrain

and so innervati on to caudate and

PFCx and thus performance on WM

PFCx

VTA/SNc

COMT: ? genotype determines ratio of

of dopamine levels in PFCx relative

to caudate and thus performance on WM

CAUDATE

68

COMT & BDNF polymorphisms

both significantly predict fronto-striatal cognition (n=291 patients)

Model Dependent Variable Explanatory Variables β coefficient P value

Multivariate Tower of London COMT genotypeBDNF genotypeAge at onset

Age at assessmentSexUPDRS MotorPremorbid IQ

Dopaminergic exposure

-0.5100.8900.010

-0.0600.090

-0.0040.030

-1.840

0.0400.0060.720

0.0700.8000.7800.110

<0.001

69

So how many types of PD are there//don’t know but one possible classificatio n

(excluding PD+ syndromes)

TYPE

I

IIIII

IV

V

AGE

Young (<30 years)

>30>50

>50

>65

GENETICCONTRIBUTION

+++

+(+ related tophenotype)

-

-

MAJORMOTOR

SYMPTOMS

Dystonia

TremorNon-tremor

Any

Any

COGNITIVEDEFICITS

None

NoneNone or predictable

based on geneticmake-up

Frontal initially thendementia

Major from onset (inc DLB and DAT)

NATURAL HISTORY/PROGNOSIS

Slow/Good

Slow/GoodGood and slower

Faster/Poor

Fast/poor



Dr. Roger Barker


70

Middle-age pd with non-tremor dominant disease, no cognitive deficits,

known polymorphisms and not badly dyskinetic







71

There are different roads to the successful use

of cell therapies in PD.

Whilst this approach has been successful in some cases,

there is now a need

to pursue other roads with a common goal,

including those involved with better defining PD

and the complications of drug therapies in advanced disease.

CONCLUSION

72

Our work is supported by:

MRC; PDS;Wellcom e Trust & MSD

Simon Lewis

Tom Foltynie

Andy Michell

Jo Ryde

Richard Armstrong

Meena Jain

Wendy Phillips

Stan Lazic

Pam Tyers

William Kuan

Tim Harrower

Yvette Hooks

Anna Robbins

Aileen HoMain Collaborations in PD:Professor Trevor Robbins; Barbara Sahakian, Andy Blackwell and Adrian Owen (Cambridge)

Professor Danny Weinberger (NIMH)Professor David Brooks (Hammersmith Hospital)



Dr. Roger Barker


73

Cambridge Centre

for Brain Repair

and

Department of Neurology

Cambridge CB2 2PY, [email protected]

Roger Barker

74

parkinson’s disease and transplants: with what? where? and in … · 2015-12-25 · parkinson’s...

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