Nagy Habib k16z

Genes & Cells Therapy

Professor Nagy Habib Department of Surgical Oncology & Technology Hammersmith Hospital Campus [email protected]

Stem cells Embryonic

Foetal

Adult stem cell

Placenta Umbilical cord Different organs

Bone marrow MSC

MAPC

HSC

CD34+ CD34-

OmniCyte

CULTURE INITIATION 

Proliferation ­ DAY 3

HAEMOPOIETIC CELLS ­ DAY 7

MESENCHYMAL  STEM  CELLS ­ DAY 14

EPITHELIOID CELL ­ DAY 14

DENDRITE­FORMING CELL ­ DAY 14

Mean number of cells x 10e-3/plate ± sem

10000

1000

100

10

1 0

2

4

6

8 10 12 14

Day of culture

FRESHLY ISOLATED CELLS (cont)

Gene AC133 Angiopoietin­1 Angiopoietin­2 CXCR4 ICAM­2 PECAM TAL­1 VEGF VWF GATA­4 Nkx 2.5 MEF­2D Phospholamban Pdx­1 C­met

OmniCytes

Other CD34+

+ + + + + + + + + + + + + + +

­ ­ + ­ + ­ ­ ­ ­ ­ ­ ­ ­ ­ ­

Omnicytes express neuronal markers after 7 days in culture

IgG control

NeuN

24 ± 7%

Nestin

NSE

49 ± 10%

3 ± 2%

IGF1 CK19 NKX2.2 Somatostatin IAP IGF

NgN3 Somatostatin Beta cellulin

Pax6 Pax4 Islet1 PDX1 Insulin 1kb DNA ladder

Glut2 Amylase exendin4 Nkx6.1 NeuroD

Day 0 PANCREAS MARKERS

Clinical protocol Insulin

C-peptide

Co-localised insulin and c-peptide

42.0 37.0 32.0 27.0 22.0 17.0 12.0 7.0

Da y3 Da y5 Da y Da 9 y1 Da 9 y2 Da 8 y3 Da 3 y3 Da 7 y4 Da 5 y5 Da 0 y5 Da 5 y5 Da 9 y6 Da 4 y6 Da 8 y7 3

Blood glucose mmol/l

Insulincyte, Blood Glucose, mmol/l Stem cells injected 01-Dec-2005

Stem Cells PBS

Mouse No.

Treatment

Human Insulin mU/l

Human C-Peptide pmol/l

Mouse Insulin Ug/l

Serum Blood Sugar

2

Stem cells

0,00

nd

0,60

9,6

3

Stem cells

22,47

nd

0,12

9.9

4

Stem cells

21,72

1134

1,49

9,7

5

Saline

0,00

nd

2,80

15.8

6

Saline

0,00

nd

0,26

32,2

OmniCytes DAPI-nuclei; pkh26-membrane

Neuroblastoma (dapi-nucleous; pkh67-membrane)

Fusion OmniCytes and neuroblastoma

Growth of fibrosarcoma(RHT14-2) in single culture and in coculture with CD34+ SC 600

Number of cells (x10 4)

500 400 Cocultx104 300

Fibro cultx104 Fibro in cocultx104

200 100 0 0

2

4

6

Num ber of days in culture

8

Non transfected cells

Cells transfected with IFNγ10% expressing IFNγ

% cells expressing IFNgamma

IFNgamma expression 12 10 8 6 4 2 0

no transfection

10

8

10

9

10

10

adn-IFNgamma concentraction

10

11

CK18

HNF4 alpha

AFP

Transferrin

1kb DNA ladder

HNF3 beta

Cyto p450

HGF

Vimentin

Nestin

Albumin

Glucokinase

C MET

LDLR

Αlpha1 A trypsin

Day 14 LIVER MARKERS

HUMAN CELLS IN MOUSE LIVER SECTION - DAY 8

MOUSE LIVER SECTIONS STAINED FOR HUMAN CYTOKERATIN 18 ONE DAY POST TRANSPLANT Negative control Transplanted animal                        Day 0 Day 1 Day 8

Human nuclei staining Negative control

Nu301, Human nuclei staining Mouse sacrificed 2 days after transplantation

ENGRAFTMENT IN LIVER DAMAGE MODEL

Patient 5

20-04-2005

27-06-2005

Conclusions • No side effects or procedure-related complications. • Subjective improvements reported • Objective improvements in 3/5 patients.

190 170 150 130 110 90 70 50

37 32 27 22 Pre

WK1` weeks

WK2

Albumin (g/L)

Bilirubin (umol/L)

3 Alcoholic cirrhosis

BILIRUBIN ALB

70

50

60 50

40 30

40 30

20

20 10

10

0

0 Pre

WK2

WK6 Weeks

WK10

WK14

Albumin (g/L)

Bilirubin (umol/L)

Cirrhosis ( Autoimmune Hepatitis)

Bilirubin Albumin

Glycogen Storage Disease Father to six year old daughter ALT

350 300 250 200

ALT

150 100 50 0

AST

300 250 200 150 100 50 0

AST

Clinical Trials • Phase 1 Liver study successfully completed Phase 2 study started (liver) • Studies planned in 2006 (MHRA approved): Heart, Stroke, Diabetes, Phase III liver • Others: inflammatory bowel disease, peripheral vascular disease, fracture tibia, back pain, osteoarthritis of the knee t, chronic renal failure, chronic pain, MS