Hyung Min Chung

Konkuk University, College of Medicine,Seoul, Korea

2014 MFDS Meeting2014. 7. 9~10.

2014 MFDS Global Biopharmaceutical Forum

Ideal Cell Sources for Stem Cell Therapy???

Ideal Cells for Application

Embryonic Stem CellsSCNT-derived ESCsParthenogenic embryo-derived ESCsSingle blastomere-derived ESCs

Induced Pluripotent Stem CellsDirect Conversion

Mid brain tissue from aborted fetus

Autologous Adult Stem CellsAdipose-derivedPeripheral blood-derivedBone marrow-derivedTesticular stem cells (HTSC)

Allogeneic Adult Stem CellsCord blood-derivedPlacenta-derivedWharton’s jelly

HLA typing of 38 CHA-hESC lines vs 6740 donated cord bloodsfor simulation of stem cell transplantation

050

100150200250300350

A, B antigen and DRB1

allele level (2 mismatch)

A, B and DRB1 allele level

(2 mismatch)

Full match 1 mismatch 2 mismatch

A, B antigen and DRB1 allele level 0.3% 2.43% 22.4%

A, B and DRB1 allele level 0.21% 1.74% 14.21%

Cell Transplantation, 2010

56 hESCs established Successful derivate SCNT-hESC linesProduction of Single Blastomere derived hESCDevelopment of 100 clinical grade hESCs establishment

Human ES Cell Bank : overcome histocompatability

It was estimated that our 38 CHA-hESC lines can provide a coverage for 27% and 45% of the Korean population with A, B, DR allele level and A, B antigen/DR allele level matches, respectively.

National Stem Cell Bank

Human ES Cell Product – RPE Program for Blindness

2009 2010, 2011, 2012

Products Company Tg Diseases Status

OPC(Oligodendrocytesprogenitor cells)

Geron Spinal Cord Injury

Phase I (2009.01)

Discontinued(November, 2011.11)

hES-RPEAdvanced Cell

Technology, Inc.SMD

Dry-AMDPhase I/IIa (2010.11)Phase I/IIa (2011.01)

hES-RPE CHABIO&DIOSTECH, Inc.

SMDDry-AMD

MMD

Phase I (2011.05)Phase I/IIa (2012.05)Phase I/IIa (2013.11)

hES-RPE Pfizer AMD Expected in Europe

hES-RPE Cell cure Neuroscience AMD Expected in Europe

iPS-RPE RIKEN AMD Approval of IND (2014)

Blindness

Accumulation of Drugen Cause by death of perivascular mural cell

RPE Therapy PVPC Therapy

Stargardt Macular DystrophyAge related Macular Degeneration Diabetic/Ischemic Retinopathy

Human ES Cell Therapy : Blindness

Dry AMD(90%) Wet AMD(10%)

PROVIDE nutrients and growth factors- photoreceptors see no blood

RECYCLES vitamin A - maintains photoreceptor layer

DETOXIFIES photoreceptor layer MAINTAINS Bruch’s membrane

- natural antiangiogenic barrier - immune privilege of retina

ABSORBS stray light/protects from UV

Retinal Pigment Epithelium

1. Target Disease : Blindness (Age related Macular Degeneration, Retinitis Pigmentosa, Stardgart’s disease

2. Clinical study process is ongoing

- November 19, 2009 : pre-clinical activities in preparation of the first IND filing with the FDA (USA)

- March 2010: Granted Orphan Drug Status from US FDA for Treatment of Stargardt’s Macular Dystrophy

- May 2011: Approval of clinical trial in KFDA for SMD (Phase I)

- May 2012 : Approval of clinical trial in kFDA for Dry-AMD (Phase I/IIa)

- Nov 2013: Approval of clinical trial in kFDA for MMD (Phase I/IIa)

Blindness (Stargardt’s Disease, Age-Related Macular Degeneration)

Dry AMD(90%) Wet AMD(10%)

EB-DM (8주) RGM- RGMM (4주)MEF-GM & hES-GM (2주)

FM(유효기간 3개월) IP-Process (4시간 이내)

MEF 세포배양 &

hES 세포의 증식EB의 형성과성장 hRPE세포의 증식과 분화 hRPE세포의 증식과 분화

수집, 충전 동결보존 임상의약품 출하 임상시술

Operation

Generation of RPE from hESCs

CHA-RPE lot #-001 CHA-RPE lot #-002 CHA-RPE lot #-003

ZO-1 MITF BestrophinPax-6

Purity and Gene Expression

OCT4

Nanog DAPI

RPE

hES-

MA

09

Sir T

1D

API

MEF RPE

Removal of Stemness Cells & Mouse MEF cells

0.001

0.1

10

1000

Fold

(2^

-ddCT)

RPE Lot 2RPE Lot 1

Gene expression

Storage and COA제 품 개 요

임상시험용 401제 품 명: MA09-hRPE제품번호 :RPE-0006(CRP120921)용 량 : 2 x 106 Cells/mL유효 기간: 2013. 01. 23

○ 완제품 보관장소

완제품 보관실에 비치된 vapor type LN2 탱크(<-135℃ )

○ 제품성적서

ERG response at P60: Amplitude (uV)

α-wave (outer)

β-wave (inner)

β-cone wave

NegativeControl 5 38 28

Test Group 35 110 59

ERG(Electroretinogram)

Test Product

Cycles/degree

NegativeControl

untreated 0.21 ± 0.03

sham 0.29 ± 0.03

Test Group hES-RPE 0.42 ± 0.03

Positive Control

(Normal)untreated 0.6

Optomotor Test

TestProduct

2.7log Unit

NegativeControl

untreated 0 %

sham 18 %

Test Group hES-RPE 52 %

Luminance Threshold

Preclinical Outcomes (1)

Histology Analysis

Test Product ONL LyersPositive Control

(Normal rat)untreated 10-12 deep

Test Group hES-RPE 5-7 deep

Negative Control untreated Single deep

A. Test Group

B. Negative Control C. Test Group: human nuclear marker

1. Proof of Concept Study: RCS rat

Preclinical Test : RP, AMD model

RPE Engraftment – Mouse Model

Human hES-RPE cells engraft and align with mouse RPE cells in mouse eye

For each set: Panel (C) is a bright field image

and Panel (D) shows immunofluorescence

with anti-human bestrophin (green) and anti-

human mitochondria (red) merged and

overlayed on the bright field image.

Magnification 400x

Lancet, 2012

Optomotor Test Results

GroupUn-treated

sham 5K 20K 50K 75K 100K

Normalrat

Cycle/degree 0.16 0.18 0.24 0.28 0.44 0.41 0.43 0.6

Preclinical Test : Dose Finding Data

Luminance Threshold Test

DoseMedium

only 20K 50K 75K 100K

2.2log Unit 3% 28% 45% 40% 65% (Non

significant)

Blue Line: RPERed Line: sham

20K

100K75K

50K

GLP InstituteSinclair Research (non-GLP)Wuxuasse, MO, USA

Study Product hES-RPE, (100,000 cells)

Route for Injection Subretinal injection

Animal Model NIH-III nude mice

Duration of Study 6 months

Exp. Design

Test Results No observation of tumor (including teratoma) formation in hES-RPE injection group

Group Duration Test cells No. of mice

12 mo 99.99% hRPE cells/

0.01% hES

8

6 mo 8

22 mo 99.9% hRPE cells/

0.1% hES

8

6 mo 8

32 mo 99% hRPE cells/

1% hES

8

6 mo 8

42 mo

100 % hES8

6 mo 8

Preclinical Test : Tumorogenicity

WeeksTeratomaFormation hESC RPE

4 Malignant4/6

(66.67%)0/6(0%)

12 Malignant 5/7(71.43%)

0/7(0%)

40 Malignant8/9

(88.89%)0/11(0%)

Teratoma formation

P<0.0001

Biodistribution

3

4

2

1

▶ Pathologies not observedAll eye slides were reviewed by a board certified veterinary pathologists

Only typical retinal morphology

No ectopic tissue or abnormal pathology

Histological examination: the presence of human-specific nuclear marker

Negative staining for human-specificproliferating cell nuclear antigen (PCNA)

Preclinical Study - Safety

Phase I/IIa Clinical Trial Design : Dry-AMD

12 patients for each trial, ascending dosages of 50K, 100K, 150K and 200K cells. - For each cohort, 1st patient treatment followed by 6 weeks DMSB review

before remainder of cohort.

Patients will be monitored weekly – including high definition imaging of retina

High Definition Spectral Domain Optical Coherence Tomography (SD-OCT) Retinal AutofluorescenceAdaptive Optics Scanning Laser Ophthalmoscopy (AOSLO)

Permit comparison of RPE and photoreceptor activity before and after treatment

50K cells 100K cells 150K cells 200K cells

DSMB review

First Human Trials in AMD & SMD

21 SMD Patients Treated

12 patients (50K cell cohort) treated – US, UK & Korea Trials

8 patients (100K cells cohort) treated – US & UK Trials

1 patients (150K cells cohort) treated – US Trials15 Dry-AMD Patients Treated

8 patients (50K cell cohort) treated – US & Korea Trials

5 patients (100K cells cohort) treated – US Trials

2 patients (150K cells cohort) treated – US Trials

Designate the Orphan Drug at 2014 !

Jules Stein Eye InstituteSteven D. Schwartz, MD

BascomPalmer Eye Institute

Byron L. Lam, MD Wills Eye InstituteCarl D. Regillo, MD

Bascom Palmer Eye InstitutePhilip J. Rosenfeld, MD PhD

Massachusetts Eye and Ear Infirmary

Dean Eliott, MD

Edinburgh Royal InfirmaryBaljean Dhillon Bmed Sci, BM Bs, FRCS

Moorfields Eye Hospital James Bainbridge, MA MB Bcir PhD FRCOphth

London

Cha Hospital Won-kyung Song, MD PhD

Other Human ES Cell Products and Related Technologies

Stem Cell Differentiation into Perivascular Progenitor Cells (PVPCs)Cardiac Muscle Cells (CMs)

In vivo POC

Perivascular Progenitor Cell Program

Accumulation of Drugen Cause by death of perivascular mural cell

RPE Therapy PVPC Therapy

Stargardt Macular DystrophyAge related Macular Degeneration

Diabetic/Ischemic Retinopathy

Concept of PeriVascular Progenitor Cell

• Multipotent MSC-like cellmesodermal multipotent cell, perivascular cell, vascular MSC

PeriVascular Progenitor Cell

• PVPCs , principally pericytes, were identified in multiple human organs including sketal muscle, pancreas, adipose tissue and placenta.

• Pericyte density has been described for neural tissues, in particular the retina.

• PVPCs perform an important role in blood vessel homeostasis.

• PVPCs have no pan marker, but long term cultured PVPCs stably expressed NG2, CD146 and PDGFR.

• They express all makers of MSC such as CD44, CD73, CD90, CD105.

• In contrast, they do not express the markers of hematopoietic, endothelial and myogenic cell.

hESC-PVPC Derivation

Derivation of PVPC

• Non-sorted cell derivation – natural selection

• Matrix-dependant single cell attachment- induced CD44 expression, - natural selection of PDGFR-b- population

• CHA-3, CHA-5, CHA-9, CHA15, CHA11, H9 hESC-PVPC

• Up to 35th passage, stable, single layer

calcein dye transfer assay

Functional Gap Junction Formation between hESC-PVPCs and Other Vascular Cells

hESC-PVPC Recruitment during Vasculogenic Tube Assembly

In vitro 3D collagen matrix models of endothelial lumen formation during vasculogenesis

In vivo localization of hESC-PVPC in mouse STZ-diabetic retinopathy model

• Smooth Muscle like Cells

Differentiation Potentials

adipogenesis

osteogenesis

Oil-red staining

Von Kossa’s staining

• Adipose, Chondrocyte, Osteoblast cells

vSMC (vascular smooth muscle cell) Differentiation

Two different types of SMCcontraction

Derivation of SMPC in vivo vasclugenesis

0min 30min

Carbachol (10-5M)

hESC-BFC (brown fat cell) & Myocyte

BFC vs. WFC Myocyte

Cell sheet using Poly-NIPAAM coated plate

Cardiomyocyte sheetor cardiac patch

transplanted to ischemic hearts as cardiac patches

Osteocyte differentiated from hESC-PVPC

Myocyte differentiated from hESC-PVPC

Improvement of Blood Vessel Permeabilization

normal mouse retina STZ diabetic mouse retina(12week) hESC-PVPC injected-STZ diabetic mouse retina(12week)

Intravitreous injection of MSCs in STZ diabetic mice

BN-STZ 6wk

BN-CTL 6wk

BN-STZ 6wk (PVPC 5x104 T.P. 4wk)

Improvement of Blood Vessel Permeabilization

Dextran DiI

Transplanted hESC-PVPCs take on Characteristics of Retinal Pericyte in STZ-diabetic Mouse Model

CTL OIR-CTL

OIR-PVPC

isolectin

Isolectin DiO

isolectin

Isolectin DiO

isolectin

Transplanted hESC-PVPCs injected at Mouse OIR Model

Transplanted hESC-PVPCs injected at STZ-diabetic Retinopathy Model survived within Perivascular Region

Cardiomyocytes Program

hESC-Cardiomyocyte

Synchronization of Contraction under Bio-fule System

+ -

Ion channel changes in cardiomyocytes stimulated by enzyme conductivity

Ca2+

Myocardial infarction model

Before operation After operation

Int’l J Cardiology, 2013

Int’l J Cardiology, 2013

Thank to ..

Lee DR CHA Stem Cell Institute of CHA UniversityKim SJ Samsung Medical CenterKim SJ Seoul National Univ. School of MedicineKim JH Korea University Kang SW Korea University Ansan Hospital Kim HB Hanyang Univ. School of MedicineCho SW Yonsei UniversityKim HH Dankook University

Research Collaborators

Total 151 papers published in SCI-journal 68 papers published during recent 4 years (‘2010-’) in stem cell fieldPatent registration: 48 (domestic) cases, 6 (USA) cases Grants: \ 6,892,000,000 during recent 3 years (‘2010-’)

Representative Research Product