Blindness and cardiovascular disease are targeted areas for stem cell therapy. For blindness, clinical trials are ongoing using human embryonic stem cell-derived retinal pigment epithelial cells to treat age-related macular degeneration and Stargardt's disease. Perivascular progenitor cells may also help diseases like diabetic retinopathy. For cardiovascular applications, stem cell-derived cardiomyocytes and brown fat cells are being studied. Preclinical studies show stem cell treatments improve vascular permeability and function in disease models.

1. Hyung Min Chung
Konkuk University, College of Medicine,
Seoul, Korea
2014 MFDS Meeting
2014. 7. 9~10.
2014 MFDS Global Biopharmaceutical Forum

2. Ideal Cell Sources for Stem Cell Therapy???
Ideal Cells for
Application
Embryonic Stem Cells
SCNT-derived ESCs
Parthenogenic embryo-derived ESCs
Single blastomere-derived ESCs
Induced Pluripotent Stem Cells
Direct Conversion
Mid brain tissue from aborted fetus
Autologous Adult Stem Cells
Adipose-derived
Peripheral blood-derived
Bone marrow-derived
Testicular stem cells (HTSC)
Allogeneic Adult Stem Cells
Cord blood-derived
Placenta-derived
Wharton’s jelly

3. HLA typing of 38 CHA-hESC lines vs 6740 donated cord bloods
for simulation of stem cell transplantation
0
50
100
150
200
250
300
350
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 lines
Production of Single Blastomere derived hESC
Development 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

4. Human ES Cell Product – RPE Program for Blindness

5. 2009 2010, 2011, 2012

6. Products Company Tg Diseases Status
OPC
(Oligodendrocytes
progenitor cells)
Geron
Spinal Cord
Injury
Phase I (2009.01)
Discontinued
(November, 2011.11)
hES-RPE
Advanced Cell
Technology, Inc.
SMD
Dry-AMD
Phase I/IIa (2010.11)
Phase I/IIa (2011.01)
hES-RPE
CHABIO&DIOSTE
CH, Inc.
SMD
Dry-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)

7. Blindness
Accumulation of Drugen Cause by death of perivascular mural cell
RPE Therapy PVPC Therapy
Stargardt Macular Dystrophy
Age related Macular Degeneration
Diabetic/Ischemic Retinopathy

8. 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

9. 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%)

10. EB-DM (8주) RGM- RGMM (4주)MEF-GM & hES-GM (2주)
FM(유효기간 3개월) IP-Process (4시간 이내)
MEF 세포배양 &
hES 세포의 증식
EB의 형성과성장 hRPE세포의 증식과 분화 hRPE세포의 증식과 분화
수집, 충전 동결보존 임상의약품 출하 임상시술
Operation

11. Generation of RPE from hESCs
CHA-RPE lot #-001 CHA-RPE lot #-002 CHA-RPE lot #-003
ZO-1 MITF BestrophinPax-6

12. Purity and Gene Expression
OC
T4
Nanog DAPI
RPEhES-MA09
SirT1DAPI
MEF RPE
Removal of Stemness Cells & Mouse MEF cells
0.001
0.1
10
1000
Fold(2^-ddCT)
RPE Lot 2
RPE Lot 1
Gene expression

13. Storage and COA
제 품 개 요
임상시험용 401
제 품 명: MA09-hRPE
제품번호 :RPE-0006(CRP120921)
용 량 : 2 x 106 Cells/mL
유효 기간: 2013. 01. 23
○ 완제품 보관장소
완제품 보관실에 비치된 vapor type LN2 탱크
(<-135℃ )
○ 제품성적서

14. ERG response at P60:
Amplitude (uV)
α-wave
(outer)
β-wave
(inner)
β-cone
wave
Negative
Control
5 38 28
Test
Group
35 110 59
ERG(Electroretinogram)
Test
Product
Cycles/
degree
Negative
Control
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
Test
Product
2.7
log Unit
Negative
Control
untreated 0 %
sham 18 %
Test Group hES-RPE 52 %
Luminance Threshold
Preclinical Outcomes (1)

15. Histology Analysis
Test Product ONL Lyers
Positive 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

16. 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

17. Optomotor Test Results
Group
Un-t
reat
ed
sham 5K 20K 50K 75K
100
K
Nor
mal
rat
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
Dose
Medium
only
20K 50K 75K 100K
2.2
log Unit
3% 28% 45% 40%
65% (Non
significant)
Blue Line: RPE
Red Line: sham
20K
100K75K
50K

18. GLP Institute
Sinclair 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
1
2 mo 99.99% hRPE cells/
0.01% hES
8
6 mo 8
2
2 mo 99.9% hRPE cells/
0.1% hES
8
6 mo 8
3
2 mo 99% hRPE cells/
1% hES
8
6 mo 8
4
2 mo
100 % hES
8
6 mo 8
Preclinical Test : Tumorogenicity
Weeks
Teratoma
Formation
hESC RPE
4 Malignant
4/6
(66.67%)
0/6
(0%)
12 Malignant
5/7
(71.43%)
0/7
(0%)
40 Malignant
8/9
(88.89%)
0/11
(0%)
Teratoma formation
P<0.0001

19. Biodistribution
3
4
2
1
▶ Pathologies not observed
All 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-specific
proliferating cell nuclear antigen (PCNA)
Preclinical Study - Safety

20. 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 Autofluorescence
Adaptive 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

21. 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 Trials
15 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 !

22. Jules Stein Eye Institute
Steven D. Schwartz, MD
Bascom
Palmer
Eye Institute
Byron L. Lam, MD Wills Eye Institute
Carl D. Regillo, MD
Bascom Palmer Eye Institute
Philip J. Rosenfeld, MD PhD
Massachusetts
Eye
and Ear Infirmary
Dean Eliott, MD
Edinburgh Royal Infirmary
Baljean Dhillon Bmed Sci, BM Bs, FRCS
Moorfields Eye Hospital
James Bainbridge, MA MB Bcir PhD FRCOphth
London
Cha Hospital
Won-kyung Song, MD PhD

23. Other Human ES Cell Products and Related Technologies
 Stem Cell Differentiation into
Perivascular Progenitor Cells (PVPCs)
Cardiac Muscle Cells (CMs)
 In vivo POC

24. Perivascular Progenitor Cell Program
Accumulation of Drugen Cause by death of perivascular mural cell
RPE Therapy PVPC Therapy
Stargardt Macular Dystrophy
Age related Macular Degeneration
Diabetic/Ischemic Retinopathy
Concept of PeriVascular Progenitor Cell
• Multipotent MSC-like cell
mesodermal 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.

25. 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

26. calcein dye transfer assay
Functional Gap Junction Formation between hESC-PVPCs and Other
Vascular Cells

27. 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

28. • Smooth Muscle like Cells
Differentiation Potentials
adipogenesis
osteogenesis
Oil-red staining
Von Kossa’s staining
• Adipose, Chondrocyte, Osteoblast cells

29. vSMC (vascular smooth muscle cell) Differentiation
Two different types of SMC
contraction
Derivation of SMPC in vivo vasclugenesis
0min 30min
Carbachol (10-5M)

30. hESC-BFC (brown fat cell) & Myocyte
BFC vs. WFC Myocyte
Cell sheet using Poly-NIPAAM coated plate
Cardiomyocyte sheet
or cardiac patch
transplanted to
ischemic hearts as
cardiac patches
Osteocyte differentiated from hESC-PVPC
Myocyte differentiated from hESC-PVPC

31. Improvement of Blood Vessel Permeabilization

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

33. Intravitreous injection of MSCs in STZ diabetic mice

34. BN-STZ
6wk
BN-CTL
6wk
BN-STZ
6wk (PVPC
5x104 T.P.
4wk)
Improvement of Blood Vessel Permeabilization

35. Dextran DiI
Transplanted hESC-PVPCs take on Characteristics of Retinal
Pericyte in STZ-diabetic Mouse Model

36. CTL OIR-
CTL
OIR-PVPC
isolectin
Isolectin DiO
isolectin
Isolectin DiO
isolectin
Transplanted hESC-PVPCs injected at Mouse OIR Model

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

38. Cardiomyocytes Program

39. hESC-Cardiomyocyte

40. Synchronization of Contraction under Bio-fule System
+ -

41. Ion channel changes in cardiomyocytes stimulated by enzyme conductivity
Ca2+

42. Myocardial infarction model
Before operation After operation
Int’l J Cardiology, 2013

43. Int’l J Cardiology, 2013

44. Thank to ..
Lee DR CHA Stem Cell Institute of CHA University
Kim SJ Samsung Medical Center
Kim SJ Seoul National Univ. School of Medicine
Kim JH Korea University
Kang SW Korea University Ansan Hospital
Kim HB Hanyang Univ. School of Medicine
Cho SW Yonsei University
Kim HH Dankook University
Research Collaborators
Total 151 papers published in SCI-journal
68 papers published during recent 4 years (‘2010-’) in stem cell field
Patent registration: 48 (domestic) cases, 6 (USA) cases
Grants: 6,892,000,000 during recent 3 years (‘2010-’)
Representative Research Product