Retinal cell therapy using iPS cells · Retinal cell therapy using iPS cells Center for Developmental Biology, RIKEN ... Masayo Takahashi MD, PhD. embryonic tissue. neural stem cell

Retinal cell therapy using iPS cells

Center for Developmental Biology, RIKENLaboratory for Retinal Regeneration

Kobe City Medical Center General HospitalOphthalmology department

Institute of Biomedical Research and Innovation HospitalOphthalmology section

Masayo Takahashi MD, PhD

embryonic tissueneural stem cell

Regeneration of central nervous system using stem cells

intrinsic neural stem cells

dubious

２. Transplantation

A Replacement therapy

B Trophic effect

１. . Regeneration from intrinsic stem cells

Trophic factorImmature cellSmall amount

Functional cellMature cellEnough amount

Mitotic factorDifferentiation facotr

Preclinicalresearch

Clinical research(Act on the safety of regenerative medicine) forced on Nov 25, 2014

Regulation for cell therapy in Japan

Clinical trial(revised Pharmaceutical affairs act)

forced on Nov 25, 2014【Regenerative medicine 2014.11.25~ 】

※Earlier Patient Access !

Informed Consent and Post Market Safety Measures

Marketing(further confirmation

of efficacy and safety)

Adaptive Licensing with

conditionClinical

Research

Approval orExpiration ofprovisional

approval

MarketingClinical Trial(confirmation of

probable benefit* and safety**)

Application

* Probable benefit: Confirmation of efficacy with small patient population.** Safety: Earlier detection and evaluation of adverse events.

Enforcement 2015.11.25~

Retina

Peripheral retina（rod）

fovea（cone）

Optic disc

Retinal pigment epithelium （RPE）

Fundus

Φ2㎜

Macula

Technology ~retinal cell transplantation~

2017/6/7 5

RPE cells (for AMD)Started clinical study (Safaty)

IssueVariety of cell formsEfficacy

Photoreceptor (for Retinitis pigmentosa)Issue

Structure of photoreceptor cellsPurificationFunctional connection with the host’s neurons

Retinal cell transplantation


2017/6/7 Retina Institute Japan 6






Dry-type Chronic

AMDDrusenRPE atrophy

Neovascularization retinaRPE Basement

membrane

Choroid

Age-related macular degeneration （AMD)

Wet –typeSubacute

Current treatment（anti-VEGF drug injection）

RPE transplantation

edema / subretinal fluid

exduatehemorrhage

Treatment for age-related macular degeneration (AMD) with hiPS-RPE cell sheets

Patients with AMD

Pure RPE cells

Skin fibrobrasts

(Reprogramming)

Transplantation

8

iPS cells 24 line → ６ line

4 Mo 4 Mo

３ line

2 Mo

Differentiation

RPE cell sheets

Plasmid （Glis1）

Φ4mm skin incision

無菌試験室

iPS細胞室皮膚細胞処置室機器室分化調製室

着衣室1

エアーロック室1

女子更衣室

男子更衣室

廃棄物保管庫

エントランス

クリーン廊下

準備室2着衣室2

脱衣室2

滅菌室

準備室1

脱衣室1

パスルーム

保管室 QC室

居室

一般廊下

準備室3

脱衣室3

脱衣室4

エアーロック室2

収納庫2

収納庫1

着衣室3

着衣室4

気流方向

Grade AGrade BGrade CGrade D一般空調

清浄度区分

Skin harvesting from the patient

Cell culture in the cell processing facility

Safety cabinet

Incubator

Skin trephine

iPS-derived RPE sheet

Cell Processing Facility (CPF)

Quality & quantityof hiPSC-RPE cell-sheets

Collagen 4 DAPI Laminin DAPI ZO-1 DAPI

Hiroyuki Kamao

6w

PEDF

VEGF

apical

basal

Monitoring points : RPE function

PhagocytosisColor

PEDF Growth factors VEGF

Cluster analysisof gene expression profiles

12

RPE & Tumors

• No report of metastatic tumor ever in the history• Even in the familial tumor patients

=various cancer formation with hereditary oncogene (ex. p53) mutations

only hyperplasia of RPE occurs• PEDF (pigment epithelial derived factor)

= strong antitumor factor

• Eye ball is full of Retinoic Acid = antitumor factor

Cells

Environment

Summary of tumorigenicity test

donor Vector # of animal tumor

１st2010. 11～2011. 9

RPE cellRetrovirus

Sendai virusPlasmid A

57 0

2nd2011. 2～2012 4

RPE cell Plasmid A 11 0

RPE cell：OCT3/4 remnant+ Plasmid A 7 0

3rd2012. 3～10

RPE cell Plasmid B 27 0

4th2012. 12～2013 7

RPE sheet（subcutaneous・subretinal）

Plasmid B 16 0

（Kanemura et al.2014 PlosONE)

Purified hiPS-RPE did not generate tumors

NOD-SCID mice, subcutaneous, 1ｘ10＾5 cells in Matrigel

The most important thing is ….

(even with genetic changes)

Safety issue ( risk size) will be different depending on

15

1. Final cell types 2. Treatment procedure (i.v. or surgery)3. Host environment (diseases)

Hazard Level

1. The protocol that sometimes makes tumor in vivo2. Purity: contamination of tumor cells

16

↑ Actual hazard difficult to prove negative ↓ Possible hazard easy to find

3. Genetic change: SNV, Indel, CNV、Plasmid remnant4. Epigenetic change …..

Regulatory Science VS Basic Science

Quality 2: Purity

Quantity: Small amount of cells

Quality 1: function

OCT ; fine examination

Requirements for replacement therapy

Safety: no tumor formation

OCTOCT

Kanemura et al. 2013 Scientific report

Kuroda et al. 2012 PlosONE

Clinical application

SURGERY ROOM（SEP. 12. 2014）

19

iPS-RPE sheet transplantation

iPS-RPE sheet transplantation

CNV removal

vitreous

retina

lensInjectionneedle

injection

subretinalinjectionneedle

refluxfluid

Wet AMD

2mm

1mm

Fundus photograph after the surgery

3 month 6 month 1 year

1 week 4 week3 day

Animal experiment

rejection

0.3 0.3

Anti-VEGF injection

Visual acuity before & after the surgery

13 times injection into the eyeball

0.01

0.1

1

-240 -216 -192 -168 -144 -120 -96 -72 -48 -24 0 24 48 72 96 120

preoperative BCVA postoperative BCVA

postoperative weeks

BC

VA

surgery

preoperative weeks

1 year

NEI VFQ-25 (QOL)

術前W

Pre

The patient, who has remained anonymous, told The Japan Times:

‘I am glad I received the treatment. I feel my eyesight has brightened and widened.’

*

B B’3 days 1 year

1 week

1 year

pretreatment

1 year results： OCT (transvers section)

Fixation tests using MP-1

March, 2017

EDITORIALPolar Extremes in the Clinical Use of Stem Cells

George Q. Daley, M.D., Ph.D.N Engl J Med 2017; 376:1075-1077

March 16, 2017

Preclinicalresearch










conditionClinical

Research


approval



Application



HLA 6 loci homozygous

iPS line

HLA types CiRA

SheetSurgery risk: Big

SuspensionSurgery risk: Small

AutoCost: Big

Auto/Sheet8.2013～（Clinical research）

Auto/SuspensionApplication in preparation

AlloCost : Small

Allo/SheetApplication in preparation

Allo/SuspensionApproved by the ministry

2.3.2017

Types of hiPS-RPE cell transplantation

HLA 6 loci match 17% of Japanese population

MHC homo iPS-RPE transplantation into hetero matched monkey eye: no immune rejection

MHC Homo (HT-1)

MHC Homo iPS cells

MHC Hetero

MHC Hetero(MHC Matched)

Control

MHC (MHC mismatched)

iPS-RPE cells(MHC Homo：1121A1) Subretinal transplantation w/o immunosuppression

MHC mismatched monkey → rejection

MHC matched monkey → no rejection

Retina intact

Retinal inflammatory findingsOCT 2M

OCT 2M

RPE graft sheet RPE graft sheet

Cystic edemaDisappearance of outer retinal layers

Immune reaction to the MHC matched or unmatched iPS-RPE(in normal monkey )

The scheme of new clinical study using allograft

A*24:02-B*52:01-C*12:02-DRB1*15:02-DQB1*06:01-DPB1*09:01 Cell suspension

Superdonor blood cells

Frozen → culture → graft

Japanese man is first to receive 'reprogrammed' stem cells from another person

World-first transplant, used to treat macular degeneration, represents a major step forward in movement to create banks of ready-made stem cells.

David Cyranoski 3/28/2017


2017/6/7 Retina Institute Japan 32






厚生労働科学研究費科学研究費補助金難治性疾患克服研究事業網脈絡膜・視神経萎縮症に関する研究平成17年度総括・分担研究報告書 p.p.263-267 (2006)Health Labour Sciences Research Grant for Research on Measures for Intractable Diseases

Causative diseases of visual impairment in Japan (2005)

Graph1

glaucoma

diabetic retinopathy

retinitis pigmentosa

AMD

high myopia

cataract

others

causative disease of defect in vision

AMD; 9%

cataract; 3%

others; 27%

20.7

19

13.7

9.1

7.8

3.2

26.5

Sheet1

causative disease of defect in vision

glaucoma20.7

diabetic retinopathy19

retinitis pigmentosa13.7

AMD9.1

high myopia7.8

cataract3.2

others26.5

グラフのデータ範囲の大きさを変更するには、範囲の右下隅をドラッグしてください。

Photoreceptor degeneration

Retinitis pigmentosa•More than 30000 patients in Japan•No treatment

After birth（normal）

photoreceptor

Self-organizing optic-cup morphogenesis in three-dimensional culture

Mototsugu Eiraku, Nozomu Takata, Hiroki Ishibashi, Masako Kawada,Eriko Sakakura, Satoru Okuda, Kiyotoshi Sekiguchi, Taiji Adachi, Yoshiki Sasai (CDB, RIKEN)

April 7, 2011

http://www.nature.com/nature/journal/v472/n7341/full/nature09941.html

Retinal tissue derived from mouse ES cells in vitroSensory retina RPE

緑：神経網膜

OS

Green:photoreceptor

IS

Nucleus

Dr. Eiraku

Outer segment

Host retina

Donor sheet

RPE

Outer segment

Photoreceptor

Michiko MandaiMD, Phd

Mouse retinal sheet transplantation

AssawachananontMD, Phd

Stem Cell Reports 2014

Graft

Host retina

Host-graft synaptic contact in direct contact pattern


MED probe (MED-P5155)1.2mm

Electrode size: 50x50µm 1.2mm

Evalutation of Function after transplantation

Grafted cell inSubretinal space

Glass Micropipette

GCL

INL

ONL

Dim light stimulation

MED64 Amp

MED probe (64 channels)

Graft

Light intensity:750cds/m2Stimulus duration:10ms1Hz low cut filter

X-axis:Time (500ms/DIV)Y-axis:Amplitude(0.05mV/DIV)

microERG

光刺激のタイミング

on

Response to the light stimuli after transplantation

stimuli

ElectricalShock

５s

Training with light & sound

Within 2w (avoidance ratio:70%

Host with good transplants（post OCT selection）

Light only wild type rd1

After transplantation….

Rd1-2J

wt

light

Light +beep

Graft-

beep

light

Electric shock

5 sec

light

wt

Donor and host photoreceptors engage in material transfer following transplantation of post-mitotic photoreceptor

precursors R.A. Pearson1,*, A. Gonzalez-Cordero1, E.L. West1,**, J.R. Ribeiro1,**, N. Aghaizu1,**, D. Goh1, R.D. Sampson1, A. Georgiadis1, P.V. Waldron1, Y. Duran1, A. Naeem1, M. Kloc1, E. Cristante1, K.

Kruczek1, K. Warre-Cornish1,w, J.C. Sowden2, A.J. Smith1 & R.R. Ali1,3,*

Retinal transplantation of photoreceptors results in donor–host cytoplasmic exchange

Tiago Santos-Ferreira1,*, S ́ılvia Llonch1,*, Oliver Borsch1,*, Kai Postel1, Jochen Haas1 & Marius Ader1

Published 4 Oct 2016 It was cytoplasmic exchange !!

Graft

Host retina

Host-graft synaptic contact in direct contact pattern


Effect of Regenerative medicine

1. Donor cells

2. Host environment

Glial scar

Rod degeneration Cone degeneration Bipolar degenration

Iba1

ONL

Red: Iba1Microglia

ONLINL

INL

ONL

INLONL

3w

4w2w ３w

Immflamaion

Red: GFAPMuller glia

4w2w 7w 12w

Time window

Host environment and transplantation（rd mice：rapid photoreceptor degeneration model mice）

Retinitis Pigmentosa

ONL

ONL (Photoreceptor cell layer) +

Transplantation

In human patients, various stages exist in one eye

Patient-derived iPSCs studies

Photoreceptor

RPE

Retina

RGC

Photoreceptor dysfunction/degeneration

RGC dysfunction/degenerationLeber hereditary optic neuropathy

Normal tension glaucoma

・1 in 4000～8000 individuals・~50 causal genes identified・~60% are sporadic/unknown

(Mofiried from Nat Rev Neurosci.)

Retinitis Pigmentosa

⑨ MT-ND4 ComplexI

・1 in ~50000 individuals ・80% of Japanese LHON patients

・70~90% in Japanese patients・Causal genes characterized (MYOC WDR36 OPTN) ⑩ NTG (uncharacterized)

① RHO outer segment② RP1 clila③ PRPH2 outer segment④ RP9 splicing factor⑤ PRPH2 outer segment⑥ RP2 cilia⑦ EYS outer segment/cilia⑧ PRPF31 splicing factor/cilia

Preclinicalresearch










conditionClinical

Research


approval



Application



Lab Member

Project LeaderMasayo Takahashi

Deputy Project Leader

Michiko Mandai

Sunao Sugita

Research ScientistAkishi Onishi

Akiko Maeda

Genshiro Sunagawa

Ojos Matsuyama

Yuki Arai

Akiko Yoshida

Student TraineeShiniciro ItoSatoshi IrahaKaori UedaSo GotoKenichi MakabeWataru KobayashiCody KimeAnouk GeorgesRyutaro Akiba

CPC teamHiroyuki KitajimaNoriko SakaiYumiko ShibataMotoki TeragdaMitsuhiro Nishida

Retinal cell therapy using iPS cellsスライド番号 2スライド番号 3Retina Technology ~retinal cell transplantation~Technology ~retinal cell transplantation~スライド番号 7 Treatment for age-related macular degeneration (AMD) with hiPS-RPE cell sheetsスライド番号 9Quality & quantity� of hiPSC-RPE cell-sheetsMonitoring points : RPE functionCluster analysis �of gene expression profiles RPE & Tumors Summary of tumorigenicity testスライド番号 15Hazard Levelスライド番号 17スライド番号 18iPS-RPE sheet transplantationFundus photograph �after the surgeryVisual acuity before & after the surgeryNEI VFQ-25 (QOL)スライド番号 23スライド番号 24スライド番号 25スライド番号 26スライド番号 27スライド番号 28スライド番号 29The scheme of new clinical study using allograftスライド番号 31Technology ~retinal cell transplantation~スライド番号 33スライド番号 34Self-organizing optic-cup morphogenesis �in three-dimensional cultureスライド番号 36スライド番号 37スライド番号 38スライド番号 39スライド番号 40スライド番号 41スライド番号 42スライド番号 43スライド番号 44スライド番号 45スライド番号 46スライド番号 47スライド番号 48スライド番号 49スライド番号 50

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Retinal cell therapy using iPS cells · Retinal cell therapy using iPS cells Center for Developmental Biology, RIKEN ... Masayo Takahashi MD, PhD. embryonic tissue. neural stem cell