Supplementary appendixThis appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors.

Supplement to: Schwartz SD, Regillo CD, Lam BL, et al. Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt’s macular dystrophy: follow-up of two open-label phase 1/2 studies. Lancet 2014; published online Oct 15. http://dx.doi.org/10.1016/S0140-6736(14)61376-3.

SUPPLEMENTARY MATERIAL

Supplementary Table 1. Inclusion and exclusion criteria

Detailed Methods

The hESC and hESC-derived RPE were generated as described (1). Briefly, vials of hESC-MA09

were thawed and expanded on mitotically-inactivated mouse embryonic fibroblasts (MEF) using

current Good Manufacturing Practices. Since the hESCs were co-cultured with animal cells, the

differentiated derivatives are classified as a xenotransplantation product and subject to FDA

guidelines for donor animal and product processing, testing, and archiving, as well as patient,

monitoring and registration. After hESC expansion, the cells were sequentially induced to form

embryoid bodies followed by cellular outgrowth and localized differentiation into pigmented RPE

patches (2). The pigmented patches were isolated with collagenase, and after purification and

trypsinization, the dissociated cells were seeded, grown to confluence, and induced to redifferentiate

for a total of three serial passages (P0, P1, P2)(1). Passage 2 hESC-RPE were cryopreserved and

served as the starting material for formulating cells for clinical use.

The hESC-RPE cells were assessed for safety and characterized for a number of RPE-specific

attributes at various times, including in-process testing and testing performed after thaw and final

product formulation. Safety assessment for pathogens, karyotyping, DNA fingerprinting,

immunohistochemical staining, and fluorescence in situ hybridization (FISH) were performed as

described (19). Assessment of purity and the extent of differentiation were based on the percentage

of bestrophin, Pax6, ZO-1 and/or MITF stained cells. Screening to confirm the absence of

pluripotency markers was performed by staining for OCT-4 and Alkaline Phosphatase.

Phagocytosis (potency assay) was assessed by quantitative fluorescence activated cell sorting

(FACS) analysis of hESC-derived RPE cultures exposed to PhRodoTM

(Invitrogen) fluorescent

bioparticles. Quantitative reverse transcription (q-RT) PCR assays were performed to confirm up-

regulation of RPE-specific genes (RPE-65, PAX-6, MITF, bestrophin) and down-regulation of

hESC-specific genes (OCT-4, NANOG, SOX-2).

On the day of transplantation, vials were thawed, formulated, and assessed for suitable viability and

the absence of microbial contaminants by Gram staining prior to delivering to the operating room

(1). Pars plana vitrectomy including surgical induction of posterior vitreous separation from the

optic nerve to the posterior border of the vitreous base was carried out. A volume of 150uL of

reconstituted RPE was injected through a MedOne PolyTip® Cannula 23/38 or 25/38 delivering the

targeted dose of viable RPE cells into the subretinal space in sites centered over a “transition zone”

(the area between atrophic photoreceptor/RPE/choriocapillaris and relatively healthy post-equatorial

retina) as assessed with AF and OCT imaging. Transplantation sites were chosen carefully based

on the presence of native, albeit compromised, RPE and similarly compromised overlying

photoreceptors, to optimize the chances of transplant integration and potential for photoreceptor cell

rescue.

The systemic immunosuppression regimen included low-dose tacrolimus (target blood levels 3-7

ng/mL) and mycophenolate mofetil (MMF ranging 0.5g - 2g orally/day) one week prior to the

surgical procedure and continued for a period of 6 weeks. At week 6, the regimen called for

discontinuation of tacrolimus and a continuation of the MMF for an additional six weeks or longer

(at the discretion of the investigator). Two patients (1 AMD, 1 SMD) had tacrolimus and MMF

discontinued after 12 to 15 days of administration due to adverse events. A second AMD patient

could not tolerate MMF, which was discontinued on day 50, and the tacrolimus continued until

week 12. One investigator electively continued the MMF for 47 to 55 weeks for 6 patients and >66

weeks for 1 patient (3 AMD, 4 SMD). Per protocol, local steroid drops were withheld until

postoperative day 4.

Transplanted patients were followed by serial ophthalmic examinations including best-corrected

visual acuity (BCVA), visual field testing, slit lamp biomicroscopy, ophthalmoscopy, optical

coherence tomography, fluorescein angiography, autofluorescence imaging and electroretinography.

Systemic monitoring included cancer screening, hematologic and serologic testing as part of both

the immunosuppression protocols and safety studies. Visual acuity was assessed as the number of

correct letters read with the subject turning his or her head to obtain the best score. A visual acuity

score of 0 letters (20/2000) was assigned to Count Fingers and a score of -50 letters (20/20,000) was

assigned to Hand Motion (3).

References

1. Schwartz SD, Hubschman JP, Heilwell G, et al. Embryonic stem cell trials for macular

degeneration: a preliminary report. The Lancet 2012; 379:713-20.

2. Lu B, Malcuit C, Wang S, et al. Long-term safety and function of RPE from human

embryonic stem cells in preclinical models of macular degeneration. Stem Cells 2009; 21,

2125-2135.

3. This is based on the assumption that fingers are approximately the size of the elements of a

200 letter. The value is conservative because the contrast is lower for count fingers than for

the ETDRS chart. The value for hand motion is based on low vision studies showing hand

motion is ten times worse than count fingers (i.e., detection of hand motion at 20 feet has

approximately 20/20,000 Snellen visual acuity equivalent (Holladay, J.T. Proper Method

for Calculating Average Visual Acuity. Journal of Refractive Surgery 1997; 13: 388-

391). Light perception is not a visual acuity measurement, but rather the detection of a

stimulus. Light perception assessments were excluded from the analysis and instead the last

observation was carried forward.

4. Early Treatment Diabetic Retinopathy Study Research Group. Photocoagulation for diabetic

macular edema: Early Treatment Diabetic Retinopathy Study report number 1. Arch

Opthalmol. 1985;103:1796-1806.

Supplementary Table 2. Duration of immunosuppression

Duration of Administration

Comments

(Weeks)

Indication Site ID

Subject ID

Dose Group MMF Tacrolimus

AMD

JSEI 201

50,000

7.1 12

Could not tolerate MMF; related adverse events of shortness of breath (severe), fatigue (moderate), diarrhea (moderate) and stomach ache (severe)

JSEI 202 50,000 52.9 5.6

JSEI 203 50,000 52.3 8

Wills 204 100,000 12.9 7

MEEI 205 100,000

JSEI 206 100,000 2.1 1.6 Stopped due to adverse event - UTI

JSEI 207 150,000 46.9+ 5.7 MMF ongoing

MEEI 208 150,000

BP 209 150,000

SMD JSEI 101 50,000 54.4 7.3

JSEI 102 50,000 52.3 7.3

JSEI 103 50,000 53.3 7.3

Wills 104 100,000 12.9 6.9

Wills 105 100,000 13.9 5.7

JSEI 106 100,000 66.1+ 7.1 MMF ongoing

BP 107 150,000 8

Wills 108 150,000 1.7 1.7 Stopped due to endopthalmitis

BP 109 150,000

Supplementary Table 3. Summary of all adverse events by time of onset

System Organ Classification/ Adverse Event Term

SMD AMD

Time of Onset Time of Onset

<6 Months

≥6 Months

<6 Months ≥6

Months

N=9 N=8 N=9 N=8

n n n n

With Any Event 8 4 9 3

Cardiovascular Disorders

1 1 2 0

Edema Peripheral

1

Hypertension 1

Irregular Heart Rate 1

1

Eye Disorders 6 2 7 1

Anterior Surface Pigment 1

Blurry Vision 1

Cataract 2 1 1

Charles Bonnet Syndrome 1

Conjunctival Hemorrhage

2

Corneal Abrasion

1

Dry Eye 1 1

Epiretinal Membrane 1

Eye Dilated 1

Eye Pain 2 2

Eye Watery 1 1

Floaters 2 2

Foreign Body Sensation

2

Hyperemia 1

Inferior Folds 1

Posterior Vitreal Detachment 1

Ptosis 1 1

Retinal Vascular Leakage 1

Suconjunctival Hemorrhage @ Injection Site 1

Suture Granuloma 1

Vitreous Strand 1

General Disorders 2 1 6

1

Anxiety 1

Chest Pain 1 1

Decreased Appetite

2

Dizziness 1 2

Ear Wax 1

Fatigue 1

Hair Loss 1

Stiffness 1

Sweating 1

Tooth Ache 1

Weakness 2

Gastrointestinal Disorders 4 2 6

2

Appetite decreased

2

Blockage of Salivary Duct 1

Constipation 1 2 1

Diarrhea 3 2

Dry Mouth 1 1

Gas 1

Gum Pain 1

Hemorrhoids 1

Nausea 1 3

Stomach Pain 1 1

Vomiting 1 1

Infections 4 1 4 2

Bronchitis 1 1

Endophthalmitis 1

Fever 1

Shingles 1

Sinus Infection 1 1

Sore Throat 2

Thrush 2

Upper Respiratory Tract Infection

2

Urinary Tract Infection 1

2 1

Injury 0 0 2 0

Broken hip 1

Shin Injury 1

Investigations 1 0 1 0

Increased Intraocular Pressure 1 1

Musculoskeletal Disorders 1 2 3 0

Chest Wall Pain 1

Knee Pain 1

Lower Back Pain 1

Muscle Cramp 1

Osteoarthritis 1

Shoulder Pain 1

Torn Gelnoid Labrum

1

Neoplasms 0 0 3 1

Basal Cell Carcinoma 1

Cyst 1

Squamous Cell Carcinoma 1

Nervous System Disorders

4 0 4 1

Headache 2 1

Hemiparesis 1

Insomnia 2 1

Syncope 1

Tremor 1

Psychiatric Disorders 1 0 2 1

Altered Mental Status

1

1

Confusion 1

Depressed Mood

1

Respiratory Disorders 3 0 2

1

Allergic Rhinitis 1

Basilar Crackles 1

Hoarseness 1

Nasal Polyps 1

Shortness of Breath 1

Sinus Congestion

2

Sinus Problems 1

Basilar Crackles

Skin Disorders 0 0 2 0

Dry Skin 1

Hyperpigmented Cheeks Bilateral 1

Spots on Scalp/Face 1

Supplementary Table 4. Serious adverse events

System Organ Classification/ Serious Adverse Event Term

SMD AMD

N=9 N=9

n n

With Any Serious Adverse Event 2 4

General Disorders 1 0

Chest Pain 1

Infections 1 1

Endophthalmitis 1

Urinary Tract Infection 1

Injury 0 1

Femoral Neck Fracture 1

Neoplasms 0 1

Basal Cell Carcinoma 1

Squamous Cell Carcinoma 1

Nervous System Disorders 0 2

Hemiparesis 1

Syncope

1

Psychiatric Disorders 1

Mental Status Change 1

Supplementary Table 5. Systemic AEs Likely Related to

Immunossuppression

SMD AMD N=9 N=9

System Organ Classification/ Adverse Event Term n n Cardiovascular 1 0 Heart Palpitation 1 Central Nervous System 8 7 Altered Mental Status Secondary to UTI 1 Headache 3 3 Mental Fogginess 2 Confusion 1

Dizziness Post-Anesthesia w/ Blood Pressure Drop 1

Feels Hot, as if Blushing 1 Increased Tremor 1 Insomnia 1 Off Balance 1 Gastrointestinal 6 16 Decreased Appetite 1 4 Diarrhea 1 3 Increased Gas 1 Nausea 1 4 Dry Mouth 1 Mouth Sore Inner Gum (Left) "Tori" 1 Stomach Ache 1 4 General 2 6 Fatigue 3 Weakness 3 Burning Sensation in Her Palms and Feet 1 Fevers 1 Sweating 1 Genitourinary 1 0 Microscopic Hematuria 1 Hematology 0 4 Elevated Neutrophil 1 Elevated WBC 1 Low Lymphocyte 1

Low Platelet Count 1 Infection 2 6 Acute on Chronic Sinusitis 1 Shingles 1 Thrush (tongue) 1 Sore Throat 1 Urinary Tract Infection 1 3 Metbolic 1 3 Elevated Potassium 1 Elevated AST and ALT 1 High/Increased Creatinine 2 Musculoskeletal 0 2 General Stiffness 1 Tight Left Quad Muscle/Cramp 1 Psychiatric 0 1 Depressed Mood 1 Respiratory 1 3 Hoarse Voice 1 Shortness of Breath 3 Skin 0 2 Cyst on Outer Right Chest Wall 1 Cystic lesion on back 1

Supplementary Table 6. Change in best-correct visual acuity (BCVA) in SMD Patient 101 and AMD

Patient 207

*Patient 101 developed a cataract during the first year that was removed.

Supplementary Figure 1. Autofluorescence images of a patient with increasing pigmentation

consistent with transplanted hESC-RPE. Autofluorescence images (A-D) of an SMD patient, which

correspond to the color fundus photographs in Figure 1G-I. Preoperative autofluorescence imaging shows

hypo-autofluorescence centrally with small satellite lesions of hypo-autofluorescence, and a surrounding rim

of hyper-autofluorescence (A). Two weeks (B) and 3 months (C) after surgery. There is speckled hyper-

autofluorescence in the upper half of the central atrophic lesion (B and C, arrows) that diminishes by 1 year

(D).

Supplementary Figure 2. National Eye Institute Visual Function Questionnaire 25 scores for

AMD and SMD patients during first year. National Eye Institute Visual Function Questionnaire

25 (VFQ-25) scores for mental health and vision subscales, including general vision, peripheral

vision, near activities, and distance activities. Scores at baseline (left hand column) and net change

relative to baseline <3 months (3-8 weeks; light blue bars) and 3-12 months (12-52 weeks; dark

blue bars)(right hand column; N=8 in both groups, there was no data available for 1 AMD and 1

SMD patient). Data are medians (error bars show 25-75th percentiles)