2015 bioa klimanskaya_irina

BIOASSAY DEVELOPMENT FOR HUMAN EMBRYONICSTEM CELL-DERIVED RETINAL PIGMENTEPITHELIUM: PROGRESS AND CHALLENGES

Irina Klimanskaya, Ph.D.

March 23, 2015

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Retinal Pigment Epithelium (RPE)

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Presentation Notes

-- is a highly specialized tissue

Structure of the Retina

photoreceptor

RPE

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Presentation Notes

It forms a barrier between the subretinal space and the blood vessels, underlying the photoreceptor. retina is a layered structure with several layers of neurons interconnected by synapses. The only neurons that are directly sensitive to light are the photoreceptor cells. outer nuclear layer contains several strata of oval nuclear bodies; they are of two kinds, viz.: rod and cone granules, so named on account of their being respectively connected with the rods and cones of the next layer. There is no direct blood supply to the photoreceptor layer.

Visual Physiology

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Photoreceptor has no direct blood supply and relies on the RPE for homeostasis including delivery of vitamins and nutrients, maintenace of ions and removal of its waste products.

Visual Physiology: Macular Degeneration

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When RPE begins to malfunction or dies, the photoreceptor follows. This leads to retinal degenrations resulting in such diseases and age-related macular degeneration (AMD) affecting over one million of patients each year in the US.

Photo by Vlad Chertikhinchertphotography.com

Age Related Macular Dissociation

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This is how the world looks to a patient with AMD – the central vision is lost, and this dark area spreads out, eventually resulting in full blindness

Photo by Vlad Chertikhinchertphotography.com

Normal Vision

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And this is what the picture hsould look like

RPE Transplantation

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Ocata Therapeutics has initiated clinical trials for treatment of AMD and another RPE-related retinal degeneration, Stargardt’s disease, using transplantation of RPE derived from himan embryonic stem cells

Thesis of RPE Transplantation: Restoration of Anatomy & Function

Neural signal restored

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When hESC-derived RPE engraft they can take over the function of the lost patient’s RPE and maintain surviving photoreceptor health, thus preserving the vision

Generation of RPE from Human ES CellsA blastomere was removed from a morula stage embryo

hESC MA09 line was established

hESC differentiate to RPE

Pigmented clusters are isolated

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RPE Transdifferentiation in Culture

Adapted from: B. A. Pfeffer and N. J. PhilpExp. Eye Res.,126 (2014) 1-4

dissociation

dissociation

pigmented cluster cell culture

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Presenter

Presentation Notes

RPE is known for its ability to “transdifferentiate” in culture, going back to its developmental roots – retinal and neural progenitor phenotype. The cells lose epithelial morphology and melanin, but after they reach confluence and mature overtime, they re-differentiate. The cycle continues until the cells get senescent

RPE undergoes dedifferentiation during culture expansionand matures after reaching confluence

freshly dissociated clusters Day 1

early confluence mature

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Post-Thaw Survival is Higher in Less Pigmented Cells

21 h post plating 3 days in culture

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Presentation Notes

Less pigmented cells have a better survival in culture after cryopreservation

RPE Cells are Harvested Before They Fully MatureGrowing stage Polarized but not fully mature

mature

cryopreservationin vitro – assessment

in vivo – post-transplantation

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Presentation Notes

This is why RPE cells for transplantation are harvested and cryopreserved BEFORE they fully mature. However, each lot is subsequently thawed and cultured to maturity, so the cells can be tested for function-related properties

In Vitro vs. In Vivo Limitations

in vitro2D surfaceformulated extracellular matrixformulated mediaformulated growth factorspassaging – enzymes, proliferation

in vivo3D environmentphysiological matrix with growth factorsphysiological microenvironmentsignals from blood and surrounding cellsno or very slow renewal throughout life

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Presenter

Presentation Notes

There are always limitation of cell culture system compared to in vivo environment such as…

In Vitro vs. In Vivo Limitations

in vitro2D surfaceformulated extracellular matrixformulated mediaformulated growth factorspassaging – enzymes, proliferation

in vivo3D environmentphysiological matrix with growth factorsphysiological microenvironmentsignals from blood and surrounding cellsno or very slow renewal throughout life

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Presentation Notes

a function can improve once the cells fully mature and feel at home but maybe it can only be detected under artificial “optimized” conditions and cannot be sustained in vivo? Or maybe some functions will improve and some get worse?

RPE Multitasking

adapted f from O. Strauss Physiol Rev 85: 845–881, 2005;doi:10.1152/physrev.00021.2004

bestrophin 1MCT3

claudin 19 ZO-1

barrier

melanintyrosinase

STRA6RPE65 LRAT

αvβ5 integrinMFG-E8CD36MerTKFAKGas6Myosin II and VIIa

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Presenter

Presentation Notes

The most obvious function (from looking at its morphology) is light absorption. For a long time this function was believed to be the only one by the RPE. Now we know of a great many other functions of the RPE. Blood retina barrier – mediated by tight junctions. Epithelial transport – both direction, blood to retina – glucose, retinal, fatty acids. Retina to blood -- Mainly water, ions and metabolic end products. Spatial buffering of ions for neural signal transduction of visual information. Metabolism of vitamin A. Secretion of PEDF (which has angiostatic properties) on the retina side and VEGF on the choroid side for regulation of vasculogenesis

as a Potency Assay for a Relevant Physiological Function of hESC-RPE

Phagocytosis

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Phagocytosis by RPE

particle

from wikipedia.org

binding of OS

engulfment, formation of phagosomes

fusion of phagosomes with lysosomes

degradation of OS

recycling

excretion

DHAVitamin A

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docosahexaenoic acid (DHA) – component of the retinal membrane. It is also important that the transport goes form apical to basal, with excretion under the RPE, not into the subretinal space

Phagocytosis: Heavy Work Load and High Pace

From B.M. Kevany and K.Palczewski Physiology (Bethesda). 2010 February ; 25(1): 8–15

rod cone

RPE cells apical surface microvilliEach RPE cell serves approximately 25-30 photoreceptor cells, each of which sheds ~7% of its outer segment mass per day

Each RPE cell must completely dispose of 25,000 to 30,000 OS disksevery day

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this high pace heavy work needs to be perfectly orchestrated

1st Generation Potency Assay: Phagocytosis of Latex Beads by Electron Microscopy

DBlatex beads

melanosome

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not QC friendly, artificial particles – good luck digesting them. This assay shows the general ability of hESC-RPE to bind and engulf particles – but what’s next?

Current Phagocytosis Assay: Quantitative pHrodo-Bioparticle Uptake

37oC phagocytosis

4oC controlRPE phagocytosis with pHrodo

bioparticles

pHrodo fluorescence is pH-sensitive

control

neutral acidic

4oC37oC

bioparticles only

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we developed another assay using fluorescently labeled bioparticles, fragments of bacterial wall, labeled with pH-sensitive dye. this dye becomes highly fluorescent in acidic environment, so it allows to visualize only internalized particles after they fuse with lysosomes. This is our current assay, and we can see that a large population of RPE shows a shift in fluorescent vs. 4oC control. It is not 100% on this picture, and we think it is because there could be a different rate of phagocytosis in more and less differentiated cells

pHrodo Bioparticles Demonstrate the Completion of Several Phagocytosis Steps

particlebinding of OS

engulfment, formation of phagosomes

fusion of phagosomeswith lysosomes

degradation of OS

recycling

excretion

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Presenter

Presentation Notes

Phagocytosis with bioparticles confirms that RPE cells are capable of at least, the first several steps of phagocytosis, and it looks like lysosomal pH is acidic

OS Phagocytis clearance: Recognition and Binding

αVβ5

FAK

photoreceptor OS

CD36MerTK

MFG-8

Gas6

RPE apical surface

OS phagosome

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OS Recognition and Binding Defects

αVβ5

CD36MerTK

MFG-8

Gas6

RPE apical surface

debris in subretinal space photoreceptor damage

XFAK

photoreceptor OS

XX

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OS Phagocytis clearance: Degradation

OS phagosome

RPE apical surface

RPE basal surface

OS phagolysosome

lysosome

lysosomal pHcathepsin Dprotease activity

Myosin VIIa

excretion26

OS Degradation Defects

OS phagosome

RPE apical surface

RPE basal surface

OS phagolysosome

lysosome

lysosomal pHcathepsin Dprotease activity

Myosin VIIa

drusen

X

XX

lipofuscin incomplete OS digestionlipofuscin accumulationdrusen formationretinal degeneration

X

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Vision Diseases Related to Phagocytosis Defects

retinal degeneration Retinitis pigmentosaStargardt’s disease Age Related Macular Degeneration

Loss of the synchronized phagocytosis and diurnal cycle

Disruption of OS internalization

Disruption of phagosometrafficking to the basal surface

Impaired degradation of OS and lipofuscin accumulation

Accumulation of undigested phagosomesand lipofuscin

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with pHrodo OSpHrodo-OS

Phagocytosis Assay Using Bovine Photoreceptor OS –The Next Generation

control4oC

37oC

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Presenter

Presentation Notes

OS labled with FITC have been a gold standard, and we started developing this assay, so we could compare the behavior of our cells with the literature data. It will allow to get more RPE-specific readings, such as OS binding and engulfment (avb5, MFG-8, MerTK, Myopsin II and VIIa, cathepsin D The limitations are FITC is also pH-dependent, but in the opposite way, so the double peak probably reflects a drop in fluorescence when the phagosomes fuse with lysosomes, but we can detect other statges – binding, early phagosomes ABCA4 mutation -- accumulation of all-trans retinal and N-retinylidene-PE on the OS membrane leading to lipofuscin accumulation Stargardt’s disease The further development of this assay will integrate the best of both – OS labeled with pHrodo: we can visualize the mostly phagolysosomes or mostly non-phagolysosomes

RPE cells continue to mature after injection

Sclera

photoreceptorcells

choroid

RPE RPE

Sclera

photoreceptorcells

choroid

RPE

Mouse retina 9 months after RPE transplantation

anti-human mitochondria anti-human bestrophin merged

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Presenter

Presentation Notes

When hESC-RPE cells were injected in mouse eyes, they continued to mature in vivo

Vision Rescue in RCS Rats

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Apreservation of photoreceptor ONL

area with loss of ONL

anti-human nuclei

anti-human nuclei

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Presentation Notes

Moreover, the histology showed preservation of the photoreceptor outer nuclei layer where human RPE cells engrafted

Vision Improved and Sustained At 1 Year

Persistent signal of efficacy in treated eyes & lack of improvement in untreated eyes

SMD

• *1 subject excluded due to cataract formation• *1 subject excluded due to <6 months follow-up

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Days After Transplant

8 Subjects with 12 Months Follow-up*

Treated Eye Untreated Eye

*5 subjects excluded due to cataract formation; 1 subject excluded due to <6 months follow-up

AMD

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Human Clinical Trials Show Vision Improvement

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Last year the results of two clinical Phase1/2 studies (RPE transplantation for AMD and Stargardt’s disease) were published in Lancet, and these results showed that there was a significant vision improvement

AMD: BCVA Improved and Sustained At 1 Year


• *1 subject was excluded due to cataract formation

• *Month 6 BCVA result was carried forward to Month 12 for 2 subjects

December 2014Lancet publication: May 2014


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Difference Between Eyes

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SMD: BCVA Improved and Sustained At 1 Year


December 2014May 2014 - The Lancet publication

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CONCLUSIONS AND FURTHER DEVELOPMENT

1. RPE phagocytosis of photoreceptor outer segments (OS) is a physiologically relevant potency assay; QC friendly, relatively simple, and quantitative.

2. hESC-RPE are capable of OS phagocytosis and show normal progression through the stage of phagolysosome formation.

3. Mechanism of Action can be addressed by examining specific steps in the cascade of phagocytic events including: OS binding processing, and export.

1.How does the stage of maturity/pigmentation affect the phagocytosis rate? 2.Which rate of phagocytosis is physiologically appropriate? 3. Can we set quantitative parameters?4. What is the OS phagocytosis capacity of hESC-RPE?5. is there OS clearance and lipofuscin accumulation in cultured RPE?6. How to correlate the in vitro data with anticipated in vivo performance of these cells?

CONCLUSIONS

OPEN QUESTIONS

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Presenter

Presentation Notes

during the assay development we learned a few thigns and we still have open questions

Aknowledgements

Ocata Therapeutics

Robert LanzaEddy AngladeRoger GayEd Mickunas

Julie CarsonTong LiRutika PradhanDeb PeakBryan PadovanoJen ShepardJudson Ratliff Lucy VilnerChris MalcuitLinda Lemieux

Casey Eye Institute, Oregon Health and Science University/Cedars-Sinai Medical Center

Ray LundShaomei WangBin LuSergej GirmanToby HolmesYves Sauvé

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Molecular Markers in RPE Transdifferentiation and Re-Differentiation

βIII tubulin

βIII tubulin HMCPax6 merged

mergedphasePax6dividing cells – early after passaging

mature culture

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Presenter

Presentation Notes

The top panels shows early RPE cultures with dividing cells – they are flat, elongated, no pigmentation, and neural and retinal progenitor markers beta tub II and Pax6 are in every cells. Several weeks later, after the confluence and reversal to original cobblestone pigmented morphology, these markers of RPE progenitor stage are lost from the majority of the polulation and only remain in small islets of still immature cells.

Function-Related Molecular Markers of RPEMorphology and pigmentation

up-regulation of RPE genes and down-regulation of hESC genes

best Pax6 MITFRPE65

Sox-2Oct-4NANOG

bestrophin – late marker, transport

Polarity/tight junctions: ZO-1

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Presenter

Presentation Notes

morphology evaluation – do all cell exhibit epithelial morphology, forming a cobblestone monolayer? Any senescent cells? Are they pigmented? Tight junctions and polarization – Zo-1 immunostaining. Bestrophin, a late RPE marker involved in transport – specific organization pattern. Pax6, the marker of immature RPE is still present in most cells because they are not fully mature. Other RPE markers such as MITF, RPE65 (visual cycle) are upregulated, and hESC markers and absent

Key signature proteins for mature functional RPE.

Function Protein Distribution Testlight absorption melanin melanosomes Morphology, absorbance

visual cycle STRA6RPE65

basolateral membranecytoplasmic

Q-PCR, enzyme activity, immunostaining

barrier claudin 19 ZO-1

apical tight junctional complex

transepithelial resistance,immunostaining

transport bestrophin 1MCT3

basolateral Transwell assay,immunostaining

metabolism ubiquinol-cytochrome c reductase core protein II

mitochondria Q-PCR, enzyme activity, immunostaining

phagocytosis MERTK αvβ5 integrin

apical membrane light and Electron Microscopy, FACS

melanogenesis tyrosinase melanosome light and Electron Microscopy

secreted PEDF extracellular, apical ELISA, Western blot

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Presenter

Presentation Notes

The presence of certain molecular markers and their localization points to the ability of the cells to perform specific functions, for instance (melanin, RPE65, Zo-1, bestrophin, PEDF) – easily tested by Q-PCR+IF+ELISA. But the most important RPE function, phagocytosis, is very complex, can be disrupted in multiple spots, so we chose it for the development a potency assay.

Preservation of Vision in Elovl4 Mouse

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Vision Rescue in RCS Rats – Functional Tests Luminance threshold

hESC-RPE, 100K untreated eye

- - - - - - - - - -- 1.9 1.7 2.0 - - 4.0 4.0 3.9 4.1

1.4 1.2 1.0 1.6 - - 3.9 3.5 3.7 3.81.6 1.1 0.8 0.9 - - 3.6 2.3 3.2 3.41.3 0.9 0.7 1.0 - - 3.0 3.1 2.9 3.3

- - - - - - - - - -- 2.6 2.4 2.5 - - NR NR NR -

2.1 1.9 2.1 1.6 - - NR NR NR NR2.4 1.6 1.4 1.9 - - 4.1 4.4 4.6 NR2.1 1.3 1.0 1.2 - - 4.6 4.7 4.8 NR

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age 187days

age 98 Days

functional sensitivity to light across the visual field of the eye. The topographical map depicts the luminance threshold responses(measured in log units) relative to background illumination

<2.0 log units >2.3 log units

<2.6 log units >4.6 0 log units

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Presenter

Presentation Notes

and in RCS rats, which normally become blind at the age of 3 months, these cell preserved vision for over 200 days. Luminance threshold – how much light it takes to induce response in a particular region of the brain, different regions can be mapped Data are point-by-point comparisons of % of collicular area showing thresholds equal to or greater than designated threshold.

Vision Improved and Sustained At 1 Year


SMD


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