RPE Human

What is the Retinal Pigment Epithelium (RPE) and what are its primary functions in human vision?

The RPE is a monolayer of cells located between the neural retina and choroidal blood supply that is critically important for photoreceptor function. It forms the outer blood-retinal barrier and supports photoreceptor health through multiple mechanisms. During light/dark transitions, the RPE undergoes physiological changes that maintain the chemical composition and volume of extracellular spaces separating the apical membrane/photoreceptor and basolateral/Bruch membrane interfaces . The RPE performs several key functions including:

• Regulating nutrient and waste transport between photoreceptors and choroid

• Participating in the visual cycle

• Phagocytosing shed photoreceptor outer segments

• Secreting growth factors and cytokines in a polarized manner

• Absorbing scattered light to improve visual acuity

Damage to the RPE with aging and disease can lead to vision loss, as seen in age-related macular degeneration (AMD), Best's disease, retinitis pigmentosa, and other retinal degenerative diseases .

What types of human RPE models are currently used in research?

Research on human RPE utilizes several distinct models, each with specific characteristics:

• Native human RPE (nhRPE): RPE cells obtained directly from donor eyes, representing the natural state but limited by viability issues after death .

• Fetal human RPE (fhRPE): Primary cultures from fetal tissue that exhibit many functions of native RPE and generally maintain better differentiation in culture .

• Adult human RPE (ahRPE): Cultures derived from adult donors that may better represent mature tissue physiology but historically have been prone to dedifferentiation .

• RPE stem cell-derived RPE: Cultures generated from a subset of adult RPE cells that can self-renew and produce large numbers of new RPE cells .

• Stem cell-derived RPE: RPE generated from human embryonic or induced pluripotent stem cells, offering potential for patient-specific disease modeling .

Each model presents advantages and limitations for specific research questions, with recent protocols improving the preservation of native characteristics in culture systems .

What are the key methods for isolating and culturing adult human RPE while preserving native physiology?

Recent advances have improved the culture of adult human RPE with preserved native physiology. Based on the research results, successful protocols include:

• Isolation from donor tissue: Careful dissection techniques to obtain RPE from the posterior pole of human donor eyes, with special attention to the RPE stem cell population that can be activated in vitro to self-renew .

• Culture conditions that prevent dedifferentiation: Methods that reduce the likelihood of RPE permanently changing while fostering the preservation of native RPE physiology, including specific media formulations and growth factors .

• Establishing confluent monolayers: Creating conditions that allow the formation of cobblestone-like RPE monolayers that maintain polarity and functional characteristics .

• Cell expansion: The protocol described in the research allows obtaining approximately 5-6 × 10^6 RPE cells per donor globe pair, which can undergo 6-8 population doublings during passages 0 and 1, resulting in at least 6.2 × 10^7 RPE cells per donor .

This approach has successfully yielded RPE monolayer cultures even from aged donors (including nonagenarians) and patients with conditions like AMD, diabetic retinopathy, and glaucoma .

How can researchers verify that cultured human RPE maintains native characteristics?

Verifying the maintenance of native characteristics in cultured human RPE involves multiple assessment methods:

• Morphological evaluation:

  • Observation of typical cobblestone morphology

  • Electron microscopy to confirm polarized features including microvilli, tight junctions, and melanosomes

• Immunohistochemical analysis:

  • Verification of polarized expression of RPE-specific markers

  • Assessment of tight junction proteins (e.g., ZO-1)

• Electrophysiological measurements:

  • Transepithelial potential (TEP)

  • Transepithelial resistance (RT)

  • Membrane voltage responses to ion concentration changes

• Functional assessments:

  • Fluid transport capabilities

  • Polarized secretion of cytokines and growth factors

• Gene expression profiling:

  • Comparison of expression patterns with native RPE

  • Verification of RPE signature genes

The study reported mean transepithelial potential of 1.19 ± 0.24 mV (apical positive) and mean transepithelial resistance of 178.7 ± 9.9 Ω·cm² in cultured adult human RPE, which helps establish baseline values for comparison .

What are the key differences between fetal and adult human RPE culture models?

Key differences include:

• fhRPE maintains higher transepithelial resistance compared to both ahRPE and native tissue

• ahRPE shows membrane voltage and resistance properties more similar to native tissue

• fhRPE exhibits stronger responses to ionic changes

• Developmental transcription factors are more highly expressed in fhRPE

• Regulatory transcription factors MITF and OTX2 are more highly expressed in ahRPE cultures

These differences suggest that while fhRPE cultures may represent a more immature RPE state, ahRPE cultures may better reflect the mature native tissue for certain applications .

How can human RPE models be used to study retinal degenerative diseases?

Human RPE models offer valuable platforms for studying retinal degenerative diseases through multiple approaches:

• "Disease in a dish" modeling:

  • Creating in vitro models of RPE-related diseases using patient-derived cells

  • Particularly valuable for studying age-related macular degeneration (AMD), Best's disease, and retinitis pigmentosa

• Comparative studies:

  • Comparing RPE characteristics between healthy donors and diseased donors

  • Analyzing age-related changes in RPE function by comparing young and aged donors' RPE

• Drug screening platforms:

  • Using well-characterized RPE cultures to test therapeutic compounds

  • Assessing drug effects on specific RPE functions like fluid transport, barrier integrity, and cytokine secretion

• Mechanistic investigations:

  • Studying specific disease mechanisms through targeted manipulations

  • Examining how specific mutations or environmental factors affect RPE function

• Three-dimensional culture models:

  • Bioengineering complex models that incorporate RPE with retinal neurons

  • Creating more physiologically relevant systems to study disease progression and treatment

The ability to obtain RPESC-derived RPE monolayer cultures from patients with conditions like AMD, diabetic retinopathy, and glaucoma enables direct comparison between healthy and diseased states in controlled laboratory conditions .

What role does autophagy play in RPE health and disease, and how can it be studied?

Autophagy plays a critical role in RPE health and disease:

• Function in healthy RPE:

  • Autophagy is a cellular "housekeeping" process that removes damaged organelles and protein aggregates

  • It maintains RPE homeostasis by preventing accumulation of toxic byproducts from photoreceptor phagocytosis

  • Constitutive autophagy is essential for RPE function and photoreceptor support

• Role in age-related macular degeneration (AMD):

  • Changes in autophagy are implicated in AMD pathogenesis

  • Reduced autophagic capacity may contribute to lipofuscin and drusen formation

  • Autophagy dysfunction can lead to RPE degeneration and subsequent photoreceptor loss

• Research approaches:

  • Comparing autophagy markers and activity between young and aged RPE

  • Examining how autophagy changes might relate to AMD development

  • Investigating whether enhancing autophagy could reverse RPE degeneration

• Methodological considerations:

  • Using fluorescent markers to visualize autophagic flux in RPE cells

  • Employing electron microscopy to identify autophagosomes

  • Measuring expression of autophagy-related genes and proteins

  • Testing autophagy modulators as potential therapeutic agents

Current research is exploring how the process of autophagy might be exploited to reverse degeneration of the RPE, particularly in late-stage disease where other therapeutic approaches have shown limited efficacy .

What are the current approaches to bioengineering RPE tissues for transplantation and drug testing?

Current approaches to bioengineering RPE tissues include:

• Two-dimensional monolayer cultures:

  • Traditional approach using RPE cells grown on transwell membranes

  • Allows polarized growth and functional assessment

  • Limited in recapitulating the complete native environment

• Three-dimensional culture models:

  • Incorporating RPE with retinal neurons derived from human stem cells

  • Better mimics complex cellular interactions in the retina

  • Provides more physiologically relevant platforms for drug screening

• Scaffold-based approaches:

  • Using biocompatible substrates to support RPE growth and transplantation

  • Developing materials that mimic Bruch's membrane properties

  • Enhancing integration and survival of transplanted cells

• Co-culture systems:

  • Studying interactions between retinal and RPE progenitors

  • Developing superior engineered tissues through understanding developmental cues

  • Optimizing conditions that promote proper differentiation and function

• Transplantation strategies:

  • Early transplantation of RPE or retinal progenitors to slow retinal degeneration

  • Investigating whether engineered tissues can restore vision in late-stage disease

  • Examining long-term integration and function of transplanted tissues

Research shows that in mouse models, early transplantation of RPE or retinal progenitors slows retinal degeneration, but later transplantation has limited efficacy in reversing existing degeneration. Current work aims to determine whether specialized engineered tissues could overcome these limitations .

What electrophysiological parameters should be measured to assess human RPE function in vitro?

Key electrophysiological parameters for assessing human RPE function include:

Researchers should also test for the presence of specific ion channels that may be lost or gained during culture, such as L-type Ca²⁺ channels (typically lost after 9 days in culture), low-voltage gated Ca²⁺ channels, and TTX-sensitive Na⁺ channels (which may indicate transdifferentiation toward a neural phenotype) .

How should researchers approach gene expression analysis in human RPE studies?

Researchers should approach gene expression analysis in human RPE studies with these methodological considerations:

• RPE signature gene identification:

  • Focus on the unique signature set of genes (approximately 154) that distinguish RPE from other cell types

  • These are defined as genes expressed at least 10-fold higher in RPE compared to other tissues

  • Be aware that important RPE genes may be missed if they are expressed at low levels or are widely expressed in other tissues

• Comparative analysis approaches:

  • Compare expression profiles between:

    • Native RPE (nhRPE)

    • Cultured adult RPE (ahRPE)

    • Fetal RPE (fhRPE)

    • RPE cell lines (e.g., ARPE-19)

  • Consider age-related and disease-related differences

• Time-sensitive considerations:

  • Be aware that native RPE RNA is typically harvested 24-40 hours post-mortem, which may affect RNA quality

  • This delay may not accurately reflect the gene expression of healthy native RPE in vivo

  • Further research is needed to evaluate how time, procurement method, and storage affect RPE physiology

• Functional gene categories to examine:

  • Visual cycle genes

  • Transcription factors (e.g., PAX6, LHX2, SOX9, CRX, MITF, OTX2)

  • Epithelial markers and polarity genes

  • Ion channels and transporters

  • Cytokine and growth factor genes

• Advanced genomic techniques:

  • For studies requiring tens of millions of cells, techniques like chromatin immunoprecipitation sequencing may be employed

  • The described culture method can produce sufficient cell numbers (5 × 10⁸ RPE cells/donor with two passages) for such approaches

What are the best practices for evaluating polarized cytokine secretion in human RPE cultures?

Best practices for evaluating polarized cytokine secretion in human RPE cultures include:

• Experimental setup:

  • Culture RPE cells on permeable transwell inserts to allow access to both apical and basolateral compartments

  • Ensure confluence and mature tight junctions (verified by transepithelial resistance)

  • Collect conditioned media separately from apical and basolateral chambers

• Key cytokines to measure:

  • Vascular Endothelial Growth Factor (VEGF): Expect preferential basal secretion (approximately 2582 ± 146 pg/mL/day basally vs. 1548 ± 162 pg/mL/day apically)

  • Pigment Epithelium-Derived Factor (PEDF): Expect preferential apical secretion (approximately 1487 ± 280 ng/mL/day apically vs. 864 ± 132 ng/mL/day basally)

• Quantification methods:

  • Enzyme-Linked Immunosorbent Assay (ELISA) for precise quantification

  • Multiplex assays for screening multiple cytokines simultaneously

  • Western blotting for semi-quantitative analysis

• Experimental controls:

  • Include permeability controls to verify barrier integrity

  • Use polarized epithelial cells with known secretion patterns as positive controls

  • Include non-polarized cells as negative controls

• Analysis and interpretation:

  • Calculate secretion rates normalized to time and surface area

  • Compare directional ratios (apical vs. basolateral) rather than absolute values

  • Assess how polarized secretion changes with experimental manipulations

• Verification methods:

  • Confirm cytokine receptor distribution on appropriate surfaces

  • Correlate secretion patterns with gene expression data

  • Verify functional consequences of polarized secretion

The polarized secretion of these factors is critical for maintaining the health of photoreceptors (apical to RPE) and the choroidal vasculature (basal to RPE), and aberrant secretion patterns may contribute to retinal pathologies .

What defines human subjects research in the context of RPE studies?

In the context of RPE studies, defining human subjects research requires careful consideration of regulatory frameworks:

• Definition of research involving human subjects:
  An activity is considered research if it is both:

  • A systematic investigation (where data is collected and analyzed to answer a specific research question)

  • Designed to contribute to generalizable knowledge (i.e., applied to populations outside of the specific study population or designed to contribute to knowledge in your field)

• Application to RPE research:

  • Studies using primary human RPE cells obtained from donor eyes typically qualify as human subjects research

  • Research using established, de-identified cell lines may not require full IRB review

  • Studies involving patient-derived RPE cells for disease modeling would likely qualify as human subjects research

• IRB requirements:

  • If a project meets both criteria above, it requires IRB review

  • If it does not meet both criteria, it is not considered human subjects research requiring IRB review

  • Researchers should consult with their institutional IRB for specific guidance

• Special considerations for RPE research:

  • Source of tissue (cadaveric vs. living donors)

  • Consent processes for donor tissue use

  • Privacy and confidentiality concerns when linking cells to specific donor information

  • Additional protections when using RPE cells derived from vulnerable populations

Researchers should carefully evaluate whether their RPE studies constitute human subjects research under applicable regulations and seek appropriate IRB oversight before proceeding .

What ethical considerations should researchers address when obtaining human eye tissue for RPE studies?

Researchers must address several ethical considerations when obtaining human eye tissue for RPE studies:

• Informed consent:

  • Ensure proper consent processes for postmortem donation of eyes/tissues

  • Clearly communicate the research purposes in consent documents

  • Consider whether consent allows for future, unspecified research use

  • Address commercial potential and intellectual property considerations

• Privacy and confidentiality:

  • Implement procedures to protect donor identity

  • Consider whether genetic or other identifying information will be maintained

  • Establish protocols for secure data storage and limited access

• Tissue procurement timing:

  • Recognize that native human RPE is typically processed 24-40 hours after death

  • This timing affects RNA quality and may not accurately reflect in vivo RPE conditions

  • Balance research needs with respect for the deceased and family bereavement

• Equitable selection:

  • Ensure representative diversity in donor tissue

  • Consider whether certain populations are over/underrepresented

  • Balance research focusing on specific disease populations with potential biases

• Cultural and religious considerations:

  • Respect diverse cultural and religious perspectives on tissue donation

  • Accommodate specific handling requests where possible

  • Ensure dignified treatment of all donor tissues

• Return of research results:

  • Establish policies regarding incidental findings

  • Consider whether certain findings should be returned to families

  • Address potential clinical significance of research discoveries

Researchers should work closely with institutional ethics committees, eye banks, and tissue procurement organizations to ensure all ethical considerations are appropriately addressed throughout the research process.

How might advanced bioengineering approaches enhance our understanding of human RPE pathophysiology?

Advanced bioengineering approaches offer promising avenues to enhance our understanding of human RPE pathophysiology:

• Complex three-dimensional tissue models:

  • Bioengineering three-dimensional culture models incorporating RPE and retinal neurons derived from human stem cells

  • Creating models that better recapitulate the retina/RPE/choroid complex

  • Allowing investigation of intercellular interactions in a more physiologically relevant environment

• Microfluidic organ-on-chip systems:

  • Developing dynamic culture systems with controlled fluid flow

  • Mimicking the blood-retina barrier with appropriate compartmentalization

  • Allowing real-time monitoring of cellular responses to stimuli

• Patient-specific disease modeling:

  • Using induced pluripotent stem cells (iPSCs) from patients with RPE-related diseases

  • Creating "disease in a dish" models to study pathophysiology and test interventions

  • Enabling personalized medicine approaches for retinal diseases

• High-throughput screening platforms:

  • Developing automated systems for testing multiple therapeutic agents

  • Screening potential therapeutic compounds in physiologically relevant models

  • Accelerating drug discovery for RPE-related diseases

• Integration of advanced imaging and sensing technologies:

  • Incorporating real-time imaging of cellular processes

  • Monitoring metabolic activities and transport functions

  • Measuring electrical properties under various conditions

These approaches will enable more comprehensive investigation of RPE pathophysiology, potentially leading to novel therapeutic strategies for conditions like age-related macular degeneration and other retinal degenerative diseases .

What research questions remain unanswered regarding the interaction between aging and RPE dysfunction?

Several critical research questions remain unanswered regarding the interaction between aging and RPE dysfunction:

• Molecular mechanisms of age-related RPE changes:

  • How do epigenetic modifications change with age in RPE cells?

  • What triggers the transition from normal aging to pathological states?

  • How do transcription factors like MITF and OTX2 regulation change with aging?

• Autophagy dynamics in aging RPE:

  • How does autophagic capacity change with age in human RPE?

  • Can enhancement of autophagy reverse age-related RPE dysfunction?

  • What is the relationship between autophagy changes and drusen formation?

• Mitochondrial function and oxidative stress:

  • How do mitochondrial dynamics change in aging RPE?

  • What is the relationship between oxidative damage accumulation and RPE dysfunction?

  • Can targeting mitochondrial function prevent age-related RPE degeneration?

• Extracellular matrix remodeling with age:

  • How do changes in Bruch's membrane composition affect RPE function?

  • What role do RPE-secreted matrix metalloproteinases play in age-related changes?

  • Can interventions targeting ECM remodeling preserve RPE function?

• Comparative biology of RPE aging:

  • Why do some individuals maintain healthy RPE into advanced age while others develop dysfunction?

  • What protective factors might prevent age-related RPE pathology?

  • How do environmental factors interact with genetic predisposition in RPE aging?

• Translational research challenges:

  • Can ahRPE cultures from elderly donors accurately model age-related changes?

  • What interventions might effectively reverse established age-related RPE dysfunction?

  • How can findings from culture models be translated to effective clinical interventions?

The ability to culture RPE from aged donors, including nonagenarians, offers unprecedented opportunities to study these questions in relevant human models, potentially leading to interventions that target the aging component of RPE-related diseases .

How might gene editing technologies advance RPE disease modeling and therapeutic development?

Gene editing technologies offer transformative potential for RPE disease modeling and therapeutic development:

• Precision disease modeling:

  • Creating isogenic cell lines that differ only in disease-causing mutations

  • Introducing specific genetic variants associated with RPE diseases like AMD

  • Enabling controlled studies of genetic contributions to disease phenotypes

• Mechanistic studies of monogenic RPE diseases:

  • Using homologous recombination to create models of single-gene disorders

  • Investigating the molecular pathways disrupted by specific mutations

  • Understanding how genetic defects lead to RPE dysfunction

• Modeling complex polygenic diseases:

  • Introducing multiple genetic risk factors to model diseases like AMD

  • Studying gene-environment interactions in controlled settings

  • Creating models that reflect the genetic diversity of patient populations

• Gene correction approaches for therapy:

  • Developing ex vivo gene editing strategies for autologous RPE transplantation

  • Testing correction of disease-causing mutations before cell transplantation

  • Evaluating functional restoration after genetic correction

• In vivo gene editing for RPE diseases:

  • Developing delivery systems targeting RPE cells in the retina

  • Testing direct correction of mutations in the native RPE layer

  • Evaluating safety and efficacy of in vivo approaches

• Challenges and considerations:

  • Off-target effects of gene editing technologies

  • Delivery methods for reaching the RPE effectively

  • Ethical implications of germline vs. somatic editing approaches

While fetal human RPE paired with gene editing has already advanced understanding of RPE physiology, the development of adult human RPE culture methods provides additional opportunities to study age-related and multifactorial diseases like AMD that have both genetic and epigenetic components .