DEV3032 L16 PDF - Epidermal Research & Regeneration Lecture Notes

🥽 Lecture 16: Epidermal research and regeneration Lecture 1: Define Epithelial Stem Cells (ESC) Epithelial/Epidermal Stem Cells Definition: Epidermal stem cells are undifferentiated cells with the...

🥽 Lecture 16: Epidermal research and regeneration Lecture 1: Define Epithelial Stem Cells (ESC) Epithelial/Epidermal Stem Cells Definition: Epidermal stem cells are undifferentiated cells with the ability to self-renew and differentiate into specialized cell types within the epidermis. ESCs location in different epithelial tissues: Limbus Area of the Cornea Crypts in the Intestine Terminal Ends of Mammary Ducts Bulge Region of Hair Follicles Interfollicular Stem Cells in the Skin Lecture 16: Epidermal research and regeneration 1  Issues with identifying ESCs: Lack of molecular markers exclusively expressed by stem cells. Reliance on multiple phenotypes and criteria instead of a single marker to identify stem cells. Epithelial stem cells characteristics Slow Cycling in Their Native Niche: Lecture 16: Epidermal research and regeneration 2  Remain slow cycling under normal conditions. High Proliferative Capacity Upon Activation: Become highly proliferative when: Activated during wound healing. Removed from their native niche. Undifferentiated Nature: Remain undifferentiated in their native state. Pigmentation Characteristics: Limbus and Interfollicular Stem Cells: Pigmented. Follicular Epidermal Stem Cells (Bulge Area): Do not express melanin (not pigmented). Not all epithelial stem cells are pigmented. Skin Architecture The three major layers - epidermis, dermis, endodermis: Lecture 16: Epidermal research and regeneration 3  Epidermis: A stratified, differentiated layer. As cells differentiate, they move upwards through distinct layers: Spinous Layer Granular Layer Terminally Differentiated Stratum Corneum Includes appendages such as: Hair follicles Sweat glands Dermis: Separated from the epidermis by the basement membrane. Rich in extracellular matrix. Composed of two layers: Upper Papillary Layer Lower Reticular Layer Contains various cell types and structures: Fibroblasts Sensory nerves Arrector pili muscles (cause goosebumps) Blood vessels Adipocytes Immune cells Subcutaneous Tissue (Fat Layer): Shown in yellow. Considered a separate layer with distinct structure and function, although not marked as a separate layer in the diagram Lecture 16: Epidermal research and regeneration 4  Green's Method: Key Points 1. Developed in the 1970s by Professor Green and Dr. Rainwald. 2. Discovery: Basal epidermal cells expand in culture with murine fibroblasts as feeder cells. 3. Technique: Combination of feeder cells and specialized media supports epidermal cell growth. 4. Challenges: At the time, stem cell markers and expansion methods were unknown. 5. First Success: Cultured epidermis healed a patient’s wounds in the 1980s, revolutionizing burn treatment. 6. Significance: Pioneered epithelial cell therapy, laying the foundation for advancements in regenerative medicine. Epithelial Cell Culture - The Green’s Method - Three types of epidermal colonies, categorised based on size and proliferative capacity when cultured on fibroblasts: Lecture 16: Epidermal research and regeneration 5  1. Holoclones Largest colonies. Highest proliferative capacity. Contained 95% terminal cells upon replating. Rarely formed new colonies when replated, and cells divided fewer than 15 times in culture. Lecture 16: Epidermal research and regeneration 6  Terminal cells - final stage of cell development, cells become specialised Cell Surface Markers on Basal Adult Keratinocytes Keratinocytes - the most prominent cell within the epidermis Holoclones from interfollicular stem cells (SC) Meroclones from transient amplifying cells (TA) Paraclones from early differentiating cells (ED) Separation of Epidermal Cell Populations using FACs FACS (Fluorescence-Activated Cell Sorting): Technique: Labels cell surface antigens with fluorescent antibodies. Passes cell suspension through a narrow stream to measure fluorescence on individual cells. Each dot on the graph represents a single cell. Dot Plot Analysis: X-Axis: Alpha-6 integrin expression. Y-Axis: CD71 expression. FACS enables researchers to: Precisely isolate and study specific subpopulations of keratinocytes. Uncover functional differences between these populations. Advance therapies for burns, wounds, and other skin disorders by targeting or utilizing the correct cell types. Lecture 16: Epidermal research and regeneration 7  Subpopulations of Basal Keratinocytes 1. Stem Cells (SC): High alpha-6 integrin expression. Low CD71 expression. High clonogenic capacity. 2. Transient Amplifying Cells (TA): High alpha-6 integrin expression. Also express CD71. Progenitor cells, dividing and differentiating further. 3. Differentiating Cells (Early Differentiating) (ED): Do not express alpha-6 integrin. Limited proliferative capacity, involved in terminal differentiation. Hierarchical Model of Skin Regeneration Lecture 16: Epidermal research and regeneration 8  Colony Formation Assay: Supports the relationship between alpha-6 integrin expression and clonogenicity. Location in Epidermis: Interfollicular stem cells reside in the basal layer of the epidermis. Stem cells divide asymmetrically, producing: A new stem cell (to maintain the pool). A transient amplifying cell (to continue dividing and differentiating). Differentiation Process 1. Transient amplifying cells divide and begin to differentiate. 2. As differentiation progresses: Cells stratify and move upward through the epidermal layers. Fully differentiated keratinocytes are shed from the surface. Skin Layers and Marker Expression Collagen IV & Laminin 511 These proteins are expressed at the basement membrane, separating the epidermis from the dermis. Lecture 16: Epidermal research and regeneration 9  K5 & K14 Expressed in basal keratinocytes Location: Basal layer Function: Proliferate and self-renew Differentiation: Undifferentiated, stem-like cells Keratin 10 & Involucrin Expressed in super basal differentiating keratinocytes Location: Above the basal layer (spinous and granular layers) Function: Start differentiating and producing skin proteins Differentiation: Differentiating, transitioning to terminal keratinocytes Ki-67 Marks dividing keratinocytes, particularly visible in the basal layer as brown staining. Lecture 16: Epidermal research and regeneration 10  QUESTION: What is a common characteristic of epithelial stem cells in hair follicles, epidermis and cornea? highly proliferative abundant quiescent pigmented Explanation: The common characteristic of epithelial stem cells in hair follicles, epidermis, and cornea is that they are quiescent (inactive) and rare under normal conditions. Although they have a high proliferative capacity, they only start to proliferate when under stress, such as in response to a wound or when removed from their niche. Interfollicular stem cells and limbal stem cells contain melanin pigments, but follicular stem cells do not. Lecture 2: Apply ESC therapies to treat wounds Potential uses of cultured epithelial stem cells in regenerative medicine CEA - cultured epidermal/epithelial autograph: a tissue grown from one's own skin cells for use in placing on the person's own body Current application of cultured epidermal stem cells Animal studies: Lecture 16: Epidermal research and regeneration 11  Regeneration of urethra Limbal stem cell deficiency Regeneration of other epithelia Clinical studies: Stable vitiligo Epidermolysis Bullosa Clinical use: Chronic wounds Burns Cultured Epithelial Autografts (CEA): The Green’s Method Ie. a tissue grown from one's own skin cells for use in placing on the person's own body INSTEAD of the standard split skin grafting (a surgical procedure that involves removing a thin layer of skin from a healthy part of the body and using it to cover damaged or missing skin) Manufacturing Process of CEA 1. Biopsy Collection: Small biopsy (~2x2 cm) taken from burns patients. Preferred sites: armpits or groin. 2. Enzymatic Digestion: Basal human adult keratinocytes (HAKs) isolated. High expression of keratin 14 observed (marker for stem and progenitor epidermal cells). 3. Cell Seeding and Expansion: HAKs seeded on mitotically inactive feeder cells. Irradiated (to stop cell division) 3T3-J2 strain (feeder cells) are used 3T3 cells: A line of fibroblast cells originally derived from mouse embryos. Lecture 16: Epidermal research and regeneration 12  J2 strain: A specific sub-strain of 3T3 cells, widely used in cell culture because of its reliability in supporting the growth of other cells, such as keratinocytes. They nurture the keratinocytes without dividing themselves, enabling efficient growth and expansion of keratinocyte colonies. Seeding refers to placing the isolated keratinocytes on top of the feeder cell layer. Day 3–4: Keratinocyte colonies appear. Day 8: Cells reach 70–80% confluence (Passage 1). Passage 2: Reseeding expands cells further; confluence achieved by Day 7. Total duration: 20–40 days. Cultured Epithelial Autografts (CEA) on Fibrin Gel Use of Fibrin Gel as a Carrier for CEA: Lecture 16: Epidermal research and regeneration 13  Fibrin gel created by combining thrombin and fibrinogen to promote clot formation. Mimics natural blood clot formation: Thrombin: Activates platelet aggregation. Fibrinogen: Forms fibrin clots. Basal keratinocytes seeded on fibrin gel: Expanded keratinocytes attach and form colonies. Confluent CEA ready for transplantation. Advantages of CEA 1. Reduced Donor Site Requirement: Minimizes need for split skin grafting. 2. Permanent Wound Closure: Take rate reported up to 85%. FOR MY OWN UNDERSTANDING Seeding and Growth Process Basal keratinocytes (the specific type of skin cells, isolated from the biopsy) are seeded onto the fibrin gel. The fibrin gel acts as a supportive structure or "carrier" for the cells. The cells grow and multiply on the fibrin gel, eventually forming a confluent sheet (a continuous layer of cells). Lecture 16: Epidermal research and regeneration 14  What is CEA? CEA (Cultured Epithelial Autografts) refers to the entire cell sheet that has been cultured. This includes: The keratinocyte cell layer (the actual living cells). The fibrin gel carrier (used in the modified method). When the cells have grown to confluence (a full, continuous layer), this cell sheet (CEA) is ready to be transplanted onto the wound or burn site. Visualizing the Process Think of the fibrin gel as the "base" (like a scaffold), and the keratinocytes as seeds that are "planted" on it. Over time, the "seeds" grow into a dense, living layer that can function as skin, which is then transplanted to cover and heal the wound. So, in summary, CEA is the combination of the cultured keratinocytes (cells) and the fibrin gel (carrier), which together form the transplantable product. Acellular Skin Substitutes (Scaffolds): Commercially available or in Clinical Trial Cells often require support from an extracellular matrix (ECM)or scaffold: Materials designed to mimic the structural and functional properties of the dermal layer of the skin. They are acellular, meaning they do not contain living cells but provide a supportive framework for cell attachment and tissue regeneration. They are stripped of cellular components to reduce the risk of immune rejection and inflammation. Purpose of the ECM/Scaffold: Stabilise the cells. Lecture 16: Epidermal research and regeneration 15  Keep cells alive. Provide the correct architecture for skin tissue. Enhance functionality. Examples: Integra, Alloderm, Hyaluromatrix, BiTemporising matrix Temporary Cellular Skin Substitutes: Commercially available or in Clinical Trial Certain cell therapy products use allogeneic (from a donor of the same species) or xenogeneic (from a different species) cells. These products function primarily as dressings because they cannot integrate into the wound → not for engraftment, but instead they enhance natural wound healing Key Characteristics Primary Role: Serve as temporary wound coverings. Limitation: Unable to integrate into the wound tissue. Examples: Cadaver allograft, porcine xenograft, Apligraf, Stratagraf, Dermagraf Lecture 16: Epidermal research and regeneration 16  Permanent Cellular Skin Substitutes: Commercially available or in Clinical Trial Composite Skin: Combines autologous fibroblasts and keratinocytes (from the patient’s own cells). Designed for permanent wound closure. Composite Skin Structure: Composed of at least two layers: 1) Dermis 2) Epidermis Production: More challenging to create compared to epidermis-only products. Advantages: Provides significantly greater functionality than epidermis alone. Examples: Epicel, Recell, Reconstructed skin, Engineered skin substitute, Cultured composite skin (CCS) Lecture 16: Epidermal research and regeneration 17  Engineered Skin Substitute (ESS)- In vitro Structure of ESS Matrix composition: collagen fibres with pores to allow cells to invade and integrate Double-layer design: Dermis: Formed by seeding fibroblasts into the collagen matrix. Fibroblasts populate the matrix to create a functional dermal layer. Epidermis: Formed by seeding keratinocytes and melanocytes on top of the dermis. In vitro development → ESS is first developed and optimised in a lab Lecture 16: Epidermal research and regeneration 18  Engineered Skin Substitute (ESS) - In vivo In vivo testing - tested in animals to assess safety & functionality - necessary step before progressing to clinical trials for patient treatments. Human-derived, making it a xenograft when used on mice. Requires immune-compromised mice (thymic mice) to prevent rejection. Procedure Steps 1. Preparation of Wound Bed: Illustrated in Photograph A. 2. Application of the Graft: ESS is placed on the prepared wound bed. 3. Dressing the Graft: The graft is secured with a dressing to hold it in place. Results Outcome: ESS grafted reasonably well in thymic mice. Next Step: Based on the success, ESS progressed to clinical trials. Lecture 16: Epidermal research and regeneration 19  Engineered Skin Substitute (ESS)- Clinical Data ESS Application on Patient: The images demonstrate the surgical application of ESS on a young burn patient. Postoperative Images: Postoperative images show the wound after the graft. The tissue displayed: A mature epidermis. A well-vascularized dermis. Wound Closure: ESS successfully closed the wound, which was the main goal of the trial. Discoloration: The discoloration (pale color) observed is due to the lack of melanocytes in the ESS at that time. Melanocytes were not included in the skin substitute, leading to a lack of pigmentation. Lecture 16: Epidermal research and regeneration 20  Clinical Trial Results of ESS on Children with Massive Burns PLOT A: Ratio of Closed Wound Area to Donor Skin Although the total body surface area closed by ESS was lower compared to autografts, the use of much less donor skin was a significant advantage. Ie. ESS has a much higher ratio compares to autograft (AG) → a high ratio of closed wound area to donor skin indicates that a small amount of donor skin is able to cover a large area of the wound. PLOT B: Mortality Percentage ESS Mortality Rate: 6.25% (1 out of 16 patients). National Burn Repository Mortality Rate (2012): 30%. Key Finding: ESS therapy significantly reduced mortality compared to the national average in 2012. PLOT C: Percentage of Engraftment ESS Engraftment Rate: Just over 80%. Autograft Engraftment Rate: 100%. Key Finding: ESS showed a strong engraftment rate, though autografts had a slightly higher success rate. Lecture 16: Epidermal research and regeneration 21  Application of Human plasma in Skin Tissue Engineering: A novel method The issue: Collagen matrix (eg. Integra) serves as a foundation of the dermal layer - they are highly porous and not suitable for double-layer skin/composite skin construction Though the porosity makes an ideal scaffold for fibroblasts to populate. HOWEVER - having composite skin (a two-layer skin substitute with both epidermis and dermis) offers significant advantages over just a single layer of skin: More natural structure Improved wound healing Better integration Enhanced durablity Lecture 16: Epidermal research and regeneration 22  How to fix the issue → the addition of plasma clot (ie. plasma clotting technique) The addition of plasma clot to Integra helps to stabilize and modify the dermal structure, making it more conducive to forming a stable composite skin. Technique for Composite Skin Construction Plasma Clotting: Plasma clotting is applied to porous matrices before keratinocyte seeding. Objective: Create a stable, functional matrix for composite skin. Cell Expansion: Fibroblasts and keratinocytes are isolated and expanded from the patient. Matrix Testing: The matrix used for testing is Integra, a collagen-based scaffold. Human Skin Equivalent (HSE): HSE is cultured in media containing protonin to inhibit clot degradation. Plasma Clot Formation Part A: Clot Failure Condition: Plasma and calcium chloride alone were insufficient to form a plasma clot. Result: The matrix could not form a stable clot without the presence of fibroblasts. Part B: Clot Formation with Fibroblasts Condition: Lecture 16: Epidermal research and regeneration 23  A visible clot forms when fibroblasts are cultured directly on the matrix surface and incubated with human plasma and calcium chloride. Result: The clot was stable with the presence of fibroblasts. Part C: Unstable Clot Condition: When plasma is added to fibroblasts in Integra, the clot forms but collapses when exposed to shear force. Result: Clot is unstable and not ideal for further use in composite skin. Part D: Stable Clot Formation Condition: Calcium chloride added to plasma soaking Integra populated with fibroblasts. Result: Stable clot formation achieved, making it more suitable for skin construction. CONCLUSION: Both fibroblasts and calcium are required for stable plasma clot formation, which is crucial for building composite skin. The technique developed ensures stability and functionality of the composite skin for clinical application. Lecture 16: Epidermal research and regeneration 24  Human Skin Equivalent using Integra and Plasma Histological Analysis of Human Skin Equivalent (HSE) Expression of Specific Markers: Histological analysis of the human skin equivalent (HSE) shows the expression of specific markers that indicate the success of engineered skin. Comparison to Native Skin: The top panel shows HSE, and the bottom panel shows native skin. Three layers of the epidermis can be identified in the HSE: Stratum basale (basal layer) Stratum spinosum (spiny layer) Stratum corneum (outermost layer) Proliferative Cells: In the HSE, Ki67 (a marker of cell proliferation) can be seen in the basal layer, indicating active cell division and proliferation. Effect of Aprotinin and Calcium on Clot Formation: With Aprotinin and Calcium: The combination of clotted human plasma and aprotinin creates a smooth surface for epidermization. Lecture 16: Epidermal research and regeneration 25  The HSE formed in this condition closely resembles native skin in structure and function. Without Aprotinin (Degraded Clot): When aprotinin is not added, the plasma clot degrades prematurely, leading to the keratinocytes invading the entire matrix and differentiating in clusters, rather than forming a structured epidermis. Calcium and Aprotinin Importance: Both calcium and aprotinin are essential for the formation of a stable plasma clot, which is crucial for proper epidermal development. Basement Membrane Formation in the Novel HSE Collagen IV and Laminin 5.11: These are markers of the basement membrane, which plays a critical role in the dermal-epidermal junction. The continuous red line marks the basement membrane, which is essential for skin integrity and keeps the dermis and epidermis connected. Importance of Basement Membrane: The basement membrane is crucial for the grafting and integration of the epidermis, as it helps hold the epidermis and dermis together. Without a functional basement membrane, epidermal grafting cannot occur effectively. Lecture 16: Epidermal research and regeneration 26  Animal Study Results: Testing of HSE in Atomic Mice Two Scaffolds Tested: Integra HSE: Collagen-based matrix. BTM HSE: Fully synthetic matrix. Results: BTM Supported Human Keratinocytes Better: BTM supported human keratinocytes significantly higher than Integra, as shown by the presence of grafted human epidermis. Vascularization: CD31 (a marker for endothelial cells) was used to measure vascularization of the grafts. The results showed promising vascularization. Next Steps: Phase One Clinical Trial: The next step is to test the Human Skin Equivalent (HSE) in a Phase One clinical trial for wound treatment. Lecture 16: Epidermal research and regeneration 27  Challenges in Creating Fully Skin-Like Substitutes Current Progress in Composite Skin: Composite skin has shown promise in permanent wound closure. Barrier function, epidermal layer, and dermal-epidermal junction have been successfully reproduced. Missing Features in Engineered Skin: Hair follicles (absent in current substitutes). Sebaceous glands (not yet present). Sweat glands (currently not included). Melanocytes (can be added for pigmentation). Neurons (not present in current engineered skin). Fat (currently lacking in engineered skin). Conclusion: While fully skin-like substitutes are not yet a reality, essential functions like the barrier function have been successfully reproduced. The development of fully functional skin substitutes is still a work in progress. Lecture 16: Epidermal research and regeneration 28  QUESTION: Name one type of wound in which epithelial cell therapy has been trialed and shown to be beneficial. Surgical wound Burns Diabetic foot ulcer Pressure Wounds QUESTION: What are the components of Engineered Skin Substitute for burns treatment? Answer: Autologous Fibroblasts, autologous keratinocytes and Collagen-GAG scaffold. Lecture 3: Apply combined ESC and gene therapy to treat Epidermolysis Bullosa Junctional Epidermolysis Bullosa (JEB) Overview: JEB is a recessive genetic disorder that affects children. Life Expectancy: The average life expectancy is only 20 to 30 years. High Mortality: Over 40% of children with JEB die before adolescence. Nickname: Babies born with JEB are often called butterfly babies due to their very fragile skin. Even a simple hug can cause blisters. Genetic Cause: JEB is caused by a mutation in a single gene, affecting proteins that are essential for skin integrity at the dermal-epidermal junction. The mutated genes include: Laminin 332 - (LAMB3 specifically, which encode the laminin 332 protein) Integrin alpha 6, beta 4 Collagen 17 (XVII) Lecture 16: Epidermal research and regeneration 29  These proteins are part of the protein complex in the basement membrane, which helps basal keratinocytes attach to the basement membrane, crucial for skin integrity. Potential for Gene Therapy: Since JEB is caused by a single gene mutation, gene therapy is a feasible approach for correction. Treatment of Junctional Epidermolysis Bullosa Patient: 7-year-old boy with a G2A mutation in laminin 332 (LAMB3) Procedure Overview: 1. Skin Biopsy: A small skin biopsy was taken from the patient. 2. Isolation and Expansion of Basal Keratinocytes: Basal keratinocytes were isolated from the biopsy. These keratinocytes were expanded on fetal feeder cells. 3. Gene Correction: LAMB3 gene correction was carried out using a retrovirus vector. The retrovirus introduced the corrected gene, allowing the keratinocytes to express the correct laminin 332 protein. 4. Keratinocyte Expansion: Lecture 16: Epidermal research and regeneration 30  The transduced keratinocytes were expanded and grown on fibroblast gel (similar to CEA production in the lab). 5. Grafting Procedure: The corrected keratinocytes were grafted over three surgeries: First surgery: Grafting on the limbs. Second surgery: Grafting on the trunk. Third surgery: Grafting on smaller areas. 6. Postoperative Monitoring: Punch biopsies were taken at: 1 to 4 months 8 months 21 months These biopsies were used for clonal analysis and gene tracing to monitor the success of the treatment. Clonal Analysis and Gene Tracing in JEB Treatment Marking of Grafted Areas: Left leg: Marked as 4 MC (4 months post-graft) and 8 MC (8 months post-graft). Lecture 16: Epidermal research and regeneration 31  Left arm: Marked as 8 MC (8 months post-graft). Pre-graft Cultures (PG) and Post-graft Samples: PG refers to pre-graft cultures. 4 MC and 8 MC refer to post-graft samples taken 4 months and 8 months after the graft. Analysis Results: 1. Next Generation Sequencing (NGS): Pre-graft cultures (PG): Correct Laminin-311 (LAMB3) gene was integrated in a high number of transduced keratinocytes in pre-graft cultures. Post-graft (4-8 months): The number of integrated gene copies decreased significantly in grafted keratinocytes compared to pre-graft cultures. 1. Explanation for Decrease in Gene Integration: High integration levels in the pre-graft cultures may lead to instability of the transduced cells. Only cells with lower integration survived and contributed to the grafts, resulting in a lower number of integrated genes in Lecture 16: Epidermal research and regeneration 32  the grafts after 4 to 8 months. 2. Gene Integration Locations: 40% of integration sites were located in introns. 5% of integration sites were in exons. This suggests that the integration of the gene into introns is less likely to interfere with gene translation, making it less disruptive to the overall genome. Conclusion of study: The results show that gene integration occurred successfully in the pre-graft cultures, but a drop in gene integration in grafted cells was observed over time, likely due to instability caused by high integration levels. Epithelial Stem Cells (holoclones) Were Responsible for Regenerating the Patient’s Skin Genetic Tracing and Cell Contribution to Skin Regeneration: Short-Term Contribution: Paraclones and meraclones contributed to the formation of the new dermis. Long-Term Contribution: Epidermal stem cells (referred to as holoclones) were primarily responsible for regenerating the patient's skin over time. Lecture 16: Epidermal research and regeneration 33  Clonal Tracing of Transgenic Epidermis Two Hypotheses in Stem Cell Biology: 1. Asymmetric Division Hypothesis: Stem cells divide unevenly, creating one stem cell (which remains a stem cell) and one progenitor cell (which differentiates into specialized cells). This allows for long-term stem cell maintenance and tissue regeneration. Lecture 16: Epidermal research and regeneration 34  2. Symmetric Division Hypothesis: Stem cells divide evenly, producing two progenitor cells that both differentiate into specialized cells. There are no "special" stem cells, and regeneration is maintained by progenitor cells rather than a dedicated stem cell population. Clonal Tracing and Results: Clonogenic progenitor cells (shown in blue) and their numbers were compared in both: Original skin (pre-treatment). Transgenic epidermis (post-treatment). Findings: The number of holoclones (stem cells) in the primary cultures and the predicted number of integrations based on NGS sequencing showed consistency with Hypothesis 1. In four to eight months post-graft, the number of transplanted holoclones was consistent with the predicted numbers in Hypothesis 1. Conclusion: Clonal tracing indicated that the human epidermis is sustained by a limited number of long-lived stem cells (holoclones). These holoclones have the ability to self-renew both in vitro and in vivo, generating progenitor cells and replenishing differentiated keratinocytes. 2 Year Post Treatment: No Blisters in the Grafted Area & No Evidence of Cancer This slide shows the application of gene-corrected autologous keratinocytes on fibrin gel for treating a wound. Panel A: Shows the preparation of the wound bed with a healthy, pink dermis. Panel B: Displays the engraftment of the genetically corrected epidermis on the wound. Lecture 16: Epidermal research and regeneration 35  Panel C: After one month, it shows complete epidermal regeneration of the arm, with no blisters in the grafted area, and the graft remains intact after two years of follow-up. There is also no evidence of cancer. This contrasts with previous concerns about retrovirus-based gene therapy, where random insertion of genes into the genome could cause cancer. For example, in the early 2000s, five children in a retrovirus- based gene therapy trial for severe combined immunodeficiency developed leukemia. However, this current study showed no evidence of genomic instability or the insertion of genes affecting cancer-related pathways. QUESTION: What gene was corrected and using what type of vector was used for the treatment of JEB LMB3 using a lentivirus vector Col XVII using a retrovirus vector LMB3 using a retrovirus vector a6 Integrin using a retrovirus vector Lecture 4: Evaluate corneal epidermal cell and tissue therapy Cornea Structure Corneal Epithelium: The outermost layer, about 5-7 cells thick and less than 10% of the total corneal thickness. Epithelium cells are constantly produced and shed in the tear layer, similar to skin. The entire corneal epithelium renews every week. Lecture 16: Epidermal research and regeneration 36  Bowman’s Layer: A very thin but dense fibrous sheet of connective tissue located beneath the epithelium. It is similar to the basement membrane of the skin and forms the transition between the epithelium and the underlying stroma. Stroma: The middle part of the cornea, making up about 90% of its thickness. Composed of collagen fibers arranged uniformly and parallel, giving the cornea its clarity. Descemet’s Membrane: Separates the stroma from the corneal endothelium. Corneal Endothelium: The innermost layer, composed of a single layer of endothelial cells. Limbal stem cells: located in the basal layer of the limbus, where they divide into transient amplifying cells, similar to skin. Lecture 16: Epidermal research and regeneration 37  These stem cells also self-renew to maintain the stem cell population. Differentiated cells form the superficial layer of the cornea as they move upward and inward, also similar to skin differentiation. Identification of P63 as the Limbal Stem Cell Marker for the Cornea Part A: Immunohistochemistry for P63 Expression Cultured epidermal cells from the cornea were stained with a P63- specific antibody. P63 expression is confined to the basal cell layers. P63-positive cells are located at intervals, either as single cells (marked with arrowheads) or in clusters. Part B: Expression of P63 in Primary Epidermal Cultures Lecture 16: Epidermal research and regeneration 38  Primary epidermal cultures from two different donors (K71 and K100) were analyzed. Cell extracts were prepared from cultures generated by holoclones, meroclones, and paraclones. P63 expression was: Highly expressed in holoclones (stem cells), Pretty much absent in paraclones, Present at low levels in meroclones. The expression of housekeeping genes remained constant across all cell types, confirming that P63 expression is specific to stem cells. Lecture 16: Epidermal research and regeneration 39  Restoration of the corneal epithelium in patients with complete unilateral limbal stem cell deficiency Case 1: 20-Year-Old Woman (Thermal Burn Injury) Injury: Right eye thermal burn in 2001 Initial Condition: The corneal surface was covered by vascularized epithelium, and visual acuity was severely reduced to hand movement. Treatment: In July 2005, a 2 mm biopsy was taken from her left limbus (uninjured eye) for minimal culture preparation. Grafted the cultured limbal cells to the injured right eye. Outcome: Within a week after grafting, the corneal surface was covered with a transparent epithelium. No severe scarring allowed the limbic cultures to restore corneal integrity and improve visual acuity. The image shows the condition 1.5 years after grafting. Case 2: 43-Year-Old Male (Chemical Burn Injury) Injury: Right eye chemical burn in 1998, treated unsuccessfully with amniotic membrane. Initial Condition: The corneal surface was covered with fibrovascular tissue, and visual acuity was reduced to hand movement. Treatment: In May 2004, a small biopsy was taken from his left limbus (uninjured eye) for secondary limbic culture preparation. Grafted the cultured cells to the injured right eye in July 2004. Outcome: Lecture 16: Epidermal research and regeneration 40  Within a week after grafting, the corneal surface was covered with a transparent epithelium. Due to scarring, his vision was affected, but visual acuity improved to 0.7. The image shows the condition 4 years after grafting. Key Points: Both treatments used autologous therapies. Cultured cells were sourced from the uninjured eye, which allowed the restoration of corneal integrity in the injured eye. Clinical Trial for Unilateral Limbal Stem Cell Deficiency Treatment Participants: 112 patients treated for unilateral limbal stem cell deficiency. Key Marker: P63 Bright Cells were used to identify stem cells in the primary cultures. Lecture 16: Epidermal research and regeneration 41  Percentage of P63 Bright Cells was calculated and correlated with graft outcomes. Results: Panel A: The box plot shows a relationship between the percentage of P63 bright cells and the outcome of the graft. Higher expression of P63 was associated with successful grafts. Panel B: Cultures containing more than 3% P63 bright cells were successful in 78 cases. Conclusion: A clear correlation exists between the presence of stem cells (identified by P63 expression) and the success of the clinical outcome. QUESTION: Where do the epithelial stem cells reside in the cornea, and what marker can distinguish them? In corneal epithelium and they are marked with high levels of P63 expression In corneal endothelium and they are marked with high levels of P61 expression In border of the cornea and the sclera and they are marked with high levels of P63 expression Lecture 16: Epidermal research and regeneration 42  In Bowman’s layer of cornea and they are marked with high levels of P61 expression SUMMARY (of whole lecture) Epithelial stem cells exist in the limbus area of cornea, in the crypt of intestine, in the terminal end bud at the end of mammary duct, lung, uterus, the bulge region of the hair follicles and basal layer of skin. CEA: Cultured Epithelial Autograft, first reported in 1975, was the birth of skin cell therapy. Adult basal keratinocytes in presence of feeder cells, formed three types of epidermal colonies: Holoclones, with the greatest proliferation capacity (over 15 passages), paraclones with very limited proliferative capacity (3-4 passages) and meroclones that had intermediate proliferative capacity. Later these colonies were identified to be generated from stem cells, early differentiating cells and transient amplifying cells, respectively. Engineered composite skin with the matrix plus dermal and epidermal components are currently on trials for burns treatment. Junctional Epidermolysis bullosa or JEB caused by a point mutation in LMN311 has been shown to be treated with a combined gene and cell therapy by transducing the patient’s keratinocyte to express the correct LAMB3 gene. The cultured holoclones generated from transduced stem cells contributed most to the long term regeneration of the skin. Limbal stem cells, similar to skin stem cells, have been successfully cultured and applied for treatment of unilateral limbal stem cell deficiency Lecture 16: Epidermal research and regeneration 43