Lecture 16: Epidermal research and regeneration

Epidermal Research and Regeneration

• Epithelial/Epidermal Stem Cells (ESCs) are undifferentiated cells with the ability to self-renew and differentiate into specialized cells within the epidermis.
• ESCs location in various epithelial tissues: Limbus area of the cornea, Crypts in the intestine, Terminal ends of mammary ducts, Bulge region of hair follicles, and Interfollicular stem cells in the skin.
• Issues with identifying ESCs: Lack of specific molecular markers, reliance on multiple criteria (phenotypes, instead of a single marker).

Epithelial Stem Cell Characteristics

• Slow cycling: high proliferative potential
• High proliferative capacity: highly proliferative when activated during wound healing or removed from their native niche.
• Relatively undifferentiated phenotype: Remain undifferentiated in their native state.
• Specialized stromal niche: essential for maintaining and supporting properties.
• Pigment protection in exposed area: various areas have different pigment properties.

Slow Cycling in Their Native Niche

• Remain slow cycling in normal conditions.
• Become highly proliferative when activated during wound healing.
• Become highly proliferative when removed from their native niche.
• Undifferentiated nature: Remain undifferentiated in their natural state.

Pigmentation Characteristics

• Limbus and Interfollicular Stem Cells: Pigmented.
• Follicular Epidermal Stem Cells (Bulge Area): Not pigmented.
• Not all epithelial stem cells are pigmented.

Skin Architecture

• The three major layers are epidermis, dermis, and endodermis.

Green's Method

• Developed in the 1970s by Prof. Green and Dr. Rainwald.
• Discovery: Basal epidermal cells expand in culture with murine fibroblasts as feeder cells.
• Technique: Combination of feeder cells and specialized media supports epidermal cell growth.
• Challenges: At the time, stem cell markers and expansion methods were unknown.
• First Success: Cultured epidermis healed a patient's wounds in the 1980s, revolutionizing burn treatment
• Significance: Pioneered epithelial cell therapy, laying the foundation for advancements in regenerative medicine.

Epithelial Cell Culture

• Three types of epidermal colonies based on size and proliferative capacity when cultured on fibroblasts: Holoclones, Meroclones, and Paraclones.
  • Holoclones: Largest colonies, highest proliferative capacity, contain less than 5% terminal cells upon replicating
  • Meroclones: Intermediate size and proliferative capacity, contain 5-9% terminal cells upon replicating
  • Paraclones: Smallest colonies, limited proliferative capacity, contain more than 95% terminal cells upon replicating
  • Rarely formed new colonies when replated, and cells divided fewer than 15 times in culture.

Cell Surface Markers on Basal Adult Keratinocytes

• Keratinocytes: the most prominent cell type in 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

• Technique: Labels cell surface antigens with fluorescent antibodies. Passes cell suspension through a narrow stream.
• Dot plot analysis X-axis: Alpha-6 integrin expression Y-axis: CD71 expression
• Enables researchers to: Precisely isolate and study specific subpopulations of keratinocytes, Uncover functional differences (between populations), Advance skin therapies (e.g., burns, wounds).

Subpopulations of Basal Keratinocytes

• Stem Cells (SC): High alpha-6 integrin expression, low CD71 expression
• Transient Amplifying Cells (TA): High alpha-6 integrin expression, high CD71 expression, progenitor cells.
• Differentiating Cells (ED): Do not express alpha-6 integrin, limited proliferative capacity.

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

• Transient amplifying cells divide and begin to differentiate
• 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.

K5 & K14, Keratin 10 & Involucrin, and Ki-67

• Expressed in basal/various keratinocytes
• Function and location: Proliferation, differentiation.

Summary of Differences in Markers

• The summary of markers shows expression in native and cultured skin.

Apply ESC Therapies to Treat Wounds

• Potential uses of cultured epithelial stem cells in regenerative medicine:
• CEA - cultured epidermal/epithelial autograph Current applications of cultured epidermal stem cells, Animal studies

Cultured Epithelial Autografts (CEA): The Green's Method

• Cultured tissue from one's own skin.
• Manufacturing process: Biopsy, Enzymatic digestion, Cell seeding and expansion;
• Preferred sites (biopsy): armpits and groin.

Seeding and Growth Process for CEA

• Basal keratinocytes (from biopsy) are seeded onto fibrin gel.
• Fibrin gel: supportive structure or "carrier" for the cells.

What is CEA?

• CEA refers to the entire cell sheet that has been cultured.
• Includes keratinocyte cell layer (the actual living cells) and the fibrin gel carrier.

Acellular Skin Substitutes (Scaffolds)

• Materials designed to mimic structural and functional properties of the dermal layer.
• They are acellular (don't contain living cells but provide a framework).
• Purpose of ECM/scaffold: Stabilize cells

Skin Substitutes (Temporary and Permanent)

• Composition and indications for use for various skin substitute types (temporary and permanent).

Engineered Skin Substitute (ESS)

• In vitro and in vivo testing, human tissue, safety, functionality.
• Preparing wound bed and applying graft.
• Wound closure and tissue regeneration.

Application of Human plasma in Skin Tissue Engineering

• Collagen matrix (Integra) as a scaffold for fibroblasts to populate
• Two-layer skin substitutes (dermis and epidermis) are better than only a single layer.
• Improvement in wound healing, integration, and durability.

How to fix the issue of collagen matrix?

• Adding plasma clot to Integra helps to stabilize dermal structure and makes it more conducive to forming a stable composite skin

Human Skin Equivalent (HSE)

• Histological analysis of human skin equivalent(HSE), the expression of specific markers shows the procedure success in engineered skin.
• Three layers of epidermis are identified in the HSE.

Animal Study Results

• Testing of HSE in atomic mice.
• BTM (Biologically treated mesenchymal) supported human keratinocytes better than Integra.
• Shows presence of grafted human epidermis and promising vascularization measurements (CD31).

Challenges in Creating Fully Skin-Like Substitutes

• Focuses on what is missing:
  • Hair follicles, sebaceous glands, sweat glands, melanocytes, and neurons

Corneal Epithelial Cell and Tissue Therapy

• Corneal structure and corneal epithelium.
• Differentiated cells of cornea move upward similar to skin.

Identification of P63 as the Limbal Stem Cell Marker for the Cornea

• Part A (Immunohistochemistry), Cultured cornea cells, expression in basal layers, as single cells or clusters Part B. Expression of P63 in primary epidermal cultures, Primary epidermal cultures from donors, cell extracts, and holoclones, meroclones, and paraclones.

Restoration of Corneal Epithelium

• Case studies, biopsies from uninjured limbus, grafting techniques.
• Outcomes, results, and key points.