Lecture 16: Epidermal research and regeneration

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Questions and Answers

Which type of epidermal colony is characterized by the highest proliferative capacity?

Transient amplifying cells

Meroclones

Holoclones (correct)

Paraclones

What is the primary role of FACS in epidermal cell research?

To isolate specific keratinocyte subpopulations (correct)

To enhance the growth of all epidermal cells

To measure the size of colonies over time

To differentiate between terminal cells and stem cells

Which cell type is considered to have low CD71 expression and high alpha-6 integrin expression?

Temporary amplifying cells

Terminal cells

Early differentiating cells

Stem Cells (correct)

What percentage of terminal cells do Holoclones contain upon replating?

95% Signup and view all the answers

What is the expected behavior of Paraclones when replated?

They rarely form new colonies Signup and view all the answers

Which colony type is associated with transient amplifying cells?

Meroclones Signup and view all the answers

In dot plot analysis during FACS, what does the X-axis represent?

Alpha-6 integrin expression Signup and view all the answers

What is the primary difference between the Asymmetric Division Hypothesis and the Symmetric Division Hypothesis?

Asymmetric division maintains a dedicated stem cell population, whereas symmetric division relies on progenitor cells for regeneration. Signup and view all the answers

Which finding supports the Asymmetric Division Hypothesis in the study of transgenic epidermis?

The increased number of holoclones indicated a population of stem cells. Signup and view all the answers

What do the results of the clonal tracing suggest about the human epidermis?

It derives from a limited number of long-lived stem cells. Signup and view all the answers

How did the results of the longitudinal study on holoclones compare to the predictions made by Hypothesis 1?

They aligned with the predicted numbers based on NGS sequencing. Signup and view all the answers

What role do holoclones play in the epidermis as indicated by the research?

They provide a continuous source of progenitor cells and replenish keratinocytes. Signup and view all the answers

What characteristic is most associated with Transient Amplifying Cells (TA)?

High alpha-6 integrin expression Signup and view all the answers

Which statement is true regarding the differentiation process of cells in the epidermis?

Cells stratify and move upward as they differentiate Signup and view all the answers

What role do interfollicular stem cells play in skin regeneration?

They maintain the stem cell pool through asymmetric division Signup and view all the answers

Which of the following proteins are expressed at the basement membrane?

Collagen IV and Laminin 511 Signup and view all the answers

How do Differentiating Cells (Early Differentiating) differ from Transient Amplifying Cells?

They are involved in terminal differentiation Signup and view all the answers

What is a key characteristic of the asymmetrical division performed by stem cells?

One daughter cell becomes a TA cell and the other remains a stem cell Signup and view all the answers

What is the primary function of Colony Formation Assay in epidermal research?

To support the relationship between clonogenicity and alpha-6 integrin Signup and view all the answers

Which cell type is primarily involved in terminal differentiation within the epidermal hierarchy?

Differentiating cells Signup and view all the answers

What distinguishes the function of keratinocytes that have fully differentiated?

They are shed from the surface of the skin Signup and view all the answers

What are the key advantages of using composite skin over a single layer of skin?

Natural structure and improved healing Signup and view all the answers

Which technique is utilized to enhance the stability of composite skin?

Plasma clotting Signup and view all the answers

What is the purpose of adding plasma clotting to porous matrices?

To create a stable functional matrix Signup and view all the answers

Which cells are isolated and expanded from the patient for composite skin construction?

Keratinocytes and fibroblasts Signup and view all the answers

What matrices are commonly used for testing in composite skin construction?

Integra, a collagen-based scaffold Signup and view all the answers

What condition led to clot failure in the plasma clot formation process?

Absence of fibroblasts Signup and view all the answers

What is the role of protonin in the cultivation of Human Skin Equivalent (HSE)?

To inhibit clot degradation Signup and view all the answers

What effect does the addition of a plasma clot have on the composite skin's characteristics?

Enhanced wound healing and natural structure Signup and view all the answers

What is the significance of using composite skin in medical applications?

It leads to more natural healing processes Signup and view all the answers

Which statement is true regarding the construction of composite skin?

Composite skin includes both epidermis and dermis Signup and view all the answers

Which type of wound has been shown to benefit from epithelial cell therapy?

Surgical wound Signup and view all the answers

What components make up the Engineered Skin Substitute for burns treatment?

Autologous fibroblasts, autologous keratinocytes, and collagen-GAG scaffold Signup and view all the answers

What is the nickname given to babies born with Junctional Epidermolysis Bullosa (JEB)?

Butterfly babies Signup and view all the answers

Which protein is specifically mutated in Junctional Epidermolysis Bullosa (JEB)?

Laminin 332 (LAMB3) Signup and view all the answers

What is the average life expectancy of children diagnosed with JEB?

20 to 30 years Signup and view all the answers

What critical role do the proteins affected by JEB play in skin integrity?

Facilitating attachment of basal keratinocytes to the basement membrane Signup and view all the answers

Which of the following is NOT a genetic cause of Junctional Epidermolysis Bullosa?

Fibroblast growth factor mutation Signup and view all the answers

What is a potential treatment approach for Junctional Epidermolysis Bullosa due to its genetic nature?

Gene therapy Signup and view all the answers

What is the characteristic feature of the skin of children with Junctional Epidermolysis Bullosa?

Presence of extensive blisters Signup and view all the answers

Study Notes

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.

Studying That Suits You

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Description

Test your knowledge on epidermal cell types, their characteristics, and the role of various research methodologies like FACS. This quiz covers topics such as Holoclones, transient amplifying cells, and division hypotheses in epidermal studies. Perfect for those interested in cellular biology and dermatological research.