Stem cells Flashcards
Tissue eng
Tissue engineering is an interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function.
Three general strategies for the creation of new tissue
Three general strategies have been adopted for the creation of new tissue
1) Isolated cells or cell substitutes.
2) Tissue-inducing substances.
3) Cells placed on or within matrices.
ECM
- The biochemical and biophysical properties of the extracellular matrix (ECM) dictate tissue-specific cell behaviour
- The molecules that are associated with the ECM of each tissue, including collagens, proteoglycans, laminins and fibronectin, and the manner in which they are assembled determine the structure and the organization of the resultant ECM.
- The product is a specific ECM signature that is comprised of unique compositional and topographical features that both reflect and facilitate the functional requirements of the tissue
ECM collagen
- Small, modular subunits form homopolymers and heteropolymers that become supramolecular assemblies with highly specialized organization
- Collagens are the major proteins of the ECM. The structural hallmark of all collagens is the triple helix, which is a right-handed helix of three polypeptide α- chains (homotrimers and heterotrimers), each of which contains one or more regions that are characterized by the repeating amino acid motif Gly-X-Y, where X and Y can be any amino acid
pluripotent stem cells
Can make all types of specialized cells in the body
Embryonic stem cells are pluripotent
Multipotent
Can make multiple types of specialized cells, but not all types Tissue stem cells are multipotent
Organogenesis
The series of organized integrated processes that transform an amorphous mass of cells into a complete organ in the developing embryo.
small, heterogeneous, variable complexity in cellular composition
lack stromal, vascular and immunological components
4 elements influencing organoid culture
Cell source
soluble factors
matrix
physical cues/ integrating cues
What do you do to cells after collection to make organoids
Enzymatic tissue dissociation -> dissolve ECM
Mechanical tissue dissociation
4 R’s of Term
Repair
Regenerate
Replacement
Restoration of impaired function
What was the first organ transplant
mass gen kidney transplant1954
Isolated Cells or Cell Substitutes, def, pros, cons
Transplantation of specific cells to replace malfunctioning or absent cells in a patient, avoiding surgical complications and focusing on replacing only the needed cells.
Benefits: Less invasive, avoids surgery, allows pre-manipulation of cells, targets therapy specifically.
Limitations: Potential failure to maintain function in the recipient, risk of immunological rejection.
Tissue-Inducing Substances, def, needs, ex.
Biologically active molecules like growth factors used to stimulate the body’s own tissue repair mechanisms. Tissue-inducing substances can influence chemically, mech and bio the growth and development of a stem cell
Key Needs: Purification and large-scale production of signal molecules; development of precise delivery methods to target sites.
Example: Bone Morphogenetic Proteins BMPs for bone healing
Cells Placed on or Within Matrices
Embedding cells in natural or synthetic matrices to support tissue formation or repair, used in both closed (immunoprotected) and open (integrated) systems.
Closed Systems: Semi-permeable membranes isolate cells from the immune system, used for implants or extracorporeal devices.
Open Systems: Cells in matrices are implanted to integrate with bodily tissues, using materials like collagen or synthetic polymers.
Immunological Strategies: Use of immunosuppressive drugs or autologous cells to prevent rejection.
Ross G. Harrison (1910) innovations
Achieved the first successful ex vivo cell culture, proving nerve fibers originate from nerve cells, not secreted by other tissues. Utilized a solution containing clotted frog lymph for structural support, overcoming challenges with previous methods.
Introduced the hanging drop technique, adapting from microbiology to embryology, facilitating unrestricted cell growth.
Overcame bacterial infection in cultures by establishing sterile working conditions.
Alexis Carrel (1873–1944) tissue culture
Nobel Laureate who pioneered cell culture techniques, growing endothermal animal cells ex vivo using clotted plasma as a scaffold, foundational for modern tissue culture.
Tissue Culture Established (1912): Carrel developed methodologies for mass cell culture, leading to the establishment of tissue culture as a key experimental tool in biology and medical sciences.
Carrel’s Laboratory Practices: Technicians in Carrel’s lab at the Rockefeller Institute wore distinctive full-length black, hooded gowns, reflecting the unique work culture and the era’s scientific practices.
Alan Turing and Morphogenesis
Introduced the first computational model for biological phenomena, demonstrating how reaction-diffusion systems could explain pattern formation in biological development through mathematical models.
ECM Influence on Cell Behavior
The biochemical and biophysical properties of the extracellular matrix (ECM) are crucial in dictating tissue-specific cell behavior, guiding how cells grow, differentiate, and interact.
Composition and Assembly ECM
ECM is composed of collagens, proteoglycans, laminins, and fibronectin. The specific assembly and organization of these molecules create a unique ECM signature for each tissue, reflecting its structural and functional needs.
- The extracellular matrix (ECM) is a dynamic structure that is constantly remodelled to control tissue homeostasis
- The ECM in mammals is composed of around 300 proteins, known as the core matrisome
Tissue-Specific ECM Roles
Each tissue features a specialized ECM that provides structural support, transmits forces, and filters macromolecules
Different classes of ECM molecules are designed to interact with each other, producing unique physical and signaling properties essential for tissue health and activity.
Structural Composition and triple helix of collagen
Structural Composition: Collagen, the primary protein in the ECM, forms through the assembly of small, modular subunits into homopolymers and heteropolymers, creating supramolecular assemblies with a highly specialized organization.
Triple Helix Hallmark: The defining structural feature of collagen is its triple helix configuration, comprising three polypeptide α-chains (either homotrimers or heterotrimers) that wind into a right-handed helix. This structure is characterized by the repeating amino acid motif Gly-X-Y, where X and Y can be any amino acid(proline and hydroxyproline), allowing for varied composition and properties among collagen types.
Complex Assembly Process collagen
The transformation from the initial translational product of collagen into a mature fibrillar structure involves intricate intracellular and extracellular post-translational modifications. These steps ensure the collagen fibrils are properly formed and capable of withstanding tensile forces.
collagen peptides are synthesized in the endoplasmic reticulum as procollagen, which includes additional peptide sequences at both ends. These procollagen molecules are then transported to the Golgi apparatus, where they are processed and packaged before being secreted outside the cell. Once outside, the extra peptide sequences are cleaved off, allowing the collagen molecules to assemble into triple helices and form fibrils. These fibrils then aggregate to form mature collagen fibers, providing structural support and tensile strength to tissues.
Mechanical Properties Collagen source
The unique mechanical strengths of fibrillar collagen, essential for tissue integrity and function, are directly attributable to its triple helical structure. This relationship between the collagen’s three-dimensional protein structure and its role in the ECM highlights the critical interplay between molecular configuration and ECM functionality.
Types and Locations ECM proteoglycans
Heparan Sulfate Proteoglycans (HSPGs): Found in the basement membrane, crucial for cellular signaling and filtration.
Chondroitin Sulfate Proteoglycans (CSPGs): Located in cartilage and neural ECMs, they play key roles in structural integrity and cell signaling.
Proteoglycan def and Functions
- The function of proteoglycans derives from the biochemical and hydrodynamic characteristics of the glycosaminoglycan (GAG) components of the molecules
- Long, negatively charged, linear chains of disaccharide repeats that bind water to provide hydration and compressive resistance.
Laminin
Laminins: Large, mosaic glycoproteins in basal lamina and mesenchymal compartments, mediating cell-ECM interactions through integrins and dystroglycan.
– modular domains within the laminin molecule like RGD
Fibronectin
A multidomain glycoprotein that forms fibrillar structures around cells, essential for cell adhesion and matrix assembly.
Basal lamina Definition and Role
The basal lamina is a specialized layer of the extracellular matrix that provides structural support to epithelial, endothelial, muscle, fat cells, the central nervous system, and Schwann cells.
It plays a crucial role in
- segregating tissues,
- acting as a macromolecular filter, and
- serving as a site for cell adhesion.
- shields tissues from disruptive biochemical and biophysical stresses
- Mediates communication among cells in the tissue and between cells and their external environment
Composition and Specificity basal lamina
Comprises tissue-specific isoforms of laminin and collagen IV, alongside various proteoglycans and accessory proteins, granting unique properties to different locations.
Predominantly interacts with cells through laminins, which attach to cell surface receptors and sulphated glycoproteins, activating intracellular signaling pathways crucial for cell polarity and differentiation.
Structural and Functional Contributions basal lamina
Anchors epithelium to the dermis, guides development and differentiation, and functions as a mechanical barrier.
Deposited early in development to establish polarity.
Tightly regulated expression of ECM components within the basal lamina influences cell fate determination.
ECM MMPs
- Metalloproteinases are the main endopeptidases responsible for ECM degradation
- These enzymes can also generate ECM fragments with different bioactive properties than their full-length proteins
- These matrikines regulate many processes such as migration, adhesion and differentiation.
Important scaffolds characteristics
- Surface chemistry
- Matrix topography
- Cell organization, alignment
- Fiber alignment -> tissue development
- Rigidity
* 5-23 kPa - Porosity
- Large interconnected * small disconnected
Biologically-derived Materials for scaffolds
- Polymers
- Collagen
- Laminin
- Fibrin
- Matrigel
- Decellularized matrix
- Ceramics
- Hydroxyapatite
- Calcium phosphate * Bioglass
Advantages of ECM scaffolds
FDA Approval
“Off the Shelf” Availability: ECM-derived scaffolds are readily available, facilitating their use in medical applications without extensive preparation time.
No Need for Exogenous Additives: Their use eliminates the necessity for adding external growth factors or cells, simplifying the regulatory approval process for clinical applications and reducing potential complications. (GAGs)
- GAGs in the ECM-derived material present growth factors to cell surface receptors in biologically relevant ratios and 3D conformations
- Non-chemically cross-linked ECM-derived materials degrade and release biochemical signals at a similar rate to native tissue ECM (bioinductive properties)
Bioinductive Properties of ECM-Derived Materials
Inductive Template: Acts as a template for site-specific functional tissue formation.
Mechanisms:
Release of growth factors, cytokines, and cryptic peptides with biological activities, including antimicrobial.
Chemoattraction of endogenous stem/progenitor cells (examples SIS and urinary bladder submucosa).
Modulation of the host immune response towards an M2/Th2 phenotype -> repair response
ECM hydrogels and ECM mimics
- Hydrogels derived from ECM-derived materials occupy a specific niche as a diverse class of biomaterials
- Hydrogel products constitute a group of polymeric materials, (hydrophilic structure renders them capable of holding large amounts of water in their three-dimensional networks)
- ECM mimics: Hydrogels from naturally derived materials containing one or more molecules of the extracellular matrix (e.g., collagen, agarose, alginate, chitosan, fibrin, gelatin, and hyaluronic acid)
Matrigel: reconstituted basement membrane extract from Engelbreth–Holm–Swarm tumor, and gels from decellularized tissue
Whats that special mouse
auriculosaurus mouse was a nude mouse implanted with a scaffold seeded with cells to form an ear shaped implant
What special collagens make up most of humans collagen
Collagen types I, II and III make up 80– 90% of the collagen in the human body
What factors make collagen mechanical properties vary
- Measurable physical properties make the structure of collagen
– Quantity of collagen
– Quantity of crosslinks
– Spatial distribution and homogeneity of the collagen network
Binding domains of basal lamina
Many of its components contain binding domains that allow the basement membrane to bind and to retain growth factors and chemokines, which have crucial roles in directing differentiation and guiding tissue repair and regeneration
Focal adhesions
Cell-ECM adhesion sites.
Components: Integrins, anchor proteins
Functions: Anchor cells to ECM, facilitate signal transduction.
Antimicrobial bioinductive properties of ECM scaffold
ECM scaffolds have been proven to resist bacterial infections due to antimicrobial peptides (AMPs)/polypeptides that are present in the tissue and act as a first line of defense in the innate immune response to pathogens
- At least 18 AMPs have been identified in porcine tissues which are the primary source of Small Intestinal Submucosa and Urinary Bladder Matrix
- Multiple antibacterial molecules are present in ECM degradation products -> aid regen
Measurable mechanical characteristics of cartilage
– Tensile stiffness
– Viscoelastic measures (deform in response to stress and then return to its original shape, such as the aggregate modulus -> stiffness under compressive loads)
– Lubricity of the surfaces (cartilage, etc.)
ECM cartilage
Heterofibrils of different collagen types compose the cartilage extracellular matrix (ECM) structure.
- Hyaline (holds our noses
together, Collagen organized by zonal regions) - Fibrocartilage (cushions
our joints and/or tendons)
Collagen cartilage and development
- Different collagens are involved in the process of cartilage formation during embryonic development.
- Collagen type I is expressed by mesenchymal stem cells.
- Collagen type VI accumulates during condensation and is then replaced by collagen type
II and minor collagens as the cells differentiate and deposit chondrogenic ECM
What are successful examples of scaffold tissue
Small intestinal submucosa for vasculature, Decell recell rat heart and liver, cartilage and skin
Roles of basal lamina
basal lamina directly contributes to its functions within tissues: to anchor the epithelium to the dermis, to guide development and differentiation and to function as a mechanical barrier to cells and macromolecules.
highly crosslinked and functions as an efficient barrier to both cellular and molecular traffic between distinct layers of tissue. However, it is also thin enough to permit transit in certain conditions, such as when immune cells infiltrate during inflammation
