p3 Flashcards
organoid def and source
self-organized 3D tissue that is typically derived from stem cells (pluripotent, fetal, or adult) and mimics the key functional, structural, and biological complexity of an organ
Cells comprising organoids can be derived from induced pluripotent stem cells (iPSCs) or tissue-derived cells (TDCs), including normal stem/progenitor cells, differentiated cells, and cancer cells
Organoid cultures exhibit significant heterogeneity and variable complexity in cellular composition
lack stromal, vascular and immunological components
Organogenesis
The series of organized integrated processes that transform an amorphous mass of cells into a complete organ in the developing embryo.
How do starting cells influence organoids
Starting cell population
* Can affect the variability and heterogeneity
* Can affect the function (quality of isolated starting cells)
How is tissue dissociation done
enzymatic (to dissolve the ECM) and mechanical
iPSC maintenance
- iPSCs can be maintained and expanded as undifferentiated clonal populations over many generations.
- Undifferentiated human iPSCs are maintained on feeder cells or defined ECM substrates.
TDCs sources
- TDCs from solid organs are derived from either biopsy samples or surgical resections
- TDCs cells can also be isolated from liquid samples such as peripheral blood, ascites, and pleural effusions
What is matrigel made of
Matrigel is mainly composed of laminin, collagen IV, entactin, perlecan and growth factors
chemically heterogeneous and has a poorly defined
composition
Matrix for organoids
- Natural matrices can be recombinantly produced from proteins or polysaccharides
- Synthetic hydrogels allow independent manipulation of biochemical and biophysical matrix properties
- The ideal organoid matrix is stress-relaxing and dynamic
- Dynamic hydrogels based on polyethylene glycol (PEG) demonstrate how tunable hydrogel properties control stemness and differentiation in cultured organoids
What are soluble factors
Largely, proteins such as growth factors, or small-molecule drugs, which can activate or inhibit signalling pathways
Physical cues for organoids
- Nutrient supply and waste removal, which are diffusion-dependent, become less efficient during organoid growth into larger tissue structures
- Can be partially resolved using shaking cultures, spinning bioreactors or suspension under continuous agitation, or in continuously stirred bioreactors
- These bioreactors can monitor pH, temperature, oxygen and glucose levels to maximize mass transfer while minimizing shear stress
Microheterogeneity in Stem Cells
Definition: Variation in characteristics such as protein and RNA expression among neighboring cells within homogeneous stem cell colonies. Indicates diversity in gene expression and cell identity even in seemingly uniform cell populations.
Characterization of Stem Cells via RNA Analysis
Key Methods: RT-PCR, microarray, RNA-sequencing (RNA-seq).
Purpose: To quantify gene expression levels and identify the cell’s state or identity by analyzing specific RNA molecules.
Epigenetic Modifications in Stem Cells
Definition: Stable alterations in gene expression without changes to the DNA sequence, including DNA methylation, histone modification, and chromatin remodeling. Crucial for cell type differentiation and identity.
Embryonic Stem Cells (ES Cells)
Location: Derived from the inner cell mass of the blastocyst.
Properties: Pluripotent, capable of differentiating into all cell types.
Challenges: Ethical concerns, potential for immune rejection, and tumor formation.
Tissue Stem Cells (Adult Stem Cells)
Definition: Multipotent cells found in specific tissues, capable of differentiating into a limited range of cell types relevant to their origin, e.g., hematopoietic stem cells in bone marrow.
Induced Pluripotent Stem Cells (iPS Cells)
Method: Genetic reprogramming of adult cells to a pluripotent state.
Advantages: Avoids ethical issues associated with ES cells, provides patient-specific therapies.
Stem Cell Niches
Definition: The microenvironment surrounding stem cells that provides signals for self-renewal and differentiation. Includes both direct cell contacts and soluble factors.
Tissue renewal cell type roles
stem cell:
- self renew - divide rarely - high potency - rare
committed progenitors:
- “transient amplifying cells” - multipotent
- divide rapidly
- no self-renewal
specialized cells:
- work
- no division
Mesenchymal stem cells:
Bone cartilage fat stem cells, from the bone marrow
Haematopoetic stem cells
Immune cells blood cell stem cells, from bone marrow
4 Types of Tissues
Categories: Epithelial, connective, nervous, and muscle tissue.
Epithelial tissue
Epithelial tissue, also referred to as epithelium, refers to the sheets of cells that cover exterior surfaces of the body, lines internal cavities and passageways, and forms certain glands
Epithelial tissue characteristics
- Some epithelial cells are secretory (hormones, mucus, sweat, digestive enzymes)
- Some epi’s have cilia to transport substances over surfaces or through tubes
- Some have protective functions (create boundaries bt inside and outside)
- Can have receptors that transduce nervous system stimulation (smell, taste, mechano, etc…)
- Have distinct inner and outer surfaces
- Inner surfaces = basal end of the epi (rests on basal lamina) * Outer surfaces = apical ends of the cell
Connective tissue
Connective tissue, as its name implies, binds the cells and organs of the body together and functions in the protection, support, and integration of all parts of the body
Connective tissue uses
- Support and reinforce other tissues
- Dispersed populations of cells embedded in an ECM typically of
* Collagen (~25% of total protein mass)
* Elastin (wrinkles with aging) - Cartilage provides rigid structural support * Collagen fibers embedded in flexible matrix
- Bone provides rigid structural support
- Collagen fibers hardened by Calcium phosphate
- Adipose tissue * Stored energy
Nervous tissue def and uses
- Nervous tissue is also excitable, allowing the propagation of electrochemical signals in the form of nerve impulses that communicate between different regions of the body.
uses:
* Process information
* Neurons communicate via electrical and chemical signals * Glial cells support neurons (more glial cells than neurons)
Muscle tissue def and char
Muscle tissue is excitable, responding to stimulation and contracting to provide movement, and occurs as three major types: skeletal (voluntary) muscle, smooth muscle, and cardiac muscle in the heart.
- Contract and cause motion * Elongated cell morphology * Most abundant tissue type
- When active (contracting) uses most of the energy produced by the body
Feeder cells
Feeder cells are a layer of support cells used to grow or maintain pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), in an undifferentiated state. These feeder cells are typically inactivated, meaning they cannot divide, but they can produce nutrients and secrete factors essential for the growth and maintenance of pluripotent stem cells
How to form aggregates
Centrifugation, low adhesion medium (hydrophilic hydrogel), hanging drop aggregation
How to deliver soluble factors
Soluble cues in vivo are typically presented to cells by the ECM or nearby cells, coordinated in time and space
growth factors can be encapsulated within nanoparticles, and conjugated onto cell surfaces for controlled release
Whats dif between iPSC, tissue and embryonic
iPSC and embryonic can differentiate to any type of specialized cell, tissue only specific types
Can progenitor cells self renew
Nay, they only divide rapidly to eventually specialize
Mesenchymal polarity and adhesion vs epithelial
Epithelial cells adhere together by tight junctions and adherens junctions localized near the apical surface. Epithelial cells also have a basal surface that rests on a basal lamina (ECM).
Mesenchymal cells in contrast do not have well-defined cell-cell adhesion complexes,
Mesenchymal cells have front-end/ back-end polarity instead of apical/basal polarity,
Mesenchymal cells are characterized by their ability to invade the basal lamina
