MSE 104 MT 1 (copy)

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Tissue Engineering

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142 Terms

1

Tissue Engineering

Use of a combination of cells, engineering, materials, and suitable biochemical factors to improve or replace a biological function

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2

What scientist pioneered tissue engineering?

Bob Langer

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3

Autologous

Donor of the cells is the recipient (grafting)

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Allogenic

Donor and the recipient are the same species (whole organ transfers)

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Syngeneic

Donor is genetically identical to the recipient (twins)

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Xenogenic

Donor and recipient are different species (porcine valve transfer)

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7

What are all of the different cell processes?

Active and Passive Transport

Autophagy

Adhesion/Cohesion

Cell movement

Cell Signalling

Cell Division

DNA Repair

Metabolism

Transcription

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Autophagy

Process by which cells eat their own internal components or invaders

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9

Signal Transduction

The process by which an extracellular signal stimulates an intracellular change without crossing into the cytosol.

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10

What is one of the biggest challenges of Tissue Engineering?

Culturing enough cells at a high enough density to build an organ.

  • Cells that form tissues that make up organs in the human body pack together at such a high density that it is almost impossible to grow enough cells at that high of density.

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Hayflick Limit

Limitation on the number of cell divisions a cell can undergo due to the shortening of telomeres with each replication.

  • Because of this typical cell cultures reach a maximum density of about 10 million cells per mL

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Primary Cells

Finite cells that have a limited amount of divisions they can undergo.

  • Usually have a limit of about 20-50

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13

What are some examples of how cells are used as therapeutic agents?

Bone marrow transplants

Blood Transfusions

Skin replacements

Liver cells used in liver assistance devices

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14

What is decellularizing?

Decellularizing the the practice of removing all cells from an organ and leaving only the abiotic filaments and extracellular fiber matrix.

  • This is used for reseeding an organ structure with autologous cells

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15

What are the levels of tissue organization from smallest to largest?

  • Cells

  • Tissues

  • Functional Units

  • Organs

  • Organ Systems

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16

What are tissues?

Tissues are a collection of cells with a related function and their extracellular matrix consisting of protein fibers

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17

What is the Extracellular Matrix?

The ECM is an interconnected network of proteins, and polysaccharides that cells connect to and receive information about their external environment from

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18

What are some types of proteins present in the extracellular matrix?

  • Laminin

  • Fibronectin

  • Enactin

  • Collagen

  • Perlecan

  • Elastin

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19

What are integrins?

Integrins are a class of cell-surface receptor proteins that can both anchor a cell to the extracellular matrix through connection to the cytoskeleton and protein fibers in the ECM, and relay information to the cell about its external environment

  • There are different types of integrins specific to each type of protein filament in the ECM that it attaches to.

    • They can impact cell differentiation, adhesion, shape, apoptosis, and more

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20

Why do integrins cluster sometimes?

The clustering of integrins makes for a stronger force of adhesion.

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21

What processes do the ECM play a role in, in tissues.

Adhesion, Spreading, Shape, Apoptosis, and Differentiation

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22

What are the 2 main categories of cell types?

Epithelial Cells and Mesenchymal Cells

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23

Epithelial Cells

Category of adherent cell types that have polarity, are tightly connected to one another, polygonal in shape, and form 2D sheets in culture.

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What does it mean for a cell to have polarity?

Polar cells, such as epithelial cells have defined apical and basal sides with each side displaying different characteristics. The basal side is attached to the ECM while the Apical side usually faces the lumen or the outside.

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Mesenchymal cells

Loosely associated cells that grow in suspension cultures and are bipolar in shape (long)

  • Surrounded by extracellular matrix and usually make extracellular matrix

  • Present only in fillers between epithelial tissues.

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Why can’t epithelial cells form multiple layers?

Due to contact inhibition, when epithelial cells form a confluent monolayer and make physical contact with other cells in all directions around them, they stop growing.

  • Some types of cancer cells result from a lack of contact inhibition.

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27

Epithelial tissues with multiple layers are able to form because _____?

Each layer consists of a different cell type so each cell type forms only a monolayer

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Cadherins

Cell-Cell binding proteins on the membrane that allow cells to identify each other through the binding of SIMILAR CADHERINS.

  • Allow for the formation of complex tissue structures between different cell types that have the same class of cadherin.

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29

What are some of the purposes of connective tissue?

To provide mechanical support (bones and ligaments)

Aid in the immune response

Store fluid

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30

What does connective tissue primarily contain?

Fibroblasts and protein fibers secreted by the fibroblasts

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31

Types of connective tissue

Loose and Dense

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32

Loose connective tissue usually consists of________ and fibroblasts.

Elastin or Collagen

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Dense Connective tissue consisting mostly of _________ fibers and fibroblasts, form structures such as ________

collagen 1; tendons and ligaments

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34

What are the 3 dynamic states of tissues?

A tissue is always in 1 of 3 dynamic states:

Formation of the tissue

Homeostasis (normal steady state)

  • Tissue maintains the needs of the cells

Repair: Wound healing

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What are the 5 dynamic states of cells?

Within each of the 3 dynamic states of a tissue, the cells within the tissue are always in at least 1 of the 5 states:

  • Cell Proliferation

  • Cell Differentiation

  • Cell Interaction

  • Cell movement (Chemotaxis)

  • Apoptosis

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What tissue is the most dynamic (replaces cells the fastest) and how often do they do so?

Bone marrow; replaces cells in the tissue every 2-3 days

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37

Wound healing in out-of-the-womb humans is a multistep process involving?

Immediate: Platelet aggregation (blood clotting)

Within hours the wound is cleaned by white blood cells and other immune response cells, as well as inflammation

Within a week a scar of dense fibers secreted by fibroblasts forms the scab as new epidermal cells generate and endothelial cells regenerate the capillaries in the area

Within a few weeks the scab is replaced by new epidermis and scar tissue is generated by fibroblasts.

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How is wound healing in fetuses different than that of born humans?

Instead of forming a scab and replacing it with scar tissue, the layers of cells surrounding the wound slowly replicate and move in closing the wound, resulting in no scarring.

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In reference to the replacement of matrix (healing rate = ts) and discarding matrix (degradation rate = td), minimal scarring is achieved when?

When ts and td are equal in magnitude (td/ts = 1), minimal scarring is achieved since if the rate that the matrix is replaced by the fibroblasts matches the rate of proliferation of dermal tissue, there will be minimal scarring.

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40

Self-Organization involves what 3 processes?

Self-patterning

Self-Assembly

Self-Driven Morphogenesis

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41

Self-Assembly

The assembly of already differentiated character types int oa specific pattern

  • Formation of complex pattern through the reorganization of relative cell positions (cell sorting)

  • EX: Viral Shell Formation

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Self-Patterning

The formation of a complex structure from a homologous cell aggregate through spatiotemporal control of cell status(i.e. differentiation) by local or external cues, autonomously

  • Homogenous sheet of cells to a patterned heterogeneous aggregate of cells

  • Formation of plant root or stalk from a callous.

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Self-driven Morphogenesis

The formation of complex structure through the spatiotemporal control of intrinsic tissue dynamics such as stiffness, stress, constriction, etc. which occur autonomously without spatial constraints.

  • Formation of the optic-cup and neural retina through control of activated myosin contraction

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What are some examples of self-organization in 3-D cultures

  • Formation of Rathke's pouches through self-patterning

    • Formation of a gut organoid from a small intestinal stem cell creating cells that provide its own niche and cells that make up the organoid

  • Formation of a tooth germ through the culturing of oral ectodermal cells on top of cultured mesodermal tissue.

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45

What is the Turing Pattern

The Turing Pattern refers to a mathematical model used to describe the possibilities of self-patterning in organisms in which there is an activator and inhibitor and the pattern formed is dependent on the rates of diffusion of both molecules.

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46

What does self organization refer to?

Self-Organization refers to situations in which an internal system is composed of definable elements that have no prepatterns (are not originated from preexisting patterns).

  • Processes are only considered to be self-organization if the complexity of the final system is greater than that of the sum of its parts.

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47

What was Alan Turing’s contribution to the filed of morphogenesis?

Created a mathematical algorithm from diffusion equations that was able to model the different types of patterns tissues can make in self-organization.

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48

What is Gastrulation

The formation of the 3 primary germ layers resulting from a series of cell migrations.

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49

What does the Ectoderm form?

Outer Layer

  • Skin

  • Brain

  • Eyes

  • Ears

  • Hair

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50

What does the Endoderm form?

Inner layer (linings of body cavities)

  • Liver

  • Pancreas

  • Gall Bladder

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What does the Mesoderm form?

Middle Layer between Endo and ectoderm.

  • Muscle

  • Bone

  • Blood

  • Vascular System

  • Lungs

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52

What is induction?

Induction is a signal that begins some process of morphogenesis

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53

Negative Induction

When a cells signals restrict the action of another cell

  • “Do Nothing”

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Instructive Induction

When a cells signals instruct another cell to take a particular action

  • “Do something specific”

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Permissive Induction

When a cell contains all of the necessary signals to change and do different things

  • “Cell telling itself to start dividing”

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Reciprocal Induction

Both cells signal each other and tell the other to do something.

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57

What are some types of cell adhesion molecules?

Cadherins & Integrins

Desmosomes

Cell Adhesion Molecules (CAMs)

PECAM, VCAM, NCAM

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58

What are tight junctions?

Tight junctions are areas where 2 cells plasma membranes join together forming a virtually impenetrable barrier to fluid

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59

What are gap junctions?

Gap junctions are intercellular connections that connect the cytoplasm of 2 cells allowing various molecules to pass between them with ease.

  • Formed by Connexin proteins

    • Heart Muscles have lots of gap junctions to allow for high signal transfer ability

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60

What are focal Adhesions?

Focal adhesions are a macromolecular complex that are responsible for how the extracellular matrix is able to convey information about a cells external environment to the cell.

  • Regulatory signals and mechanical forces are transferred through the focal adhesion from the ECM to tell the cell about its environment.

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61

Give an example of why mesenchymal and epithelial cell interactions are vital to homeostasis?

In the bone marrow, the stem cell niche of the hematopoietic stem cells (epithelial cells), stromal support cells (mesenchymal cells) interact closely and participate in the control of the stem cells behavior and cell fate

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How does morphogenesis begin and end?

Morphogenesis begins with an induction signal that is usually a destabilization signal in tissue structures.

  • Requires this to destabilize the current formation so that the cells re-organize and self-assemble into a new different pattern

Forces involved in cell migration, division, and differentiation govern the process of morphogenesis through self-assembly, self-patterning, or self-driven morphogenesis

Morphogenesis can ONLY END when a stable conformation is reached.

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63

What are the different ways that cells can migrate?

Chemotaxis: Cells move along a soluble concentration gradient

Haptotaxis: Cells move along an insoluble gradient (attached to an insoluble matrix)

Galvanotaxis: Move along electrical currents

Contact Guidance: Cells migrate along other cells, using surface topology of other cells like walking.

Contact Inhibition: Cells move to an area where they are completely surrounded by cells and stop dividing.

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Branching Morphogenesis

The formation of a new structure from the cells of an existing structure of the same type.

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65

Cell Proliferation Equation

X(t) = X0e^(ut)

where

u = ln(2)/doubling time

t = time

X0 = initial cell count

X(t) = Final cell count

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Equation to calculate the total # of cells possible from a type of cell.

Xf = X0 × 2^n

where

n = # of population doublings

X0 = starting # of cells

Xf = Final # of cells

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67

What are the different phases of cells in culture?

Lag Phase, Log Phase, Stationary Phase, and Death Phase

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68

What are the different types of biological contamination?

Bacteria

Yeast

Mold

Viruses

Mycoplasma

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69

How are cells identified and separated?

Cells are identified and separated based on:

Morphology, Cell Size, Cell Density, Light Scattering, and Biochemical properties such as cell surface markers.

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70

What are the 3 different categories of mammalian cell morphology

Fibroblastic: Long and bipolar - Adherent

Epithelial: Cuboidal, Tight Packing, and Polar - Adherent

Lymphoblast-like: Spherical, lack polarity and loosely associated - Suspension

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71

What information does forward scatter and side scatter provide?

Forward scatter provides information about cell size while side scatter provides information on granularity and inner cell complexity

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72

On a Forward scatter side scatter graph, the Y-axis represents_______ while the X axis represents ______

Side scatter with simplicity of cells increasing as y values decrease; Forward scatter with size of cells increasing as x values increase

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How are cells separated based on cell size and density?

Differential discontinuous density centrifugation

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How are cells separated based on their biochemical properties?

Cells are tagged with antibodies or some other type of molecule that attaches to cell surface markers of specific cells.

  • The tagged cells are then either subject to Flow cytometry or magnetic cell sorting

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Cell Separation Yield Equation

Yield = (Cells out/ Cells in)

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Cell Purity Equation

Purity = (Desired Cells out/ Total Cells out)

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77

What are the conditions for a stem cell?

Undifferentiated

Self-Renewing

Potential for Differentiation

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Totipotent Stem Cells

Capable of giving rise to all cell types of the organism

→ Differentiate into Pluripotent Stem Cells

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Pluripotent Stem Cells

Capable of giving rise to all cell types of the 3 germ layers

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Multipotent Stem Cells

Capable of differentiating into several different specialized cells but not all the cells from the 3 germ layers

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Unipotent Stem Cells

Only capable of giving rise to 1 type of specialized cell

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82

What is the difference between therapeutic cloning and reproductive cloning?

In therapeutic cloning an enucleated oocyte is implanted with a nucleus extracted from an autologous cell, and is then allowed to grow into a blastocyst from which the inner cell mass is taken and cultured to produce pluripotent embryonic stem cells which are then modified and transferred back into the patient

In reproductive cloning, the same process is performed but instead of taking out the inner cell mass and culturing it, the blastocyst is implanted back into the uterus and the fetus is allowed to grow, forming a clone.

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83

Is cloning efficient?

No. It has a very low chance of creating a successful organism that survives (10%)

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84

Animal cloning can be beneficial if used for what situations?

Bringing back extinct species

Saving endangered species

Promoting disease resistance

Transgenics: manipulating DNA of animal cells to produce human proteins

Cloning high yield animals

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85

What 2 scientists won the nobel prize for reprogramming cells into iPSCs

Gurdon and Yamanaka

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86

Before the discovery of ________, the best cells to use for experiments in vitro were ________

induced pluripotent stem cells; embryonic stem cells

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87

What is one of the disadvantages of using iPSCs?

When iPSCs differentiate, they are phenotypically unstable and may change into a different cell type

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88

What is the stem cell niche

The stem cell niche is the physical location where a stem cell type resides in the body that control the stem cells behavior, differentiation, proliferation, through secreted and cell surface molecules.

  • Different stem cell niches exist for different types of stem cells

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89

What are the possible components present in a stem cell niche?

  • The stem cell

  • Stromal support cells

  • Soluble Factors

  • ECM (Mechanical Signals)

  • Neural Signals

  • Vascular Network

  • Cell Adhesion

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90

What are some examples of stem cells and their niches?

Hematopoietic Stem Cells : Niche = Bone Marrow

Intestinal Stem Cells : Niche = Crypts of the intestinal lining

Satellite Cells : Niche = Embedded in the Muscle

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91

What behaviors of the stem cell do stem cell niches control?

Stem cell differentiation, Self-renewal, asymmetric cell division, and other behaviors

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Symmetric self-renewal

Stem cell divides into 2 other stem cells

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Asymmetric self-renewal

Stem cell divides into one stem cell and one differentiated cell

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94

What are the mechanisms of asymmetric cell division?

Localization of apical and Basal determinants

Asymmetric spindle assembly and function

Extrinsic regulation from the stem cell niche

Signals and cues from noncanonical signaling pathways

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95

Describe the localization of Apical Determinants

During interphase in a stem cell, the proeins aPKC, PAR6 and L(2)GL localize apically and form a complex with one another which is then phosphorylated by Aurora A. L(2)GL is then phosphorylated by the activated aPKC and is replaced by PAR3.

The replacement of PAR3 leads to Numb inactivation by phosphorylation which is a Notch signaling suppressor

Because notch is an activator of stemness retention in cells, the inactivation of Numb increases Notch signalling resulting in the daughter cell that arises fro mthe apical side of the stem cell, retaining stemness.

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96

What is Notch?

Notch is a well known stemness retention activator

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97

Describe the localization of the Basal Determinants

During interphase, in the basal side of the stem cell, due to the absence of aPKC/PAR6/L(2)GL complex, activated Numb localizes and accumulates basally.

The accumulated activated Numb hen inhibits Notch signaling basally, preventing the retention of stemness.

The proteins Miranda, Prospero, and Brat localize basally and form a complex that remains inactive until the eventual degradation of Miranda, releasing Prospero and Brat which then act to induce differentiation.

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98

Describe the Asymmetric Spindle Formation

The asymetric spindle forms with the apical localization of Inscuteable, Pins, Gai, and other proteins to establish the aical/basal orientation.

  • Also aids in the localization of the basal determinants Prospero and Miranda.

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99

How can the stem cell niche impact asymmetric cell division?

  • Attachment to some other element in the stem cell niche can influence asymmetric cell division

    • The apical side of the stem cell reamins bound to the niche element while the basal side faces away from it.

    • Cell that loses contact with the niche element becomes differentiated

  • High Wnt Signalling side of the cell retains stemness.

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100

What molecules/ proteins in stem cells are important for maintaining stemness?

Notch and Wnt

Both Notch ad Wnt promote retention of stemness

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