Stem Cell Class - Test 2

Cre-Lox lineage tracing

Cre-ER, Loxp, and tamoxfin work together to activate LacZ in order to express β-gal and track cell lineage

How is the cre-lox system controlled in time?

cre-ER fusion protein makes Cre and Lac only expressed when tamoxifen is present

cre-ER fusion protein

Cre active only when estrogen receptor (ER) receives tamoxifen

regenerative medicine

process of creating living tissue to repair or replace tissues, including organs due to age, disease, or damage

immunosuppression

immune systems rejecting injected cells

Autologous transplant

Stem cells inserted derived from host’s cells

Allogeneis transplant

Stem cells inserted derived from not host’s cells

keratinocyte stem cells

found in the skin that play a crucial role in maintaining and repairing the epidermis, the outer layer of the skin

Bulge stem cells

region at the base of hair follicles that houses hair follicle stem cells

Epidermal autographs

a type of skin graft used to cover large burn wounds, sheets of autologous keratinocytes

extracellular matrix

scaffold for organization of tissues, proteins, and glycoproteins

Apligraf

FDA approved, allogeneic, off the shelf, bilayered cell-based ski graft

Stem cell niche

area where there is a balance of self-renewal and differentiation of stem cells in body

small intestine crypt

Trough in between small intestinal villi

small intestine villi

tiny, finger-like projections that increase surface area, aiding in nutrient absorption from digested food

Crypt Base Columnar Cell

intestinal stem cells found in crypts of the small intestine that generate all other epithelial cell types

Lineage tracing

process of identifying and documenting the ancestry or developmental history of cells, organisms, or groups

What did Cre-Lox lineage tracing prove?

That Crypt Base Columnar Cells were the ancestor to all crypt and villi intestinal cells

Islets of Langerhans

Type 1 Diabetes 

body's immune system mistakenly attacks and destroys the insulin-producing cells in the pancreas (β-cells), leads to a large amount of glucose in blood that body can’t use,

Type 2 Diabetes 

insulin resistance, 90% of diabetes patients have this kind, difficult for stem cell treatment

β-cells

single-hormone positive (only secrete insulin), insulin storage granules, sense glucose and respond with insulin

Type 1 Diabetes stemm cell treatment (Paglivca et al.)

create β-cells from stem cells

Light pathway in Retna

light → ganglion cell → bipolar cell → rod → retinal pigment epithelium →

Retinal Pigment Epithelium

Retinal maintenance and support

Macular degeneration

most common form of blindness in developing countries

microglia cell

Look for cell damage then trigger immune response to deal with it

Drusen deposit

small, yellow or white deposits that accumulate between the retina and the retinal pigment epithelium (RPE) in the eye

Drusen deposit effects

RPE layer breaks causing blood vessels growing in RPE layer and circulatory fluid leaking into retna

Dry Macular Degeneration

RPE and photo receptor degeneration, thinning macula

Wet Macular Degeneration

Bruch’s membrane ruptures and blood vessels infiltrate

macula

a small, oval-shaped area in the center of the retina, the light-sensitive tissue at the back of the eye

Macular degeneration Stem cell treatment (Schwartz et al.)

Create iPS derived RPE sheets as well as transplant photo receptors above RPE layer

Cell types in spinal chord

neurons, glial cells

neurons

send nerve impulses up and down tracks

glial cells

cells that support and protect neurons in the central and peripheral nervous systems (oligodendrocytes, astrocytes, macroglia, ependymal)

oligodendrocytes

deposit myelin sheaths, insulating axons

astrocytes

key support cells for neurons, supply nutrients, maintain ion balance, responsible for wound response and scarring

macroglia

macrophages of nervous system, remove damaged cells, combat infection

ependymal cells

propel CSF, post-injury regenerative capacity, line central canal of spine

Primary Spinal chord injury

trauma-induced neuronal damage and axon disruption

Secondary spinal chord injury

defects are caused by inflammation and glial scar formation

Pros of glial scarring

Good for sealing wound site, prevents further immune interference

Cons of glial scarring

creates physical and molecular barrier for repair

Are there robust stem cells in the adult spinal chord?

No

Stem Cell repair treatment for spinal chord injury

MSC transplant with growth factors, trophic factors, and anti-inflammatories

Stem Cell repair treatment for spinal chord injury cons

MSCs must be injected within 8 weeks of injury, MSCs are not naturally in spine, MSCs tend to differentiate into many different cells

Stem Cell support treatment for spinal chord injury

deliver oligodendrocyte progenitor cells into injured area to remethylate axons

Stem Cell support treatment for spinal chord injury cons

OPCs must be inserted within at least 7 days after injury, only works when axons are demethylated, not destroyed

Stem Cell replace treatment for spinal chord injury cons

If spinal chord is severed, insert graft of NSCs (Neural Stem Cells), trophic factors in fibrin matrix creates neural relays

Parkinson’s Disease

The loss of dopaminergic neurons in the substantia nigra, reduced input to striatum

DA neuron in-vitro synthesis

LSB/S/F8/CHIR protocol

DA neuron Rodent Assay

Mice used in assay only have DA neurons in nigra but not in striatum, when give anfetamine, mice will turn in circles since DA neurons only on one side

DA neuron Rodent Assay success

Mice can function normally under influence of anfetamine since DA neurons are now in both striatum and nigra

DA neuron Rodent Assay fail

Mice keep turning in one direction due to DA neuron imbalance

Cancer stem cells can

self renew, give rise to differentiated tumor growth, drive tumor growth

stem cell origin of cancer

tumor-initiating cells may correspond to altered adult stem cells or more differentiated cells (progenitors) with enhanced self-renewal capacity

Stochastic tumor property model

transforming events create malignant cell of origin and malignant, self-renewing (predicts all tumors are tumorigenic)

Cancer stem cell tumor property model

transforming events create malignant cell of origin and mostly non-malignant, non-self renewing (predicts only some cells are tumorigenic)

Xenograft Assay

1) take piece of tumor

2) dissociate cells (categorize cells into cancer stem cells and non-cancer stem cells via cell sorting)

3) insert into mouse, group that grows tumor has cancer stem cells, group that doesn’t grow tumor does not

Wnt pathway

When Wnt binds to cell surface receptor, β-catenin survives and binds to TCF, This allows mRNA production in nucleus increasing cell proliferation and differentiation

When Wnt was activated in CBC

Huge tumors, massive overgrowth

When Wnt was activated in more superior cells of the crypts

tumors were benign

Steps in metastasis

1. primary tumor turns into invasive carcinoma

2. these cells circulate body and find secondary tissue

3. infiltration

4. latency

5. colonization, initiating formation of secondary tumor

metastasis

the spread of cancer cells from their original (primary) tumor to other parts of the body

Factors for preferred sites of metastasis

• capillary anatomy and factors affecting extravasation (exit from blood vessel)

• adherence between migrating CSCs (cancer stem cells) and target tissues

• ability of CSCs to establish a supportive novel niche

Transdifferentiation / Direct Cell Conversion

Skipping pluripotency phase and directly differentiating a cell into cell of interest

advantages of transdifferentiation

1. no pluripotency so no risk of teratoma

2. no multi-step differentiation protocol to optimize

3. fewer steps, less time

4. pluripotent cell-derived tissues are typically embryonic and immature

SCNT (Somatic Cell Nuclear Transfer)

take fully differentiated cell → isolate nucleus → revert to pluripotency → redifferentiate → differentiated cell type

Pioneer Transcription Factor

can open chromatin for cell specific gene expression, now other transcription factors can drive expression since pioneer has opened chromatin

Chemically Induced Transdifferentiation

cardiac progenitor cells produced and expanded with defined factors in-vitro without viral vectors

C. elegans (round worms)

Have undifferentiated stem cell population in gonads

How do C. elegans stem cells stay undifferentiated?

Notch signaling pathway and distal tip cells organize germ line stem cells

Fruit fly stem cells

found in ovaries

mouse intestinal crypt niche

paneth cells (located right next to CBC) signal to CBCs to differentiate or maintain multipotency

common features of lineage tracing

1. inducible system delivers a pulse of site-specific recombinase activity

2. recombinase targets specific DNA recognition site to produce stable genomic rearrangement in parental cells

3. genomic rearrangement activates expression of report/marker gene that is used to trace cell lineage

4. larger marked clones are obtained over time

How to control Cre-lox lineag tracing in space

1. Breed mouse with cre function and mouse cre not functions

2. Make mouse with both

FLP-FRT lineage tracing

1. Activate FLP with heat shock (turn up heat)

2. FLP binds to FRT target sites

3. Now LacZ gene is active and can be used as marker

• not controllable in space