1 V5: Inducible cellular switching Cellular ProgramsWS 2010 – lecture 5 Rudolf Jänisch,...

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V5: Inducible cellular switching

Cellular ProgramsWS 2010 – lecture 5

www.wikipedia.org

Rudolf Jänisch, Alexander van Oudenaarden

*1942, MIT *1960, MIT

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Elite cells vs. all cells?

Models to account for the reprogramming process fall into two categories.

‘Deterministic’ models:

Within a donor population either ‘all’ or only a subset of ‘elite’ or ‘stem-like’

cells have the potential to generate iPS cells and are reprogrammed with a

fixed latency.

‘Stochastic’ models:

Within a donor population most if not all (model iii) or only a subset of ‘elite’

somatic cells (model iv) have the potential to generate iPS cells, albeit with

different latencies.


Hanna et al. Nature 462, 595 (2009)

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Models of progressing to a pluripotent stateduring direct reprogramming

Input: stimulated expression of Oct4,

Sox2, Klf4 and c-Myc reprogramming

factors in somatic cells.

Paper 5 tests 4 different models to

account for the latency of donor

somatic cells in progressing towards

the iPS cell state.

Latency can be measured in units of

absolute time or cell divisions until

the first iPS cell is generated from a

monoclonal population.


www.wikipedia.org

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Experimental system

Questions:

(1) what are NGFP1 cells?

(2) what are B cells

(3) how does reprogramming with ES media and DOX work


www.wikipedia.org

Trick: differentiatedcells include fluorescentmarker for Nanogexpression (marker forES and iPS state).

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Answers (1) NGFP1 cell line

The Stemgent® Primary iPS NGFP1 cell line is one of 3 induced pluripotent

stem (iPS) cell lines that were generated by reprogramming Nanog-GFP; rtTA

mouse embryonic fibroblasts (MEFs) using the 4 Dox-inducible transcription

factors Oct4, Sox2, Klf4, and c-Myc delivered by lentiviral vectors.

The Nanog-GFP; rtTA MEFs used to generate these NGFP cell lines contain a

constitutively expressed reverse tetracycline transactivator (rtTA) driven by the

ROSA26 promoter (R26-M2rtTA) as well as GFP knocked-in to the endogenous

Nanog locus (Nanog-GFP).

This NGFP1 iPS cell line harbors both rtTA and GFP as well as the Dox

inducible transcription factors Oct4, Sox2, Klf4, and c-Myc (with uncharacterized

proviral integration copy numbers) during lentiviral transduction.


www.miltenyibiotec.com

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Answers (3) Tet-On system


www.wikipedia.org

Tetracycline-Controlled Transcriptional Activation is a method of inducible

expression where transcription is reversibly turned on or off in the presence of

the antibiotic tetracycline or one of its derivatives (e.g. doxycycline).

In nature, the Ptet promoter expresses TetR, the repressor, and TetA, the

protein that pumps tetracycline antibiotic out of the cell.

The two most commonly used inducible expression systems for research of

eukaryote cell biology are named Tet-Off and Tet-On.

They consist of a fusion of the Tet repressor and a VP16 activation domain to

create a transcriptional activator protein (transactivator) rather than a repressor.

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Answers (3) Tet-On system


www.wikipedia.org

Gene expression is activated as a result of binding of the Tet-Off or Tet-On

protein to tetracycline response elements (TREs) located within an inducible

promoter.

The difference between Tet-On and Tet-Off is not whether the transactivator

turns a gene on or off; rather, both proteins activate expression.

The difference relates to their respective response to doxycycline; Tet-Off

activates expression in the absence of Dox, whereas Tet-On activates in the

presence of Dox.

In the Tet-On system, the rtTA protein is capable of binding the operator only

when bound by doxycycline. Thus the introduction of doxycyline to the system

initiates the transcription of the genetic product.

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Doxycycline

Doxycycline is a member of the

tetracycline antibiotics group and is

commonly used to treat a variety of

infections.


www.wikipedia.org

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NGFP1 cell line

When cultured under standard mouse embryonic stem (ES) cell culture conditions, the

morphology of NGFP1 iPS cells is identical to that of mouse ES cells. The cells express

both GFP from the endogenous Nanog locus as well as the pluripotency marker SSEA-1.

Stemgent® Mouse Primary iPS cells-NGFP1 can be used to generate chimeric mice from

which cells can be isolated for secondary reprogramming studies.

Chimera:animal that has two or more different populations of genetically distinct cells that originated in different zygotes

involved with sexual reproduction. Chimeras are formed from four parent cells (two fertilized eggs or early embryos

fused together). Each population of cells keeps its own character and the resulting animal is a mixture of tissues.

The simple addition of Doxycycline (Dox) to the growth medium used to propagate the

isolated cell types from the chimeric mouse results in the upregulation in expression for

the 4 transcription factors implicated in the reprogramming process.

The Stemgent® Mouse Primary iPS cells-NGFP1 were generated in the lab of Dr. Rudolf

Jaenisch, M.D. at Whitehead Institute-MIT. Dr. Jaenisch is a recognized leader in the

study of epigenetic regulation of gene expression with numerous publications focused on

ES and iPS cellular mechanisms and methodologies.


www.miltenyibiotec.com

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NGFP1 cell line

Derivation of iPS Cells Using

an Inducible Lentiviral System

(A) Somatic cells harboring a

GFP reporter driven by the

endogenous Oct4 or Nanog

promoters were infected with

tet-inducible lentiviral vectors

carrying the cDNAs of Oct4,

Sox2, Klf4, and c-Myc.


Brambrink et al. Cell Stem Cells 2, 151 (2008)

(B) The addition of dox after infection induced lentiviral expression and

subsequently led to reprogrammed, GFP-positive iPS cell colonies.

(C and D) The iPS cells were pluripotent and contributed to viable

chimeras after injection into BALB/C host blastocysts as indicated by coat

color. White mice are nonchimeric BALB/C animals, whereas mixed coat

color mice are chimeric.

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What are B cells?

B cells are lymphocytes that play a large role in the humoral immune response (as

opposed to the cell-mediated immune response, which is governed by T cells).

The principal functions of B cells are to make antibodies against antigens, perform

the role of antigen-presenting cells (APCs) and eventually develop into memory B

cells after activation by antigen interaction.

B cells are an essential component of the adaptive immune system.


www.wikipedia.org

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Development of B cells

Immature B cells are produced in the bone marrow of most mammals.

After reaching the IgM+ immature stage in the bone marrow, these immature B cells migrate

to the spleen, where they are called transitional B cells, and some of these cells differentiate

into mature B lymphocytes.

B cell development occurs through several stages, each stage representing a change in the

genome content at the antibody loci.

An antibody is composed of two identical light (L) and two identical heavy (H) chains, and the

genes specifying them are found in the 'V' (Variable) region and the 'C' (Constant) region.

In the heavy-chain 'V' region there are three segments; V, D and J, which recombine

randomly, in a process called VDJ recombination, to produce a unique variable domain in the

immunoglobulin of each individual B cell. (Random mutations also occur).

Similar rearrangements occur for light-chain 'V' region except there are only two segments

involved; V and J.


www.wikipedia.org

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Development of B cells


www.wikipedia.org

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p53 pathway

p53 (also known as protein 53 or tumor

protein 53), is a tumor suppressor

protein that in humans is encoded by the

TP53 gene.

p53 is important in multicellular

organisms, where it regulates the cell

cycle and, thus, functions as a tumor

suppressor that is involved in preventing

cancer.

P53 is called "the guardian of the

genome", referring to its role in

conserving stability by preventing

genome mutation.


www.rcsb.org

The name p53 refers to its apparent molecular

mass: It runs as a 53-kilodalton (kDa) protein on

SDS-PAGE. But the mass of p53 is actually only

43.7 kDa. The difference in speed on the gel is

due to the high number of proline residues in the

protein, which slow its migration on SDS-PAGE,

thus making it appear heavier than it actually is.

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p53 signaling pathway

p53 is a transcription factor who's activity

is regulated by phosphorylation.

The function of p53 is to keep the cell

from progressing through the cell cycle if

there is damage to DNA present.

It may do this in multiple ways from

holding the cell at a checkpoint until

repairs can be made to causing the cell to

enter apoptosis if the damage cannot be

repaired.

The critical role of p53 is evidenced by the

fact that it is mutated in a very large

fraction of tumors from nearly all sources.


www.biocarta.com

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p53:MDM2 complex

In a normal cell p53 is inactivated by its

negative regulator, mdm2.

Upon DNA damage or other stresses,

various pathways will lead to the

dissociation of the p53 and mdm2

complex.

Once activated, p53 will induce a cell

cycle arrest to allow either repair and

survival of the cell or apoptosis to

discard the damaged cell.

How p53 makes this choice is currently

unknown


www.wikipedia.org

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Apoptotic signaling in response to DNA damage

The cellular activation of the caspase cascade

resulting in cell death is triggered by chemical

damage to DNA.

This stimulates a sequence resulting in the

cleavage of Bid in a manner similar to the binding

of so called “death-receptors” or directly initiates

the permeability transition of the mitochondrial

membrane. The permeability transition releases

several factors including cytochrome c, AIF and

other factors in to the cytoplasm.

Cellular ProgramsWS 2010 – lecture 5www.biocarta.com

Cytochrome c, a key protein in electron transport, is released from mitochondria in response to

apoptotic signals, and activates Apaf-1, a protease released from mitochondria. Activated

Apaf-1 activates caspase-9 and the rest of the caspase cascade. The caspases are a class of

cysteine proteases that includes several representatives involved in apoptosis. The caspases

convey the apoptotic signal in a proteolytic cascade, with caspases cleaving and activating

other caspases that then degrade other cellular targets that lead to cell death.

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Cell cycle: G1/S checkpoint

The G1/S cell cycle checkpoint controls the

passage of eukaryotic cells from the first 'gap'

phase (G1) into the DNA synthesis phase (S).

Two cell cycle kinases, CDK4/6-cyclin D and

CDK2-cyclin E, and the transcription complex

that includes Rb and E2F are pivotal in controlling

this checkpoint.

During G1 phase, the Rb-HDAC repressor

complex binds to the E2F-DP1 transcription

factors, inhibiting the downstream transcription.

Phosphorylation of Rb by CDK4/6 and CDK2

dissociates the Rb-repressor complex, permitting

transcription of S-phase genes encoding for

proteins that amplify the G1 to S phase switch and

that are required for DNA replication ...


www.biocarta.com

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Cell cycle: G2/M checkpoint

The G2/M DNA damage checkpoint prevents the cell

from entering mitosis (M phase) if the genome is

damaged.

Cdc2-cyclin B kinase is pivotal in regulating this

transition. During G2 phase, Cdc2 is maintained in an

inactive state by the kinases Wee1 and Mt1. As cells

approach M phase, the phosphatase Cdc25 is

activated. Cdc25 then activates Cdc2, establishing a

feedback amplification loop that efficiently drives the

cell into mitosis.

DNA damage activates the DNA-PK/ATM/ATR

kinases, initiating two parallel cascades that inactivate

Cdc2-cyclin B. The first cascade rapidly inhibits

progression into mitosis: the CHK kinases

phosphorylate and inactivate Cdc25, which can no

longer activate Cdc2.

Cellular ProgramsWS 2010 – lecture 5www.biocarta.com

The second cascade is slower. Phosphorylation of p53 dissociates it from MDM2, activating its DNA binding activity. Acetylation

by p300/PCAF further activates its transcriptional activity. The genes that are turned on by p53 constitute effectors of this second

cascade. They include 14-3-3s, which binds to the phosphorylated Cdc2-cyclin B kinase and exports it from the nucleus;

GADD45, which apparently binds to and dissociates the Cdc2-cyclin B kinase; and p21Cip1, an inhibitor of a subset of the

cyclin-dependent kinases including Cdc2 (CDK1).

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Here: p53 was inhibited

p.597:

„Recently, p53 inhibition has been shown to enhance the efficiency of iPS cell

formation from fibroblasts ... by reducing apoptosis after initial transgene

infection.

...

NGFP1 iPS cells were infected with a constitutively expressed lentiviral vector

encoding a short interfering RNA (siRNA) hairpin for p53.

Infected cells were injected into hosts blastocysts and NGFP1-p53 knockdown

B cells were single-cell sorted and cultured in doxycycline.


www.wikipedia.org

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Summary

Systematic study to understand mechanism of reprogramming.

Distinguish models for „elite cells“ vs. „all cells can do“.

Experiments started from an artificially generated „only little developed“ B cell.

This explains high success rates for reprogramming.


www.wikipedia.org

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