1 V6: direct cell reprogramming Cellular ProgramsWS 2010 – lecture 6 National Post Mickie Bhatia, McMasters Univ. Ontario/Canada Canadian researchers transform

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V6: direct cell reprogramming

Cellular ProgramsWS 2010 – lecture 6

National Post

Mickie Bhatia,McMasters Univ.Ontario/Canada

Canadian researchers transform skin into blood, National Post. When scientists at Hamilton’s McMaster University noticed that a petri dish full of long, thin skin cells now also had a quite different occupant — distinctive, round blood cells — they suspected they were on to something big. ...The team under Dr. Mick Bhatia ... devised a process that transforms skin cells directly into blood, a mind-bending breakthrough that one expert suggests may turn cell biology “upside down.”The discovery opens the door to creating healthy blood from a mere patch of skin. For leukemia sufferers unable to find a bone-marrow donor — and other patients — the innovation could one day prove life saving.

The discovery began as Dr. Bhatia’s team were trying to create iPS cells from skin. Eva Szabo, a post-doctoral student, noticed one day what looked like blood cells in the dish of skin cells, an observation soon borne out by testing. The team then manipulated 3 different types of “factors” – proteins that turn on or off genes within cells – until they could intentionally convert the skin into blood “progenitors” or precursors, which in turn become blood cells.

Eva Szabo, Born inTransylvania

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Direct reprogramming


Modified after National Post

Dr. Bhatia’s discovery appears to go a major step beyond indirect cellular reprogramming involving a middle stage of creating induced pluripotent stem (iPS) cells

Directly reprogramming skin into other types of cell has a number of advantages:(1) Just deriving stem cells from skin can take 5 or 6 months; reprogramming them into other types of cell is another laborious task beyond that. In contrast, Dr. Bhatia’s lab can get from skin to blood within a few weeks.

(2) The produced adult blood cells are much more useful for clinical applications like transfusing into a cancer patient. Stem cells tend to produce fetal blood cells. “One of the most important things is, you’re actually making the right cells,” said Dr. Bhatia. More fundamentally, the findings suggest that even cells that have differentiated into a particular type — be it skin, heart or liver — are not frozen in that state and can be directly reprogrammed to do other things, ...

And the process was not overly complex. It just required a single transcription factor.

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Medical perspective: fighting leukemia


National Post

Dr. Bhatia’s lab is now working on converting skin into cells other than blood, as well.

The blood process, though, could eventually prove crucial to the many leukemia patients who linger on waiting lists for bone marrow transplants, where stem cells from the marrow are given to the patient to rebuild a blood system decimated by cancer.

Compatible donors are often few and far between. Instead, patients would be transfused with blood derived from healthy skin cells, bearing the person’s own genetic make-up.

Big hurdles remain, such as “scaling up” the system to produce enough blood, conducting trials and winning regulatory approval.

Dr. Bhatia said he hopes that this could be achieved in a couple of years. Dr. Dunbar predicted it would be 5 years at the earliest, and more likely 10 to 15, assuming the lab’s early results are borne out under more testing.

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Fibroblasts

A fibroblast is a type of cell that synthesizes the

extracellular matrix and collagen, the structural

framework (stroma) for animal tissues, and plays a

critical role in wound healing.

Fibroblasts are the most common cells of connective

tissue in animals.

The main function of fibroblasts is to maintain the

structural integrity of connective tissues by

continuously secreting precursors of the extracellular

matrix.


www.wikipedia.org

NIH/3T3 Fibroblasts in cell culture

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Haematopoiesis

Haematopoiesis (from

Ancient Greek: αἷμα,

"blood"; ποιεῖν "to make") is

the formation of blood

cellular components.

All cellular blood

components are derived

from haematopoietic stem

cells. In a healthy adult

person, approximately 1011–

1012 new blood cells are

produced daily in order to

maintain steady state levels

in the peripheral circulation.


www.wikipedia.org

Development of different blood cells from haematopoietic stem cell to mature cells

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Experimental strategy


www.nature.com

To make blood progenitor cells, Bhatia and his team collected skin fibroblasts from several volunteers. They infected the cells with a virus that inserted the gene OCT4, and then grew them in a soup of immune-stimulating proteins called cytokines.

Cytokines (Greek cyto-, cell; and -kinos, movement) are small cell-signaling protein

molecules.

They are secreted by the glial cells of the nervous system and by numerous cells of the

immune system and are a category of signaling molecules used extensively in

intercellular communication.

Why cytokines?

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CD45

The protein encoded by this gene is a

member of the protein tyrosine phosphatase

(PTP) family.

PTPs are signaling molecules that regulate

a variety of cellular processes including cell

growth, differentiation, mitotic cycle, and

oncogenic transformation.

This PTP contains an extracellular domain,

a single transmembrane segment and two

tandem intracytoplasmic catalytic domains,

and thus belongs to receptor type PTP.


www.wikipedia.org

Protein tyrosine phosphatase, receptor type, C also known as PTPRC is an enzyme that, in humans, is encoded by the PTPRC gene. PTPRC is also known as CD45 antigen (CD stands for cluster of differentiation).

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CD45CD45 is specifically expressed in hematopoietic cells and is an essential regulator

of T- and B-cell antigen receptor signaling.

It functions either through direct interaction with components of the antigen

receptor complexes or by activating various Src family kinases required for the

antigen receptor signaling. CD45 also suppresses JAK kinases, and, thus,

functions as a regulator of cytokine receptor signaling.

Four alternatively spliced transcripts variants of this gene, which encode distinct

isoforms, have been reported.

CD45 is a type I transmembrane protein that is in various forms present on all

differentiated hematopoietic cells except erythrocytes and plasma cells that assists

in the activation of those cells (a form of co-stimulation).


www.wikipedia.org

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Cytokine cocktail

Cytokines can be classified as proteins, peptides, or glycoproteins.

The term "cytokine" encompasses a large and diverse family of regulators

produced throughout the body by cells of diverse embryological origin.

The term "cytokine" is sometimes used to refer only to the immunomodulating

agents, such as interleukins and interferons.

Biochemists disagree as to which molecules should be termed cytokines and

which hormones.

As we learn more about each, anatomic and structural distinctions between the

two are fading.


www.wikipedia.org

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Cytokines vs. hormones

Classic protein hormones circulate in nanomolar (10-9 M/l) concentrations that

usually vary by less than one order of magnitude.

In contrast, some cytokines (such as IL-6) circulate in picomolar (10-12 M/l)

concentrations that can increase up to 1,000-fold during trauma or infection.

The widespread distribution of cellular sources for cytokines may be a feature that

differentiates them from hormones.

Virtually all nucleated cells, but especially endo/epithelial cells and resident

macrophages (many near the interface with the external environment) are potent

producers of IL-1, IL-6, and TNF-α.

In contrast, classic hormones, such as insulin, are secreted from discrete glands

(e.g., the pancreas).


www.wikipedia.org

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Response to cytokines

Each cytokine has a matching cell-surface receptor.

Subsequent cascades of intracellular signalling then alter cell functions.

This may include - the upregulation and/or downregulation of several genes and their TFs, resulting

in the production of other cytokines, - an increase in the number of surface receptors for other molecules, - or the suppression of their own effect by feedback inhibition.


www.wikipedia.org

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Erythrocyte differentiation pathwayStem cells in the bone

marrow produce a variety

of hematopoietic cell

types from common

progenitor cells under the

influence of cytokines and

growth factors.

CFU-GEMM cells are a

key intermediate in the

differentiation of granulo–

cytes, erythrocytes,

monocytes and

megakaryocytes.

Cellular ProgramsWS 2010 – lecture 6www.biocarta.com

Erythropoietin (EPO) is a cytokine produced in the kidneys that, along with other cytokines, induces red

blood cell (erythrocyte) differentiation in the bone marrow from CFU-GEMM cells.

As the erythrocyte lineage progresses, cells lose their nuclei, and move out of the bone marrow into

circulation. The ability of EPO to selectively induce red blood cell differentiation has allowed extensive

therapeutic use of the recombinant form of this cytokine to treat anemias. EPO is also used for doping!

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IGF-2

Insulin-like growth factor 2 (IGF-2) is one of three protein hormones

that share structural similarity to insulin.

In human and mouse, Igf2 is imprinted, with expression resulting

favourably from the paternally inherited allele.

IGF-2 exerts its effects by binding to the IGF-1 receptor.

IGF2 may also bind to the IGF-2 receptor (also called the cation-

independent mannose 6-phosphate receptor). IGF2R functions to

clear IGF2 from the cell surface to attenuate signalling

The major role of IGF2 is as a growth promoting hormone during

gestation.


www.wikipedia.org

IGF2, 1igl.pdb

Insulin, 2g4m.pdb

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IGF-1 receptor

Two alpha subunits and two beta subunits

make up the IGF-1 receptor.

In response to ligand binding, the α

chains induce the tyrosine autophos–

phorylation of the β chains.

This triggers a cascade of intracellular

signaling that, while somewhat cell type

specific, often promotes cell survival and

cell proliferation.


www.wikipedia.org

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IGF-1 signalling pathwayInsulin like growth factor 1 (IGF-1) and its

receptor (IGF-1R) provide a potent

proliferative signaling system that

stimulates growth in many different cell

types and blocks apoptosis.

In vivo IGF-1 acts as an intermediate of

many growth hormone responses, and

may stimulate the growth of some types

of cancer.

IGF-1 also provides a mitogenic signal to

act as a growth factor for many tissue

culture cells.

Cellular ProgramsWS 2010 – lecture 6www.biocarta.com

One component of IGF-1 mitogenic signaling is association of the receptor tyrosine kinase with Shc, Grb2, and Sos-1 to

activate ras and the Map kinase cascade (raf, Mek, Erk). An endpoint of the Map kinase pathway is modification of

transcription factor activity, such as activation of ELK transcription factors. Serum response factor (SRF) and AP-1 contribute

to mitogenic signaling by many factors. Phosphorylation of IRS-1 and PI3 kinase activation are also involved in IGF-1

signaling, similar to insulin signaling.

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Stem cell factor

Stem Cell Factor (also known as SCF, kit-ligand, KL,

or steel factor) is a cytokine that binds to the c-Kit

receptor (CD117).

SCF can exist both as a transmembrane protein and

a soluble protein. The soluble and transmembrane

forms of the protein are formed by alternative splicing

of the same RNA transcript.

This cytokine plays an important role in

hematopoiesis (formation of blood cells),

spermatogenesis, and melanogenesis.


www.wikipedia.org

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Stem cell factor

In particular, SCF plays an important role in the hematopoiesis during embryonic

development. Sites where hematopoiesis takes place, such as the fetal liver and bone

marrow, all express SCF.

Mice that do not express SCF die in utero from severe anemia.

Mice that do not express the receptor for SCF (c-Kit) also die from anemia.

Non-lethal point mutants on the c-Kit receptor can cause anemia, decreased fertility, and

decreased pigmentation

SCF may serve as guidance cues that direct hematopoietic stem cells (HSCs) to their stem

cell niche (the microenvironment in which a stem cell resides), and it plays an important role

in HSC maintenance.


www.wikipedia.org

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Stem cell nicheSCF plays a role in the regulation of HSCs in the stem cell

niche in the bone marrow.

SCF increases the survival of HSCs in vitro and contributes

to the self renewal and maintenance of HSCs in-vivo.

HSCs at all stages of development express the same levels

of the receptor for SCF (c-Kit).

The stromal cells that surround HSCs are a component of

the stem cell niche, and they release a number of ligands,

including SCF.

In the bone marrow, HSCs and hematopoietic progenitor

cells are adjacent to stromal cells, such as fibroblasts and

osteoblasts, see figure.


www.wikipedia.org

Diagram of a hematopoietic stem cell (HSC) inside its niche. It is adjacent to stromal cells that secrete ligands, such as stem cell factor (SCF).

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Summary

Direct reprogramming of somatic cells avoids complications of iPS cell formation.

Cytokines involved in intracellular communication have an important role in cell

reprogramming.

Oct4 appears to be the major „reset“ button of differentiation.

New medical applications? To treat leukemia?

Mickie Bhatia: red blood cells created from stem cells do not make the adult form of

haemoglobin. „Those cells, because they think they are embryonic, make embryonic and

fetal blood.“

Ian Wilmut: „Because the progenitor cells bypass pluripotency, there is little risk of them

forming tumours when implanted into patients.“


www.nature.com

Documents

1 V6: direct cell reprogramming Cellular ProgramsWS 2010 – lecture 6 National Post Mickie Bhatia, McMasters Univ. Ontario/Canada Canadian researchers transform