Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

GENOMIC AND OTHER CELL TRACING

APPROACHES,

REPROGRAMMING

Dr. Péter Balogh and Dr. Péter Engelmann

Transdifferentiation and regenerative medicine – Lecture 5

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

Animal cloning

• 1952: Tadpole

• 1963: Carp

• 1986: Mice

• 1996: Sheep

• 2000: Monkey

• 2001: Cattle, cat

• 2003: Rat, horse, mule

• 2005: Dog

• 2008: Human

Stem Cell Potential

Type Description Examples

Totipotent Cells develop into a new individual

Cells of 1-4 days old embryos

Pluripotent Cells form any cell type Some cells of blastocyst (5-14 days old)

Multipotent Differentiated cells, but can form other tissues

Fetal tissue, cord blood, and adult stem cells

+ Oct4, Sox2, Klf4, Myc

reprogramming

Blastocyst

Embryonic stem cells Pluripotent Zygote

Totipotent

Adult Epiblast

(post-implantation)

Epiblast stem cells Pluripotent

Late embryo/

early foetus

Embryonic germ cells Pluripotent

Adult stem cells Multipotent or

unipotent

Skin

Central nervous system

Bone marrow

Other

Induced pluripotent stem cells Pluripotent Inner cell mass Epiblast Primordial

germ cells Fate

decision

Fate decision

Conventional Sources of Stem Cells

1 Somatic stem cells

• Harvested from mature organs or tissues (bone marrow)

• Multipotent, may be tissue specific, pluripotent?

• Many established clinical uses 2 Embryonic stem cell

• Derived from ICM of blastocyst

• Pluripotent, differentiate to all cell lineages

• Encumbered by technical and ethical issues

• May be induced from adult tissues

Origins of ES Cell Lines

1 Excess IVF embryos

2 Therapeutic Cloning (somatic cell nuclear transfer)

• Donor oocyte + somatic cell nucleus

• Cells have characteristics of nuclear donor

• Lines representing different diseases

• Individualized lines: non-immunogenic to donor

Somatic Cell Nuclear Transfer

• Challenging: In cloned cell lines, about 4% of genes function abnormally, owing to departures from

normal activation or expression of certain genes - Imprinting, methylation state

• Limited success: ~25 percent of nuclear transfers

led to a blastocyst; 35 percent of blastocysts led to

establishment of cell lines

Micromanipulation equipment

• Inverted microscope (fluorescent)

• CO

incubator

• Thermal / heatable stage

• Holding pipette (inner diameter 10 µm)

• Injection pipette ( inner diameter 7 µm)

Chromosome removal (‘Enucleation’)

• Chemical enucleation: using specific inhibitors

• Mechanical enucleation:

1 The egg is immobilized on the holding pipette with the chromosome–spindle.

2 The zona pellucida is penetrated by the injection pipette and the injection pipette is pushed against the

chromosome–spindle complex for aspiration.

3 Aspiration of the chromosome–spindle complex.

4 Complete removal of the chromosome–spindle complex and exit of injection pipette.

5 Release of chromosome–spindle complex.

Nuclear injection

• Electrofusion

• Microinjection:

1 Penetration of the egg’s zona pellucida by the injection pipette.

2 Aspiration of small amount of cytoplasm to facilitate re- sealing of the egg’s plasma membrane.

Egg activation

• Mammalian eggs are arrested in metaphase stage during ovulation.

• SCNT is unable to reinitiate / trigger the cell cycle, PLCζ enzyme is missing, resulting in abolished Ca²⁺ influx.

• During egg activation Ca²⁺ rise is essential, which can be evoked by strontium chloride (SrCl₂).

• SrCl₂ treatment is more effective than EtOH or ionophores.

• One hour after nuclear injection happened, egg activation can be performed in specialized conditions.

formation

• ESCs can be derived from eight cell embryos or from morula stage, however the most efficien

scenario, when blastocysts are used.

• By the 5th or 6th day after plating, an inner cell mass outgrowth is usually observed.

• For the culture of ESC cells feeder cells are essential.

• Four to five days later ESC colonies should appear

at the side of culture dishes.

Stem cell characterization I

• Characterization: test the cells to see whether they exhibit the fundamental properties that make them embryonic stem cells

• Growing and subculturing the stem cells for many months microscope inspection for the healthy and undifferentiated of cells.

• Using specific techniques to determine the presence of

surface markers that are found only on undifferentiated cells

• Presence of Oct4 a transcription factor, which helps turn genes on and off at the right time for the processes of cell differentiation and embryonic development.

Stem cell characterization II

• Determining whether the cells can be subcultured after freezing, thawing, and replating

• Testing whether the human embryonic stem cells are pluripotent by:

– allowing the cells to differentiate spontaneously in cell culture

– manipulating the cells so they will differentiate to form specific cell types

– injecting the cells into an immunosuppressed mouse to test for the formation of a benign tumor called a teratoma

• Teratomas typically contain a mixture of many differentiated or partly differentiated cell types

• An indication that the embryonic stem cells are capable of differentiating into multiple cell types.

• When embryoid bodies are formed they begin to differentiate

spontaneously/They can form muscle cells/nerve cell/another types.

Stem cell markers I

• Oct4: octamer-binding transcription factor 4 homeodomain tr. molecule is coded by POU5F1 gene and marks ES cells and undifferentiated,

maternal factor active in oocyte and in embryos.

• Sox2: or SRY (sex determining region Y)-box 2 HMG factor act as a transcriptional activator after forming a protein complex with other proteins (Oct4, Pax6). Essential for iPSc formation.

• SSEA3/4: stage specific embryonic antigens are of five to six

monosaccharides attached to a ceramide lipid tail. Their presence rapidly increasing during differentiation. SSEA-3 and SSEA-4 were recently shown not to be essential for the maintenance of hESC pluripotency

Stem cell markers II

• TRA-1-60, TRA-1-81: tumor rejection antigens widely used markers for stem cell characterization. They can recognize a keratan-sulfated proteoglycan (KSPG) in neuraminidase-

sensitive and neuraminidase-insensitive fashion.

• Alkaline phosphatase is a hydrolase enzyme, it is also

essential to identify stem cells and verify their functionality.

Cell tracing in stem cell biology:

non genomic

• BrdU (bromodeoxyuridine) incorporation

• Fluorescent dyes:

– CM-DiI – CFSE

– Hoechst 33342

– PKH26

genomic I

1 GFP

• 27 kDa protein (isolated originally from jellyfish)

• popular reporter system in tissue after cloning gene of interest

• different GFP variant 2 Lac-Z

• lac operon gene from E. coli

• histochemical reporter using X-gal substrate

Cell tracing in stem cell biology:

genomic II

3 Y chromosome marker

• The detection is a relatively simple process

compared to gene cloning and expression based methods (GFP, LacZ)

• FISH analysis

• High labeling efficiency

• Widely used stem cell transplantation

approaches (cardiac-, intestine disease)

In vivo imaging for cell tracing

• New development of time lapse and two-photon microscopy gave boost for live cell imaging

including cell tracing.

• Stem cells can be imaged at various time points and locations to generate time-lapse movies, and automated image analysis and statistical analyses are used to quantify the dynamic cells’ behaviour.

• Together with cell migration, changes in cell shape and changes in proliferation kinetics can be

monitored.

Cell tracing in stem cell biology

z

y

x

Single-cell fate analyses

Migration Proliferation Cell-shape change

t₁ t₂

t_n

Automated image analyses

and

statistical analyses

Reprogramming

Somatic cells can be dedifferentiated into stem cells, so called induced pluripotent stem (iPS) cells using certain aprroaches.

• Cell-fusion based

• Nuclear extract based

• Transfection of pluripotent factors

• Somatic cell nuclear transfer

Molecular mechanisms of self-renewal

G2

G1 M

S

Cell-cycle regulation

Prevention of differentation Sox2 Nanog

Oct3/4

Klf4 Tbx3

STAT3

Akt MAPK

Jak

PI3K Grb2

Lif

Cdx

2 Gata4

c-Myc b-Myb

Genes involved in reprogramming

• Nanog:The nanog cDNA consists of 2184 nucleotides (nt) and contains a single open reading frame encoding a poly-peptide of 305 amino acids.

– the role in pluripotency of both inner cell mass (ICM) and embryonic stem (ES) cells

– the ability to maintain ES cell self-renewal.

• Klf4: Krüppel-like factor, interact with CREB transcription factor, expressed in ESC and MSCs.

• Lin28: a cytoplasmic mRNA binding protein, binds to IGF-2 mRNA,

enhance the efficiency of the formation of iPSc from human fibroblasts, marker of undifferentiated human embryonic stem cells, able to bind let- 7 miRNA and inhibit it.

• Oct4: see previously

• Sox2: see previously

Telomerase activity I

• Telomeres are ribonucleoprotein heterochromatic structures at the ends of chromosomes that protect them from

degradation and from being detected as double-strand DNA breaks.

• When Dolly was cloned using SCNT, reliable question was raised about the age of her cells? Telomere was shorter, by approximately 20%, when compared with age-matched

controls.

• After some conflicting results concluded that shortened telomeres of somatic donor cells could be indeed re-

elongated during reprogramming, although the degree of elongation was quite variable, underscoring the complexity of telomere length control.

Telomers in iPS cells

• High levels of Tert (reverse transcriptase component of

telomerase) and high telomerase activity were described in iPS cells.

• iPS reprogramming of normal cells (mice and human) results

telomerase activation and restoration of telomeres, like setting the clock, to a length and chromatin state that is similar to that found in ES cells.

• Telomerase activation during iPS reprogramming is associated with upregulation of TERT, but also TERC (Tel. Associated rNA component) become activated. Moreover OCT4 and NANOG bind to the TERC gene reg. element, which may explain why these

components are upregulated in iPS cells

Summary

• ES and iPS cell fates can be monitored with a branch of fluorescent vital dyes (non-genomic/

genomic) using in vivo imaging techniques.

• Restoration of pluripotency factors and self-renewal specific genes (Oct4, Sox2, Klf4) can exhibit the

reprogramming for iPS cells.

• iPS cell generation can be good candidates for

regenerative medicine, however there are still

several unanswered questions/ concerns in

reprogramming.