Physiology of Regeneration · realtime PCR (Heatmap builder) Pluripotency Epiblast Germ-line...

Physiology of Regeneration

Topics:

• Stem cell biology

• Sources of stem cells for regenerative medicine

– embryonic and nonembryonic

• Pluripotent stem cells

• Very Small Embryonic Like Stem Cells (VSELs)

isolated from the adult tissues

Right concept – Wrong design……

DREAMS

People always dreamed to achieve

longevity, but….

-Ambrosia is a food

of gods and not of

ordinary people,

- Holly Grail was

never found by

Knights of Templar,

and…

?

Lucas Cranach (The Elder) (1546)

The Fountain of Youth….

- a legendary spring that restores the youth of anyone who

drinks of its waters

Olive Tree

Urodels - Salamander

blastema

Why we do not regenerate like

salamander?

• Regeneration – risk of tumorogenesis?

• Different philosophy to adjust to the

environment ?

• Longevity = Regeneration = Stem Cell Renewal

• Senescence = Decrease in Regeneration =

Decrease in number/function of Stem Cells?

Would Stem Cells become our Ambrosia?

I want

Ambrosia !!!!

We are regenerating ourselves

continuously

• Intestine epithelium – 2 days

• Epidermis – 14 days

• Erythrocytes – 150 days

• Leucocytes – 4-7 days

1. Turtle 188 years

2. Human 122 years

3. Sturgeon ~ 100 years

4. Wale fish ~ 80 years

5. Elephant 77 years

Longevity records:

Jeanne Louise Calment

Tui Malila

Clinical applications of stem cells

• Currently:

• Hematopoietic Stem Cells

• Epidermal Stem Cells

• Mesenchymal Stroma Cells

• New potential applications:

• Heart Infarct

• Stroke

• Parkinson's disease

• Spinal cord injuries

• Diabetes

• Myopathies

• Liver damage

Komórki macierzyste – “panaceum

na wszystko” ?

Properties of Stem Cell

• Self-renewal

• Quiescence

• High resistance to radio-chemotherapy and

cytostatic drugs

• Characteristic morphology (e.g., large nucleus,

euchromatin)

Zygote

More than 200 different cell

types (totally~1014 cells)

Fertilization

Commitment

Fertilized

egg cell

(Zygote) Embryonic

Stem Cells

Tissue Committed

Stem Cells

blood

muscles

neural

epidermis

liver

other

Ectodermal SC

Mesodermal SC

Endodermal SC

Totipotent

Pluripotent

Multipotent

Unipotent

Hierarchy of stem cells

Totipotent stem

cells

They give onset to both the body of an embryo and

tissues of placenta. Under normal conditions

totipotent cells are fertlized oocyte ( zygote) and initial

blastomers. Under artificial conditions totipotency is

retained by zygote equivalent so-called clonote created as

a result of nuclear transfer of nucleus of somatic cell to

the oocyte.

Pluripotent stem

cells

They give onset to cells of all three germ layers.

Pluripotent cells are cells of internal mass of blastocyst

and cells in epiblast.

Monopotent stem

cells or tissue

specific stem cells

They include so-called tissue specific cells which give

onset to one line of cells. Examples include stem cells of

intestinal epithelium, hemopoietic stem cells, epidermal

stem cells, neural stem cells, hepatic stem cells and stem

cells of skeletal muscles.

Embryogenesis

Zygote Morula Blastula (Blastocyst) Gastrula

Tissue contribution of three germ layers

Ectoderm Brain, sympathetic ganglions, peripheral

nerves, eye, epidermis, skin appendices,

pigment cells

Mesoderm Hemato/lymphopoietic cells,

endothelium, skeletal muscles, heart,

adipocytes, connective tissues (bone,

tendon, cartilage), smooth muscles,

tubule cells of the kidney

Endoderm liver, pancreas, lung, gut, thyroid gland

SYMETRIC ASYMETRIC

STEM CELL DIVISION

Organ/tissue stem cell niches

• Bone Marrow

• Skeletal muscles

• Myocardium

• Brain

• Liver

• Intestine

• Epidermis

• Kidney

Hormones

Bone Marrow

Epidermis Intestinal epithelium

Sites of adult neurogenesis (rodent studies) compared with appropriate human brain regions. Neurogenesis has

been confirmed in two regions of the adult brain: the subventricular zone (SVZ) of the anterior lateral ventricles (the

site of origin for olfactory bulb neurons) and the dentate gyrus of the hippocampus (a brain region involved in

learning and memory). In the SVZ, progenitor cells migrate to the olfactory bulb, where they differentiate into

neurons. In the dentate gyrus, cells divide along the subgranular zone (also see the figure in the sidebar, below) and

migrate into the granule cell layer before terminally differentiating into granule cells.

Brain

ETHICS

• Due to scientific advances, at the beginning of the third millennium, human beings have reached out for technologies available, till now, only to the supreme beings.

- the development of physics has enabled us to explore

nuclear energy,

- the development of biology and genetics has explained the mystery of the creation of new organisms and their regeneration thus leading humanity into the fascinating world of stem cells

Innowacyjne metody wykorzystania komórek macierzystych w medycynie

Zygote

200 different types of stem cells

Oocyte - fertilization

Clonote

2. Therapeutic

Cloning

Oocyte Somatic Cell

+

Oocyte

Sperm

PLURIPPOTENT STEM CELL

Zygote

1. Fertilization Blastocyst

Isolation form

Embryos

Therapeutic Cloning

Clonote

2. Therapeutic

Cloning

Oocyte Somatic Cell

+

Oocyte

Sperm


Zygote


Cloning Mammoth?

Clonote

2. Therapeutic

Cloning

Oocyte Somatic Cell

+

Oocyte

Sperm


Zygote


?

Reproductive cloning???

Fertilization Cloning

Clonote Clonote Zygote

Adult Stem cells

Pluripotent Stem Cells

Isolation from Adult

Tisuses Gene Transduction

Somatic Cell (e.g., fibroblast)

Oct-4, Nanog, c-myc, Klf-4

Adult Stem Cell Induced Pluripotent Stem

Cell (iPS)

Various potential sources of pluripotent stem cells (PSC)

PSC isolated from

embryos obtained

by fertilization and

stored in embryo

banks

PSC isolated from

embryos obtained

by formation of

clonote (therapeutic

cloning)

PSC obtained as a

result of

transformation of

somatic cells

(induced PSC)

PSC isolated from

adult tissues

Risk of developing

teratomas + + + -

Histo -

incompatibility

problem

+ - - -

Required donor of

ovum + + - -

Ethical

reservations

yes yes/no* no no

* This problem is differently considered by various major religions of the world. A number of religions

potentially accept therapeutic cloning (for example Islam, Buddhism, Judaism) but unquestionable majority

of them reject reproductive cloning.

Teratoma

Somatotrophic factors, stem cells,

regeneration, aging and cancer

- The Rosetta Stone is an ancient Egyptian granodiorite stele inscribed with a decree issued at

Memphis in 196 BCE on behalf of King Ptolemy V.

- The decree appears in three scripts: the upper text is Ancient Egyptian hieroglyphs, the middle

portion Demotic script, and the lowest Ancient Greek.

- Because it presents essentially the same text in all three scripts (with some minor differences

between them), it provided the key to the modern understanding of Egyptian hieroglyphs

?

http://en.wikipedia.org/wiki/Egyptian_language

http://en.wikipedia.org/wiki/Egyptian_hieroglyphs

http://en.wikipedia.org/wiki/Demotic_(Egyptian)

http://en.wikipedia.org/wiki/Ancient_Greek


- The Rosetta Stone is an ancient Egyptian granodiorite stele inscribed with a decree issued at

Memphis in 196 BCE on behalf of King Ptolemy V.

- The decree appears in three scripts: the upper text is Ancient Egyptian hieroglyphs, the middle

portion Demotic script, and the lowest Ancient Greek.

- Because it presents essentially the same text in all three scripts (with some minor differences

between them), it provided the key to the modern understanding of Egyptian hieroglyphs

http://en.wikipedia.org/wiki/Egyptian_language


http://en.wikipedia.org/wiki/Demotic_(Egyptian)

http://en.wikipedia.org/wiki/Ancient_Greek


Concept of trans-dedifferentiation (plasticity) of HSC

Concept of trans-dedifferentiation (plasticity) of HSC

?

PARACRINE EFFECTS OF STEM CELLS

Adult Stem Cell Therapies

Ratajczak M.Z. et al. Leukemia 2012, 26, 1166 -- 1173.

Adult Stem Cell Therapies – Paracrine Effects


Adult Stem Cell Therapies – Heterogeneity of Stem Cells


Part I

Of Pluripotent Stem

Cells in Adult

Tissues…

Sca-1+lin-CD45+

Sca-1+lin-CD45-

Kucia et al. Leukemia 2006,20:857-869

Very Small Embryonic/Epiblast Like (VSEL) Stem Cell Hematopoietic Stem Cell (HSC)

0 200 400 600 800 1000 FL2-A

M1

VSEL are diploid

SS

EA

-1

Oc

t4

Na

no

g

7-AAD

VSEL exclude 7-ADD

TEM of murine Sca-1+lin- CD45- cells (VSELs)

Kucia et al. Leukemia 2006,20:857-869

A

B

Scenario 1 Scenario 2

PSC

TCSCs

PSC

TCSCs + quiescent

“locked in” PSCs

Embryo

VSELs are present in different murine tissues (A) and their

number decreases with age (B)

Zuba-Surma et al. Cytometry 2008, 73, 1116-1127.

24

Co

nte

nt

of

VS

ELs [

%]

Age of mice [months]

2 4 7 10 12 18 36

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

Ratajczak et al. Exp. Gerontology 2008, 43, 1009-1017.

A B

Heat-Map Analysis

for Ct Value from

realtime PCR

(Heatmap builder)

Pluripotency

Epiblast

Germ-line

Specification

Germ-line

Development

DNA

methylation

C/T antigen

Imprinted

genes

Cell Cycle

VSEL HSC MNC ESC-D3

Shin et al. Leukemia 2010, 24, 1450–1461

1 mm 1 mm 1 mm

1 mm 1 mm 1 mm

UCB - VSELs

mPB - VSELs

TEM of human CD133+lin- CD45- cells (VSELs)

• In vitro PSC criteria

- Undifferentiated morphology, euchromatin, high nuclear/cytoplasm ratio

- Express PSCs markers (e.g., Oct-4, Nanog, SSEA) and bivalent domains, female PSCs reactivate X chromosome

- Differentiate into cells from all three germ layers (meso-, ecto- and endoderm) – multilineage differentiation

• In vivo PSC criteria

- Complementation of blastocyst development

- Teratoma formation

Pluripotent Stem Cell (PSC) - Criteria

Part II

Of Germline and

Imprinted Genes….

+

Zygote

Morula

ICM

Epiblast

? ?

PGC

Trophoectoderm Trophoectoderm

Mesoderm

Ectoderm

Endoderm

Mesoderm

Ectoderm

Endoderm

Germ Cells

Oocyte Sperm

Ratajczak et al. Leukemia 2007, 21, 860-867.

1. Inflammation, tissue injury

2. Epigenetic changes

3. Reestablishment of the

somatic imprint

SDF-1 gradient

CXCR4+ epiblast derived cells/PGC (VSEL)

Legend:

Embryo

extra-embryonic

ectoderm

EPIBLAST

(primitive ectoderm)

PGC

precursors

Bone Marrow Fetal Liver

Muscles

Myocardium

Genital Ridges

Adrenal Glands

Nervous System

Activation

Quiescence

1. Ectopic niche

2. Inhibitory factors

3. Erasure of the somatic

imprint

VS

EL

sta

tus

Germ line as origin and “skeleton/scafold” of

stem cell system in adult body

Ratajczak et al. J Autoimmunity 2008:30;151-162.

Germ Cells

+

Zygote

Morula

ICM

Epiblast

? ?

PGC

Trophoectoderm Trophoectoderm

Mesoderm

Ectoderm

Endoderm

Mesoderm

Ectoderm

Endoderm

Oocyte Sperm

Erasure of

somatic

imprint ?

Erasure of

somatic

imprint ?


Somatic imprinting – regulates expression of some developmentally important genes

Primordial germ cells



VSEL?

Igf2-H19

Rasgrf1

VSEL HSC MSC ESC-D3

7.6 % 43.8 % 66.1 % 52.7 %

10.4 % 52.5 % 40.0 % 64.6 %

VSEL HSC MSC ESC-D3

Epigenetic changes in imprinted genes

Shin et al. Leukemia 2009, 23, 2042-2051.

78.6 %

VSEL HSC MSC ESC-D3

88.8 % 36.7 % 44.7 %

Igf2R

VSEL HSC MSC ESC

Oct-4

Igf2-H19 N N

Rasgrf1 N N N

Igf2R N N

KCNQ1 N N

Peg1/Mest N N N

SNRPN N N N N

Legend - : hypermethylation, : hypomethylation, N : normal status

Methylation status of crucial somatic imprinted genes

VSEL: Increase in mRNA for proliferation repressing genes - H19, p57Kip2 , Igf2R

Decrease in mRNA for proliferation promoting genes - IGF2, Rasgrf1. Igf1R


VSEL HSC MSC ESC

Oct-4

Igf2-H19 N N

Rasgrf1 N N N

Igf2R N N

KCNQ1 N N

Peg1/Mest N N N

SNRPN N N N N

Legend - : hypermethylation, : hypomethylation, N : normal status

Methylation status of crucial somatic imprinted genes


VSEL: Increase in mRNA for proliferation repressing genes - H19, p57Kip2 , Igf2R

Decrease in mRNA for proliferation promoting genes - IGF2, Rasgrf1. Igf1R

CELL PROLIFERATION METABOLIC FUNCTIONS

I NITIATION OF EMBRYOGENESIS

PROLIFERATION OF PLURIPOTENT STEM CELLS

INSULIN-INSULIN LIKE GROWTH FACTORS SIGNALING

M

P

H19 Igf2

DMR1

H19 Igf2

M

P

CTCF

H19 Igf2

DMR1

H19 Igf2

Igf2-H19 – imprinted

“master” locus

H19 Igf2

DMR1

H19 Igf2

M

P

CTCF

Proper Somatic Imprint

Paternally Imprinted Locus Igf2-H19 –

master regulator of VSELs quiescence

H19 Igf2

DMR1

H19 Igf2

M

P

CTCF

Jin-Yang locus



H19 Igf2

DMR1

H19 Igf2

CTCF

CTCF

M

P

Erasure of Imprint



H19 Igf2

DMR1

H19 Igf2

CTCF

CTCF

M

M

H19 Igf2

DMR1

H19 Igf2

M

M

CTCF

Development of viable bimaternal mouse individual from a reconstructed oocyte containing two haploid sets

of maternal genome, was made possible by the appropriate expression of Igf2 and H19 genes.

“Kaguya” a bimaternal mouse grew

up a normal adult with reproductive

competence

+

Kono et al. Nature 2004, 71, 1560-1567.

Part III

VSELs –

Somatotrophic

Signaling, Aging and

Cancerogenesis….

VSELs and Aging


24

Co

nte

nt

of

VS

ELs [

%]


2 4 7 10 12 18 36

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08


VSELs and Aging


24

Co

nte

nt

of

VS

ELs [

%]


2 4 7 10 12 18 36

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08


Extension of life

span?

GH/Insulin/IGF-1 axis in Aging

Calorie

Restriction

Growth Hormone

Insulin

Insulin-like growth

factor-1

Calorie

Restriction

Growth Hormone

Insulin

Insulin-like growth

factor-1

EXTENSION OF LIFESPAN

LOWER RISK OF CANCER


GH/Insulin/IGF-1 axis

High

Calorie Diet

Growth Hormone Insulin-like growth

factor-1 Insulin

Calorie

Restriction

Growth Hormone

Insulin

Insulin-like growth

factor-1



GH/Insulin/IGF-1 axis in

Aging

High

Calorie Diet

Growth Hormone Insulin-like growth

factor-1 Insulin

Calorie

Restriction

Growth Hormone

Insulin

Insulin-like growth

factor-1



DECREASE OF LIFESPAN

HIGHER RISK OF CANCER


Insulin/Insulin like growth factor molecular pathways are

involved in regulation of lifespan

Down-regulation of glucose-dependent signaling by mutations in the

RAS2, CYR1/PKA or SCH9 genes extend life-span up to 300%

Down-regulation of DAF-2, AGE-1 and AKT-1/AKT-2 proteins extend

survival up to 300%

Mutations that decrease the activity of the insulin/IGF-1-like pathway

cause dwarfism but nearly double longevity

Mice homozygous for mutation in GH-R, GH, Prop-1 or Pit-1 genes

are dwarfs but live 25 to 65% longer, Long living mice are also RasGRF1 mutants.

Short Living Mice – (higher risk of cancer)

- bGH transgenic mice

- Igf-2 transgenic mice

Long Living Mice (lower risk of cancer)

- Laron dwarfs

- Ames dwarfs

- GH -/- mice

- PAPP-A -/-

- RasGRF1-/- mice


Increase in number of VSELs (left panel) and HSCs (right

panel) in 2 month old and 2 year old Laron dwarf mice (GHR-/-)

as compared to their normal littermates (GHR+/-).

2 months old 2 years old

VS

EL

ra

tio

No

. o

f S

ca-1

+li

n- C

D45

- cell

s

per

10

6 B

MM

NC

/bo

dy w

eig

ht

2 months old 2 years old

HS

C r

ati

o

No

. o

f S

ca-1

+li

n- C

D45

+ c

ell

s

per

10

6 B

MM

NC

/bo

dy w

eig

ht

GHR+/- GHR-/- GHR+/- GHR-/- GHR+/- GHR-/- GHR+/- GHR-/-

*

* *

*

Kucia M. et al. Age 2012 (in press)

VS

EL

ra

tio

No

. o

f S

ca

-1+li

n- C

D4

5- c

ell

s

pe

r 1

06 B

MM

NC

/bo

dy w

eig

ht

HS

C r

ati

o

No

. o

f S

ca

-1+li

n- C

D4

5+ c

ell

s

pe

r 1

06 B

MM

NC

/bo

dy w

eig

ht

normal bGH normal bGH normal bGH normal bGH

6 months 1 year 6 months 1 year

Reduced number of VSELs and HSCs in 6 months and 12

months old high circulating IGF-1 level bovine Growth

Hormone (bGH) Transgenic Mice

Kucia M. et al. Age 2012 (in press)

Kucia M. et al. Leukemia 2011, 25, 1370–1374.

Aging…..

I have plenty

of VSELs !!!

Lucky you !!!

Beckwith-Wiedemann Syndrome (fetal overgrowth + pediatric sarcomas)

High level of Igf-2 due to LOI

H19 Igf2

DMR1

H19 Igf2

M

P

CTCF

Normal Somatic Imprint Loss of Imprint (LOI)

H19 Igf2

DMR1

H19 Igf2

M

P

H19 Igf2

DMR1

H19 Igf2

M

P

Loss of Imprint

Beckwith-Wiedemann Syndrome (fetal overgrowth + pediatric sarcomas)

High level of Igf-2 due to LOI

Embryonal rest theory of cancer development

1829 – J. Recamier; 1854 – R. Remak; 1858 – R. Virchow

Proposed that cancer arises from embryo-like cells.

1874 – F. Durante; 1875 – J. Cohnheim – Suggested that adult tissues contain

embryonic remnants that normally lie dormant, but can be activated to

become cancerous.

1910 - Wright - Proposed germinal cell origin of Willm’s tumor (nephroblastoma)

1911 – J. Beard – Tumors arise from displaced trophoblast or activated germ cells

Julius Cohnheim Rudolf Virchow John Beard

• Presence of “classical” germ line tumors

Seminomas, ovarian tumors, yolk sac tumor, mediastinal-, brain-germ cell tumors, teratomas, teratocarcinomas,

• Expression of Cancer Testis (C/T) Antigens in cancer cells

C/T antigens (~40 identified) are encoded by genes that are normally expressed only in the human germ line, but C/T antigens are also expressed in various tumor types (e.g., gastric-, lung-, liver-, bladder-carcinomas, melanomas, medulloblastomas, pediatric sarcomas, germinal tumors).

• Expression of chorionic gonadotropin (hCG) and/or carcinoembryonic antigen (CEA)

Several types of cancer contain either the beta subunit of hCG or its fragments and/or CEA.

• Expression of Oct-4 by several tumors

Oct-4 is a marker of germ line pluripotent stem cells an is also expressed in various tumor types (e.g., gastric-, lung-, bladder-, oral mucosa-carcinomas, germinal tumors).

Data supporting theory of cancer formation in germ cell

compartment

VSEL HS

C

MNC

0

50

100

150

200

250

Fold

diffe

renc

e (m

RNA)

MAGE-A2

VSEL HS

C

MNC

0

100

200

300

400

500

Fold

diffe

renc

e (m

RNA)

MAGE-A10

VSEL HS

C

MNC

0

50

100

150

200

Fold

diffe

renc

e (m

RNA)

MAGE-B3

VSEL HS

C

MNC

0

10

20

30

40

50

60

Fold

diffe

renc

e (m

RNA)

Ssbx1

VSEL –

express

several C/T

Antigens

Ratajczak et al. Am J Pathology 2009, 174, 1985-1992.

Germ line/epiblast-derived Oct-4+ VSEL as tumor initiating cells?

Tumor types

Potential Mechanisms

Teratomas, Teratocarcinomas Persistent somatic imprint in Oct-4+

cells (epiblast-derived?), additional

mutations

Germinomas, Seminomas, Teratomas,

Dermoid cyst, Hydatidiform mole,

Cells left along PGC migratory

route, persistent somatic imprint,

additional mutations?

Pediatric Sarcomas (rhabdomyosarcoma,

neuroblastoma, Ewing sarcoma, Willms

tumor-nephroblastoma)

Mutated Oct-4+ cells in various

peripheral tissue locations?

Other malignancies (e.g., Helicobacter

pylori – related stomach cancer, smoke -

related lung cancer)

Circulating Oct-4+ cells

incorporated at the wrong time to

the wrong place, additional

mutations? Fusion – chromosomal

instability?


Laron Dwarfs – Liver GH receptor deficiency –

Low level of circulating IGF-1

Problems still to be addressed

- Human counterparts of VSELs are not characterized yet extensively at molecular level.

- Efficient ex vivo expansion of VSELs is still a problem.

- Does number of VSELs correlates also with longevity in humans?

- Does calorie restriction and physical activity positively regulate number of VSELs in adult tissues?

- The involvement of VSELs in development of malignancies still needs a direct evidence

year 1925 year 1930

Pablo Picasso – “Study of the women head”

• If you have a horse run with it

• If you have a bird fly with it

• If you have a dream follow it……

Physiology of Regeneration · realtime PCR (Heatmap builder) Pluripotency Epiblast Germ-line...

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