• Nem Talált Eredményt

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

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(1)

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

(2)

STEM CELL TYPES, THEIR

MAINTENANCE AND HOMEOSTASIS

Dr. Péter Balogh and Dr. Péter Engelmann

Transdifferentiation and regenerative medicine – Lecture 2

in the Teaching Material of

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

(3)

different origins and developmental spectra

ES:

• Embryonic stem cells from the ICM (inner cell mass)

• Primordial Germ Cells (PGCs) → Embryonic Germ (EG) cells

iPS: non-embryonic somatic cells developed by the introduction of specific key transcription factors: Oct4, Sox2, c-myc, Klf4

MSC: mesenchymal stem cells present in bone marrow, adipose tissue, umbilical cord blood, amniotic fluid, placenta, dental pulp, tendons, synovial membrane and skeletal muscle, capable of self- renewal and differentiation in vitro into a variety of cells of the

mesenchymal lineages such as osteoblasts, chondrocytes, adipocytes and myoblasts

(4)

Sources of embryonic stem cells (ESCs)

Morula Early blastocyst

Inner cell mass (ICM)

Late blastocyst

Epiblast

Egg cylinder stage

Primitive

ectoderm Germ cell lineage

Trophectoderm Blastocyst cavity

Primitive endoderm

Parietal endoderm

Visceral endoderm

Extraembryonic ectoderm

Somatic cell lineages Ectoderm

Mesoderm Endoderm

Proamniotic cavity

Oct3/4

Cdx2

Gata6 Nanog

(5)

Stem cell sources in the mouse embryo

• Preimplantation embryo: inner cell mass (ICM) of the blastocyst (early blastocyst stage).

• Late blastocyst stage: formation of epiblast

• Postimplantation embryo: formation of primitive

ectoderm with restricted pluripotency → the germ

cell lineage and somatic lineages of the embryo.

(6)

Characteristics of ES cells

• Derivation from the preimplantation or periimplantation embryo

• Prolonged undifferentiated proliferation,

• Stable developmental potential to form derivatives of all three embryonic germ layers even after prolonged culture

• EC cells: teratocarcinoma-derived pluripotent embryonal carcinoma cells generating cells of all three germ layers

Cartilage (mesodermal)

Intestinal glands (endodermal)

Epidermis (ectodermal)

(7)

Cell membrane markers for ESCs

Sia Gal Glc

Man GlcNAc

GlcA IdoA

Fuc Xyl GalNAc Tra 1-60 (KSPG)

NG2 and 473HD (CSPG)

Lewis X

PSA-NCAM

CD34

SSEA-3 SSEA-4

(8)

Structure of glycoantigens characteristic for ES cells

• SSEA-3 and SSEA-4: 5–6 monosaccharides attached to a ceramide lipid tail, forming the

globoseries glycosphingolipids GL-5 and GL-7, their expression is reduced upon differentiation.

• The TRA (tumor rejection antigens) TRA-1–60 and

TRA-1–81 keratan sulfated proteoglycan (KSPG)

epitopes , probably associated with podocalyxin, a

heavily sialylated membrane protein structurally

similar to CD34.

(9)

Characteristics of CD-defined antigens for ES cells

• CD34: HSC/endothelial shared antigen expressed hemopoietic stem cells/progenitors

• CD133: Five transmembrane domain cell-surface

glycoprotein, expressed by neural stem cells

(10)

Main regulatory mechanisms of stem cells – external and internal effects

External

Interactions with the matrix proteins, soluble

factors and other cell types in stem cell niches, direct interactions with ECM proteins, complex signaling feedback from adjacent ESC niche cells (stromal/differentiated).

Internal

TF network regulating pluripotency or

differentiation

(11)

Stem cell niches in various organs

Germarium of the ovary

Terminal filament

Cap cell

Cystoblast BL

GSC

Inner sheath cell SSC

16-cell cyst

Follicle cells

Egg chamber

The apex of the testis

Hub cells

BL GSC SSC

Spermatogonia

The subventricular zone (SVZ) of the brain

Neuroblast Astrocyte

Lateral ventricle BV

BL

Transit- amplifying Ependymal cells

The bone marrow

Bone marrow

Osteoblast Stromal cell

Multipotent SC HSC

Lymphoid Myeloid

The crypt of an intestinal villus

Enteroendocrine cells

Villus Goblet cells

Crypt

BL

Transit amplifying Stem cells

Paneth cells

The bulge of the hair follicle

Hair shaft

BL

Hair bulb Matrix

Dermal papilla Muscle

Sebaceous gland

Bulge SC

Meiosis Spermatocytes Gonialblast

Cyst cell

(12)

Stem cell environment – examples for stem cell niche

• Germanium region of the ovary and the apex of the testis (germ-line stem cell and somatic stem cell)

• Subventricular zone in the brain (neural stem cell)

• Bulge of hair follicle (epithelial stem cell)

• Crypt of intestinal villi (endodermal stem cell)

• Bone marrow (hemopoietic stem cell)

(13)

Multiple interactions involved in stem cell homeostasis

ESC regulators Oct4 Nanog Tbx3

Wnt signaling Tcf3 Tle1 Fzd5

Epigenetic regulators Jarid2 Phc1 N-myc

RNA binding protein

Dppa5

Telomere associated

Rif1

Tumor suppressor

Trp53bp1 Oct4

Nanog Tcf3

Pluripotency Differentation

Oct4 Nanog

Tcf3

Oct4 Oct4

Sox2 Sall4

GCNF LRH1

(14)

Antagonistic regulatory circuits between differentiation and

pluripotency

• ESC/iPS regulation – hierarchic transcription factors

• Wnt signaling

• Epigenetic regulators

• RNA binding

• Telomere associated effectors

• Tumor suppression

• Cell cycle regulation

(15)

mRNA regulation of stem cell gene expression

Other factors Oct4

Sox2 Nanog

mRNAs

AAAAA AAAAA

Alternatively spliced mRNAs

AAAAA AAAAA

Intergenic spliced mRNAs

AAAAA

AAAAA siRNAs?

Other RNAs?

miRNAs

Intergenic transcripts

Antisense transcripts

(16)

TF regulation for

self-renewal/differentiation

• Oct3/4, Nanog, Sox2, Stat3: maintenance of proliferation

• Cdx2: Inhibitory cross-interaction with Oct3/4

(17)

Reprogramming: Induction of pluripotency in iPS cells

Target genes

Epigenetic modifiers Transcription factors Sox2

Oct3/4 Klf4

c-Myc

(18)

Reprogramming: Lineage shift in differentiated cells

• Reprogramming of B-cell lineage into macrophages – role of C/EBPa

• Induction of neuronal commitment from fibroblasts – Ascl1, Brn2 and Mytl1

(19)

Sequential maturation and regeneration of pluripotency

Ectoderm Mesoderm

Endoderm

Pluripotent cell Pluripotent cell

Ectoderm progenitor

Neuronal progenitor

Mature neuron

Pluripotent cell

(20)

Differentiation-associated

commitment and reversibility

Differentiation is coupled with

• commitment and loss of pluripotency/transdifferentiation capacity BETWEEN lineages

• Requirement for continuous stimulation for promoting specification WITHIN a lineage.

Reversal: Introduction of iPS-associated multilineage

differentiation is associated with LOWERING of pleiotropic induction requirement and ELEVATION of differentiation signal threshold

(21)

Summary

• Depending on their origin and developmental spectra, stem cells are quite heterogeneous, where their homeostasis is determined by their (a) endogenous programming with

various levels of regulating gene expression and (b)

external factors, including cytokines and adhesion proteins binding to extacellular matrix an other cell comprising the stem cell niche.

• Stem cell commitment and differentiation are not

irreversible, as differentiated cell can be modulated to regain multipotency.

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