Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

STEM CELL TYPES, THEIRMAINTENANCE AND HOMEOSTASIS

Dr. Péter Balogh and Dr. Péter EngelmannTransdifferentiation and regenerative medicine – Lecture 2

Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

TÁMOP-4.1.2-08/1/A-2009-0011Sources and types of stem cells: different origins and developmental spectraES: • 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

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Sources of embryonic stem cells (ESCs)

Morula Early blastocyst

Inner cell mass(ICM)

Late blastocyst

Epiblast

Egg cylinder stage

Primitiveectoderm Germ cell lineage

Trophectoderm

Blastocyst cavity

Primitiveendoderm

Parietalendoderm

Visceralendoderm

Extraembryonic ectoderm

Somatic cell lineagesEctodermMesodermEndoderm

Proamnioticcavity

Oct3/4

Cdx2

Gata6

Nanog

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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.

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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)

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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-3SSEA-4

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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.

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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

TÁMOP-4.1.2-08/1/A-2009-0011Main regulatory mechanisms of stem cells – external and internal effectsExternalInteractions 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). InternalTF network regulating pluripotency or differentiation

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Stem cell niches in various organs

Germarium of the ovary

Terminalfilament

Cap cell

Cystoblast

BL

GSC

Inner sheathcell SSC

16-cellcyst

Folliclecells

Eggchamber

The apex of the testis

Hub cells

BLSSCGSC

Spermatogonia

The subventricular zone (SVZ) of the brain

NeuroblastAstrocyte

Lateral ventricle

BV

BL

Transit-amplifying

Ependymal cells

The bone marrow

Bone marrow

Osteoblast

Stromal cell

Multipotent SCHSC

LymphoidMyeloid

The crypt of an intestinal villus

Enteroendocrine cells Villus

Goblet cells

Crypt

BL

Transit amplifyingStem cells

Paneth cells

The bulge of the hair follicleHair shaft

BL

Hairbulb

MatrixDermal papilla

MuscleSebaceou

sglandBulge SC

MeiosisSpermatocytes

GonialblastCyst cell

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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)

TÁMOP-4.1.2-08/1/A-2009-0011Multiple interactions involved in stem cell homeostasis

ESC regulators

Oct4 Nanog Tbx3

Wnt signaling

Tcf3 Tle1 Fzd5

Epigenetic regulators

Jarid2 Phc1 N-myc

RNA bindingprotein

Dppa5

Telomereassociated

Rif1

Tumorsuppressor

Trp53bp1

Oct4Nanog Tcf3

Pluripotency Differentation

Oct4

Nanog

Tcf3

Oct4

Oct4

Sox2Sall4

GCNFLRH1

TÁMOP-4.1.2-08/1/A-2009-0011Antagonistic 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

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mRNA regulation of stem cell gene expression

Other factors

Oct4Sox2 Nanog

mRNAs

AAAAAAAAAA

Alternatively spliced mRNAs

AAAAAAAAAA

Intergenic spliced mRNAs

AAAAAAAAAA siRNAs?

Other RNAs?

miRNAs

Intergenic transcripts

Antisense transcripts

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TF regulation forself-renewal/differentiation• Oct3/4, Nanog, Sox2, Stat3: maintenance of

proliferation• Cdx2: Inhibitory cross-interaction with Oct3/4

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Reprogramming: Induction of pluripotency in iPS cells

Target genes

Epigenetic modifiers

Transcription factorsSox2Oct3/4 Klf4

c-Myc

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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

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Sequential maturation and regeneration of pluripotency

EctodermMesodermEndodermPluripotent cell

Pluripotent cell

Ectodermprogenitor

Neuronalprogenitor

Mature neuron

Pluripotent cell

TÁMOP-4.1.2-08/1/A-2009-0011Differentiation-associated commitment and reversibilityDifferentiation 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

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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.