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
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
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
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
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.
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)
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
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.
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
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
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
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)
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
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
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
TF regulation for
self-renewal/differentiation
• Oct3/4, Nanog, Sox2, Stat3: maintenance of proliferation
• Cdx2: Inhibitory cross-interaction with Oct3/4
Reprogramming: Induction of pluripotency in iPS cells
Target genes
Epigenetic modifiers Transcription factors Sox2
Oct3/4 Klf4
c-Myc
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
Sequential maturation and regeneration of pluripotency
Ectoderm Mesoderm
Endoderm
Pluripotent cell Pluripotent cell
Ectoderm progenitor
Neuronal progenitor
Mature neuron
Pluripotent cell
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
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.