Cell Differentiation

Advanced Molecular and Cellular Biology

Institute of Biology and Anatomy

國防醫學院
生物及解剖學科
徐佳福 助理教授
Room 5363; TEL: 18717
 Cell Differentiation
• A complex multistep process of cell
specialization that begins with the installation of
a genetic programme, named determination,
specific for a cell lineage.
• Development of the differentiation programme
includes the cell-type specific silencing of some
genes and the expression of other genes, that
regulate the biological functions associated with
the cellular type and that distinguish the
specialized cells.
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 Cell Differentiation
• Terminal differentiation is the end stage of this
process where the cells irreversibly lose their
proliferative capacity and which represents a
form of negative control of growing.
• Regulating molecules interact to produce the
correct balance between cellular multiplication
and differentiation during embryogenesis and
the normal behavior of an adult.

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 Cell Differentiation
• Cancer is a process in which changes in
regulating circuits are produced, such as
– proliferation control, the balance between
cellular survival and programmed cell death
(apoptosis),
– the communication with neighbouring cells
and with the extracellular matrix,
– angionenesis, and finally,
– the migration of tumoural cell, the invasion
and metastasic dissemination.

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 Cell Differentiation
• Embryology
• Stem cells
• Niche
• Epithelial-mesenchymal transition
• Dedifferentiation, transdifferentiation
• Reprogramming
• Tissue engineering

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Development of the blastocyst lineages

(a) Summary of gene expression patterns

during blastocyst formation.

(b) Developmental potentials of mouse

embryonic stem cells in vitro.
Nanog maintains self-renewal of mouse embryonic
stem cells and represses primitive endoderm fate.
Forced expression of Gata6 (in red) induces
differentiation to primitive endoderm.

(c) Developmental potentials of human

embryonic stem cells in vitro.
Treatment with BMP4 (in red) promotes
differentiation to trophectoderm. Nanog maintains
self renewal of human embryonic stem cells and
represses extra-embryonic fate (trophectoderm and
primitive endoderm). Upon overexpression (in red),
Nanog promotes differentiation to primitive ectoderm.

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Specification of primary
germ layers and
their derivatives

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Ways of stem cell speciation and differentiation. Solid arrows indicate experimentally
proven and Dashed arrows hypothesized ways of cell speciation and/or differentiation

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Schematic representation of stem cell
Differentiation pathways

(A) and (B) depict classical linear

Differentiation pathways from a
multipotent stem cell to differentiated tissue
progeny.

(C) Depicts the process of de-differentiation

when a presumably terminally differentiated
cell can revert to a less-differentiated cell type
and then give rise to new mature progeny.

(D) Depicts the process of trans-

differentiation, when a presumably
terminally differentiated cell can give
rise to other related and, in some
instances, unrelated mature cell types

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 Restoring stemness

The hypothesis envisions taking cells from a patient in

need of tissue replacement, exposing them in vitro to
factors that reprogram them to a state of multipotency,
and subjecting the reprogramed cells to differentiation protocols
designed to generate specialized cells. This protocol, if
successful, could provide autologous transplants for the patient.

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 Cell Differentiation

Bone

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Transcriptional control of osteoblastic, chondrocytic, adipocytic and myocytic differentiation

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 Differentiation factors in chondrocytic and osteoblastic differentiation

1. Sox9, together with Sox5 and

Sox6, regulates differentiation
of chondrocytes.
2. Runx2 and Osterix control
osteoblastic differentiation.
3. Maturation of chondrocytes to
hypertrophy is controlled
positively by Runx2 and
negatively by Sox9. As Runx2
has a role in both chondrocytic
and osteoblastic differentiation.
4. Osterix is likely the factor
that provides specificity in
osteoblastic differentiation.

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Bipotential adipo-osteoprogenitor cells
• Osteoblasts, differentiated to the point of already
expressing OCN, are able to undergo rapid
differentiation events that lead to essentially 100% of the
formerly osteoblastic cells expressing adipogenesis
when they are transfected with the nuclear receptor
family member peroxisome proliferator-activated
receptor γ2 or PPARγ2
• Although osteocalcin (OCN) is a very late marker of
osteoblast maturation, data are consistent with
osteoblastic cells being able to transfifferentiate into an
adipogenic phenoty, an outcome of significant clinical
interest in osteoporosis and aging or immobilized
skeleton.
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Control of Osteoblast Development
• Cbfa1/Runx2:
– A member of the runt homology domain transcription factor
family, play a crucial role in osteoblast development.
– Regulate OCN.
– Haploinsufficiency in mice and humans leads to the cleidocranial
dysplasia phenotype.
– The earliest of osteoblast differentiation markers currently known.
– Up regulated in cultures treated with bone morphogenetic
proteins (BMP).
– Not a master gene, in contrast to MyoD and PPARg2.
– Necessary but not sufficient to support differentiation to the
mature osteoblast phenotype

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 PPARγ2
• CCAAT/enhancer-binding (C/EBP) protein
family, play a key role in adipocyte
differentiation.
• Is able to transdifferentiate osteoblastic
cells to adipocytes, may do so via its
ability to downregulate Cbfa1

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 Sox9
• essential for chondrocyte differentiation,
expression of various chondrocyte genes,
and cartilage formation

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 Indian hedgehog (Ihh)
• regulates chondrocyte differentiation
• not normally expressed in
intramembranous bones
• osteoblast differentiation occurs normaly in
these bones in Ihh -/- animals
• may be regulator of Cbfa1 and osteoblast
development, but in a skeletal site-specific
manner
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 Osteoprogenitor Cells and Regulation of
Osteoblast Differentiation and Activity
• Osteoprogenitor Cells
– Cell-specific macromolecules: type I collagen, OCN,
OPN, BSP. Transcription factors: Cbfa1, AP-1 family
members, Msx-2, Dlx-5.
– Estimates by limiting dilution have indicated that
these osteoprogenitor cells are relatively rare in cell
populations digested from fetal rat calvaria (i.e., < 1%)
and rat and mouse bone marrow stroma (i.e., 0.5-1 x
10-5 of the nucleated cells of unfractionated marrow or
< 1% of the stromal layer) under standard isolation
and culture conditions.
– Plasticity between mature cell phenotypes normally
considered indicative of terminal differentiation can
contribute to osteoblast pools, at least in vitro
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 Differentiation of Osteoprogenitor
Cells to Osteoblasts
• Bone nodule formation can be subdivided into three
stages: (i) proliferation, (ii) extracellular matrix
development and maturation, and (iii) mineralization.
• ALP increases and then decreases when mineralization
is well progressed;
• OPN peaks twice during proliferation and then again
later but prior to certain other matrix proteins, including
BSP and OCN;
• BSP is expressed transiently very early and is then
upregulated again in differentiated osteoblasts forming
bone;
• OCN appears approximately concomitantly with
mineralization

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 Differentiation of Osteoprogenitor
Cells to Osteoblasts
• All these osteoblast-associated marker are
upregulated prior to the cessation of proliferation
in osteoblast precursors except OCN, which is
upregulated only in postproliferative osteoblasts
• Differentiation is well progressed before
osteoblast precursors leave the proliferative
cycle
• 12 markers, seven transitional stages
• There is heterogeneity among osteoblast
developmental pathways and/or the resulting
osteoblasts

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 Differentiation of Osteoprogenitor
Cells to Osteoblasts
• Only two markers of nine sampled, ALP and PTH1R,
appeared to be “global” or ”ubiquitous” markers
expressed by all osteoblasts in vivo
• All other markers analyzed were expressed differentially
at both mRNA and protein levels in only subsets of
osteoblasts, depending on the maturational state of the
bone, the age of the osteoblast, and on the environment
• Not all mature osteoblasts develop via the same
regulatory mechanisms nor are they identical molecularly
or functionally. The makeup of different parts of bones
may be significantly different

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Transcription Factor, Hormone, Cytokine, and Growth Factor Regulation of

CFU-F and Osteoprogenitor Cell Proliferation and Differentiation

• Regulaiton by Transcirption Facotrs

– At least three and perhaps more Cbfa1
isoforms that have been described.
– Cbfa1 regulates OCN expression and
osteoblast differentiation.
– Cbfa1 is regulated by BMPs, but not directly.
Cbfa1 regulates itself directly via binding on
its own promoter

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 Endochondral bone formation

a, Mesenchymal cells condense.

b, Cells of condensations become chondrocytes (c).

c, Chondrocytes at the centre of condensation stop

proliferating and become hypertrophic (h).

d, Perichondrial cells adjacent to hypertrophic

chondrocytes become osteoblasts, forming bone
collar (bc). Hypertrophic chondrocytes direct the
formation of mineralized matrix, attract blood vessels,
and undergo apoptosis.

e, Osteoblasts of primary spongiosa accompany

vascular invasion, forming the primary spongiosa (ps).

f, Chondrocytes continue to proliferate, lengthening the

bone. Osteoblasts of primary spongiosa are
precursors of eventual trabecular bone; osteoblasts of
bone collar become cortical bone.

g, At the end of the bone, the secondary

ossification centre (soc) forms through cycles of
chondrocyte hypertrophy, vascular invasion and
osteoblast activity. The growth plate below the
secondary centre of ossification forms orderly
columns of proliferating chondrocytes (col).
Haematopoietic marrow (hm) expands in marrow
space along with stromal cells.

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Indian hedgehog (Ihh)/parathyroid hormone-related protein
(PTHrP) negative-feedback loop
1. PTHrP is secreted from perichondrial
cells and chondrocytes at the ends of
long bones.

2. PTHrP acts on receptors on

proliferating chondrocytes to keep the
chondrocytes proliferating and,
thereby, to delay the production of Ihh.
When the source of PTHrP production
is sufficiently distant, then Ihh is
produced. The Ihh acts on its receptor
on chondrocytes to increase the rate of
Proliferation.

3. through a poorly understood

mechanism, stimulates the production
of PTHrP at the ends of bones.

4. Ihh also acts on perichondrial cells to

convert these cells into osteoblasts of
the bone collar.

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 Osteoclastogenesis

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Hormonal control of bone resorption

a, pro-resorptive and calcitropic factors

b, anabolic and anti-osteoclastic factors

1) RANKL expression is induced in
osteoblasts, activated T cells, synovial
fibroblasts and bone marrow stromal
cells, and subsequently binds to its
specific membrane-bound receptor
RANK, thereby triggering a network of
TRAF mediated kinase cascades that
promote osteoclast differentiation,
activation and survival.
2) Conversely, OPG expression is
induced by factors that block bone
catabolism and promote anabolic effects.
OPG binds and neutralizes RANKL,
leading to a block In osteoclastogenesis
and decreased survival of pre-existing
osteoclasts.
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 Leptin signalling pathways

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 Cell Differentiation

Kidney

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 The Kidneys
• Intermediate mesoderm (中間中胚層)
• Nephrogenic cord (腎索)
• 原腎(Pronephros): 發展不完全且不具功能，相當於魚類
的腎臟，在胚胎時期第四週出現，然後不久便退化，但大
部分的原腎管仍然存在，且成為中腎的一部分。
• 中腎(Mesonephros): 發展完全，但只具短暫功能，相當
於兩棲類的腎臟，在胚胎時期第四週末出現。過渡時期腎
臟(Interim kidney):具有腎絲球和中腎小管[mesonephric
tubules，形成睪丸的輸精管(efferent ductules of the
testes)]，再第一個三月期末退化。中腎管(Mesonephric
duct):在男性形成性腺管，而女性的輸卵管
(paramesonephric or Mullerian duct)則與腎臟的胚胎發育
無關。

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 腎臟和輸尿管
• 後腎(Metanephros): 成為後來真正的腎臟，在胚
胎時期第五週出現，然後四週後開始有功能，由
中胚葉(mesoderm)發育而來。後腎胚質
(Metanephric blastem) 發育為腎元，後腎憇室
(metanephric diverticulum) 發育為輸尿管、腎
盂、腎盞、和收集管。
• 胚胎時期腎臟由許多腎錐體融合而成，故外表有
許多個隆 (Lobulation)，出生後腎臟變大主要為近
側曲小管增長和，間質組織增加所導致。

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Stages of renal branching morphogenesis and nephron formation

Ureteric bud outgrowth

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Stages of renal branching morphogenesis and nephron formation

Ureteric bud branching and nephron induction

Ureteric bud branching and nephron
formation

Nephron arcade formation

Induction of metanephric mesenchyme by

the branched ureteric bud

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Stages of renal branching morphogenesis and nephron formation

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A model for cell lineage during ureteric
bud (UB) branching, based on the
behavior of wild-type green fluorescent
protein (GFP)+cells and Ret-/- cells in
chimeric kidneys.

(A) A UB branch contains trunk (green) and tip (blue)

domains.
(B), The trunk first elongates without acquiring tips cells,
while the tip grows, stimulated by GDNF/Ret signaling, to
form the ampulla.
(C) As branching ensues (C and D), the ampulla and the
adjacent trunk epithelium are remodeled by hypothetical
forces (arrows in C), causing the trunk epithelium to form
the proximal side of two new trunks (asterisks in E), while
tip cells form the two new tips and the distal epithelium of
the new trunks (black bracket).
(F), Summary of lineage relationships between tip and
trunk cells; tip cells generate both tip and trunk cells, while
trunk cells generate only more trunk cells during terminal
branching (but presumably can form tip cells during lateral
branching).

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 Cell Differentiation

Pancreas

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Schematic diagram of pancreatic development in the mouse

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A simplified hierarchy of transcription factor expression in the developing pancreas

purple, bHLH;
green, Maf proteins;
orange, Hnfs;
gray, unidentified factors.

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Morphogenesis of the mouse pancreas and the expression of key genes during embryonic development

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 A simplified overview of pancreas cell lineage determination.

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Thank you !