厦门大学细胞生物学课件
<< CELL BIOLOGY >>
--Embryo Development and
Cell Differentiation
袁立
教授
（厦门大学生命科学学院）
Sections of this chapter
• Introduction
• Processes of embryonic development
å Mechanisms of cell differentiation
å Modern research implicated in
Embryology and Cell Biology
3. Mechanisms of cell differentiation
Cell differentiation occurs in
multicellular organisms
Human:
1015 cells,
>265 cell types
3.1 An overview on cell differentiation
Number and type of cells in several species
Species
Volvox (团藻)
Porifer (海绵)
Hydra (水螅)
Planaria (涡虫)
Human
Cell numbers
102
103
105
109
Cell Types
2
5-10
10-20
100
1015
265
Cells type: functionally and morphologically
specialized
Cell differentiation
¾ Definition:
A developmental process in which structures
and functions become increasingly specialized.
¾ Stem cells:
Cells that have the ability to divide for indefinite
periods in culture and to give rise to specialized
cells.
Self-renew or Differentiation
Cell differentiation in monocellular organisms
Mating: n + n t 2n
e.g. Cell differentiation in yeast Spore formation: 2n t n + n
Differences of monocellular organisms
from multicellular organisms
¾ The former is mostly to adapt to their living
environment; and the later is to construct
tissues and organs to carry out various
functions.
¾ The process and the mechanism are much
less complicated in the former than in the
later.
Cell differentiation in multicellular organisms
A developmental process in which structures
and functions become increasingly specialized.
• In this process, young, immature cells take on individual
characteristics and reach their mature form and function. The
process is thus often marked by a change in the microscopic
appearance, or morphology, of the cell.
• Cell differentiation is a stepwise process. Cells go through a
series of progressive restrictions of developmental potential and
get into commitments to certain differential pathways.
• Once differentiated, these specialized cells are usually terminal
and nondividing, although some may be induced to divide
following injury (dedifferentiation), and transdifferentiation may
happen under certain abnormal settings.
• Change of gene expression is the main molecular base of cell
differentiation. --Tissue-specific genes need to be activated.
Technical terms related to cell differentiations
• Restriction - lost of developmental potential of a cell (occurs
with the process of differentiation)
• Determination - commitment of a cell to a particular path of
differentiation. (Early stage of differentiation-generally
irreversible, but in the case of marginal discs of drosophila,
transdetermination may occur)
• Induction - qualitative changing of a tissue induced by
signals from other tissues (cell differentiation in a group level)
• Intercellular communication - signals exchanging among
cells (to ensure cell differentiation in a concordance)
• Specialization - the structural adaptation of some body part
for a particular function (‘cell differentiation’ in the developing
embryo)
Any questions on these terms?
3.2. Stem Cells
Cells that have the ability to divide for indefinite
periods and to give rise to specialized cells.
Defining properties:
1. It is not itself terminally
differentiated (that is, it is not at the
end of a pathway of differentiation).
2. It can divide without limit (or at
least for the life time of the animal).
3. When it divides, each daughter has
a choice: it can either remain a
stem cell, or it can embark on a
course that commits it to terminal
differentiation
3. Mechanisms of cell differentiation
Potentiality of stem cells
¾ Stem cell:
Totipotent: Can form
every cell type, including
germ cells
Pluripotent: Can form
many different
derivatives, but not
germ cells
Multipotent: Can form a
few different cell types
Toti > Pluri > Multi
全能>多能>专能
Totipotent changes during embryonic
development
Totipotent cell
Pluripotent cell
(Inner cell mass)
Are hemapoitec cells pluripotent or multipotent? Why?
Cell Determination
Totipotent- pluripotent – determined – differentiated.
3.3. Mechanisms of cell differentiation
Asymmetry of cell division
August Weismann proposed that nucleus
of the zygote contained a number of
special factors – determinants, and that as
the zygote underwent cell division， there
would be an unequal distribution of these
determinants in the daughter cells.
(1834-1914)
Mosaic development
According to Weismann, the egg is a mosaic of
discrete localized determinants, and through
asymmetrical cell division, the daughter cells
progressively become different from each other.
1888, Wilhelm Roux’s experiment to investigate
Weismann’s theory of mosaic development.
(1850-1924)
“Development of the frog is based on a mosaic mechanism,
the cells having their character and fate determined at each
cleavage.” ---- Autonomous specification
Conditional specification
Regulative development :
---Cell specification is determined by signals
from it’s environment or other cells. --1892
(1867-1941)
Hans Driesch’s
Experiment confirmed
“Regulative development”
Ability of the embryo
to develop normally
even when some
portions are removed
or rearranged
Natural examples of regulative development
Nemoria arizonaria
Araschnia levana
Summer
Spring
Changes with day length & temp.
Changes with food in-take
Any one gives more examples of regulative development ?
Three mechanisms of cell differentiation
Differentiation of gene loss and amplification
• Differential gene loss and Selective gene amplification
are first revealed in lower organisms; In higher animal
like human, there are few cases.
• In higher animal like human, the differential ‘gene loss’
could happen during the cleavage, and there are
actually by no means of real gene loss, since we know
now the genome in all somatic cells is the same, thus
the ‘asymmetry of cell division’ even in the cleavage is
mainly implicated in cytoplasmic contents.
• In higher animal selective gene amplification could
happen temporarily, e.g., rDNA in Xenopus (1000x);
however, this has no important impact for developing
different cell types.
• In human, both gene loss and amplification are mainly
implicated in differentiation of immunity cells
‘Selective/differential gene expression’, appears
to be the dominant mechanism for cell to
differentiate into different types, due to the fact
that various cell types contain same genome but
have different expression profiles.
Anyway, all the three mechanisms modifies gene
expression that drives some cells different from
others in the same organism.
3.4. Differential gene expression
All cells have the same DNA!
Why do we have so many different cell types?
♦ Genes could be switched “on” or “off”
Different cells transcribe different sets of genes
A
B
C
D
E
F
G
H
I
J
Skin cell
Blood cell
Brain cell
Same genome, different transcriptome.
House-keeping and luxury genes
Human cells contains about 22,500 genes,
only 1/10~1/5 are expressed in a functional cell.
9House-keeping genes:
Expressed in all cell types, essential for all cells, responsible
for the routine metabolic functions (e.g. respiration).
9Luxury genes:
Tissue-specific genes, expressed in special cells, making
one cell type different from another cell type.
♦ Differential gene expression is more than
“switch on and off”
It can be different genes (off/on), expression level (up/down-regulation) or different isoforms of a gene.
Gene expression is regulated at multiple levels
Transcription
¾Structure of chromosome - e.g., condensation
¾Chemical modifications - e.g., methylation
¾Regulatory proteins -bind to control regions in DNA
¾External signals - hormones and growth factors
RNA processing
¾Splicing isoforms - function variations of gene products
Translation
¾mRNA longevity - 3’UTR ( e.g., AU - rich elements)
¾Poly A tail
¾Hormones (e.g. Prolactin to casein gene, 2x, 25x)
Post-translation
¾Cleaving some domains - e.g. proinsulin
¾Removing protecting proteins - e.g., Dorsal
¾Localization - e.g. membrane proteins
¾Assembly with other proteins-e.g., hemoglobin
¾Binding to ions-e.g. calmodulin
¾Chemical modifications - e.g.. phosphorylation
RNAs play roles at all level of gene regulation
nuclus
1
DNA
Trascription 2
hnRNA
Splicing
3
ncRNA
4
cytoplasm
mRNA
5
Protein
The miRNA seem to be key
administers of our gene, and
involved in the process of
cell differntiation!
6
Degradation
7
Degradation
A type of non-coding small
RNA, named micro RNA
(miRNA) is recently fund to
function specially as
“regulatory RNAs”
Questions
What are stem cells, their characteristics
distinguish from fully differentiated cells?
What are mechanisms of cell differentiation,
and their evidence?
How would you integrate autonomous and
regulative specification together in the
course of cell differentiation?
How gene function could be regulated in
different cells?
4. Modern research implicated in
Embryology and Cell Biology
• Planting
• Embryo splitting
• Animal cloning
• Human cloning
• On stem cell
4.1. Planting
Plant cell is totipotent
4.1. Planting
Tissue culture
1.取材
2.材料消毒与接种
4.1. Planting
Growth induction
3.愈伤组织培养
5.诱导根生长
4.诱导芽生长
6.炼苗
4.1. Planting
Transplanting
7. 移栽
Obtaining big number
4.2. Embryo splitting
4.2 Embryo splitting
Hupothetical human and horse
clones
4.3 Animal cloning
-- Newt
Newt Somatic Cell Nuclear Transfer (Cloning)
Hans Spemann，Germany Embryologist，1869-1941
1928年
The first nuclear transfer
experiment
1935年 Nobel prize -embronic induction
1938年 Predicting the possibility of
advanced animal cloning
Newt nuclear transfer experiment
蝾螈和青蛙蟾蜍皆属于两栖纲，但属于不同的目。
4.3 Animal cloning
-- Tadpoles cloning (1952)
Robert Briggs
(1911-1983)
Thomas King
(1921-2000).
blastula cell
4.3 Animal cloning
-- Frog cloning (1962)
John Gurdon
(1933- )
had used the nucleus of
fully differentiated adult
intestinal cells to clone
South African frogs.
4.3 Animal cloning
--Higher animal cloning
In 1979, Karl Illmensee claimed to have cloned three
mice ,however, a succession of failed cloning attempts
were beginning to convince biologists that the cloning
of a mammal was impossible.
In 1986, Steen Willadsen cloned a cow using
differentiated, one week old embryo cells.
In 1996, Ian Wilmut and Keith Campbell cloned
the first organism ever to be cloned from adult
cells, Sheep-Dolly
4.3 Animal cloning
--Dolly
Removing the maternal
nucleus before nuclear transfer
Dolly: A lamb with no father
July 5,1996 ~ February 14,2003
Totipotent somatic
cell nucleus!
9 Dolly （1996）
Of the 277 adult
udder cells that they
used to perform
nuclear transfers , 29
grew into developing
embryos. Of these 29
embryos, one turned
into a successful
pregnancy, and on
July 5, 1996, Dolly,
the world's first
mammal cloned from
adult cells, was born.
9Background of Dolly
Scotland PPL Therapeutics
Ian Wilmut
Roslin Institute
Edinburgh
Keith cambell
1996年克隆羊多莉和
其生母苏格兰黑绵羊
多莉和她的子女
1998年多莉产下第一只小羊
2003年2月多莉因患关节炎及
肺部感染被执行安乐死
4.3 Animal cloning
-- Upsurge
Calf，05/07/1998,Japan Mouse，05/12/1999,USA
Monkey，
14/01/2001,USA
Cat，14/02/2002,USA
Pig，05/03/2000,USA
Mule，29/05/2003,USA
4.4. Human Embryo Cloning
• 2001 – First cloned human embryos (only to
six cell stage) created by Advanced Cell
Technology (USA)
• 2004* – First human cloned blastocyst
created and a cell line established (Korea)
Woo Suk Hwang
（黄禹锡）
Ethics scandal…….
9Korean University says cloning claim faked………..
9Investigators: Hwang never produced
patient-specific stem cells…………..
9ROK pioneer apologizes for ethics scandal………..
XX
中国的克隆研究现状
1963年 生物学家童第周对金鱼、鲫鱼进行细胞核移植
1990年5月 西北农业大学畜牧所克隆一只山羊
1992年 江苏农科院克隆一只兔子
1993年 中科院发育生物学研究所与扬州大学农学院合作，克隆一只山羊
1995年7月 华南师大与广西农大合作，克隆一头奶牛、黄牛杂种牛
1995年10月 西北农大克隆6头猪
1996年12月 湖南医大克隆6只老鼠,同年，中国农科院畜牧所克隆一头公牛犊
（以上为胚胎细胞克隆研究）
1999年 中国科学院动物研究所研究员陈大元成功地培育出了大熊猫的早期胚胎。
2000年6月，西北农林科技大学成功获得成年体细胞克隆山羊
2001年~2004年初，山东莱阳农学院、中科院动物所、中国农业大学等相继在
山东、新疆等基地获得几十头体细胞克隆牛出生
4.4. Human Embryo Cloning
-- Ethics debate
9Safety
1. Low success ratio
2. Imperfect of cloning animal
3. Premature senile
9Ethics
1. Therapeutic cloning
2. Reproductive cloning
4.5 On stem cells
Where to get the stem cells?
(1) Embryonic stem cells (ES)
(2) Adult stem cells
4.5 On stem cells
Embryonic stem cells
• Derived from embryos that develop from
eggs that have been fertilized in vitro and
then donated for research purposes with
informed consent of the donors
• Not derived from eggs fertilized in a
woman's body
• Are pluripotent, i.e., can differentiate into
any body cell type
4.5 On stem cells
Adult stem cells
• Have been found in:
– Brain
– Bone marrow
– Blood vessels
– Digestive tract
– Skeletal muscle
– Skin
– Liver
– Umbilical cord
• Are multipotent, e.g., hematopoietic stem
cells form blood components
4.5 On stem cells
Differences Between Adult and E Stem Cells
EMBRYONIC
STEM CELLS
Pluripotent
Differentiation
Potential
Ease of Culture Easy
Rejection
Potential
ADULT STEM
CELLS
Multipotent
Difficult grow in
large numbers
Yes (unless use None (if cells
from patient)
Therapeutic
Cloning)
Methods to obtain stem cells
4.5 On stem cells
Stem cell- for the therapeutic cloning
9isolated from the embryo, fetus,or adult,
9defined as undifferentiated cells
9can divide without limit
9when it divides each daughter has a choice
-it can remain a stem cell
-give rise to specialized cells that make up the
tissues and organs of the body
4.5 On stem cells
9ES culture
1. Irradiated mouse
fibroblast feeder cells
4.5 On stem cells
9ES culture
2. leukaemia inhibitory
factor (LIF白血病抑制因子) ,
maintain the
undifferertiation of ES
When remove the LIF,
ES can differentiate
quickly.
- LIF
4.5 On stem cells
Promises of stem cell research
4.5 On stem cells
Generating chimeric mouse
4.5 On stem cells
Glial ES contribute to development in
central nerve system
Brustle et al., Science 285: 754 (1999)
4.5 On stem cells
Heart Muscle Repair with Adult Stem Cells.
4.5 On stem cells
Ethic debate on embryonic stem cell
Life or cell mass ?
Destroy blastocyst,
Collect stem cells
One more step
to clone a man
¾ 人胚胎干细胞研究伦理指导原则
。。。。。。。
第四条禁止进行生殖性克隆人的任何研究。
第五条用于研究的人胚胎干细胞只能通过下列方式获得：
(一)体外受精时多余的配子或囊胚；(二)自然或自愿选择流产的胎
儿细胞；(三)体细胞核移植技术所获得的囊胚和单性分裂囊胚；
(四)自愿捐献的生殖细胞。
第六条进行人胚胎干细胞研究，必须遵守以下行为规范：
(一)利用体外受精、体细胞核移植、单性复制技术或遗传修饰获得
的囊胚，其体外培养期限自受精或核移植开始不得超过14天。
(二)不得将前款中获得的已用于研究的人囊胚植入人或任何其它动
物的生殖系统。
(三)不得将人的生殖细胞与其他物种的生殖细胞结合。
第七条禁止买卖人类配子、受精卵、胚胎或胎儿组织。
。。。。。。。
《人胚胎干细胞研究伦理指导原则》科技部’卫生部2004年01月
Questions
Do you think it is possible to clone a higher
animal without any defects with nuclei of
fully differentiated cell? Why?
Do you agree with the ideas to clone an animal?
Why? And how about a man?
How do you agree the regulations for the using
the embryonic stem cells issued by our
government?