Cell differentiation is known as a process in which cells

become specialized. Pluripotency means the potential to become

any cell type and is a potential observed in stem or progenitor
cells at the beginning of the differentiation process. This potential is
slowly lost as the cell differentiates and gains specialized functions.
The differentiation process alters the cell dramatically, its shape,
size, and energy requirements. This process is not a linear and
irreversible process. Differentiation selects a subset of genetic
information to be expressed at different stages of the differentiation
process. Therefore, differentiated cells can be manipulated back to a
more primitive or stem cell-like state, by reprogramming it to
express a particular set of genes.
Cell differentiation is a result of the integration of different stimulus
in a spatiotemporal fashion. Cell differentiation is sensitive to both
mechanical and chemical stimulus from the environment. Improved
understanding of the differentiation process can allow us to
engineer the process to achieve desired outcome. This is especially
true when developing biomaterials as delivery carriers of the stem
or progenitor cells in vivo.

Role of Cytoplasm in Cell Differentiation

The importance of cytoplasm on cell differentiation has been

demonstrated in large number of experiments.

The egg of snail produces a lobe-like structure at the vegetal end

during cell differentiation which is known as Polar lobe. If this lobe
is excised, defective embryo is produced. Again, a coloured area is
produced in the amphibian egg during cell differentiation after
fertilisation. This is known as Grey Crescent. If the grey crescent is
injured it induces abnormality in the nervous system.

In case of amphibian egg, if the first cleavage is perpendicular to the

grey crescent and the resulting blastomere is separated, each
blastomere will produce a normal animal. But if the cleavage takes
place parallel to the grey crescent and if the two blastomeres are
separated—the one having grey crescent will produce normal
animal. Thus the differentiation of cell depends on the partitioning
of substances in the cytoplasm.
Another observation has been made in case of the embryonic
development of egg of the round worm, Ascaris. During the
development of embryo, the first cleavage occur perpendicular to
the animal vegetal axis.

When the animal pole of the new blastomere starts dividing, the
heterochromatic portion of the chromosome becomes degenerated.
The euchromatic portion of the chromosome is fragmented into
numerous small chromosomes by a process known as chromosome
diminution.

Thus chromosome diminution occurs during embryonic develop-

ment. Now Theodor Boveri made an experiment in which eggs are
centrifuged before cleavage in order to disturb the polarity of the
cell.

By centrifugation, the mixture of both animal and vegetal cytoplasm

occurs and the first cleavage occurs along the animal vegetal axis
not perpendicular to it. No chromosome diminution occurs—this
indicates the role of cytoplasm in chromosomal behaviour.

Again, in case of embryonic development of Drosophila, the

primordial germ cells generally arise from the posterior end of the
egg. Illmensee and Mahowald made an experiment by removing
some cytoplasm from the posterior end of the egg of Drosophila and
injected then into the anterior end of another egg. It has been noted
that germ ceils are then produced from the anterior end of the
injected egg.
Thus it can be said that the cytoplasm has an important role in
inducing differentiation in cells. Its role has also been noted in adult
cells. When the inactive nuclei of mature erythrocytes are injected
into the cytoplasm of active cells, the inactive nuclei become
transcriptionally active.

Similar effect has also been noted in other types of cells. All these
results clearly reveal that cytoplasmic factors are responsible for cell
differentiation. But in case of mammals, embryonic cells are
totipotent after the first cleavage division till the development of the
embryo.
This has been evidenced by segregating mouse or rabbit embryo at
8 or 16 cells stage. Each blastomere gives rise to blastocyst if the
proper cultural conditions are provided and these blastocysts will
form normal animal after implantation into the uterus of another
female rat. This property of totipotency of egg cell is due to the
homogeneity of its cytoplasm.

The retention of totipotency by an individual cell has been

demonstrated in case of plant systems. Any cell from any part of the
plant can be grown in culture where they form at first the mass of
undifferentiated tissue, known as callus.

This callus can be regenerated into a whole plant if the proper

concentration of the hormone is supplemented in the media. But
single animal cells after the first cleavage division will not form full
animal even if the proper nutrients and other conditions are given
in culture.

Genetic totipotency of animal cell has been demonstrated by John

Gurdon in the nuclear transplantation experiment in toad; showing
that each and every cell possesses all the genes required for the
development of the whole organism.

Still the development or differentiation into whole organism has not

been in animal system. Thus researchers thought that the pattern of
gene expression in each cell type is different, although all the genes
are present in each cell. So we see that the control of gene
expression in higher organisms, particularly animals, is very
complicated.

In other words, many types of post-transcriptional modifications of

the primary transcript of the gene are regulated in the formation of
the final functional gene product necessary for a particular cell
differentiation or development.

Two model organisms are used for the study of the specific genes
involved for a particular cellular development. One is the round-
worm, Caenorhabditis elegans, and another is Drosophila, C.
elegans.
These are used as a model in the developmental genetics
for the following reasons:

i. Short life cycle (3 days).

ii. Ease of maintenance like E. coli.

iii. Can reproduce by self or by cross- fertilization.

iv. Hermaphrodite (XX)—contains 5 pairs of autosomes and 1 pair

of X chromosomes.

v. The male (XO) contains 5 pairs of autosomes and a single X

chromosome.

vi. Haploid genome is about 8 x 107 bp.

vii. More than 600 genes have been identified.

viii. Easy to obtain homozygous populations, as self-fertilization is

possible.

ix. In-breeding is automatic in hermaphrodite population.

x. DNA transformation through microinjection at the selected stage

of development is possible in this animal.

The reproductive system of the hermaphrodite animal produces a

bilobed structure in early stages of development. The oocytes and
embryos in each lobe start developing from the distal end to the
uterus. All stages can be detected easily and thus microinjection of
DNA can be done at the selected stage of development.

Another important work on Drosophila made the discovery of new

class of genes called Homeotic genes. These genes were identified
through mutations where one part of the body is replaced by a
structure that is found somewhere else. This unusual thing occurs
during development of the embryo. The mutations of homeotic gene
(Antp) result in the formation of middle legs in place of antenna of
Drosophila.
The molecular analysis of homeotic gene has resulted in the
discovery of a 180bp sequence present in other homeotic genes.
This sequence is known as Homeobox. Since the homeobox is
present in higher animals also, gene cloning and sequencing has
been done from mice to man. The function of these genes has been
found to be the same as in Drosophila.

The homeobox genes are expressed in highly differentiated cells in

case of Amphibians and Mammals. Homeobox genes are found to
play very important role in developmental processes and they are
highly conserved during evolution.

Role of nucleus in cell differentiation

In cell and developmental biology, stem cells and their nuclei are
often described as “plastic”, referring to their ability to differentiate
into multiple lineages and activate diverse gene expression profiles.
This plasticity in gene expression in (embryonic) stem cells is
associated with distinct chromatin structure and epigenetic
modifications compared to differentiated cells. Only recently has it
become apparent that these changes are also reflected in altered
mechanical properties of stem cell nuclei.

Stem cells
While nucleosomal proteins, particularly core histones (i.e., H2A,
H2B, H3, H4), form stable complexes with chromatin in
differentiated cells, the mobility of these nucleosomal proteins is
significantly increased in embryonic stem cells, suggesting a more
accessible chromatin structure that could facilitate transcriptional
activation. Micropipette aspiration experiments on human adult
stem cells and hematopoietic stem cells demonstrate that nuclear
stiffness dramatically increases during induced neurectodermal
differentiation of these cells. Within 6 days of differentiation, the
nuclear stiffness of human embryonic stem cells increases 6-fold,
reaching values typical for fully differentiated cells such as
embryonic fibroblasts. Time-lapse imaging of mouse embryonic
stem cells and primary mouse embryonic fibroblasts reveals that
embryonic stem cells show severe dynamic fluctuations in nuclear
shape, again suggesting that stem cell nuclei also have increased
physical plasticity. Interestingly, the stiffness of human
hematopoietic stem cells, which have lower developmental plasticity
than embryonic stem cells, falls between that of undifferentiated
embryonic stem cells and fibroblasts. As discussed in more detail
below, the reduced nuclear stiffness of embryonic and
hematopoietic stem cells can most likely be attributed to two
distinct contributions: (i) Changes in lamin expression and (ii)
altered chromatin structure
Altered lamin expression in embryonic stem cells
In contrast to most differentiated cells, embryonic stem cells do not
express any A-type lamins, and hematopoietic stem cells express
only lamin C, not A. As lamins A and C are the main contributors to
nuclear stiffness, particularly lamin A, these differences in lamin
expression alone could be sufficient to explain the reduced nuclear
stiffness in embryonic and hematopoietic stem cells. Supporting
this idea is the finding that reducing lamin A/C expression in
human epithelial cells by shRNA mediated RNA interference
resulted in nuclear stiffness comparable to that of hematopoietic
stem cells.
Altered chromatin mechanics in embryonic stem cells
Embryonic stem cells have an increased mobility of core
nucleosomal proteins, possibly reflecting a more accessible
chromatin configuration. Detailed analysis of intranuclear
deformations during micropipette aspiration of epithelial cells in
which lamin A/C expression had been reduced by RNA interference
revealed that chromatin has fluidic-like viscoelastic properties
consistent with the compliance of single chromatin fibres. It will be
interesting to conduct a direct comparison between the viscoelastic
properties of embryonic stem cells and terminally differentiated
cells in future experiments.
Importantly, the contribution of lamins and chromatin organization
might not be independent factors, as loss of A-type lamins results in
loss of peripheral heterochromatin, suggesting a direct involvement
of A-type lamins in chromatin organization.
Granulocytes
While most differentiated cells express at least one of the A-type
lamins, neutrophils stand out as an example of differentiated cells
that reduce the expression of A-type lamins and other nuclear
envelope proteins to optimize nuclear shape and structure for their
cellular function. Neutrophils are recruited to sites of infections,
where they exit the bloodstream to enter the affected tissue. These
processes require neutrophils to squeeze through narrow openings
only a few micrometres wide, i.e., smaller than the nuclear
diameter. During granulopoiesis, i.e., the differentiation of
promyelocytes into mature neutrophils, cells develop increasingly
lobulated nuclei; this modulation in nuclear morphology is
associated with almost complete loss of lamin A/C expression and
increased expression of lamin B receptor (LBR). These adaptations
result in a highly malleable nucleus, making it ideally suited for
trans endothelial migration and passage through narrow tissue
spaces. Recent findings suggest that the cellular plasticity might be
further enhanced by the downregulation of cytoskeletal proteins
such as vimentin and proteins involved in nuclear-cytoskeletal
coupling. As in the case of embryonic stem cells, these changes are
also associated with alterations in chromatin structure, again
suggesting a tight mechanistic link between nuclear envelope
composition and chromatin organization.