A hypothetical
model of artificial differentiation
Felix Mircea Brehar,
MD*,
* First Neurosurgical department – “Bagdasar-Arseni” Emergency Clinic Hospital,
Bucharest, Romania
e-mail: felixbrehar@yahoo.com
Abstract
The lack of specific embryonic
signals of the adult organism drastically limits the potential of stem cells
differentiation and the possibility to create a complex array of tissues.
Therefore, it becomes clear that for synthesizing a complex structure, composed
of several different tissues, one should start with a specific population of
undifferentiated cells (stem cells for example), without using the complicate
pathways of signals specific of embryonic development. In this way, one will
shortcuts several stages of embryonic development and will reach earlier the
state of terminal tissue differentiation.
In this paper, I will present an alternative hypothetical model of differentiation
that hypothesizes the differentiation of cells from the aspects of gene
expression by two types of „interface proteins”. These proteins make complex at
the X sites of DNA. When cell division occurs, the state of protein-DNA complex
becomes asymmetric and results two cells, which express different set of genes.
Key words: stem cells,
embryonic development, differentiation.
Introduction
The purpose of regenerative medicine
is to replace those tissues damaged by different injuries. In this respect,
scientists tried to use two relative similar ways of approaching this issue. One
way is to use the embryonic stem cells. These cells are totipotent
so, they can differentiate in virtual all tissues found in an adult organism. Nevertheless,
this approach raises two different problems. First, an adult organism lacks of
those signals found in the embryo during development, which are very important
for a proper final differentiation. Second, this approach will imply a human
embryonic manipulation that raises serious ethical problems (8). Another way is
to use the mesenchymal stem cells (17, 13). These
cells are not totipotent, so they can differentiate
in a limited number of adult tissues. In both cases, the lack of specific embryonic
signals of the adult organism limits the potential of stem cells
differentiation. Therefore, it becomes clear that one have to create a complex
structure composed of several different tissues starting from a specific population
of undifferentiated cells (stem cells). Before the final commitment of the
cells, several specific pathways such Delta-Notch, wnt-Frizzled,
Hedgehog, can be used for a fine regulation of tissues architecture.
First,
I will present a hypothetical model of differentiation and how one can build
several specific structures (such somites) using this
model. Then I will propose several kinds of approaches to develop this model.
The description of the hypothetical
model
According to the proposed theory, an undifferentiated
cell will synthesize in G1 phase, two
different types of nonhistonic proteins (gene
regulatory proteins): type one and type two (Figure1). These are in
inactive state and are spread aleatory
in the entire cell. Still in this phase, cell synthesizes other two types of
proteins, called interface proteins: type A and type B, and two enzymatic systems. Each one of
these interface proteins is make up of two components: an identical fragment
for both, which is complementary with a part of DNA chain, called X site, and another fragment which is
complementary with the corresponding fragment of the other interface protein. Therefore,
these two types of protein molecules couples each other, as heterodimers,
due to the complementary regions (Figure 1). The connection between the
interface protein (as a heterodimer) and X site
depend entirely of the promoter activity of the interface protein gene. After
several normal cell divisions, the concentration of interface protein will be
high enough to connect to X site. When the synthesis of DNA (S phase) starts,
each new forming chain will have attached, at X site level, a certain type of
interface protein (the connection will be made aleatory,
however because the interface proteins are coupled as heterodimers,
type A will be attached by one newly formed DNA chain, and type B by the other DNA
chain ). After mitosis, each DNA chain will expose at the level of X site, different
surface potential interaction, because of two different parts of interface proteins
connected to X site.
After the cell division, in daughter cell A, the interface protein type A is
connected at X-sites DNA’s level. This one, by helping of complementary
fragment (which have several enzymatic sites, like protein kinase
domains), will activate one of two
enzymatic systems (the two protein kinases found aleatory in both daughter cells, just like nonhistonic proteins A and B). The result is the amplification
of the initial signal and activation (by phosphorylation)
of gene regulatory protein types 1 (although, in both daughter cells, there are both
protein types). The kinase also phosphorylates
the interface proteins and promotes its degradation by ubiquitilation, therefore
the kinase protein will be no more activated. Because
of this negative feedback loop, the interface proteins and kinases
will mutually inactivate each other, short after they activate the gene
regulatory protein. The gene regulatory protein can be
synthesized in a way to interact with a repeating x sequence, which is part of introns of a
certain set of genes. The x sequence will function as an enhancer sequence and
will activate all genes that contain that sequence (Figure 1). We choose introns, because the majority of them are nonfunctional
sequences so the gene regulatory proteins will not interact with other important
repetitive DNA sequences (other enhancers or insulators, e.g.)
In the other daughter cell (cell B), the interface protein type B is connected to the X site of DNA. This interface protein,
by helping of the other protein kinase will activate, by phosphorylation,
gene regulatory protein type 2. This
can be also synthesized in a way to interact with another repeatable y sequence (Figure 1), localized at the
level of introns of another set of genes, which will
function as a specific enhancers activating all those genes. Therefore, one can
synthesize two different cellular lines starting from an undifferentiated cell using
only genetic mechanisms, without involving, other extrinsically factors.
This process of artificial induced-differentiation
can be separate in two stages. Two types of determined cells, type A and
type B, appear in the first stage. These are not different from a morphological
point of view but only because of the potential of activating different sets of
genes (Figure 2). For instance, cell A according to the previous model,
contains gene regulatory protein type I. This will activate gene a,
which synthesize receptors of membrane (R1), and gene b, which
synthesize another type of inactive nonhistonic
protein (gene regulatory protein) – type II (Figure3).
In the following
stage, the type A cells, spread in the
cellular population (resulted from repeated divisions of the pluripotent cells), will migrate one to another due to
receptors interactions (Figure 2). As the type A
cells interaction, the activation of receptors of membrane determines the
activation, through specific mediators, of nonhistonic
protein (gene regulatory protein) initially synthesized – type
II,
(Figure3). This will activate the genes c and d, which will
synthesize the specific proteins as well the gene e, responsible by its
own synthesis. In this moment, takes place the cellular differentiation. Now
the type A cells, which are different from the cells
of type B from morphological point of view, form a distinctive tissues structure.
Therefore, the
determinate cell has two variants (Figure2):
1. As a result of
interactions with the other cells of the same type (cell A with cell
A, and cell B with cell B) the process of cellular differentiation
starts and forms a certain types of tissues.
2. If this contact will
not occur, the process of differentiation does not takes place and the
determined cell may come back at the state of pluripotency,
after some divisions, because the gene regulatory protein (type I),
synthesized in the moment of cellular determination (Figure 3), does not
activate the gene which is responsible by its own synthesis. During cellular
differentiation, gene regulatory protein II does intensify the own
synthesis (positive
feedback), so the phenomenon of cellular differentiation becomes irreversible (Figure
3).
Discussions
The
main features of this model:
1. This model can make a link
between the number of cells division and the specific time of cells differentiation.
Because there is a single X site, the interaction between this site and
interface protein will depend entirely of interface protein concentration.
Therefore, the rate of interface protein synthesis will decide how many cycle a
cell need to undergo until the moment of differentiation.
2. As one can see, the cells A and
B behave in the first stage of differentiation, as they would be determinated cells. Each type of cells expresses a specific
type of receptor, without actually being different from biochemical and histological
point of view. They will differentiate only after each cell will contact other
cells of the same type, and the receptors will activate the positive feedback
loop. The result is the cells will maintain indefinitely the state of different
genes expression. If one cell of any type (A or B) is isolated during the first
stage, it will not be able to contact other cells of the same type. After a
specific period of time (when the interface proteins and non-histonic regulatory
proteins will be proteolytically cleaved), that cell
will go back in the previous undifferentiated state. Therefore, this model can mimic
the relation between determination and differentiation.
3. One can synthesize the gene
regulatory proteins II-1 and II-2 to action exactly like in figures 3 and 4, therefore to interact with a specific DNA sequence
enhancer and activate tissue specific regulatory proteins. For example: Myo D/Myf-5 (7, 16) for cells type A (to induce muscular differentiation)
and ESX factor (3, 9, 6) for cells type B (to induce
epithelial differentiation). In this way, one will produce two types of tissues
– one cycle of differentiation. Instead of this, one can synthesize these gene
regulatory proteins in such a way to interact with another z and µ DNA sequences
found in interface protein and nonhistonic proteins
(I-3, II-3 and I-4, II-4 types) gene
regulated sequence, for each one of already two determined type of cells A and
B. This will initiate another cycle of determination-differentiation for each type
of cells: A and B. One can repeat this cycle for n times. In these intermediary
states, the cells do not actually commit the differentiation into specific
adult tissues. Instead, they will express different receptors and different
types of signaling pathways that will modulate the interaction between cells
and the rate of cells multiplication. In this way, one can modulate the cells architectures
using specific signaling pathways, like Delta-notch, wnt-Frizzled,
Hedgehog, before the final commitment into specific tissues. At the end of the
entire process, the nonhistonic protein II-n will
specifically activate, for each type of cells, the tissue specific regulatory
proteins. Therefore, one may have 2ⁿ differentiated tissues (n the number
of differentiation cycle). However, this will complicate the procedure because
we need to synthesize a very large number of gene regulatory proteins.
4. One can decouple stage one from
stage two. Therefore, the cells can use this mechanism of expressing different
surface receptors for several cycles, without actually differentiate. If one assumes
that cells will also express, at a lower lever, a common type of receptor, and
all cells will have the same mitotic spindle orientation then, after several
cell cycles, the cells will group in a number of „balls” connected each other
along an axis. The number of group of cells will be equal with 2ⁿ, when n
is the number of cell cycles. Thus, this model can mimic another embryonic
phenomenon: the occurrence of somites (Figure 4).
It is known that the somites rise from the presomitic
mesoderm during early embryonic differentiation using a mechanism of cyclic
expression of a specific type pathways and receptors such Notch signaling, Wnt/beta-catenin and tyrosine phosphatase
(1, 4,
and 5). Moreover, because of the asymmetry of cell division, each „artificial somite” (in fact a group of cells kept in contact by expression
of a common type of receptor) can finally differentiate into a different types
of tissues without needing of extra cellular signals of a specific A-P axe
pattern (Figure 5)
5.
One can create, using this model, symmetrical and asymmetrical structures.
Assuming that these cells A and B
sort out in two group of cells, and each type was instructed in two different
ways: one group (cells A) to express a signal molecule, attached to the cells
membrane and the other cells (type B) to express a receptor for that signal
surface molecule. In this way, those few B cells which are in contact with type
A cells synthesize a small molecule in response of
receptors activation. This molecule will be a soluble one and will diffuse free
in the same way in both direction toward the group A and B of cells.
If both types of cells are instructed to behave in the same way at interaction with
the soluble molecule (express the same signaling pathway) then, the cells will
form symmetrical structures (Figure 6). If the cells behave in different ways (express
two different pathways) then the cells will form asymmetrical structures
(Figure 7). As one knows, the asymmetry between left and right side of the body
depend on the movement of cilia which
primary actions at the level of node (14, 11)
6.
One can mimic, using this model, the lateral inhibition phenomenon.
Let us suppose that, after the differentiation
of stem cells into cells A and B, the cells B will develop an asymmetric
differentiation: each cell B will divide into one cell B and one different cell
C. Then, the cell C will not express the receptors need for gathering and the
interface protein inside the cells will directly activate the gene regulatory
proteins (so the cells will directly commit a differentiation process). If, in
the same time, the cells B will divide for another 2 or 3 cycles and then
differentiate, then a small number of differentiated C cells will be scattered
among a larger number of B cells (Fig 8). This is a similar situation with the initially
uniform epithelial tissues differentiating in "salt-and-pepper",
regular spacing patterns which use the lateral inhibition mechanism (mediated
through Delta-Notch pathway) to produce it (12, 15).
How one
can apply this model in a real experiment?
For making this model to become
possible, a combination of genomic, proteomic and structural biology must be used. One must integrate a complex sequence of DNA (as
a plasmid construct) in the genome of the undifferentiated cells. The target
cells seems to be the mesenchymal stem cells because
are easy to obtain and their use does not raise any ethical problems. These
cells will be stimulated to divide using specific mitogen
factors. This complex DNA sequence should contain the following elements:
-
genes which code for interface protein.
-
genes which code for two different protein kinases which phosphorylate and
activate the gene regulatory proteins and phosphorylate
interface proteins and promote their degradation.
- genes
which code for two types of receptors.
There is no need for multiple alleles
of these genes, because the same proteins can be used
for many cycles. However, the promoter of gene, which synthesizes the interface
protein (and also the protein kinases and receptors),
need to have multiple enhancer sequences in order to be able to respond to
different gene regulatory proteins synthesized at every differentiation cycle
(Figure 9).
However, the promoter should lack
the sequence that respond to the gene regulatory protein synthesized in the
final cycle of differentiation. When the final differentiation step occurs,
there is no need for synthesizing the interface protein.
- genes
which code different gene regulatory proteins. The number of genes required
will depend, in this case, of the number of differentiation cycles. Let us
suppose that one wants to develop more than one cycle of differentiation. The
promoter of the gene, which synthesizes gene regulatory protein (nonhistonic protein) during the n cycle of differentiation,
should contain two specific DNA sequences. One activating sequence that respond
to the nonhistonic protein synthesized in the
previous cycle of differentiation (n-1 nonhistonic
protein) and another inhibitory sequence which will respond to the nonhistonic protein synthesized in the next cycle of differentiation
(n+1 nonhistonic protein) (Figure 10). Therefore, the
gene regulatory proteins activated at the final cycle of differentiation will
inhibit the synthesis of nonhistonic protein synthesized
at the previous cycle of differentiation and will activate the genes which code
for tissue specific gene regulatory proteins, that promote the final differentiation
step into specific array of tissue
s.
- genes
which code tissue specific gene regulatory proteins (like Myo
D, neurogenin, ESX factor, e.g.). These will be the
last genes activated by gene regulatory proteins at the end of the entire differentiation
process.
-
gene which codes for an integrase in order to
integrate the entire sequence into chromosome, and eventually a gene which
carry the AB resistance in order to select transfected
stem cells.
This
complex sequence must be integrated in a region of the
chromosome with constitutively active cromatin (near
a housekeeping gene for example).
Another
problem is to find the hypothetical X site. Because we need a unique sequence,
the X chromosome seems to be the ideal location (since only one chromosome is active
during interphase) and especially a region located
near one of its origins of replication site. In addition, the X site should contain
a long enough sequence of DNA (8-10 nucleotides) so this sequence to be unique
for the entire X chromosome.
The different proteins should have different
intracellular locations. Therefore, the interface proteins and nonhistonic protein type II should contain the special domain:
nuclear localization signals (Lys-Lys-Lys-Arg-Lys) (2),
necessary for intranuclear transport. However, the nonhistonic protein type I should not have this sequence,
so will remain only intracitoplasmatic, until the end
of cell cycle, when will be incorporate in the newly formed nuclei. The gene
regulatory protein should have at least two domains: one domain to interact
with the desire DNA sequences (x, y, z...) and the other domain for interacting
with chromatin remodeling proteins and histone acetyltransferase complexes and consecutively activating
the target genes. (10)
Conclusions
This alternative hypothetical model of differentiation
could be used as an alternative technique for “in
vitro” tissue engineering starting from undifferentiated cells, in order to
replace the extrinsecal factors and the complex bioscaffolds needed for creating a three-dimensional array
of tissues. Even if the plasmid construct presented above seems to be very
difficult to be synthetised in present days, the fast
progresses reported in proteomics and genomics could make this model of
differentiation to be realized easier in the following
years.
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