In vitro proliferation and differentiation of human embryonic stem cells

Doctoral Dissertation uoadl:1308494 201 Read counter

Τομέας Υγείας - Μητέρας - Παιδιού
Library of the School of Health Sciences
Deposit date:
Καραγιαννίδου Αγγελική
Dissertation committee:
Καθηγητής Εμμανουήλ Καναβάκης
Original Title:
In vitro πολλαπλασιασμός και διαφοροποίηση ανθρώπινων εμβρυονικών βλαστικών κυττάρων
Translated title:
In vitro proliferation and differentiation of human embryonic stem cells
The cells deriving from the early stages of the human embryos (morula and
blastocyst) are characterized as Embryonic Stem Cells (ESCs) and are the
ancestors of all tissues of the human body. The unique properties of
pluripotency, self-renewal and high proliferative potential, characterizing the
ESCs, define them as the most immature cells of human life.
The first successful isolation and culture of ESCs was described in 1981 by the
groups of Evans and Kaufman, who isolated cells from the inner cell mass (ICM)
of mouse blastocyst and cultured them on mouse fibroblasts. A key factor for
the successful culture of ESCs was found to be the presence of a suitable cell
substrate. Seventeen years later, the isolation of the first cell line of human
embryonic stem cells (hESCs) was published by Thomson and his group. The
culture of hESCs was possible using adaptive culture substrate, which consisted
of rat fibroblasts, and culture medium enriched with factor bFGF (basic
Fibroblast Growth Factor).
Culturing hESCs without substrate under the effect of suitable growth factors
cause them to differentiate into cell types originating from all three germ
layers. Their plasticity and the potentially unlimited ability of self-renewal
makes them an important tool of cell biology for both understanding
embryogenesis, and designing a model of differentiation in various tissues in
vitro, with a future goal to be able to form the building cells for the in
vitro construction of organs and tissues, or for use in cell-regenerative
treatment of many chronic diseases such as juvenile diabetes, Parkinson's
disease, Alzheimer and other neurodegenerative diseases. For the clinical use
of these cells, however, it is necessary to develop safer and simpler
production methods.
Given the wide range of future applications of hESCs in clinical practice, our
goal was to establish the methodology for culture and handling such cellular
systems and thus the development and standardization of cultivation methods of
hESCs and production of mature cells from them, in order to study: ( a) the ex
vivo proliferation and (b) the ability of certain cell lines for in vitro
differentiation to tissues of mesodermal (hematopoietic, stromal cells),
endodermal (definite endoderm) and ectodermal (nerve cells) origin with the use
of specific growth-differentiative factors in liquid and semisolid cultures.
At first we cultured the embryonic cancer cell line 2102Ep Cl.2 / A6 P48
(GlobalStem Inc.) in order to carry out an assessment of culture materials and
factors of proliferation, since it constitutes a reliable reference material to
standardize the methods of cultivation and control of pluripotency at human
normal cell lines of embryonic cells.
Then we studied the cell lines of human embryonic cells HUES-9p18 and
HUES-7p15. A comparison of their proliferation potential, using as substrate
mixture of extracellular matrix proteins (matrigel) and human mesenchymal stem
cells (MSCs) revealed no statistically significant differences. Afterwards, we
examined their ability to maintain their pluripotency by studying the
expression of transcription factors Oct3 / 4, Nanog, Sox2, Rex-1 using the
technique of RT-PCR and the expression of cell surface antigen SSEA-4 using
flow cytometry and we noticed that cells in cultures were maintained in
undifferentiated state.
The next phase of this study involved the differentiation of these cell lines
to cell types of ectodermal (neural progenitor cells), mesodermal (progenitor
haemapoetic cells and mesenchymal stromal cells) and endodermal (cells of
difinitive endoderm) origin, by the use of suitable differentiative factors.
The differentiation into all cell types was successful. Greater efficiency was
found at the differentiation to mesenchymal stromal cells and neural progenitor
cells. Next, we determined the genetic stability of undifferentiated and
differentiated hESCs using the method of molecular karyotype (Agilent 1x244K
και 4x180K CGH and SNPs arrays). Cells from both cell lines HUES-9 and HUES-7,
after 51 and 29 passages, respectively, have a normal karyotype (46XX and 46XY
respectively) but show genetic instability (deletions and duplications) in size
ranges from 100kb to 4.3 Mb. Moreover, the genetic stability was determined in
ESC-derived mesenchymal and neural cells and was found that they maintain the
same chromosomal structural abnormalities despite some slight variation in
size. There was no characteristics of in vitro neoplastic transformation, such
as uncontrolled proliferation of mature cells or selective growth advantage of
the genetically altered population. All of the above indicate that random
structural aberrations arise as a result of the effort of immature cells to
adapt to the culture (culture adaptation) and do not constitute DNA damages
affecting the proliferative and differentiative potential of cells.
As part of this thesis, for the first time in Greece, we developed the
methodology for culture and study of hESCs and we established effective methods
of producing differentiated cells in order to be used in pre-clinical
protocols, as well. Moreover, genetic instability of these cells was examined
both in the pluripotent undifferentiated stage and in their differentiative
derivatives. Based on the results of this study a genetic screening of each
cell population derived from differentiating hESCs cells is proposed.
Embryonic, Stem Cells, Diferentiation, Proliferation, Pluripotent
Number of index pages:
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Number of pages:
vii, 153

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