Isolation and ex vivo expansion of human bone marrow stromal stem cells

Doctoral Dissertation uoadl:1305830 71 Read counter

Unit:
Τομέας Υγείας - Μητέρας - Παιδιού
Library of the School of Health Sciences
Deposit date:
2013-03-29
Year:
2013
Author:
Θεοδοσάκη Μαρία
Dissertation committee:
Χρούσος Γεώργιος, Καναβάκης Εμμανουήλ, Καττάμης Αντώνιος
Original Title:
Απομόνωση και εξωσωματικός (ex vivo) πολλαπλασιασμός στρωματικών στελεχιαίων κυττάρων από τον ανθρώπινο μυελό των οστών
Languages:
Greek
Translated title:
Isolation and ex vivo expansion of human bone marrow stromal stem cells
Summary:
SSC are produced in vitro when BM is cultured under standard conditions. They
are characterized by fibroblast-like morphology, adherent growth, specific
phenotype and capacity to differentiate into adipocytes, osteocytes and
chondrocytes.
With the aim to produce ex vivo expanded SSC, in a number sufficient to treat
GVHD, but also, the detection of the most appropriate population of BM cells
for the treatment of patients with chronic ischemic cardiomyopathy, we studied
two distinct populations of SSC (CD105+ and CD271+) and two HSC (CD34+ and
CD133+), as they considered until recently.
We obtained BM samples from 23 healthy donors. The above populations were
isolated using positive immunomagnetic selection method. The isolated cells
were placed in ex vivo expansion and differentiation cultures towards
osteogenic, chondrogenic and adipogenic lineages. Surface marker expression of
cultured SSC were analyzed by flow cytometry, while chondrogenic, adipogenic
and osteogenic differentiation were confirmed by COL2A1, LPL and ALP
expression, respectively.
All selected populations generated fibroblast-like, adherent cells which
maintain their expansion potential for about 5 weeks and 9 sequential passages.
The expansion rate and the total number of cells at the end of the culture,
does not seem to vary considerably, while, the immunophenotype (CD29+, CD90+,
CD44+, CD13+, CD105+, Class I – HLA+, CD14-, CD19-, CD31-, HLA-DR-, CD34- and
CD45-) and the ability to differentiate into adipocytes, osteocytes and
chondrocytes, as demonstrated by gene expression of LPL, ALP and COL2A1
respectively, confirm their identity as SSC.
Significant variation of the above results were observed during cultivation of
a CD271+ sample, in which spontaneous generation of diverse cell clusters after
third passage, inhibited further proliferation of SSC.
This finding led us to the choice of CD105+ cells in order to receive
sufficient numbers of SSC to treat GVHD, whereas, as far as patients with
chronic ischemic cardiomyopathy are concerned, the selection of CD34+ and
CD133+ cells provide all SC that promote myocardium regeneration.
Keywords:
Stromal Stem Cells, Human Bone Marrow, Ex Vivo Expansion
Index:
No
Number of index pages:
0
Contains images:
Yes
Number of references:
242
Number of pages:
136
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