ABSTRACTS – ORAL PRESENTATIONS - AMCA, spol. s r.o.

P82. FLOW CYTOMETRIC ANALYSIS AND MULTIPLE LINEAGE DIFFERENTIATION OF
ADIPOSE TISSUE-DERIVED STEM CELLS OF DIABETIC PATIENTS
Kollárová Z. 1,2 Kubinová Š. 1,2 , Turnovcová K. 1,2 , Syková E. 1
kollarova@biomed.cas.cz
1
Institute of Experimental Medicine ASCR, Prague, Czech Republic
2
2 nd Faculty of Medicine, Charles University, Prague, Czech Republic
INTRODUCTION
Adipose tissue is an abundant source of autologous adult stem cells that may open
new therapeutic perspectives for the treatment of type I. and type II. diabetes and
their complications. However, it is unclear whether the mesenchymal stem cells of
diabetic patients, constantly influenced by hyperglycaemia, have the same properties as
mesenchymal stem cells from non-diabetic patients. To study the differencies between
adipose tissue-derived stem cells (ATSCs) isolated from diabetic and non-diabetic
patients, cell surface markers were analyzed by flow cytometry and multiple lineage
differentiation and the expression of adipose and osteogenic specific genes were studied.
MATERIAL AND METHODS
ATSCs from patients with type II. diabetes (n=6) and type I. diabetes (n=4) were compared
to ATSCs from non-diabetic patients. The tissue samples were processed by enzymatic
digestion, and cells were cultivated in full cultivation media containing MesenCult
(Stemcell technologies, Vancouver, Canada) supplemented with 10% fetal bovine serum
(PAA Laboratories, Coelbe, Germany), and Penicilin/Streptomycin (200 µg/ml; Lonza,
Cologne, Germany). Cells from the third passage were characterized by their expression
of surface markers using fluorescence-activated cell sorting (FACS). A total of 2x10 6 cells
were resuspended in 1ml PBS and incubated with their appropriate antibodies for 20
minutes at room temperature. Antibodies against human antigens CD31, CD34, CD45,
CD29, CD105, CD73, CD90, CD235a, CD271, HLA-ABC, HLA-DR+DP, VEGFR2, CD146 and
antifibroblast were used. Analysis of surface markers was performed on a FACSAria TM
Flow Cytometer and their expression was evaluated on FACSDiVa Software.
Standard protocols to differentiate the ATSCs into adipocytes, osteoblasts and
chondrocytes were followed to confirm the cells’ multipotency. Cell differentiation into
various lineages was confirmed by specific staining and real-time q-PCR. The expression
of ACTB, GAPDH, ALPL, PPAR6, Runx2and LPL was analyzed on a real-time PCR System
StepOnePlus (Applied Biosystems, San Francisco, CA,USA).
RESULTS
Flow cytometry revealed the expression of CD29, CD73, CD90, HLA-ABC and fibroblast
markers and no expression of CD31, CD34, CD45, CD235a, CD271 or HLA-DR+DP was
found in ATSCs from either diabetic or non-diabetic patients. Also, adipose differention
potential was well-preserved in both groups of cells. However, in diabetic patients the
osteogenic differentiation potential of ATSCs was decreased, which was confirmed by
specific staining for Alizarin red and the gene expression of Runx2 and LPL.
Grant support: GA ČR P304/11/0653, GA ČR 9304/11/0731, GA ČR P304/11/P633
Analytical Cytometry VII 187