Characterization of human mesenchymal stem cells by the appearance of integrins and functional analysis of collagen I-binding integrins

Introduction: Human mesenchymal stem cells (hMSC) are easily
obtainable from bone mar-row and possess the ability to
differentiate into osteoblasts. Therefore, they have been
sug-gested as a suitable source for bone regeneration. HMSC are
equipped with a variety of in-tegrins that mediate essential
cell-matrix interactions. Collagen I represent approximately 90% of
the bone protein content. Cell attachment to collagen I is mediated
by three members of the integrin receptor family named a1b1, a2b1
and a11b1 integrins. The main aim of this doctoral thesis was to
investigate the basal expression of those integrins in hMSC and to
func-tionally analyze the knockdown effect of a single collagen
I-binding integrin on hMSC behav-ior in vitro. Materials and
methods: HMSC were cultured on collagen I-coated surface. A
lentiviral trans-fer of a1-, a2- and a11-specific shRNA was applied
for downregulation of the corresponding integrin mRNA. Quantitative
PCR and western blot analysis were used to assess the basal
ex-pression, knockdown efficiency and integrin compensation.
Colorimetric adhesion assay was used for estimation of the extent
of cells attachment. HMSC spreading and migration was ob-served by
time lapse experiments. JC-1 staining was used for investigation of
the initiation of apoptosis. Results: Quantitative PCR were used to
assess the basal expression of collagen I-binding integrins in
three hMSC donors. We found that these integrins are differently
expressed as integrin a11 had the highest and integrin a2 the
lowest expression. Next, we applied lentiviral delivery of
target-specific short hairpin RNA (shRNA) in order to knockdown
each of the collagen I-binding integrins and compared them to the
hMSC transduced with a sequence against a non-human gene
abbreviated as shRNA control. We achieved significant
downregulation (> 80%) of the collagen I-binding integrin mRNA
and protein. Subsequently to the transduction, we did not noticed
pronounce morphological cell changes, however, a clear decrease of
a2- and a11-knockdown hMSC numbers was observed during cultivation.
Using a quantitative adhesion assay, we estimated that 120 min
after plating only 30% of integrin a11-deficent cells were able to
attach to collagen I. In contrast, at the same time point, 70% of
integrin a2-knockdown hMSC were attached while integrin a1- and
shRNA control hMSC have already reached 100% cell adhesion.
Furthermore, a time lapse-based investigation showed that integrin
a1- and shRNA control hMSC need approximately 35 min to fully
spread on collagen I. In contrast, integrin a2- and a11-knockdown
hMSC took approximately double more time for spreading in
comparison to shRNA control hMSC. Additionally, we analyzed the
migration capability of the four different hMSC lines. The average
path which integrin a1- and shRNA control hMSC passed was
approximately 170 µm with mean speed of 11.5 µm/h. In parallel
integrin a2 and a11-deficient hMSC migrated to a distance of
approximately 70 µm with a velocity of 5 µm/h. Since it was
observed a lost of a2- and a11-deficient hMSC, next we performed
JC-1 staining that visualizes mitochondrial leakage, a hallmark of
apoptosis. The majority of integrin a2- and a11-knockdown hMSC
exhibited mitochondrial leakage whereas integrin a1- and shRNA
control hMSC showed intact mitochondria. Finally, we used
quantitative PCR to investigate whether there were compensatory
effects between the three integrin receptors. We detect that
knockdown of integrin a1 led to upregulation of a2 and a11.
Similarly, when integrin a2 was downregulated, integrin a1 and a11
expression increased. Interestingly, knockdown of integrin a11
caused only a slight increase in integrin a1 but not in a2
expression. We also observed that upon osteogenic stimulation,
integrin a2 and a11-deficient hMSC further reduced in number and
did not mineralize the matrix even on a single cell level.
Moreover, our preliminary investigation in hMSC-derived from
osteoporosis suffering patients showed a tremendous downregulation
of integrin a2. Conclusions: Our results strongly suggested that
integrins a2b1 and a11b1 mediate an indis-pensible signaling for
hMSC. Once these receptors were ablated from cell surface, hMSC
re-duced their spreading, adhesion, migration and survival rates.
Our integrin knockdown mod-els can be used for further
investigations and understanding of the integrins a2b1 and a11b1
importance and signaling in hMSC and hOB since we observed a strong
downregulation of integrin a2 expression in osteoporosis.