Abstract
BackgroundDifferentiation of mesenchymal stem cells to osteoblasts is widely performed in research laboratories. Classical tests to prove this differentiation employ procedures such as cell fixation, cell lysis or cell scraping. Very few studies report gentle dissociation of mesenchymal stem cells undergoing an osteodifferentiation process. Here we used this technique to reveal the presence of several cell layers during osteogenesis and to study their different properties.MethodsThrough the sequential enzymatic detachment of the cells, we confirm the presence of several layers of differentiated cells and we compare them in terms of enzymatic sensitivity for dissociation, expression of cluster of differentiation, cytosolic calcium oscillations and osteogenic potential. Adipogenic and neurogenic differentiations were also performed in order to compare the cell layers.ResultsThe cells undergoing differentiation formed one layer in the neurogenic differentiation, two layers in the adipogenic differentiation and at least four layers in the osteogenic differentiation. In the latter, the upper layers, maintained by a collagen I extracellular matrix, can be dissociated using collagenase I, while the remaining lowest layer, attached to the bottom of the dish, is sensitive only to trypsin-versene. The action of collagenase I is more efficient before the mineralization of the extracellular matrix. The collagenase-sensitive and trypsin-sensitive layers differ in their cluster of differentiation expression. The dissociation of the cells on day 15 reveals that cells could resume their growth (increase in cell number) and rapidly differentiate again in osteoblasts, in 2 weeks (instead of 4 weeks). Cells from the upper layers displayed a higher mineralization.ConclusionsMSCs undergoing osteogenic differentiation form several layers with distinct osteogenic properties. This could allow the investigators to use upper layers to rapidly produce differentiated osteoblasts and the lowest layer to continue growth and differentiation until an ulterior dissociation.
Highlights
Differentiation of mesenchymal stem cells to osteoblasts is widely performed in research laboratories
All results are expressed as the mean and Characterization of cell differentiation Before analyzing the layers formed in various Human adipose mesenchymal stem cell (haMSC) differentiations, we first confirmed that the cells had undergone every differentiation correctly
We show that haMSCs undergoing osteogenic differentiation form several layers
Summary
Differentiation of mesenchymal stem cells to osteoblasts is widely performed in research laboratories. Other methods start with the scraping or the lysis of the cells using trichloroacetic acid and SDS [9], sonication [16], trizol [19], RIPA buffer [17] or many freeze–thaw cycles [15] in order to perform DNA and calcium assays [16, 20, 21], to measure internal ALP by colorimetric assays [14, 15, 17, 21, 22] or to detect specific proteins by western blot analysis or mRNA by RT-PCR [6, 8, 9, 12] None of these techniques dissociated the living cells. Some studies [14, 23] used trypsin alone (until day 14 of osteodifferentiation, before the mineralization occurred) or trypsin–EDTA [24, 25] to harvest all of the layers in 2D cultures
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
