Preparation Of Micropatterned Surfaces And Study Of The Effect Of Cell-cell Contact On Stem Cell Differentiation

The relationship between cell communication and differentiation is a fundamental. problem of Cell Biology, which is closely related to Biomaterials and Regenerative Medicine. As cell studies are concerned, while differentiation of stem cells has been paid much attention to during the latest decade, relatively little is focused upon the relationship between cell differentiation and communication. Straightforward cell-cell contact is the most popular way of cell-cell interaction. It is, however, very hard to rule out the effects of soluble factors due to paracrine of cells and the effects of different concentrations of nutrients during cell culture, if one just examines differentiation of stem cells under a series of seeding concentration. An unambiguous research approach is thus desired.Among several types of techniques for fabricating micropatterns on different substrates, we eventually determined a micropattern which can localized cells for a long time. Based upon the micropatterning technique, the effect of cell-cell contact on lineage differentiation of rat bone marrow stem cells (rMSC) was revealed semi-quantitatively. Our methodology and results not only augment fundamental knowledge of Cell Biology, but also offer innovative means and instruments for cell research as well as for regenerative medicine.The main innovative results are listed as follows:1. To find a technique to prepare micropattern of adhesive contrast with a background resistant to cell adhesion for a long time. Considering that differentiation of stem cells must proceed for at least several days, an excellent adhesion-resistant background is necessary. We developed several methods to prepare micropatterns, including topographic PLGA film, a self-assembly monolayer (SAM) of poly(ethylene glycol) (PEG) molecules on glass surface, micropits formed with PEG hydrogel, micropattern on PEG hydrogel surface. Eventually, a micropattern prepared on PEG hydrogel surface was found to meet the requirement, which made examination of differentiation of localized stem cells available. 2. To establish a semi-quantitative analysis method to evaluate the osteogenesis level of stem cells. By using grey values on different adhesion microislands on micrographs, we established a semi-quantitative analysis of the activity of the expressed alkaline phosphatase (ALP). This method is likely to be applied in other semi-quantitative analysis of optical micrographs of stained samples.3. To put forward a unique methodology to examine the effects of cell-cell contact between stem cells per se on their lineage differentiation while ruling out the interference of soluble factors or cell seeding concentration etc. MSCs from neonatal Sprague Dawley (SD) rats are employed as demonstration. During osteogenesis, the cells are well localized on the micropatterns which prevent or ensure cell-cell contact persistently. Both osteogenic and adipogenic differentiations were found to be regulated by cell-cell contact, isolated cells exhibited less significant differentiation than paired or aggregated cells. For those stem cells in contact, extent of differentiation was fairly linearly related to extent of contact characterized by coordination number. Additionally, we revealed the existence of some unknown cues besides gap junction responsible for such effects of cell-cell contact. While cell studies based upon patterned surface have been reported in many literatures, this is, to the best of our knowledge, the first time to semi-quantitatively investigate the relationship between extent of differentiation of stem cells and extent of cell-cell contact.