Comparison of several attachment methods for human iPS, embryonic and adipose-derived stem cells for tissue engineering

Abstract

As actual stem cell application quickly approaches tissue engineering and regenerative medicine, aspects such as cell attachment to scaffolds and biomaterials become important and are often overlooked. Here, we compare the effects of several attachment proteins on the adhesion, proliferation and stem cell identity of three promising human stem cell types: human adipose-derived stem cells (hASCs), human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Traditional tissue culture polystyrene plates (TCPS), Matrigel (Mat), laminin (Lam), fibronectin (FN) and poly-L-lysine (PLL) were investigated as attachment protein surfaces. For hASCs typically cultured on TCPS, laminin resulted in the greatest cell attachment and proliferation with largest cell areas, indicating favourability by cell spreading. However, mesenchymal stem cell markers indicative of hASCs were slightly more expressed on surfaces with lowest cell attachment, corresponding to increased cell roundness, a newly observed attribute in hASCs possibly indicating a more stem cell-like character. hESCs preferred Matrigel as a feeder-free culture surface. Interestingly, hiPSCs favoured laminin over Matrigel for colony expansion, shown by larger cell colony area and perimeter lengths, although cell numbers and stem cell marker expression level remained highest on Matrigel. These data provide a practical reference guide for selecting a suitable attachment method for using human induced pluripotent, embryonic or adipose stem cells in tissue engineering and regenerative medicine applications.

Figure 1. Light micrographs and growth curves of the stem cells on each of the 5 culture surfaces

A–B) hASCs proliferated most on Lam (red line) and FN (green line), even more than on the traditional tissue culture plate (blue line). C–D) hESCs attached and proliferated well on Mat (purple line) and Lam (red line), and less well on FN (green line); hESCs did not attach to TCPS nor PLL (blue and orange lines). E) hiPS attached and grew on Mat, Lam and FN, but not on TCPS nor PLL (black arrow indicates small, singular attached stem cell; white arrow indicates previously attached cell detaching and dying); F) hiPSCs had highest cell/colony numbers on Mat (purple line), closely followed by Lam (red line); FN (green line) showed moderate iPS cell attachment initially but declined to zero by day 3; TCPS (blue line) results are hidden by PLL (orange line) results- both showed no hiPSC attachment. TCPS = tissue culture polystyrene; Lam = laminin; FN = fibronectin; Mat = Matrigel; PLL = poly-L-lysine. Scale bar = 100 μm.

Figure 2. Stem cell morphometry and relative gene expression on the different culture surfaces

Morphometric results for area, perimeter length and circularity. A) hASCs exhibited largest cell areas on laminin, closely followed by fibronectin; on surfaces where cell attachment was low, such as Matrigel, hASCs had high circularity indicating little cell spreading and low preference to that surface. B) hESCs preferred Matrigel as an attachment surface, evidenced by high cell/colony area and perimeter measurements, along with low circularity or high cell spreading. C) hiPSCs preferred laminin over the standard feeder-free culture surface Matrigel, shown by largest cell/colony areas and perimeter lengths, and lowest circularity. * indicates statistical significance, p<0.05. D) Quantitative real-time polymerase chain reaction results for stem cell gene expression for hASCs at day 5; and for hESCs and hiPSCs at day 8. hASCs were probed for gene expression of mesenchymal stem cell markers CD29 (green bars), CD90 (orange bars) and Sca-1 (purple bars). ASC gene expression of these genes on the different proteins was consistently high, except on fibronectin. hESCs and hiPSCs were probed for Nanog (blue bars), Oct4 (red bars) and Klf4 (green bars) expression. ESC gene expression was highest on laminin and Matrigel, while iPSC gene expression was highest on Matrigel and PLL. TCPS = tissue culture polystyrene; Lam = laminin; FN = fibronectin; Mat = Matrigel; PLL = poly-L-lysine. Error bars = standard deviation.