“The Good into the Pot, the Bad into the Crop!”—A New Technology to Free Stem Cells from Feeder Cells

A variety of embryonic and adult stem cell lines require an intial co-culturing with feeder cells for non-differentiated growth, self renewal and maintenance of pluripotency. However for many downstream ES cell applications the feeder cells have to be considered contaminations that might interfere not just with the analysis of experimental data but also with clinical application and tissue engineering approaches. Here we introduce a novel technique that allows for the selection of pure feeder-freed stem cells, following stem cell proliferation on feeder cell layers. Complete and reproducible separation of feeder and embryonic stem cells was accomplished by adaptation of an automated cell selection system that resulted in the aspiration of distinct cell colonies or fraction of colonies according to predefined physical parameters. Analyzing neuronal differentiation we demonstrated feeder-freed stem cells to exhibit differentiation potentials comparable to embryonic stem cells differentiated under standard conditions. However, embryoid body growth as well as differentiation of stem cells into cardiomyocytes was significantly enhanced in feeder-freed cells, indicating a feeder cell dependent modulation of lineage differentiation during early embryoid body development. These findings underline the necessity to separate stem and feeder cells before the initiation of in vitro differentiation. The complete separation of stem and feeder cells by this new technology results in pure stem cell populations for translational approaches. Furthermore, a more detailed analysis of the effect of feeder cells on stem cell differentiation is now possible, that might facilitate the identification and development of new optimized human or genetically modified feeder cell lines

Protein expression analysis of neuronal differentiation markers.

<p>Immunocytochemical analysis of microtubulin associated protein 2 (<i>MAP2;</i> cyctoplasma, green), neuron-specific protein (<i>NeuN;</i> nucleus, blue), and neuron-specific enolase (<i>NSE</i>; cytoplasam, red) at phase V in <i>f-f</i>SCs and <i>st</i>SCs.</p

Analysis of feeder cell contamination during neuronal differentiation of murine embryonic stem cells.

<p>Feeder cells were transfected with the neomycine resistance (<i>nr</i>) gene. A. RT-CR and nested RT-PCR analysis of <i>nr</i> gene expression (NR) during neuronal differentiation of murine ESCs. Displayed are images of representative results following RT-PCR analysis. GAPDH was used as a control. B. Percentage of feeder cell contamination was evaluated by <i>nr</i> gene expression analysis of predefined ratios of stem and feeder cells. Displayed are representative results of PCR products. C. Following logarithmic transformation signal density is plotted as a linear function against feeder cell number (n = 4).</p

Protein expression analysis of neuronal or cardiomyocyte differentiation markers.

<p>A. GFP expression in <i>f-f</i>EBs and <i>st</i>EBS at day 14 (GFP under the control of αMHC-promoter). B. Immunocytochemical analysis of nestin expression at phase V in <i>f-f</i>SCs and <i>st</i>SCs.</p

Gene expression analysis of neuronal differentiation markers by semi-quantitative RT-PCR.

<p>A. Displayed are images of representative results following RT-PCR analysis. B. Quantitative analysis of neuronal and cell lineage marker expression in feeder-freed stem cells (<i>f-f</i>) and stem cells grown under standard conditions (<i>st</i>) (±SEM, n = 6). <i>grey: st</i>SCs; black: <i>f-f</i>SCs.</p

Automated aspiration of stem cell colony fractions from feeder cell monolayers.

<p>Panel i.–v.: Automated documentation of a representative aspiration procedure using the CellCelector™/Soft Imaging System Cell^D. lane i.–v.: Determination of feeder cell contamination by RT-PCR expression analysis of nr mRNA.</p

Quantitative analysis of time dependent increase in cross sectional dimensions of <i>f-f</i>EBs and <i>st</i>EBs (±SEM; n = 20).

<p><i>diamant</i>: <i>f-f</i>EBs; <i>triangle</i>: <i>st</i>EBs in hanging drops; <i>square</i>: <i>st</i>EBs in V-96 wells.</p