Category: Cellular Technologies
Introduction: The role of niche configuration of embryonic stem cells (ESCs) in heterocellular culture with stromal cells on neural differentiation efficiency is unknown. We develop a heterocellular culture system containing ESC colonies of defined size microprinted on stromal cells. The resulting niche resembles embryonic development in terms of direct intercellular interactions and generates neural cells from ESCs. Using a comprehensive temporal gene and protein expression profiling approach, we show that size of ESC colonies disproportionately enhances neural cell differentiation efficiency.
Methods: Two immiscible phases of culture media were formed by dissolving polyethylene glycol (PEG) and dextran (DEX) at concentrations of 5.0% (w/v) and 6.4% (w/v), respectively. A robotic liquid handler was used to print 50 nl aqueous DEX phase drops containing mouse embryonic stem cells (mESCs) onto a mitotically-inactive layer of PA6 stromal cells immersed in the aqueous PEG phase. mESCs adhered to the stromal layer and grew to form isolated colonies. Effect of mESCs colony size on neural differentiation was investigated using a comprehensive temporal analysis of gene and protein expression of differentiating mESCs for 2 weeks using q-PCR and immunostaining, respectively.
Discussion: The high throughput non-contact microprinting approach enabled formation of nine isolated mESC colonies of similar size with a center-to-center spacing of 9 mm on stromal cells. Colonies of three different sizes were separately generated by microprinting 100, 250, and 500 mESCs in each printed DEX drop. For each colony size, a total of 360 colonies in 40 Petri dishes were simultaneously generated for subsequent gene and protein expression analyses. Expression of neural marker proteins closely agreed with the gene expression data and helped identify molecular regulators of colony size-mediated neural differentiation. For the first time, we observed a disproportionate increase in the expression of neural cell markers including Nestin, TuJ, NCAM, and TH with increase in mESC colony size, both at gene and protein levels. Therefore, unlike previous studies that attributed neural differentiation of mESCs to signaling with stromal cells, we found that increased autocrine signaling of mESCs has an additional, significant effect on neural differentiation efficiency in this niche.
Conclusions: Our novel microprinting technology enabled high throughput formation of standalone mESC colonies on stromal cells to elucidate the effect of colony size on differentiation to specific neural lineages. Protein and gene profiling coupled with biostatistical analysis allowed us to capture evolution of neural cells in this heterocellular culture system and elucidate molecular regulation of enhanced neural differentiation due to mESC colony size.
Acknowledgements: This research is supported by National Science Foundation (1264562).
Ramila Joshi– Graduate Research Assistant, The University of Akron, Akron, OH
Graduate Research Assistant
The University of Akron
I am a fifth year Ph.D. student at The University of Akron. I joined Dr. Tavana’s lab in 2013, after completing my B.S. in Biomedical Engineering from Nepal in 2011. Since 2013, I have been working on micro-engineering the embryonic stem cells (ESCs) niche to regulate their neural differentiation. Using an innovative cell microprinting technology to robotically localize ESCs on a layer of supporting stromal cells, I develop defined stem cell niches with controlled colony sizes and interspacing. I then use molecular analysis and statistical tools to identify the niche configuration that maximizes the neural cell yield, and to mechanistically understand the neural differentiation process.