Embryonic Stem Cells Cultured in Microfluidic Chambers Take Control of Their Fate by Producing Endogenous Signals Including LIF

Abstract

It is important to understand the role played by endogenous signals in shaping stem cell fate decisions to develop better culture systems and to improve understanding of development processes. In this study, we describe the behavior of mouse embryonic stem cells (mESCs) inside microfluidic chambers (microchambers) operated under conditions of minimal perfusion. mESCs inside microchambers formed colonies and expressed markers of pluripotency in the absence of feeders or pluripotency-inducing signals such as leukemia inhibitory factor (LIF), while mESCs in standard cultureware differentiated rapidly. In a series of experiments, we demonstrate that remarkable differences in stem cell phenotype are due to endogenous production of LIF and other growth factors brought upon by cultivation in confines of a microchamber in the absence of perfusion (dilution). At the protein level, mESCs produced~140 times more LIF inside microchambers than under standard culture conditions. In addition, we demonstrate that pluripotent phenotype of stem cells could be degraded by increasing the height (volume) of the microchamber. Furthermore, we show that inhibition of LIF in microchambers, via the JAK/STAT3 pathway, leads to preferential differentiation into mesoderm that is driven by bone morphogenetic protein (BMP)-4. Collectively, we demonstrate for the first time that it is possible to design a cell culture system where stem cell fate is controlled solely by the endogenous signals. Our study may help shift the paradigm of stem cell cultivation away from relying on expensive exogenous molecules such as growth factors and toward designing culture chambers for harnessing endogenous signals. STEM CELLS 2016;34:1501-1512 SIGNIFICANCE STATEMENTCell secreted molecules play an essential role in modulating self-renewal and differentiation of embryonic stem cells (ESCs). To date, ESCs have not been thought capable of secreting enough growth factors to maintain their phenotype. We investigated the role of endogenous leukemia inhibitory factor in controlling self-renewal in mouse ESCs confined to small volumes and found remarkable differences compared to cells in standard cultureware. Our study demonstrates that in a small volume endogenous signaling is sufficient to control stem cell fate, and can be selectively manipulated to direct differentiation. This may help shift the stem cell culture paradigm away from relying on expensive exogenous signals and toward harnessing of endogenous signals through optimal design of the cell culture platform.

Harnessing endogenous signals from hepatocytes using a low volume multi-well plate

“…This enhancement was shown to be the function of local volume or cell-to-volume ratio and was in part driven by the accumulation of endogenous hepato-inductive signals including HGF 12 . It is worth noting that effects of low volume cultures are not limited to hepatocytes and have been observed by us recently for stem cells as well as cancer cells 13, 14 . However, microfluidic channels used in these previous reports leave something to be desired in terms of simplicity of use, throughput and scale-up.…”

“…Our studies were informed and stimulated by previous reports in the field suggesting that microfluidic channels may provide a different way of culturing cells where endogenous signals may be manipulated and possibly enhanced 27-30 . Employing microfluidic chambers our lab has previously confirmed that accumulating endogenous signals were the key drivers of phenotype enhancement for a variety of cell types from undifferentiated embryonic stem cells to highly differentiated hepatocytes 12, 13 .…”

Ductular reaction-on-a-chip: Microfluidic co-cultures to study stem cell fate selection during liver injury

“…2a). We have previously modeled transport in a microfluidic device of a similar geometry and have determined that glucose levels were comparable to standard large volume cultures18. The oxygen tension was expected to be significantly better inside microfluidic devices because of a short distance between the cells and the gas permeable PDMS membrane comprising the roof of the device.…”

“…This is being attributed to enhanced accumulation of endogenous growth factors and autocrine signals in confined volumes of microfluidic chambers operating under diffusion dominant transport conditions (low flow regime). Such enhanced autocrine signals have been observed in stem cells182223, cancer cells24 and in primary hepatocytes1925.…”

“…Several groups, including ours, have demonstrated that microfluidic devices hold significant promise for cell cultivation and analysis1718192021. Beyond well appreciated advantages of decreasing the need for cells and reagents, such devices elicit improved cell phenotype and function.…”