In the drug discovery field, the “bottom-up” approach, based on structural considerations of known targets, has not been as fruitful as was once promised; in 2008, only 21 new drugs (i.e., small molecule) were approved by the United States Food and Drug Administration and 19 in 2009. Until recently, transformed human cell lines were used as the major cellular platform for pharmaceutical drug screening, sometimes referred to as the “top-down” approach. The major concern with the use of transformed cell lines is that compounds identified as “hits” may not have direct relevance to the normal biological processes being targeted; as a result, false leads consume resources set aside for the subsequent testing on animal models and in clinical trials. Use of normal primary human somatic cells for large-scale screening is not currently feasible because of the limited number of cells obtained from biopsy and subsequent propagation in vitro. Thus, there is a great need to develop large-scale screening platforms using nontransformed cell lines.
ES cells offer a potential solution to this bottleneck. ES cells can be grown in large numbers and maintained in a pluripotent state in vitro. They can also be induced in culture to differentiate into cells from all three germ layers in a relatively normal fashion that is faithful to development in vivo. Three properties make ES cells an ideal platform for drug discovery. First, ES cells can provide virtually inexhaustible quantities of target cells, which is necessary for screening of large numbers of molecules. Second, ES cells can differentiate into mature cells with phenotypes that mimic their counterparts in vivo. Third, compared with immortalized cell lines, ES cells and their derivatives will provide a much more accurate platform for the “top-down” drug screening approach.
Type 1 or 2 diabetes is caused by destruction of pancreatic insulin-secreting beta cells as a result of autoimmunity or functional loss from peripheral insulin resistance, respectively. Currently, relatively little is known about the cell nonautonomous regulations for beta cell proliferation and maturation. Using an ES cell platform to screen and identify growth factors, peptides or small molecule effectors that direct beta cell proliferation or maturation from their immediate progenitors has the potential to significantly impact diabetes therapy. To date, attempts to perform large-scale screening of small molecules for pancreatic-lineage cells from ES cells have only yielded chemicals that enhance the proliferation or commitment of the very early Pdx1-expressing progenitors from definitive endoderm.6
This is partially due to a lack of reliable and cost-effective assays to generate the later glucose-responsive insulin-expressing cells from ES cells.
In this study, we demonstrate that our ES cell to pancreatic differentiation protocol can provide a reproducible and cost-effective cellular assay for screening of beta cell effectors. Pancreatic-like cells derived from our system not only have correct gene expression patterns and glucose-responsive insulin secretion in vitro, but they also respond as expected in gain-of-function studies, suggesting our culture protocol produces committed beta-like cells and their immediate progenitors, which could serve as screening targets.
An important goal of the present study was to define target cell stages for future screening. The diagram in correlates the sequential timed gene activation of murine ES cells in our in vitro differentiation protocol (upper box) to development in vivo (lower box). Undifferentiated ES cells (equivalent to ICM at ~E3.5) became committed to epiblast-like cells around culture days 2–3. This was followed by the formation of mesendodermal progenitors around days 3–5. Definitive endoderm-like cells were formed around in vitro day 5 (equivalent to ~E8.5). From day 6 to 10 of culture (equivalent to ~E9.5 to 12.5), gut tube and pancreatic endoderm-like cells were present. Starting from in vitro day 11 (equivalent to ~E13.5 and thereafter), pancreatic progenitor cells emerged, followed by the appearance of various lineage-committed cells, such as exocrine, endocrine, and ductal cells (culture days 13–20).
Fig. 6. Cell stage map for our in vitro ES cell differentiation assay. Upper panel depicts stages of in vitro differentiation. The stage designation is based primarily on information obtained from kinetic studies from two murine ES cell lines in this report ( (more ...)
To screen beta cell effectors, day 16 and later culture would be appropriate targets, because cells at this stage are amenable for further maturation. This is supported by the observations that cells at this stage displayed an upward trend for a number of pancreatic genes, including those for endocrine, exocrine, ductal, and pancreatic transcription factors ( and Supplementary Fig. 1
). Additionally, insulin messages and percentage of insulin 1-EGFP+
cells continued to increase after day 20 in vitro
(). Finally, day 20 cultures gave rise to well-organized pancreatic-like cells in vivo
(). As well, cells from this stage responded to MafA induction in expressing maturation markers () and had increased secretion of C-peptide in response to glucose stimulation in vitro
(). Together, these data demonstrate that the function of beta-like cells developed in our later stage of culture can be further enhanced by maturation factors and that these cells could be suitable targets for screening of beta cell effectors.
for in vitro
differentiation of beta-like cells utilize several recombinant growth factors and expensive chemicals to stimulate the formation of definitive endoderm, gut endoderm, and pancreatic cells. Compared with these protocols, our ES cell differentiation system offers several advantages for screening experiments. First, our protocol utilizes EB formation in the early differentiation stage (first 6 days), which can be easily scaled-up for a lower cost, in contrast to the adhesion monolayer culture with microcarrier technologies, which needs to be further investigated. Second, during the specific differentiation stages, relatively inexpensive chemicals or factors were used sequentially to induce endoderm and pancreatic development, including monothioglycerol (from day 0 to 6), exendin-4, and nicotinamide (used at day 13 and thereafter). The only relatively expensive item is the recombinant activin βB (used at day 13 and thereafter), and we are currently testing whether activin βB can be omitted or replaced by small molecules in this assay system. Third, compared with the human ES cell platform, the murine differentiation system provides a shorter project turnover time and ultimately offers advantages in cost-effectiveness, because glucose-responsive insulin-secreting cells from human ES cells require a longer time period to differentiate and mature in vitro
(unpublished results). In addition, in some countries, the use of the human ES cell platform carries patent issues and political concerns that may inevitably affect the progress of research. Lastly, our culture protocol generates beta-like cells that do not coexpress glucagon () and respond dynamically to glucose stimulation in vitro
(), suggesting that functional beta-like cells were generated. Our data strongly suggest that the use of these cultures as targets for screening will result in a high likelihood of identifying positive hits for bona fide
beta-cell regulators. We recognize that the use of murine cells is a limitation of our system, given that the positive hits may not necessarily translate to a similar function in human cells. However, this may be overcome by a secondary screening assay using human cells in vitro
. In conclusion, our ES cell differentiation assay is a valuable tool that can be used as a primary screening platform, which has the potential to significantly expedite the productivity of drug discovery in the treatment of diabetes.