Development of Cell Lines from iPSCs for Bioassays

The reprogramming of differentiated somatic cells to pluripotency holds great promise for drug discovery and developmental biology. Using immortalized cell lines for drug screening assays has its limitations, such as questionable relevance; and the use of primary cells is often hindered by supply difficulties. Thanks to pioneering work by the Yamanaka, Thompson, and other groups, the feasibility of creating iPSCs has generated an opportunity to provide cell lines with stem cell properties in a virtually unlimited supply [1, 2]. These cells can be derived into different cell types for specific assays, even with patient- or genotype-specific background. Technologies are being developed to produce re-differentiated cells of a number of lineages.

Take cardiomyocytes as an example. There are a number of conventional methods for inducing stem cells into cardiomyocytes: through embryoid body (EB) formation, co-culturing with visceral endoderm-like cell line (END-2), and monolayer caridomyocyte differentiation with defined growth medium and protein factors [3]. A recent publication showed that using appropriate concentrations of BMP4 and activin-A in BSA-containing medium cardiomyocytes might be achieved from iPSCs or ESCs in about 6 days [4].

Transdifferentiation, or direct reprogramming, by introducing a group of 3 cardiomyocyte-specific factors, investigators could directly program cardiac or dermal fibroblasts into cardiomyocyte-like cells [5]. Although much refinement and characterization of these directly reprogrammed cardiomyocyte-like cells, termed iCMs, will be needed before the process can become widely used, this work raised the possibility of quicker and perhaps more efficient ways of generating cells for assays. Similar transdifferentiation has resulted in induced neuron (iN) cells, also by introducing 3 tissue-specific transcription factors [6]. Therefore, it seems that by using defined combinations of tissue-specific transcription factors it is possible to generate cells of different tissue types. It is also possible that by using different, developmental stage-specific transcription activator sets, transdifferentiation can be conducted in a stepwise way and make sure cells at each step is pure. This strategy may be particularly attractive if its efficiency can be improved by the techniques developed for iPSC creation. After all, reprogramming to pluripotency and transdifferentiation to different tissue types must share certain mechanistic steps in their respective processes.

In addition, it has been reported that by briefly overexpressing the Yamanaka iPS factors and controlling growth conditions, mouse fibroblasts could be transdifferentiated up to 40% in 18 days without reversing back to pluripotency [7]. It would be interesting to see if by transient expression of iPS factors via mRNA then switching to cardiomyocyte-specific transcription factors, we can increase the efficiency for direct reprogramming. Use of chromatin-modifying chemicals that were already shown to directly reverse and alter cell fates might also be used to assist direct reprogramming. We believe that a systematic approach for studying these reprogramming aspects should benefit the iPS fields.

1. Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663-76.
2. Yu, J., et al., Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007. 318(5858): p. 1917-20.
3. Vidarsson, H., J. Hyllner, and P. Sartipy, Differentiation of human embryonic stem cells to cardiomyocytes for in vitro and in vivo applications. Stem Cell Rev, 2010. 6(1): p. 108-20.
4. Elliott, D.A., et al., NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat Methods, 2011.
5. Ieda, M., et al., Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell, 2010. 142(3): p. 375-86.
6. Pang, Z.P., et al., Induction of human neuronal cells by defined transcription factors. Nature, 2011. 476(7359): p. 220-3.
7. Efe, J.A., et al., Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat Cell Biol, 2011. 13(3): p. 215-22.

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Tags: cardiomyocyte, didifferentiation, direct reprogramming, Gata4, iCM, iN, iPSC, Mef2c, redifferentiation, Tbx5, transdifferentiation