[세상사 논문] 최지호 Ph.D.

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Jiho Choi, Ph.D in Biological and Biomedical Sciences (BBS) program at Harvard Medical School

Article
A comparison of genetically matched cell lines reveals the equivalence of human iPSCs and ESCs

Abstract
The equivalence of human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) remains controversial. Here we use genetically matched hESC and hiPSC lines to assess the contribution of cellular origin (hESC vs. hiPSC), the Sendai virus (SeV) reprogramming method and genetic background to transcriptional and DNA methylation patterns while controlling for cell line clonality and sex. We find that transcriptional and epigenetic variation originating from genetic background dominates over variation due to cellular origin or SeV infection. Moreover, the 49 differentially expressed genes we detect between genetically matched hESCs and hiPSCs neither predict functional outcome nor distinguish an independently derived, larger set of unmatched hESC and hiPSC lines. We conclude that hESCs and hiPSCs are molecularly and functionally equivalent and cannot be distinguished by a consistent gene expression signature. Our data further imply that genetic background variation is a major confounding factor for transcriptional and epigenetic comparisons of pluripotent cell lines, explaining some of the previously observed differences between genetically unmatched hESCs and hiPSCs.

Natural Pluripotency vs. Artificial Pluripotency

Pluripotency is the developmental potential of cells to become various types of mature cells in the body. During development, a pluripotent embryo progressively differentiates to give rise to mature cell types in the organism that form major organs such as the brain, heart, and kidneys. The transient nature of pluripotent cells, however, also makes it challenging to study the very mechanisms that define pluripotency.

Pluripotent stem cells can be derived in vitro from an explanted inner cell mass (ICM), a pluripotent cell population of a preimplantation blastocyst. This pluripotent cell type, termed embryonic stem cells (ESCs), retains the capability of becoming mature cells while self-renewing indefinitely in vitro. The first mouse ESCs (mESCs) were successfully derived from mouse blastocysts by Martin Evans in 1981. These pluripotent cells have become an essential experimental system in the study of mammalian developmental biology and mechanisms of cellular differentiation in vitro and mammalian genetics in vivo.

After nearly two decades, James Thomson succeeded in isolating human ESCs (hESCs) from human blastocysts in 1998. hESCs hold enormous potential for basic research, drug screening and disease modeling. More importantly, hESCs raised the possibility of attaining unlimited source of mature cells for regenerative medicine. This therapeutic application of hESC technology is based on the idea that well-characterized hESC lines could provide mature allogeneic cells to be transplanted into patients. Although the fact that hESCs are derived from human embryos limits the application of hESC technology, the possibility of utilizing hESCs as a source of allogeneic transplantation donor cells still remains a viable option.

The advent of artificial pluripotency, however, changed the scene in stem cell biology. In 2007, a Japanese scientist Shinya Yamanaka identified a subset of genes whose overexpression was noted to induce pluripotency from mature cells. He delivered four transgenes into human skin cells and successfully generated human induced pluripotent stem cells (hiPSCs), for which he was awarded the Nobel Prize in 2012. Since hiPSCs can easily be derived from patients’ mature tissue such as skin or blood, hiPSCs can theoretically provide patients with unlimited amount of autologous cells for personalized transplantation therapy. hiPSC technology can in fact circumvent both the ethical and technical conflicts that are inherent in hESCs.

Despite the immense potential of hiPSC technology, however, there has been much debate as to whether hiPSCs are molecularly and functionally equivalent to hESCs. Initial studies suggested that hiPSCs and hESCs are fundamentally different, while other studies have concluded that the two cell types are similar.

Previous studies reported that hundreds of genes are differentially expressed between mouse iPSCs (miPSCs) and mESCs. However, our lab found that transcription profiles of genetically matched miPSCs and mESCs are identical except for a few transcripts. Based on these results, our lab decided to generate and compare genetically matched hiPSCs and hESCs in order to answer the question of whether these two cell types are equivalent or not. 

 hESCs and hiPSCs originate from embryos and adult cells, respectively. Given this difference, generating genetically matched cell lines is technically challenging. To address this issue we took a rather unique approach. We differentiated hESCs into fibroblasts and then reprogrammed these fibroblasts into hiPSCs. By doing so, we could generate two sets of genetically matched hESC and hiPSC lines. A comparison of transcriptional and epigenetic profiles of these genetically matched cell lines revealed that hiPSCs are closer to genetically matched hESCs than to unmatched hiPSCs. These results showed that transcriptional and epigenetic patterns of human pluripotent stem cells are driven by genetic background rather than cell type. In addition, we found that there are no consistent gene expression differences between hESCs and hiPSCs. Genetically matched hESCs and hiPSCs also did not show any functional differences when differentiated into neural progenitors and cells of three germ layers. These results further corroborate the idea that previously observed gene expression differences are mainly due to different genetic backgrounds of the cell lines rather than different cell types of origin. Taken together, we concluded that hESCs and hiPSCs are molecularly and functionally equivalent after controlling for genetic background.

Our approach involved in vitro differentiation of hESCs into fibroblasts, which were subsequently reprogrammed into hiPSCs. It has been well documented that different types of mature cells retain various degrees of “epigenetic memory” when reprogrammed into hiPSCs, which could have profound effects at the molecular and functional levels. Thus it would be interesting to make similar comparisons by attempting differentiation of hESCs into more mature cell types such as neurons and blood cells, to be used for the generation of hiPSCs. This would be an important validation that confirms the suitability of hiPSCs for their clinical applications.

In conclusion, we believe that our results offer an explanation as to why there has been so much debate surrounding the equivalency between hESCs and hiPSCs. We hope that our findings will help to bring hiPSCs to the clinic and to realize their full therapeutic potential.