iPSCs

What is iPSCs?

Life there and back again

In the past century, scientists always believed that the life's one-way journey also applied to cells: Once a cell has developed into a specialized cell, it would be locked at that situation, unable to return back to pluripotent stem cell.

However, this prevalent view was radically overturned in 2006, when Shinya Yamanaka, a Japanese scientist who afterward shared the 2012 Nobel Prize in Physiology or Medicine with John B. Gurdon[1], proved that introduction of four transcription factors into a differentiated cell was sufficient to return the cell into a pluripotent situation. In his experiment, what Yamakana did was introducing four genes encoding the transcription factors (Myc, Oct3/4, Sox2 and Klf4), which were selected to reinstate pluripotency in somatic cells to skin fibroblasts. These synthetical new stem cells were thereafter named by their Japanese father as iPSCs (induced pluripotent stem cells). A year later, using the same four transcription factors in the 2006 paper, Yamanka's group successfully produced human iPS cells.

A technology with many possibilities

The groundbreaking work that Yamakana and his group did in 2006 truly opened up a completely new research field to this world. It was a paradigm-shifting fundamental breakthrough, as it firstly demonstrated that an differentiated somatic cell could be reprogrammed to be pluripotent stem cell again. iPSCs technology, a new technology with many possibilities, attracts scientists and researchers all over the world, mainly for its three unique superiorities:

- No strong immunological reaction: Since iPS cells can be cultured from the patient's own cells, it is not quite possible to lead to immunological reaction when directionally differentiated iPSCs are transplanted after in vitro culture. While in 2011 several studies argued the weak immune reaction to transgene-free iPSCs, these arguments were subsided later by other scientists and Yamanaka himself, pointing out that "the most prominent study that reported the immunogenicity of the cells examined undifferentiated iPSCs, which will never be used in cell transplantation therapy"[2].

- "Thank god we don't need embryos anymore!": Simultaneously, the technology of iPSC was also hailed by many commentaries for it solved the ethical problems in stem cell research field. In the past, stem cell research relied heavily of embryonic stem cells harvested from embryos. Human embryos are not easy to come by, and many people consider research involving use of human embryonic stem cells to be ethically questionable. With no further evidence, iPSC technology not only “breaks the ethical barrier of relying on using eggs or earlier embryos for deriving stem cells, but also leads to a convenient way of obtaining patient-specific stem cells”[3].

- Wide application in different fields: Nowadays, scientists are making progress in applying iPSCs in many fields. One of the applications - which has already become reality - is to use iPS cells in both analysing disease mechanisms and investigating potential new treatments. In the future scientists hope to be able to use iPS cells to culture cells that can be transplanted into the body and replace diseased cells.

These advances above together made the iPSC to be the "star cell" which offers immense potential as a source for regenerative therapies. However, the intrinsic qualities of self renewal and pluripotency that make these cells so promising are also causing equally fundamental tumorigenic risks, and the intrinsic shortcomings of the current methods of inducing pluripotency even pose more uncontrollable risks on that. High rates of tumorigenicity has become a most crucial hurdle for clinical implementation of iPSC technology.

Challenges

Tumorigenicity as a clinical hurdle

The risks of iPSC tumorigenicity have been widely concerned over the past several years. In the study in 2009, Yamakana found out that, among the 55 mice who has been transplanted with iPS cell clones, 46 mice died or became weak within 9 weeks because of tumors. Whereas in contrast group, the number of the tumor-showing mice transplanted with ES cell clones was only 3 among 34[4]. This result indicated that the mice transplanted with iPSCs have a much higher rate of tumorigenicity than with ESCs.

- The "fantastic four"are double-edged swords: In the initial study in 2006, Yamanaka has pointed out that among the four transcriptional factors he used, two (Oct-4 and Myc) were oncogenes[5]. This led to the result that the iPSCs with the four exogenetic oncogenes were more prone to tumorigenesis. Scientists figured out that almost every reprogramming factors are oncogenes by definition. Their over-expression are associated with some types of cancer. Of particular importance is the MYC transcription factor, which has emerged as one of the fundamental genes shared by iPSCs and cancer. Ectopic activation of OCT4 in somatic cells, induces dysplastic development and features of malignancy. NANOG has a role in the self renewal of CD24+ cancer stem cells in hepatocellular carcinoma. SOX2 has been shown to drive cancer-cell survival and oncogenic fate in several cancer types, including squamous cell carcinomas of the lung and esophagus. Klf-4 has been reported to promote DNA repair checkpoint uncoupling and cellular proliferation in breast cancers by p53 suppression[6].

- Risks from delivery methods: Compared to ESCs, iPSCs are exposed to several factors that could cause oncogenic transformation, such as genomic insertion of reprogramming vectors, over expression of oncogenic transcription factors and a global hypomethylation resembling that seen in cancers[6].

Methods of diminishing the tumorigenic risks of iPSCs have mainly focused on a variety of gene delivery vectors that minimize genomic disruption. These strategies can be generally divided into two categories: integrating vectors that can be excised from the host genome and non-integrating vectors. However, all these methods can not escape their shortcomings of low transduction efficiency, or even bring about new risks. This summer, as a group of young pre-scientists, we came up with a new strategy to try to minimize this potential problem.

microRNA Silencing

MicroRNA is a short 19-22nt RNA that functions as negative regulator of gene expression. By binding to a complementary target located at the 3’UTR of gene, it either triggers degradation of the whole mRNA in complete match or just blocks down protein translation when incomplete matching. There are more than 1000 kinds of microRNAs which are predicted to regulate approximately 50% of gene expression in human cells.[7]

biogenesis of microRNAs

MicroRNAs are mostly located in introns and transcribed by PolII before splicing. The transcribed pri-miRNA is then bound by double-stranded RNA-binding protein and cut by RNase III Drosha. A 70nt hairpin pre-miRNA is thus produced and transported outside the nucleus with the assistance of Exportin 5. After that, another RNaseIII Dicer cleave the pre-miRNA to generate mature microRNA duplex. One strand is incorporated into the RNA-induced silencing complex (RISC) and the other strand is degraded. When the incorporated single strand RNA finds its target, the mRNA will be degraded. [8][9]

Figure 4. Principle of microRNA

liver-specific miR-122

MicroRNA-122(miR-122) is most abundantly expressed in normal liver cells instead of liver cancer cells or other kinds of tissue cells. It accounts for approximately 70%of all the microRNAs in normal liver cells. The detailed regulatory network and the reason for its specificity is still in researches, but we know that miR-122 can target nearly 300 genes including Hfe, CPEB, ADAM10 and indirectly regulates downstream genes including p53. For functional tests, it is believed that miR-122 can regulate lipid metabolism and assist iron metabolism as well as HCV infection. Knocking down the endogenous level of miR-122 by antisense antagomirs will not interfere normal liver functions as indicated by clinical trials on the treatment of cholesterol metabolism and HCV infection. microRNA-122(miR-122) is most abundantly expressed in normal liver cells instead of liver cancer cells or other kinds of tissue cells. It accounts for approximately 70%of all the microRNAs in normal liver cells. The detailed regulatory network and the reason for its specificity is still in researches, but we know that miR-122 can target nearly 300 genes including Hfe, CPEB, ADAM10 and indirectly regulates downstream genes including p53. For functional tests, it is believed that miR-122 can regulate lipid metabolism and assist iron metabolism as well as HCV infection. Knocking down the endogenous level of miR-122 by antisense antagomirs will not interfere normal liver functions as indicated by clinical trials on the treatment of cholesterol metabolism and HCV infection.

iPSC-derived hepatocytes

Hepatocytes are always in need for tissue transplant and hepatoxity screening. Due to the limitation of tissue origin and the difficulties for isolation and maintains, iPSC-derived hepatocytes provide us a new way to generate hepatocytes for clinical use and study tossie origin and metabolism in patient with inherent diseases. [10].

In recent study, iPSC-derived liver with certain metabolic function has been generated and tested for cell replacement therapy. It can circum the drawback of phenotypic instability, scarce tissue origin and damage to normal tissue. iPSC-derived hepatocytes can better mimics normal metabolic pathways than cancer cell line due to purer genetic background.

Figure 5. Application of iPSC-derived hepatocytes