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[3], 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"[6].

- "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”[7].

- 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[9]. 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[4]. 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[11].

- 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[11].

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

MicroRNAs are short 19-22nt RNAs that function as negative regulators of gene expression. Found in all animal and plant cells as well as in viral genome, microRNAs regulate gene expression by binding to a short 6 nt region within the 3'UTR of their target mRNAs. In human cells, more than 1000 microRNAs have been described and nearly 50% of human mRNAs are predicted to be microRNA targets. Hence, microRNAs are one of the most regulatory elements in mammals.[12]

biogenesis of microRNAs

MicroRNAs can regulate gene expression and control many important process including development, differentiation, apoptosis and proliferation. An microRNA gene is transcribed by RNA polymerase II and becomes primary miRNA(pri-miRNA). Then, the RNase III endonuclease Drosha and the double-stranded RNA-binding domain (dsRBD) protein DGCR8/Pasha cleave the pri-miRNA to produce a 2-nt 3 overhang containing the ∼70-nt precursor miRNA (pre-miRNA) in the nucleus.[13] Next, exportin-5 transports the pre-miRNA into the cytoplasm, where it is cleaved by another RNase III endonuclease, Dicer, together with the dsRBD protein TRBP/Loquacious. [14]One strand of each double-stranded RNA is degraded and the other strand can target mRNA that contains the complementary sequence. Associated with other proteins, the microRNA-protein complex prevents gene expression either by degrading the target microRNA or by blocking its translation.

Figure 4. Principle of microRNA

liver-specific miR-122

MicroRNA-122(miR-122) is a liver-specific microRNA that is most abundantly expressed in the liver as it accounts for approximately 70% of all hepatic microRNAs. Two studies could show that miR-122 appears to suppress 100–200 genes in liver tissue as demonstrated in mice.

miR-122 targets the 3'UTR of the mRNAs of cytoplasmic polyadenylation element bindingprotein (CPEB), hemochromatosis (Hfe), hemojuverin(Hjv), disintegrin, and metalloprotease family 10(ADAM10) and represses their translation. miR-122 activates the translation of p53 mRNA through the suppression of CPEB and participates in cellular senescence. Through the inhibition of Hfe and Hjv, miR-122 participates in iron metabolism. MiR-122 can also positively regulated lipid metabolism through the reduction of the mRNAs of lipid-associated proteins, and that inhibition of miR-122 expression attenuated liver steatosis in high-fat-fed mice, suggesting that miR-122 may be an attractive therapeutic target for metabolic diseases. Although Li et al. have suggested that hepatocyte nuclear factor 4 alpha (HNF4A) positively regulates the expression of miR-122, the details on the tissue specificity of miR-122 expression have not been fully elucidated yet.[15][16]

First attempts to use miR-122 as a therapeutic drug target have been already made by developing antagomirs against miR-122 and to test them as a putative treatment option for hepatitis C or for diseases associated with an aberrant cholesterol homeostasis.

iPSC-derived hepatocytes

Hepatocytes have been differentiated from various kinds of cell sources, including mesenchymal, fibroblast, and embryonic stem cells. iPS-derived hepatocytes have been generated from a variety of species including mouse, human and pig. These iPS cells can be expanded and directly differentiated into hepatocytes in vitro and are capable of many hepatic functions like albumin secretion. Also human iPS-derived hepatocytes exhibit key morphological features of differentiated hepatoctes. One of the promising applications of iPS-derived hepatocytes will be to study inherited metabolic disorders of the liver such as progressive familial gereditary cholestasis, a1-antitrypsin deficiency, glycogen storage disease type 1a, familial hypercholesterolemia, hereditary tyrosinemia and Crigler–Najjar syndrome[17].

In recent studies, iPS-derived hepatocytes have been tested to determine the potential for cell replacement therapy and can be applied to produce metabolically competent hepatic cell lines. It can circumvent the drawbacks of primary human hepatocytes of the phenotypic instability over time, scarce tissue origin and the high batch-to-batch functional variability. What's more, the function of hepatocytes in human body differs from region to region of a liver because one cell does not hold all the enzymes responsible for the complex metabolism within human body. Previously set up liver-derived cancer cell line have been transfected to express metabolic-related genes. However, engineered liver cancer cell lines are tumor origin and bring in the concerns that they could not represent a normal liver. By using iPS-derived hepatocytes, we can integrate gene circuits to express metabolic enzymes to a normal level and respond to environmental changes and better mimic how a naturally hepatocytes works.

Figure 5. Application of ips-derived hepatocytes