UPDATE 09/18/2013

iPSCs

What is iPSCs?

A one-way journey?

During the first half of the 20th century, researchers 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 into that state, and unable to return to immature, pluripotent stem cell state. Conrad Hal Waddington, a famous developmental biologist in last century, once illustrated the cellular differentiation as an epigenetic landscape in which cells are seen as marbles rolling down in valleys to reach their end-point destinations as differentiated cells. They do not normally move back towards the top of the mountain to get back to the undifferentiated state, and they are not normally crossing into other valleys to develop into unrelated cell lineages[1].

However, this prevalent view was radically overturned in 1962, when John B. Gurdon demonstrated that the nucleus from a differentiated frog intestinal epithelial cell was capable of generating a fully functional tadpole upon transplantation to an enucleated egg[2]. This discovery shattered the dogma that cellular differentiation could not be a bidirectional process, but the question remained whether an intact differentiated cell could be fully reprogrammed to become pluripotent again.

Life there and back again

In 2006, Shinya Yamanaka, a Japanese scientist who afterward shared the 2012 Nobel Prize in Physiology or Medicine with John B. Gurdon[5], proved that introduction of a small set of transcription factors into a differentiated cell was sufficient to revert the cell into a pluripotent state. In his strikingly bold experiment, what Yamakana did was introducing 24 genes encoding the transcription factors which were considered as candidates to reinstate pluripotency in somatic cells to skin fibroblasts in one step. Surprisingly, he found out that a few of these cells actually generated colonies that showed a remarkable resemblance to ES cells. Then he reduced the number of genes capable of inducing such colonies one by one, and finally, four factors (Myc, Oct3/4, Sox2 and Klf4) were picked out, whose combination were identified to be sufficient to reprogram the skin fibroblasts to pluripotent stem cells[3]. 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 factor combination from the 2006 paper (Myc, Oct4, Sox2 and Klf4), Yamanka's group successfully produced human iPS cells, whereas another laboratory, James Thomson's lab, achieved the same goal with a somewhat different transcription factors combination (Lin28, Nanog, Oct4 and Sox2)[4].

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 discovery, as it was the first demonstration that an intact differentiated somatic cell could be reprogrammed to become pluripotent. 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]. Recent work by Araki and Guha used syngeneic mouse models to demonstrate that transplanted iPSC-derived embryoid bodies, skin and bone marrow tissues engraft efficiently with almost no signs of rejection[6].

- "Thank god we don't need embryos anymore!": Simultaneously, the technology of iPSC was also hailed by many commentaries as a milestone advance that solved the ethical and political 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[8]. 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.

- Wide application in different fields: Nowadays, scientists are making progress toward applying iPSCs in many fields. One of the applications - which has already become reality - is to use iPS cells in both analysis of disease mechanisms and investigation of 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. Two examples are the dopamine-producing cells in the brain that degenerate in patients with Parkinson disease, and the insulin-producing cells that die off in patients with diabetes[13]. So far, more than 100 reports published in the past three years using disease-specific iPSCs[8].

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 therapeutically promising are also responsible for an equally fundamental tumorigenic potential, and the 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 large-scale 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 tested 55 mice transplanted with SNS (secondary neurospheres) from 11 TTP-iPS cell clones, 46 mice among them died or became weak within 9 weeks after transplantation because of tumors. Whereas in contrast group, the number of the tumor-showing mice transplanted with SNS derived from the ES cell clones was only 3 among 34[11]. 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[3]. This led to the result that the iPSCs with the four exogenetic oncogenes were more prone to tumorigenesis. However, subsequent studies successively substantiated that both the Oct-4 and Myc are not essentially required for cell self-renewal. For example, in making human iPS cells, Yu et al. used a different set of four factors (Oct-4, Sox-2, Nanog, and Lin-28) to induce pluripotent stem cells from human somatic cells. These results began to raise the scientists' hopes to find the better combination of the cocktails to induce the safer iPSC. However, it turned out to be wishful thinking.

Scientists figure out that whatever the combination is, almost every inducing/reprogramming factors remaining in the cocktail are oncogenes by definition. Their over-expression has be associated with some forms 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[7].

- Risks from delivery methods: Compared to ESCs, iPSCs are exposed to a number of factors that could promote oncogenic transformation, such as genomic insertion of reprogramming vectors, over expression of oncogenic transcription factors and a global hypomethylation resembling that seen in cancers[7].

Methods of diminishing the tumorigenic transformation 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 methods can not escape their shortcomings of low transduction efficiency, or even bring about new risks. For example, piggyBac transposition carries the risk of uncontrolled cycles of excision and integration, increasing the possibility of nonconservative deletions within coding regions of the genome. standard non-integrating strategies such as using adenoviruses suffer from extremely low transduction efficiencies (0.001%). In addition, considerable technical skills are required to carry out this type of reprogramming, and only a handful of centers have been able to successfully apply these strategies. Here is a table which concludes the oncogenic risks associated with methods of inducing pluripotency in somatic cells[7].

Table 1 Oncogenic risks associated with methods of inducing pluripotency in somatic cells

Method of induction	Strengths	Weaknesses
Lentiviral vector	Robust reprogramming efficiency	Genomic integration, reactivation of integrated transgenes
Cre recombinase	Little genomic disruption	Low transduction efficiency, integration of LoxP sites into host genome
PiggyBac transposition	Minimal risk of genomic disruption	Low transduction efficiency, risk of uncontrolled rounds of excision and integration
Adenoviral vector	Low risk of genomic integration	Low transduction efficiency, limited transgene expression
Plasmid transfection	Minimal risk of genomic disruption	Very low transduction efficiency typically requires use of oncogenes such as the SV40LT antigen for successful induction of pluripotency
Minicircle	Minimal risk of genomic disruption	Low transduction efficiency
Sendai virus	Minimal risk of genomic integration ,relatively high transduction efficiency	Risk of continuous replication of viral vector in cytoplasm, leading to aberrant silencing of pluripotency transgenes
Synthetic mRNA	No risk of genomic integration, ability to control transgene expression	Variable transduction efficiencies, high technical expertise required
Protein transduction	No risk of genomic integration, ability to control transgene expression	Very low transduction efficiency, labor intensive
microRNA transfection	No risk of genomic integration	Low reprogramming efficiency
Small molecules	No risk of genomic integration	Variable off-target effects

From the table above, we can generally conclude that these promising strategies for teratoma prevention mostly improve the safety of iPSC generation at the expense of efficiency, which is still another remaining challenge in iPSC technology.

In summary, it wouldn't be more perfect if scientists found a method which guaranteed both the safety and efficiency in iPSCs technology. Just as Andrew S Lee wrote on Nature Medicine, "Ideally, the most stringent safety regimes would utilize a flexible, combinatorial approach that may require tailoring for specific PSC lines or graft types. Should these techniques fail to adequately remove enough residual PSCs, retrospective tumor treatments may also be used, including oncologic chemotherapy, radiation and surgery or the incorporation of suicide ablation genes[7]." Admittedly, just as the saying goes, "You can't have your cake and eat it!" With our knowledge of iPSC biology, it is not likable that such a perfect regime would emerge in several coming years. However, as a group of young pre-scientists like us, maybe, it is our time to look into this problem and try to figure it out in another way.

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.

biogenesis and functioning 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. 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. 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 prevent gene expression either by degrading the target microRNA or by blocking its translation.

liver-specific microRNA-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 thesuppression 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.

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.

From iPSC to 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.

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.