Hybrid Cell Line

2 THE δ OPIOID RECEPTOR

The first clearly characterised demonstration of inverse agonism at a GPCR was the capacity of ICI174864 to cause a reduction in basal high affinity GTPase activity in membranes of the neuroblastoma x glioma hybrid cell line, NG108-15, which endogenously expresses the δ-opioid receptor (Costa and Herz, 1989). Following expression of this receptor in Rat-1 fibroblasts we were able to isolate a clone, D2, expressing some 6 pmol/mg membrane protein of this receptor. In membranes of these cells agonist ligands, such as the synthetic enkephalin [D-ala²]-leucine enkephalin (DADLE), increased high affinity GTPase activity and the binding of [³⁵S]GTPγS. Both of these effects were prevented by prior treatment of the cells with pertussis toxin (Mullaney et al. 1996) demonstrating the stimulation to represent activation of G_i-like G proteins. Direct visualisation of agonist activation of G_i-like G proteins expressed by these cells was achieved by use of an assay in which cholera toxin is able to catalyse [³²P]ADP-ribosylation only of copies of G_iα which have previously been activated (Milligan et al. 1991). These are then detected by autoradiography following resolution of membrane proteins by SDSPAGE. Because a clear signal of activated G_iα could be observed in this assay without addition of opioid agonist and because pertussis toxin-pretreatment of clone D2 cells reduced the basal high affinity GTPase activity in membranes of these cells to a value similar to that observed in membranes of untransfected Rat-1 cells (Mullaney et al. 1996) we assessed whether ICI174864 would show inverse agonist characteristics in clone D2 cells. ICI174864 reduced both basal high affinity GTPase activity and [³⁵S]GTPγS binding in a concentration-dependent manner (Mullaney et al. 1996). Furthermore it eliminated the agonist-independent cholera toxin-catalysed [³²P]ADP-ribosylation of G_i (Mullaney et al. 1996). These effects of ICI174864 were not observed in membranes of untransfected cells. In the same way that the stimulatory effects of DADLE on high affinity GTPase were prevented by pertussis toxin pretreatment of the cells, pertussis toxin treatment also eliminated the inhibitory effect of ICI174864 (Mullaney et al. 1996). A central concern in many studies of inverse agonism is that the observed effects actually represent a competition between an antagonist ligand and levels of endogenous agonist present in the preparation. The δ opioid receptor ligand H-Tyr-Tic[CH₂-NH]Phe-Phe-OH (TIPP[ψ]) was unable to regulate the function of the δ-opioid receptor either positively or negatively and is thus a neutral antagonist. This mitigated against the possibility that ICI174864 was simply competing for the binding site with an unidentified agonist. Furthermore, co-addition of (TIPP[ψ]) prevented the effects of both DADLE and ICI174864 (Mullaney et al. 1996).

One major criticism of many studies on inverse agonism is that although the effects are observed in membrane preparations equivalent effects are not easily observed on whole cells. We addressed this issue in clone D2 cells using two distinct strategies. In the first of these we performed whole cell adenylyl cyclase assays following prelabelling of the cells with [³H]adenine to allow this to be incorporated into the cellular ATP pool. Basal adenylyl cyclase activity was low in these cells and was little altered by the addition of either DADLE or ICI174864. However, addition of a range of concentrations of forskolin or cholera toxin to cause a degree of activation of the adenylyl cyclase population allowed both inhibition of this activity by DADLE and further stimulation by ICI174864 above that produced by these agents (Mercouris et al 1997). Again, this effect of ICI174864 was prevented by pertussis toxin pretreatment of the cells. This appears to imply that a tonic δ-opioid receptor-G_i activation is present in these cells in the absence of opioid agonist. However, pertussis toxin treatment alone did not mimic the capacity of ICI174864 to conditionally further stimulate forskolin-amplified adenylyl cyclase activity. These results imply an element of control of inverse agonist function which requires further analysis.

The Cytosensor™ microphysiometer records alteration in cellular metabolic activity as changes in the rate of extrusion of proteins and thus of extracellular pH. An increase in proton output in response to DADLE and a decrease in response to ICI174864 was observed in clone D2 cells (Mercouris et al. 1997).

4.2 AD Cybrid Experiments

The cybrid approach was deemed reasonable to address the specific question of why individuals with AD on average have a lower platelet mitochondria COX activity than age-matched, non-AD subjects.^69–74 Nongenetic explanations included the presence in the circulation of a factor that inhibits COX activity. Because COX contains 13 subunits, 10 of which are encoded by nuclear genes and 3 of which are encoded by mitochondrial genes, genetic explanations could alternatively implicate a nuclear DNA or mtDNA-dependent component. It was a priori hypothesized that mtDNA genes were more likely to contribute to lower COX activity in AD subjects than nuclear DNA genes, since late-onset AD (LOAD) rarely demonstrates recognizable Mendelian inheritance.²²⁶ LOAD is generally considered to show sporadic epidemiology although nevertheless with a genetic influence, and in many ways the unique genetic rules of mtDNA, including heteroplasmy, threshold, mitotic segregation, and maternal inheritance uniquely position it to play a role in otherwise apparent sporadic diseases that also demonstrate altered mitochondrial function.²²⁶

In terms of applying the cybrid technique to address this question, it was reasoned that if lower mean COX activities were caused by a circulating inhibitory factor, that factor would wash out over the course of expanding the cell lines generated using platelets obtained from AD subjects (herein referred to as “AD cybrids”). Presumably, low AD subject platelet mitochondria COX activity under this scenario would not perpetuate in culture as AD cybrid line COX activities would increase in culture to match that of the cybrid lines generated from platelets obtained from age-matched control subjects (herein referred to as “control cybrids”). It was further reasoned that a nuclear DNA-dependent feature would be unlikely to account for a relative reduction in the AD cybrid COX activity because nuclei are not routinely transferred during the procedure, or if such a transfer did occur the transferred nuclei would be unlikely to perpetuate. Similar to toxin-induced activity reductions, nuclear DNA-dependent reductions in COX activity would predictably wash out after the transfer and selection process was completed.

The first published AD cybrid study was the one by Davis et al.²²⁷ This study compared COX data from a group of 20 AD cybrid cell lines to a group of 45 control cybrid cell lines. Transferred mtDNA derived from subject platelet mitochondria, and the acceptor cell line was the SH-SY5Y ρ0 line. COX activity was ~ 20% lower in the AD cybrid group than it was in the control cybrid group. It is relevant to note that the Davis et al. report was subsequently retracted, although the reasons for the retraction were unrelated to the cybrid data that were presented.²²⁸

Later in 1997 two other AD cybrid studies were reported. In the study of Sheehan et al., platelets served as the mitochondria/mtDNA donor source, and the acceptor cell line was the SH-SY5Y ρ0 line.²²⁹ An ~ 50% lower COX activity in the AD cybrids was seen. In the other study, that of Swerdlow et al., platelets served as the mitochondria/mtDNA donor source and the acceptor cell line was an NT2 teratocarcinoma-derived ρ0 line.²³⁰ Fifteen AD cybrid lines were compared to 9 control cybrid lines, and a relative 16% reduction in the AD cybrid group COX activity was observed.

Other studies of unique AD cybrid series have focused in particular on COX activity. In the study of Cardoso et al., the authors used platelet mitochondria to generate AD and control cybrid lines on an NT2 ρ0 nuclear background and found that COX activity in the AD cybrid cell line (n = 6) group was 22% lower than it was in the control cybrid cell line (n = 5) group.²³¹ In the study of Silva et al., the authors used platelet mitochondria to generate AD and control cybrid lines on an SH-SY5Y ρ0 nuclear background and found that COX activity in the AD cybrid cell line (n = 8) group was ~ 30% lower than it was in the control cybrid cell line (n = 7) group.²³² On the other hand, the study of Ito et al. also used COX activity as a primary endpoint and found COX activity was comparable between the AD and control cybrid groups.²³³ However, there are a number of notable methodologic differences between the Ito et al. study and the positive studies thus far mentioned. The Ito et al. group used a HeLa cell ρ0 cell line to generate their cybrids, and the mitochondria/mtDNA donor source was mixed; four AD cybrid lines were prepared from platelet mitochondria, three control cybrid lines were prepared from platelet mitochondria, and two control cybrid lines were prepared from fibroblast mitochondria. Also included in the analysis were what were designated as an additional three AD cybrid lines, which were generated by mixing HeLa ρ0 cells with synaptosomes prepared from a brain that was acquired from a deceased AD subject after a 20-h postmortem interval. The authors reported they were able to identify three cell colonies from this fusion that contained mtDNA, and COX activity data ascertained from each of these three colonies were individually included in the analysis. Due to these substantial methodologic differences, it is arguably difficult to conclude that the Ito et al. negative study contradicts the positive studies.

A number of AD cybrid studies have evaluated various other aspects of mitochondrial function as well as parameters influenced by mitochondrial function.^{227,229–232,234–252} In many cases changes are reported that recapitulate changes that are seen in the brains of AD subjects themselves.⁶⁸ Relative to control cybrid cell lines, in AD cybrid cell lines oxidative stress markers are increased,^{229–231,234,236,240,241,245,248,249} inflammatory and stress signaling pathways are activated,^{234,236,239–241,245,250} Aβ levels are increased,^238,241 glucose utilization is decreased,²³² oxygen consumption is decreased,²³² there is a shift toward mitochondrial fission and a smaller average mitochondrial size,^243,247 numbers of ultrastructurally perturbed mitochondrial are increased,^243,252 PGC1α mRNA levels are reduced,²³² HIF1α protein is reduced,²³² mTOR protein is reduced,²³² SIRT1 protein is reduced,²³² and apoptotic markers are increased.^{231,238–241,245}

AD cybrids have also been used to model aspects of AD-specific, mitochondria-related function that are difficult or impossible to study in autopsy brain tissue.⁶⁸ For example, mitochondrial membrane potential analyses of AD cybrid lines show a relative degree of depolarization,^{235,238,242,252} and AD cybrid mitochondria appear to internalize less calcium and are less able to buffer calcium-mediated intracellular signaling activity than control cybrid lines.²²⁹ AD cybrid ATP levels are reduced.^231,232 It has been shown using differentiated AD cybrid cell lines that mitochondrial movement is relatively reduced.²⁴⁴ AD cybrid cells are more sensitive to Aβ toxicity than are control cybrid cells.^231,250 AD cybrids have also been used to screen the molecular effects of potential therapeutic interventions; pharmacologic inhibition of mitochondrial fission activity and antioxidants has been shown to benefit certain mitochondria-related functional parameters.^{241,245,247,248}

Three studies, one performed using an NT2 ρ0 cell background and two performed using an SH-SY5Y ρ0 cell background, have reported mitochondria-relevant functional changes (including a reduction in COX activity) between cybrid lines generated from human subjects diagnosed with mild cognitive impairment (MCI; a frequent AD precursor state) and cybrids generated from age-matched control subjects.^232,246,248 To date, over 20 cybrid studies have been published that report at least one biochemical or molecular parameter that differs between groups of AD/MCI and control cybrid cell lines.^{227,229–232,234–252} Most of these studies have in fact reported multiple divergent parameters. The only categorically negative AD cybrid study was that of Ito et al.,²³³ which evaluated just one biochemical parameter (COX activity), and which is notable for a variety of distinct methodologic differences that may have caused that study to differ from the other positive studies.

Pharmacokinetics

Potency

Threshold Effect

There is a large body of data, mainly from cell culture studies, indicating that mtDNA mutations exhibit a threshold effect. That is, a certain proportion of mutant mtDNA is required before there is a reduction in respiratory chain activity. The exact threshold does seem to vary among different mtDNA mutations. For example, most studies using hybrid cell lines indicate that the threshold is greater than 90% mutant for mtRNA point mutations and greater than 60% for large-scale deletions of mtDNA. However, there is evidence that the precise threshold may be influenced by other factors. For example, the nuclear background of a cell may change the threshold, and differences have been observed when comparing transformed and untransformed cell lines. For some mutations, it seems that virtually 100% mutant mtDNA is necessary but not always sufficient to produce a disease phenotype. For example, the common mutations that associate with LHON are generally homoplasmic in all body tissues. However, affected patients generally only develop disease in the optic nerve, and there are a significant number of patients homoplasmic for a LHON mutation who never develop disease at all. Hence, while the proportion of mutant mtDNA is likely to be an important determinant of threshold, other factors must be involved. Importantly, the mechanisms determining threshold for expression are likely to be different for different mtDNA mutations.

Potency

Effects of TIP39 in Pain Assays

There is intense labeling in the outer layers of the dorsal horn of the spinal cord and the spinal trigeminal tract by an antibody to the PTH2 receptor (Figure 7). Most primary afferent (dorsal root ganglion) neurons concerned with pain perception project to this area. In situ hybridization detection of PTH2 receptor mRNA suggests that the PTH2 receptors in this zone are contributed by both a local population of intrinsic spinal cord neurons and a population of dorsal root ganglion neurons. Microinfusion of TIP39 into a mouse paw causes a withdrawal response, consistent with the activation of nociceptive sensory neurons. Although not yet demonstrated, this suggests that PTH2 receptors are present in the peripheral terminals of some sensory neurons. Consistent with this, TIP39 stimulates cAMP accumulation in a dorsal root ganglion-derived hybrid cell line (F11) and causes an increase in cytoplasmic Ca²⁺ concentration in some of the cells in dorsal root ganglion primary cultures. Intrathecal injection of TIP39 causes a scratching, biting, and licking response that is characteristic of pro-nociceptive peptides such as substance P and CGRP. Intrathecal administration of TIP39 also potentiated the responses in several spinally mediated pain assays, including the tail-flick test, and paw withdrawal from pressure. It did not change the response in the hot plate test. These results suggest that the activation of the PTH2 receptor facilitates some aspects of pain perception. In striking congruence with this, intrathecal administration of an antibody to TIP39 increased the response threshold in the same tests as those in which the sensitivity was increased by TIP39. A simple interpretation of this observation is that TIP39 release is provoked by the stimuli used in these pain assays, that it contributes to the normal response, and that antibody sequestration of TIP39 removes this pro-nociceptive influence. However, only a small number of TIP39-containing nerve fibers have been observed in the spinal cord. Further investigation is required to determine whether TIP39 released from these fibers has a significant physiological role.

Figure 7. PTH2 receptor expression in the lower brain stem and spinal cord: (a) antibody labeling of the PTH2 receptor in the spinal trigeminal tract; (b) section of the spinal cord with an attached dorsal root ganglion. In (a), sensory fibers of the trigeminal nerve that convey information related to pain from the face have their first synapses in the spinal trigeminal tract where gold-colored labeling indicates the presence of PTH2 receptors in nerve fibers. This region is equivalent to the dorsal horn of the spinal cord. In (b), clusters of silver grains from in situ hybridization detection of PTH2 receptor mRNA indicate the cells both in the outer part of the dorsal horn of the spinal cord and in the dorsal root ganglia, as well as a few deeper spinal cord cells that synthesize PTH2 receptors. Parathyroid hormone (PTH).

PTH2 receptors are also found in several brain areas involved in pain perception, particularly in several midline thalamic nuclei that are thought to be involved in processing the emotional aspects of pain perception. The Place Escape Avoidance Paradigm (PEAP) is a test designed to evaluate an affective (emotional) aspect of nociception. Rats are placed in a chamber with light and dark halves. When they are in the dark half, which they normally prefer, an inflamed hindpaw is stimulated with a blunt probe and when they are in the normally less-preferred light half of the chamber, the noninflamed hindpaw is stimulated. When a drug is administered, its effect on the proportion of time an animal spends in the two halves of the chamber is used as a measure of the drug’s effect on the aversive quality of the stimulus. Intracerebroventricular administration of TIP39 decreased the apparent aversiveness of stimulation of an inflamed paw. This effect is opposite in direction to the pro-nociceptive effects of TIP39 observed in spinal-based tests following intrathecal administration of TIP39. Although there is not yet a physiological or mechanistic explanation for these observations, it may be relevant that TIP39-containing fibers in the spinal cord arise from the medial paralemniscal nucleus, whereas subparafascicular TIP39 neurons project to midline thalamic and other brain areas that may be involved in affective aspects of nociception. TIP39 arising from these two areas may have different biological functions.

3 MUTATIONS CAUSING ALTERATIONS IN SDH ACTIVITY

6.3.4.1 Monoclonal antibodies

In contrast to the polyclonal antibodies, monoclonal antibodies specifically target one epitope of an antigen. They are produced from the identical clones of parent B cell and thus are not variable as polyclonal antibodies.

César Milstein and Georges J. F. Köhler, in 1975, developed hybridoma technique to produce monoclonal antibodies for which they got the Nobel Prize for Medicine and Physiology in 1984. In hybridoma technology, mouse is challenged with the antigen, and immune response is induced, leading to the production of B cells generating antibodies against the antigen. The B cells producing antibodies are extracted from the mouse and fused with cancerous myeloma cells ex vivo to make a hybrid cell line called hybridoma. Hybridomas are then cultured to grow genetically identical clones of cells producing one type of monoclonal antibodies. Hybridoma technology helped in the development of murine monoclonal antibodies and later with genetic engineering advancements in the development of chimeric murine-human antibodies. However, the technique has been replaced with an antibody phage display library preparation methods to generate completely humanized antibodies. In this technology, bacteriophage M13’s plasmid called phagemids is genetically engineered to contain a repertoire of different libraries of Fab antigenic determinant region of antibodies extracted from B cells. M13 phages express the Fab region and display or present it on their phage coats surface fused with the phage surface protein. The phage display antibody library is then enriched using a selection method with the ones that can specifically bind to the antigen in vitro with higher affinity (Frenzel et al., 2017). The antigens are immobilized to the surface via direct coating or biotin-streptavidin linkage (Ledsgaard, Kilstrup, Karatt-Vellatt, McCafferty, & Laustsen, 2018).

Monoclonal antibodies have been one of the most successful and widely used bio-therapeutic modalities. The global monoclonal antibody market is expected to generate revenue of 300 billion USD by the end of 2025 (Lu et al., 2020). Up until December 2019, 79 therapeutic monoclonal antibodies have been approved by FDA and commercialized in the market (Lu et al., 2020). The highest revenue-generating mAb is adalimumab (Humira), the first completely humanized monoclonal antibody generated by the antibody phage display method. Other popular ones used for different types of cancer treatment are nivolumab (Opdivo), ipilimumab (Yervoy), and pembrolizumab (Keytruda) (Lu et al., 2020). Pembrolizumab and ipilimumab drugs are developed from the advancements in cancer immunotherapy and had a profound impact on antibody-based cancer treatments (Clift, 2019). Pembrolizumab or nivolumab is an anti-PD-1, and ipilimumab is an anti-CTALA4 immune checkpoint inhibitor. The immune response of T cells is downregulated allowing cancer cells to proliferate when its cell surface receptors PD-1 or CTLA-4 bind to PD-L1 receptors present in cancer cells or CD80/CD86 receptors present in the antigen presenting cells like dendricitic cells and macrophages respectively. The drugs pemrolizumab and ipilimumab block the interaction of PD-1 and CTLA-4, respectively, and activate the immune system of T cells to identify and attack cancer cells. James P. Allison, PhD, and Tasuku Honjo, MD, PhD, were awarded the Nobel Prize in Physiology and Medicine in 2018 for the discovery of checkpoint inhibitors CTLA-4 and interaction of PD-1/PD-L1, respectively.

MAbs targeting different viruses like Ebola virus, HIV, and SARS-Cov-2 coronavirus are currently in clinical trials. Currently, all major pharma companies are in the race to generate a human monoclonal antibody to fight against the coronavirus global pandemic of 2020. The spike glycoproteins present on the surface of SARS and MERS-Corona virus (SARS-CoV and MERS-CoV) interact with the angiotensin-converting enzyme-2 (ACE-2) cell and dipeptidyl peptidase 4 (DPP4) cell surface receptors to enter into the host cell upon viral infection. Human monoclonal antibodies are being designed to either interact with the spike protein epitope or to the cell surface receptors to block their interactions (Shanmugaraj, Siriwattananon, Wangkanont, & Phoolcharoen, 2020).

Rituximab

Rituximab is one of a host of monoclonal antibodies that are changing the approach to the treatment of autoimmune disorders. Unlike IVIg, in which a multitude of antibodies having different specificities are used, these new preparations contain a highly purified antibody that works against a single target. There is great hope that these more selective therapies will result in disease control with far fewer side effects than traditional immunosuppressive medications.

Monoclonal antibodies are derived from immortalized B cells that secrete antibodies of selected specificity. The amount of foreign immunoglobulin content in these drugs varies. Chimeric monoclonal antibodies are secreted from murine-human or other hybrid cell lines. Some monoclonal antibodies are further humanized by grafting only the portion required for antigen recognition (the complementarity-determining regions) onto a human IgG molecule. The newest antibodies are fully humanized by the use of transgenic animals that carry human immunoglobulin genes. The greater the non-human content of an antibody, the greater the risk of allergic reaction in patients. However, because of the presence of anti-idiotypic antibodies in patients, the use of even fully humanized antibodies is not without this risk.

Rituximab is the monoclonal antibody best known to neuromuscular physicians. It has been used anecdotally or in uncontrolled studies in anti-MAG neuropathy (Levine & Pestronk, 1999; Pestronk et al., 2003; Renaud et al., 2003, 2006), multifocal motor neuropathy (Ruegg, Fuhr, & Steck, 2004), CIDP (Briani et al., 2004; Kilidireas, Anagnostopoulos, Karandreas, Mouselimi, & Dimopoulos, 2006; Gorson, Natarajan, Ropper, & Weinstein, 2007), other autoimmune neuropathies (Gorson et al., 2007), inflammatory myopathies (Levine, 2005; Mok, Ho, & To, 2007), myasthenia gravis (Hehir et al., 2017; Kerkeni, Marotte, & Miossec, 2008; Topakian et al., 2019; Zebardaost, Patwa, Novella, & Goldstein, 2010), and the neuropathies associated with Sjögren syndrome (Seror et al., 2007) and Waldenstrom macroglobulinemia (Dimopoulos et al., 2002).

Rituximab is a chimeric monoclonal antibody against CD20, a molecule found on the surface of developing and mature B cells. It may be expressed on plasma cells as well. The drug was initially used in the management of B cell lymphoma. A single course of rituximab is capable of depleting B cells within several weeks and maintaining depletion for months or years. IgM levels often decline after a single treatment, but IgG levels may be unaffected, presumably because of the survival of at least some plasma cells. Although the drug’s effect on B cells and IgM levels is believed to play a significant role in its mechanism of action, the importance of other effects, such as the inhibition of cell migration, is still being considered (Kosmidis & Dalakas, 2010; Stubgen, 2008a).

Rituximab is given as an intravenous infusion of 375 mg/m² weekly for 1 month or as two infusions of 1 g every other week. Because the drug’s effects are long-lasting, repeat infusions may not be needed until several months or even years have passed; however, when necessary, the cycle can be repeated after 6 months. Rituximab is believed to be most helpful in IgM-mediated diseases, such as anti-MAG neuropathy and multifocal motor neuropathy. When used for other disorders, it is theoretically advantageous to administer rituximab along with another immunosuppressant, although this likely increases the risk of infection.

Rituximab infusions are typically well tolerated, with only cough and mild hypotension being common side effects. More severe reactions, such as bronchospasm, fever, and rigors, are more common when the drug is administered to those with high B cell counts (i.e., patients with lymphoma). Progressive multifocal leukoencephalopathy has been reported in patients treated with rituximab for lymphoma, rheumatoid arthritis, systemic lupus erythematosus, and inflammatory myopathies (Molloy & Calabrese, 2008) and other neuromuscular disorders. This disease, which is caused by reactivation of latent JC virus, is usually fatal unless the immune system can be reconstituted.

Ocrelizumab is another CD20 inhibitor that has been approved to treat multiple sclerosis (Hauser et al., 2017) and potentially may be used off-label to treat neuromuscular disorders. The drug is given in doses of 300 mg IV 2 weeks apart and then 600 mg every 6 months thereafter.

Breakthrough of the decade in the 21st century: induced pluripotent stem cells

However promising the potential of human embryonic stem cells for therapy might seem, ethical reservations associated with the use of embryos has spurred the search for alternative sources of pluripotent cells. As early as 1976 Carol Miller and Frank Ruddle from Yale University, USA, demonstrated that normal differentiated cells can actually acquire pluripotency upon artificial fusion with embryonal carcinoma (EC) cells derived from teratocarcinomas. They constructed a series of these hybrid cell lines that behaved like typical EC cells even though each cell contained two nuclei with double the amount of DNA because of the fusion. Later, Takashi Tada and colleagues in Kyoto, Japan, discovered that something similar occurred when differentiated cells were fused with ES cells. These experiments demonstrated that both EC and embryonic stem (ES) cells contain factors that can induce pluripotency in differentiated cells. It was Shinya Yamanaka though, also then from Kyoto, who identified the responsible proteins and their associated genes that apparently can “reprogram” a differentiated cell to a pluripotent state. In an ingenious and incredibly labor-intensive set of experiments he developed a system that allowed him to identify pluripotency of cells by resistance to an antibiotic drug. Introduction of the candidate genes into fibroblast cells from a mouse, obtained from small biopsies from the skin, quite remarkably revealed that a set of only four genes was enough to reprogram a differentiated skin cell to a cell type closely resembling an ES cell, albeit at very low frequency. The four genes all encoded transcription factors that are normally expressed in ES cells. These modified cells surprisingly had all of the properties of ES cells, including the ability to grow indefinitely and form different cell types, like nerve and heart cells. They are now known as induced pluripotent stem cells or iPS cells, since pluripotency was artificially “induced” (Fig. 4.11). Subsequently, Yamanaka and other groups demonstrated that many types of differentiated cells, indeed also human cells, can be transformed to pluripotent stem cells using this technique. This amazing feat caused a revolution in stem cell biology since sacrificing embryos to obtain pluripotent human cells may in the future no longer be needed. Patient-specific pluripotent stem cell lines can quite easily be derived from a simple skin biopsy, enabling scientists in countries that never allowed to carry out research on human embryonic stem cell lines to join in the search for therapeutic applications of stem cells. This work on reprogramming mature cells to pluripotency was awarded the Nobel Prize in Physiology or Medicine in 2012.

Figure 4.11. Schematic representation of the original method used to generate induced pluripotent (iPS) stem cells. Normal, differentiated skin cells are derived from a human individual and cultured as usual. Four new genes, coding for proteins that are required for reprogramming the cell to an embryonic state, are introduced (transfected) into the skin cells by infection with viruses that carry these genes. After a few weeks of culture a small fraction of transfected cells forms colonies and become morphologically similar to embryonic stem cells. Indeed it has been demonstrated in mice at least that these iPS cells are truly pluripotent and that they can differentiate to derivatives of the three germ layers ectoderm, mesoderm and endoderm.

Box 4.6

Sir John Gurdon

Sir John Gurdon is a distinguished developmental biologist from the UK who was the first to show that egg cells are able to reprogram nuclei of adult cells into a totipotent cell state. This work built on earlier studies of Briggs and King who had shown that they could transfer the nucleus of an embryonic cell from a frog into an egg from which the nucleus had been removed and the egg would behave as if it had been fertilized: that is, the first steps in development took place. When they had carried out the same experiment using nuclei from adult frogs, development did not take place. In 1952 they thus published a paper stating that reprogramming of embryonic nuclei was possible, but of adult nuclei, it was not. Gurdon used a different species, the African clawed toad Xenopus laevis, and showed firstly that he could reproduce the work of Briggs and King and obtain normal tadpoles from embryonic nuclei, but more importantly, that he could also do this using the nucleus from an adult toad. Because the results were surprising, Gurdon carried out many control experiments to prove that he was right and the paper describing this work, entitled “Adult frogs derived from the nuclei of single somatic cells,” was published in Nature in 1962. Although it took many years, it was this fundamental finding that eventually led to the cloning of Dolly the sheep (see Chapter 6, Cloning: history and current applications) (Fig. B4.6.1).

Figure B4.6.1. Sir John Gurdon.

Photograph courtesy of Nancy Witty (CEO ISSCR).

In giving lectures where he has been persuaded to talk about his life and work, Sir John Gurdon never fails to mention his school reports. During his first term of science at Eton in the UK, his schoolmaster said “He is the worst pupil I have ever had and it would be a waste of time for him to pursue science.” Gurdon turned to the study of classics but nevertheless remained interested in biology and became secretary of the Natural History Society at Eton. After Eton, he applied to Oxford University to study classics but was only admitted on conditions he studied Zoology. He graduated with a first-class honors degree and intended to carry out a doctorate in entomology but was again rejected by the professor so pursued embryology instead.

This story is always regarded as one of the most encouraging for students in the face of adversity: “do what you are interested in and it can work out on the end.” Sir John Gurdon was ultimately awarded the Nobel Prize, together with Dr. Shinya Yamanaka in 2012 (see Box 4.1 and Box 4.7)

Box 4.7

Dr Shinya Yamanaka and the Cira Institute

Interview Dr. Yamanaka

When did you get the idea to try to reprogram cells directly?

By the time when I had my laboratory for the first time in my academic career as an associate professor at the Nara Institute of Science and Technology in 2000, I became interested in how embryonic stem (ES) cells maintain their differentiation ability while rapidly proliferating. At the time, many laboratories were trying to differentiate ES cells into various functional cells. In contrast, I tried to differentiate somatic cells back to the embryonic state and made the idea the theme of my laboratory.

Were the ethical issues associated with hESC important in your motivation?

Yes, I was well aware of the ethical issues over the human ES cells and wanted to find a method to circumvent the problem. However, it is true that studying hESCs is essential to advance research on human induced pluripotent stem cells (Fig, B4.7.1).

Figure B4.7.1. Professor Shinya Yamanaka.

Photograph courtesy of Center for iPS Research and Application (CIRA), Kyoto, Japan.

How did you feel when the first colonies looking like embryonic stem cells appeared?

At first, I couldn’t believe that mouse fibroblasts turned into the embryonic state so easily. So we carefully analyzed that the colonies had similar characteristics to those of ES cells and repeated the same experiments several times. It took us nearly half a year to confirm that embryonic-like stem cells were generated from mouse fibroblasts by introducing the four transcription factors—Oct-3/4, Klf4, Sox-2 and c-Myc.

When did you realize that iPS cells could be a real alternative to hES cells?

I am hoping that iPS cells can eventually replace ES cells in the future, but it is too early to tell whether we can really do it. Many iPS cells are the same as hES cells, but not all. You need to avoid the bad ones. If you use the good ones, they are the same.

Do you feel enormously pressured by the expectations of iPS cells?

I feel pressured as I know that many patients with intractable diseases expect that iPS cells could help create new cures to their diseases. I am optimistic as many researchers around the world are working hard to advance iPS cell technology to bring it to the [patient] as early as possible. I believe that iPS cells will become an effective research tool for drug screening in a few years, but at least several years of basic research will be needed before we can overcome various obstacles, such as tumor formation, and realize regenerative medicine like cell transplantation (Fig. B4.7.2).

Figure B4.7.2. The Center for iPS Research and Application (CIRA) in Kyoto, Japan.

Photograph courtesy of Center for iPS Research and Application (CIRA), Kyoto, Japan.

How has it been since you won the Nobel Prize?

In Japan it has been hectic. There has been a lot of attention from the Japanese press. But I still wanted to finish some papers so it was very busy.

“My goals over the decade include to develop new drugs to treat intractable diseases by using iPS cell technology and to conduct clinical trials using it on a few patients with Parkinson's diseases, diabetes or blood diseases.”

Shinya Yamanaka

Box 4.8

Stem Cell Research History

Figure B4.8.1

Figure B4.8.1. Stem cell research time line.