Applying Somatic Cell Nuclear Transfer in the Creation of Dolly the Cloned Sheep. Dolly the sheep was cloned through somatic cell nuclear transfer (SCNT). An adult cell from the mammary gland of a Finn-Dorset ewe acted as the nuclear donor; it was fused with an enucleated egg from a Scottish Blackface ewe, which acted as the cytoplasmic (or egg) donor. An electrical pulse acted to fuse the cells and activate the oocyte after injection into the surrogate mother ewe. A successfully implanted oocyte developed into the lamb Dolly, a clone of the nuclear donor, the Finn-Dorset ewe. SCNT may also be used to create patient-specific stem cells with great therapeutic potential.

Human induced pluripotent stem cells: The history and biology of human iPSCs were explored previously in “Induced Pluripotent Stem Cells: A New Stem Cell Line with a Long History.” In essence, iPSCs, which were first created with mouse cells in 2006 (Takahashi and Yamanaka, 2006) and then with human cells in 2007 (Yu et al., 2007; Takahashi et al., 2007), are adult cells that have been “reprogrammed” to an embryonic stem cell (ESC) state. This reprogramming is done by forcing adult cells to express proteins that are essential to the ESC identity (by transducing the adult cells with a retrovirus vector that contains the DNA for the key proteins). Consequently, human iPSCs look and behave nearly indistinguishably from hESCs. Like hESCs, iPSCs are pluripotent (they can become any cell type) and proliferate virtually indefinitely, both features which are important for use in regenerative medicine.

However, while great improvements have been made to make this technology closer to the clinic (such as multiple approaches to create iPSCs that do not have the reprogramming genes randomly integrated into their genomes [Yu et al., 2009; Zhou et al., 2009]), and it may someday be used to generate patient-specific ESC-like cells, the technology is not quite there yet. (Other similar technologies, such as “direct reprogramming,” are also being explored for the generation of patient-specific cells, but, again, this approach also has a ways to go.)

Somatic cell nuclear transfer: SCNT technology significantly predates iPSCs, and in many ways formed the basis for the idea of iPSCs. In SCNT, the nucleus from a somatic cell (an adult cell that is not a sperm or egg, i.e. not the gametes) is implanted into an egg, which already had its own nucleus removed. The egg amazingly reprograms the nucleus to become embryonic again; it’s been found that SCNT causes some 10,000 to 12,000 genes to be expressed (turned into protein) that are normally associated only with embryonic development (Niemann et al., 2008). The newly formed embryo (technically called a blastocyst) can then be implanted into a surrogate mother, and potentially become an adult organism. The organism is a clone of the animal that donated the nucleus. Although nuclear transfer studies have been conducted since the late 1930s (primarily in amphibians using nuclei donated from embryos, not adult tissues) (Spemann, 1938), it wasn’t until 1997 that the first widely-accepted successful use of SCNT was reported: Dolly the sheep was born, and she was the first cloned animal from an adult cell, and the first cloned mammal (Wilmut et al., 1997). Since Dolly, several other animals have been successfully cloned, though many problems still remain (the frequency of successful development is relatively low, as SCNT-derived embryos usually result in about 0 to 10% live births) (Wilmut et al., 1997; Wakayama et al., 1998; Solter, 1998; McKinnell and Di Bernardino, 1999; Gurdon and Byrne, 2003, Beyhan and Cibelli, 2008).

Therapeutic cloning: While SCNT has been long-explored for its ability to create cloned animals like Dolly and others (a practice called “reproductive cloning”), SCNT has other very appealing applications that do not involve the creation of an entire animal, such as “therapeutic cloning.” The goal of therapeutic cloning is to use SCNT technology to create patient-specific embryonic stem cells for medical therapies. These much sought-after cells are being labeled nuclear transfer stem cells (NTSCs), but they are essentially ESCs. While using SCNT to clone an entire animal has been fraught with developmental challenges, using SCNT to create NTSCs may be less difficult because it only requires a very early stage embryo, a blastocyst, to be formed (and NTSCs from a blastocyst may be less compromised by developmental abnormalities than an entire animal would be) (Beyhan and Cibelli, 2008). NTSCs have now been created from many different model animals.

SCNT in the Mouse: In 2000, the first embryonic stem cell line was generated from mice using SCNT (Munsie et al., 2000). After SCNT was performed, when the embryos reached the blastocyst stage the ESCs were isolated as usual. These SCNT-derived mouse ESCs (or NTSCs) were reported to be pluripotent and behave similarly to normally-derived mouse ESCs. Not only could NTSCs be successfully derived in this way, but multiple studies in mice have shown that SCNT can also be used to repair genetic diseases. This has been done by taking the nucleus from an adult cell of a mouse with a genetic defect, performing SCNT on the nucleus to create ESC lines, fixing the defect in these cell lines (using standard genetics techniques), and then transplanting the repaired (and differentiated) cells back into the original donor mouse (for descriptions of these studies, see Saric et al., 2009). The mice are virtually cured of their genetic diseases this way

SCNT in Non-Human Primates: In 2007, two ESC (or NTSC) lines were created from a rhesus macaque using SCNT (Byrne et al., 2007). The researchers took nuclei from the skin fibroblasts of a female rhesus macaque and had these undergo SCNT in the eggs of another female macaque. Although these cells could differentiate properly and express pluripotent markers, the generation efficiency was low; 304 eggs were used to create just these two ESC lines. (This was thought to be due to incomplete reprogramming of the donor nucleus.) However, two years later the same group reported a three-fold increase in NTSC generation from rhesus macaques using improved techniques, reportedly creating NTSC lines at levels similar to those seen with the generation of ESC lines from normally fertilized embryos (Sparman et al., 2009). These NTSCs also displayed proper epigenetic reprogramming, similar to normal ES cells, which had been of concern (Sparman et al., 2009).

Humans and SCNT: Currently, a scarcity of donated human eggs is probably the largest hurdle to creating patient-specific, human ESCs through SCNT. Not only are more eggs needed, but these eggs need to have certain qualities to work most efficiently in SCNT (they must be young, high-quality, and at metaphase II stage) (Cervera and Stojkovic, 2008). There are also many technical issues that need to be optimized (such as removing the least amount of cytoplasm from the egg when enucleating it, and avoiding the use of certain nuclear stains while doing this, though reports vary on even the importance of these kinds of aspects). In 2004, Hwang and colleagues reported having successfully created hESCs from a SCNT blastocyst for the first time, but their work was proven to be fraudulent. Because of these falsified studies, subsequent researchers have had to go to great lengths to show that their SCNT data is valid, making it a more difficult field to publish in.

Because of the lack of human eggs for creating human blastocysts from SCNT, researchers have pursued using eggs donated from other animals (but nuclei from adult human cells) for SCNT. This technique is called human-animal interspecies SCNT (iSCNT). Many ethical concerns have been raised over the use of this technique, especially since an iSCNT embryo contains a human nucleus, but animal mitochondrial DNA (as well as traces of human mitochondrial DNA from the nuclear transfer). ( For more on the ethical implications of these practices, see Skene et al., 2009.)

Some reports have suggested that iSCNT embryos are not “healthy,” primarily because the donated nucleus often does not become fully reprogrammed to an embryonic state. In one such study, a group used chimpanzee somatic cell nuclei with cow eggs, but the embryos did not develop past the 8- to 16-cell stages. While epigenetic changes took place that appeared normal, it turned out that many developmentally important genes were not actually being expressed at all (Nisker et al., 2010). There have also been reports of iSCNT being performed using human and animal cells, such as with rabbit eggs and human nuclei (Chen et al., 2003), though there are still many ethical and developmental concerns surrounding iSCNT.

Despite the lack of available, donated human eggs and fraudulent work of Hwang and colleagues, several stem cell-oriented companies collaboratively published in 2008 the successful creation of human blastocysts from SCNT (French et al., 2008). In this study, 21 human eggs were obtained and each was combined with a nucleus from an adult, somatic (specifically fibroblast) cell through SCNT. From the 21 eggs and nuclei, five blastocysts were created. However, only one was rigorously and conclusively shown to have genomic and mitochondrial DNA from the nucleus donor (though two others also had partial supporting data), suggesting only one true clone was created.

The Future of SCNT: Although much work remains to be done before SCNT is a viable strategy for the production of patient-specific ESCs, there is enormous potential it could fulfill when it does reach that point. Not only may a patient have cells made that are specific to them and available as virtually any cell type they should need (such as insulin-producing beta cells for a patient with diabetes), but, for example, if they have a genetic disease this too may be cured (as was demonstrated in mice using this technology). Researchers could also create cell lines from patients to study specific diseases in the laboratory, greatly advancing our understanding of these conditions (though this is currently being explored with iPSCs). Lastly, as our ability to create ESC lines from different stage embryos and from fewer cells in these embryos, it should become easier to create NTSCs from SCNT-generated embryos (Cervera and Stojkovic, 2008). As SCNT research has come close to being completely banned in the United States, it’s important not only for researchers to take proper ethical precautions, but for everyone to keep in mind the great potential this technology holds.

References:

Beyhan, Z. and Cibelli, J. B. Prospects of Somatic Cell Nuclear Transfer-Derived Embryonic Stem Cells in Regenerative Medicine. 2008.
View Book Chapter

Byrne, J. A., Pedersen, D. A., Clepper, L. L., Nelson, M., Sanger, W. G., Gokhale, S., Wolf, D. P., and Mitalipov, S. M. Producing primate embryonic stem cells by somatic cell nuclear transfer. Nature. 2007. 450: 497-502.
View Article

Cervera, R. P. and Stojkovic, M. Commentary: Somatic Cell Nuclear Transfer – Progress and Promise. Stem Cells. 2008. 26(2): 494-495.
View Article

Chen, Y., He, Z. X., Liu, A., et al. Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes. Cell Research. 2003. 13: 251-263.
View Article

French, A. J., Adams, C. A., Anderson, L. S., et al. Development of Human Cloned Blastocysts Following Somatic Cell Nuclear Transfer with Adult Fibroblasts. Stem Cells. 2008. 26(2): 485-493.
View Article

Gurdon, J. B. and Byrne, J. A. The First Half-Century of Nuclear Transplantation.
PNAS. 2003. 100(14): 8048-8052.
View Article

King, T. J. and Briggs, R. Transplantation of Living Nuclei of Late Gastrulae into Enucleated Eggs of Rana pipiens. J. Embryol. Exp. Morphol. 1954. 2: 73-80.

McKinnell, R. G., and Di Bernardino, M. A. The Biology of Cloning: History and Rationale. BioScience. 1999. 49(11): 875-885.
View Article

Munsie, M. J., Michalska, A. E., O’Brien, C. M., et al. Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei. 2000. 10(16):989-992.
View Article

Niemann, H., Tian, X. C., King, W. A., and Lee, R. S. F. Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning. Reproduction. 2008. 135: 151-163.
View Article

Saric, T., Mehrjardi, N. Z., and Hescheler, J. Alternative Embryonic Stem Cell Sources. Stem Cell Biology in Health and Disease. 2009. 2:101-143.
View Article

Skene, L., Testa, G., Hyun, I, et al. Ethics Report on Interspecies Somatic Cell Nuclear Transfer Research. Cell Stem Cell. 2009. 5(1): 27-30.
View Article

Solter, D. Dolly is a Clone – and no Longer Alone. Nature. 1998. 394: 315-316.
View Article

Sparman, M., Dighe, V., Sritanaudomchai, H., et al. Epigenetic Reprogramming by Somatic Cell Nuclear Transfer in Primates. Stem Cells. 2009. 27(6): 1255-1264.
View Article

Spemann, H. Embryonic Development and Induction. Yale University Press, New Haven. 1938.

Takahashi, K. and Yamanaka, S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell. 2006. 126: 663–676.
View Article

Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts. Cell. 2007. 131: 1-12.
View Article

Wakayama, T., Perry, A. C. F., Zuccotti, M., Johnson, K. R., and Yanagimachi, R. Full-
Term Development of Mice from Enucleated Oocytes Injected with Cumulus Cell Nuclei. Nature. 1998. 394: 369-374.
View Article

Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J., and Campbell, K. H. S. Viable Offspring Derived from Fetal and Adult Mammalian Cells. Cloning and Stem Cells. 1997. 9(1): 3-7.
View Article

Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I., and Thomson, J. A. Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. Science. 2007. 318(5858): 1917-1920.
View Article

Yu, J., Hu, K., Smuga-Otto, K., Tian, S., Stewart, R., Slukvin, I., and Thomson, J. A. Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences. Science. 2009. 324(5928): 797-801.
View Article

Zhou, H., Wu, S., Joo, J., Zhu, S., Han, D. W., Lin, T., Trauger, S., Bien, G., Yao, S., Zhu, Y., Siuzdak, G., Schöler, H. R., Duan, L., and Ding, S. Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins. Cell Stem Cell. 2009. 4(5): 381-384.
View Article

Image of “Somatic Cell Nuclear Transfer to Create Dolly” was taken from Wikipedia and redistributed freely as it is in the public domain.

Cancerous tumors and embryonic tissues have been found to share many of the same antigens, which are detected by the immune system through antibodies. This group of antigens is called oncofetal antigens. Consequently, animals can be vaccinated with embryonic tissues/cells (most recently done with human embryonic stem cells) and develop an immune response against cancer.

Interestingly, this state of immune tolerance is similar to what happens during pregnancy, and, more specifically, it’s been found that the body’s response to a tumor is very similar to its response to embryonic tissues. While much recent research has not been published in this area, there is actually a long history of studies that show: (1) there is a significant number of antigens shared between tumors and embryonic tissues (called “oncofetal antigens”) and, consequently, antibodies made against tumors can also recognize embryonic tissues, and vice versa; (2) pregnancy confers some immunity against cancer (accompanied by antibody production against oncofetal antigens), not only against its occurrence but also against its growth; (3) similar to pregnancy, an immune response against cancer can be generated by vaccinating animals with embryonic tissues. These studies and the recent re-visitation will be explored below (for a more detailed review, see Brewer et al., 2009).

The first published suggestion that tumors may have an embryonic nature came in the early 1800s (Muller, 1838). Tumors were suspected to be tissues that had been triggered to become embryonic-like again, and it is now generally accepted that tumors are indeed more “embryonic” than the tissues they are derived from, due to the re-expression of embryonic-related genes. By the late 1800s, researchers understood cancer enough to realize that they must better understand normal development in order to better combat cancerous tumors and their embryonic-like cells (Brewer et al., 2009). In the 1880s, these studies shifted focus; the field of immunology was born (from research conducted by Louis Pasteur, at the University of Strasbourg, and Robert Koch, as a medical officer in Poland) and many researchers focused on creating vaccines to cure diseases. Cancer was no exception.

The Discovery and Establishment of Oncofetal-Antigens

As the field of immunology blossomed, in the 1920s and 1930s researchers found that tumors express antigens that are also found on embryonic tissues (an antigen is a molecule that the immune system recognizes, usually a protein on the cell’s surface). Specifically, it was found that antibodies made against tumors in the digestive track reacted with the tumors as well as tissues from embryonic and fetal gut and pancreas (Hirzfeld, 1929; Hirzfeld et al., 1932). Later in the early 1970s, several studies reported that antibodies developed against a variety of animal tumors reacted against embryos (as well as the original tumor)(Brewer et al., 2009).

While it was becoming clear that antibodies made against tumors could recognize embryonic tissues, researchers wondered whether the reverse was true: Could antibodies made against embryonic tissues react against tumors? In the 1960s, it was found that about 80% of blood sera (which normally contains antibodies) from pregnant women in the first two trimesters contained antibodies that reacted with tumors (as well as embryonic tissue) (Alexdander and Fairley, 1967). These and following studies led to the idea of universal “oncofetal antigens,” antigens that are expressed by tumors and fetal/embryonic tissues.

Pregnancy Confers Some Cancer Immunity

As the concept of oncofetal antigens developed, researchers explored a hypothesis with very practical applications: Because pregnant animals develop antibodies that can recognize tumor tissues, pregnancy may “immunize” an animal against cancer. Some early observations that supported this theory were nearly 300 years old, when celibate nuns were thought to have increased incidences of breast, uterine, and ovarian cancer (Brewer et al., 2009). While some hypothesized that this was due to a lack of “hormonal stimulation” (as has been suspected to be a factor in breast cancer), others thought it might be due to a lack of antibodies generated against embryonic tissues. Further evidence to support the latter explanation was published in the 1960s to 1980s, as multiple studies reported that women and animals that were multiparous (pregnant multiple times) not only had less spontaneous cancers, but multiparous animals also had fewer carcinogen-induced cancers and were somewhat resistant to transplanted tumors (Brewer et al., 2009). In the early 1970s, researchers further dissected this immune response and found that pregnant women produced cytotoxic T lymphocytes that could kill tumor cells but not normal, benign cells (Ambrose et al., 1971).

(As an interesting side note, a decrease in pregnancy rate has been found to correlate with the presence of antibodies against oncofetal antigens, and the effect can be replicated by immunizing with embryonic or tumor tissues. For women who have spontaneous abortions, it has been found that [at the time of a spontaneous abortion] antibodies are detectable in their sera that react against tumors; when these antibodies were injected into rats, there was a significant decrease in tumor growth [Buttle et al., 1964]. This effect has been repeated in animals; when animals were immunized with embryonic or tumor tissues, their pregnancy rate also significantly decreased [Parmiani and Della Porta, 1973].)

Embryonic Tissues Confer Some Cancer Immunity

While researchers were finding that pregnant animals have some immunity against cancer, researchers started testing whether injecting, or “immunization,” with embryonic tissues alone could similarly trigger an immune response and/or confer cancer immunity. In the late 1960s and early 1970s, many reports were published supporting this theory; anti-tumor antibodies were repeatedly found to be stimulated, and tumors sometimes even prevented, after vaccination with early embryonic tissues or cells (but not adult tissues or cells), even when tested across different species, revealing the presence of conserved antigens (Brewer et al., 2009). In 1970, researchers found that rabbits immunized with homogenized 9-day-old mouse embryos created antibodies that cross-reacted with 72 different mouse tumors from 12 different tissues of origin (created “spontaneously” or induced by viruses or chemicals). The antibodies, as expected, also reacted against embryonic tissues, and, strangely, adult skin (but no other adult tissues) (1970, Stonehill and Bendich). Human embryos also conferred an immune response; again, antibodies produced against the embryos recognized many different types of human tumors and showed no cross-reactivity with adult tissues except skin (Klavins et al., 1971). Immunization with embryonic cells (instead of homogenized embryos) had similar results; immunized mice made antibodies that recognized tumors, embryos, and, again, adult skin (Bendich et al., 1973). These immunized mice were also fairly resistant to tumor induction. Overall, these studies of the late 1960s and early 1970s strongly suggest that vaccination with embryonic tissues not only triggers an immune response against cancer, but may also prevent it to some degree.

(Interestingly, studies like these revealed that even virally-induced cancers [such as SV-40 and Rauscher leukemia virus] are caused by the re-expression of embryonic genes [and not the expression of new, viral genes]; rodents injected with embryonic tissues had an immune response against such virally-induced tumors [Brewer et al., 2009].)

The Problem of Immune Tolerance

But in order to truly harness the potential of using embryonic tissues/cells/proteins to immunize against cancer, the problem of immune tolerance must be better understood. In both patients with cancer and women who are pregnant, immune tolerance takes place in an apparently similar, transient manner. In the 1960s, a study reported that while patients with sarcomas usually did not have detectable antibodies against the tumor, after the tumor was surgically removed, anti-tumor antibodies increased, while this was not seen in patients whose sarcomas were not successfully removed (Ambrose et al., 1971b; Morton et al., 1970). Similarly, antibodies against oncofetal antigens are present in pregnant women in the first two trimesters (presumably while the embryo is seen as “non-self”), then disappear, then reappear after birth (Gold, 1967). (Younger embryos in mice have also been found to be most effective at conferring tumor immunity.) The cause for the transient immune tolerance in both cases is unclear, although “blocking factors” in serum were identified in the early 1970s. The factors most likely (1) prevent recognition of the embryo/cancer (are protective antibodies or antibody/antigen complexes) and/or (2) are immunosuppressive cytokines, e.g. TGF-beta (Brewer et al., 2009). Clearly, to better understand immune tolerance in cancer, it is necessary to better understand it during pregnancy. There are most likely many reasons in common for why pregnancy and tumors are not rejected more often than they are.

Recent Revisiting

While these studies showed many promising findings for fighting cancer, published reports significantly decreased after the mid-1970s, possibly due to decreased funding and infeasibility of progressing the studies further for obvious reasons (Brewer et al., 2009). However, the connection between cancer and embryonic tissues/cells has been revisited recently with the development of human embryonic stem cells.

While it is still largely under debate, much evidence suggests that cancer arises from cancer stem cells. Cancer stem cells share many similarities with human embryonic stem cells (hESCs) (which are stem cells isolated from the inner cell mass of a blastocyst) and induced pluripotent stem cells (iPSCs) (which are adult cells that have been reprogrammed to be hESC-like). These similarities were explored in a previous All Things Stem Cell article: “Better Understanding Cancer and Induced Pluripotent Stem Cells Through Their Similarities.” The basic similarities between iPSCs/hESCs and cancer stem cells are that they can all (1) be potentially pluripotent (or at least multipotent, having an increased potency), (2) avoid apoptosis/cell death (they are proliferative), and (3) express similar cell markers (such as some oncofetal antigens). hESCs and iPSCs can also, due to these traits, create teratoma tumors (a tumor with cells from all three germ layers) when injected into animals.

Recently, a report by professors Yi Li, Zihai Li, and colleagues (at the University of Connecticut School of Medicine) expanded upon the previous embryonic immunization studies by using an established embryonic stem cell line for the first time. This re-visitation was most likely prompted by recent interest in the promising, growing fields of cancer vaccines and embryonic stem cells. Many cancer vaccines target oncofetal antigens, which are present on hESCs and iPSCs as well, lending support to the idea of using hESCs and iPSCs to stimulate an immune response against multiple antigens on a tumor.

Li et al., 2009 reported that immunization of mice with hESCs resulted in an immune response against colon cancer (CT26). The mice were immunized twice, one week apart, with hESCs (line H9), iPSCs, and irradiated colon cancer cells (CT26), and one week later exposed to the CT26 colon cancer cells. The hESCs conferred consistent, cellular and humoral responses against the colon cancer (as did the irradiated CT26 cells, as expected), significantly reducing the tumor size. Surprisingly, the iPSC line appeared to be significantly inferior at producing immunity against the tumor, relative to the hESC and CT26 cells. No significant autoimmunity was observed, which can be a concern. (For a commentary on this report, see Zwaka, 2010.)

With this recent report by Li et al. bringing attention back to the long-standing field of cancer vaccination using embryonic tissues and cells, other groups may also revisit this potentially promising tool for fighting cancer.

References

Alexander, P., and Fairley, G. H. Cellular resistance to tumors. Br. Med. Bull. 1967. 23:86–92.

Ambrose, K. R., Anderson, N. G., and Coggin, J. H. Cytostatic antibody and SV40 tumour immunity in hamsters. Nature. 1971a. 233:321–324.
View Article

Ambrose, K. R., Anderson, N. G., and Coggin, J. H. Interruption of SV40 oncogenesis with human foetal antigen. Nature. 1971b. 233:194–195.
View Article

Bendich, A., Borenfreund, E., and Stonehill, E. H. Protection of adult mice against tumor challenge by immunization with irradiated adult skin or embryo cells. J. Immunol. 1973. 111:284–285.
View Article

Brewer, B. G., Mitchell, R. A., Harandi, A., Eaton, J. W. Embryonic Vaccines Against Cancer: An Early History. Exp. Molec. Path. 2009. 86:192-197.
View Article

Buttle, G. A. H., Eperon, J., and Menzies, D. N. Induced tumour resistance in rats. Lancet. 1964. 2:12–14.
View Article

Gold, P. Circulating antibodies against carcinoembryonic antigens of the human digestive system. Cancer. 1967. 20:1663–1668.
View Article

Hirzfeld, L. Untersuchungen über die serologischen Eigenschaften der Gewebe: über serologische Eigenschaften der Neubildungen. Z. Immun.Forsch. Exp. Ther. 1929. 64:81–113.

Hirzfeld, L., Halber, U., and Rosenblat, J. C. Verwandtschaftsreaktionen zwischen Embryonal- und Krebsgewebe; Mesenchenembryo und Menschenkrebs. Z. Immunitatsforsch. Exp. Ther. 1932. 75: 209–216.

Li, Y., Zeng, H., Xu, R., Liu, B., Li, Z. Vaccination with Human Pluripotent Stem Cells Generates a Broad Spectrum of Immunological and Clinical Responses Against Colon Cancer. Stem Cells. 2009. 27:3103-3111.
View Article

Morton, D. L., Eilber, F. R., Joseph, W. L., Wood, W. C., Trahan, E., and Ketcham, A. S. Immunological factors in human sarcomas and melanomas. Ann. Surg. 1970. 172:740–749.
View Article

Muller, J. Ueber den feineren Bau und die Formen der Krankhaften Geschwulste. Reimer, Berlin (1938).

Parmiani, G., and Della Porta, G. Effects of antitumour immunity on pregnancy in the mouse. Nat. New Biol. 1973. 241:26–28.

Stonehill, E. H. and Bendich, A. Retrogenetic expression: the reappearance of embryonal antigens on cancer cells. Nature. 1970. 228:370–372.
View Article

Zwaka, T. P. Stem Cell Vaccination Against Cancer: Fighting Fire With Fire? Molec. Ther. 2010. 18.1:8-9.
View Article

Image of “Antibody and Antigens” was taken from Wikipedia and redistributed freely as it is in the public domain.

Direct Reprogramming in the Hematopoietic System. Several different transcription factors have been found that can directly reprogram one type of blood cell into another. Changing the expression levels of GATA-1 in monocytes (red) can make them differentiate into eosinophils, erythrocytes, or megakaryocytes. Making B-cells (B lymphocytes) express C/EBP transcription factors (blue) can cause them to differentiate into macrophages. Lastly, C/EBPs can also inhibit the function of the transcription factor Pax5; when Pax5 is deleted in B-cells they differentiate into T-cells (T lymphocytes), though they first dedifferentiate into a common lymphoid progenitor.

References

Choi, J., Costa, M. L., Mermelstein, C. S., Chagas, C., Holtzer, S., Holtzer, H. MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes. Proc. Natl Acad. Sci. 1990. 87: 7988–7992.
View Article

Cobaleda, C., Jochum, W. & Busslinger, M. Conversion of mature B cells into T cells by dedifferentiation to uncommitted progenitors. Nature. 2007. 449: 473–477.
View Article

Davis, R. L., Weintraub, H., Lassar, A. B. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell. 1987. 51(6): 987-1000.
View Article

Kulessa, H., Frampton, J., Graf, T. GATA-1 reprograms avian myelomonocytic
cell lines into eosinophils, thromboblasts, and erythroblasts. Genes & Dev. 1995. 9: 1250–1262.
View Article

Laiosa, C. V., Stadtfeld, M., Xie, H., de Andres-Aguayo, L., Graf, T.
Reprogramming of committed T cell progenitors to macrophages and dendritic
cells by C/EBPa and PU.1 transcription factors. Immunity. 2006. 25: 731–744.
View Article

Nutt, S. L., Heavey, B., Rolink, A. G., Busslinger, M. Commitment to the
B-lymphoid lineage depends on the transcription factor Pax5. Nature. 1999. 401:
556–562.
View Article

Schäfer, B. W., Blakely, B. T., Darlington, G. J., Blau, H. M. Effect of cell history on response to helix–loop–helix family of myogenic regulators. Nature. 1990. 344: 454 – 458.
View Article

Takahashi, K. and Yamanaka, S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell. 2006. 126: 663–676.
View Article

Vierbuchen, T., Ostermeier, A., Pang, Z. P., Kokubu, Y., Südhof, T. C., Wernig, M.
Direct conversion of fibroblasts to functional neurons by defined factors. Nature. 2010. Advanced publication.
View Article

Xie, H., Ye, M., Feng, R., Graf, T. Stepwise reprogramming of B cells into
macrophages. Cell. 2004. 117: 663–676.
View Article

Yechoor, V. et al. Neurogenin3 is sufficient for transdetermination of hepatic
progenitor cells into neo-islets in vivo but not transdifferentiation of hepatocytes.
Dev. Cell. 2009. 16: 358–373.
View Article

Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I., and Thomson, J. A. Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. Science. 2007. 318(5858): 1917-1920.
View Article

Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J., Melton, D. A. In vivo reprogramming
of adult pancreatic exocrine cells to beta-cells. Nature. 2008. 455: 627–632.
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DNA is condensed on histones, creating a structure called chromatin. (Left) A single DNA strand (formed by a sugar-phosphate backbone and nucleotide base-pairs). (Right) Chromatin is the complex formed by histones (green) and DNA (blue); the DNA can be tightly wrapped around the histones. (DNA bound to histones may be inaccessible to the transcription machinery, preventing the transcription of these genes, while unbound DNA allows space for the machinery and the genes may be transcribed.) Chd1 may function in ESCs to maintain chromatin in an open (euchromatin) state and potentially promote pluripotency in this way.

Chromatin structure plays an important role in regulating what genes are created, or expressed, in a given cell. In eukaryote organisms (almost all large organisms, such as animals, plants, and fungi, but not bacteria), DNA forms a complex with proteins that are called histones. This complex of DNA and histones is called chromatin (see figure). Histones act as spools for the DNA to be spun around, binding to DNA and packaging it into tightly coiled units (without histones, the long DNA strands would take up a very large amount of space). Whether the histones bind to the DNA or not can be regulated through chemical modification of the histones (they can be methylated or acetylated). When histones are bound to the DNA, the chromatin is in a condensed state (called heterochromatin) and the genes are not expressed because they cannot be accessed by the gene transcription machinery. However, when the histones are not bound to the DNA, the chromatin is extended (called euchromatin), and the DNA can be accessed and these genes can be expressed.

It was previously believed that embryonic stem cells had lots of open chromatin (euchromatin), but this was not a proven theory. A study on stem cells and gene expression (Efroni et al., 2008) reported that, globally but at low-levels, more genes in ESCs are actively turned into protein than are in differentiated cells. Additionally, proteins involved in changing chromatin structure and transcribing genes were expressed at relatively high levels in ESCs too. When the function of some proteins involved in chromatin-remodeling was changed, normal ESC proliferation and differentiation was also affected. Overall, Efroni et al. suggested that the differentiation of ESCs may correlate with a loss of active transcription of the cell genome.

Chd1 (chromodomain-helicase-DNA-binding protein 1) was suspected since its discovery in 1993 to play a role in gene regulation (Delmas et al., 1993). This protein was found to be in most, if not all, mammals. It has three key protein domains: a DNA-binding domain, a chromodomain (which may bind euchromatin), and a helicase domain (which is thought to activate transcription by acting against repressing transcription effects, such as heterochromatin structure).

A few months ago, research efforts led by Dr. Eran Meshorer (of Alexander Silberman Institute of Life Sciences at the Hebrew University) and Dr. Miguel Ramalho-Santos (of the University of California, San Francisco) published findings that suggest that Chd1 regulates euchromatin in mouse ESCs (Gaspar-Maia et al., 2009). Specifically, they found that Chd1 is highly expressed in ESCs (relative to differentiated cells) and that Chd1 binds to the promoters of genes actively being transcribed in mouse ESC euchromatin. When the mouse ESCs had decreased amounts of Chd1, heterochromatin (close chromatin) formed. When the ESCs had no Chd1 at all, the cells could not differentiate into all of the three germ layers (specifically, endoderm was not detected, and a preference was found for neuronal lineages). In other words, Chd1-deficient ESCs were no longer pluripotent; Chd1 appears to be necessary for this key trait of ESCs.

Interestingly, the authors also reported that Chd1 may play a key role in reprogramming cells into induced pluripotent stem cells (iPSCs); when Chd1 was downregulated in fibroblast cells and researchers tried to reprogram these cells into iPSCs, significantly fewer iPSCs resulted. These findings indicate that Chd1 may not only be important in maintaining pluripotency in ESCs, but also in creating pluripotency in cells that are not stem cells.

Overall, Chd1 definitely merits further investigation as a possible key regulator of stem cell potency and differentiation. Currently, although it is known that Chd1 binds to euchromatin, the exact mechanisms of how Chd1 counters heterochromatin formation are unclear; it may act to prevent the spread of heterochromatin areas to euchromatin. Additionally, it will be important to see whether this role of Chd1 as a regulator of stem cell potency is maintained in human ESCs and other stem cell types. With a better understanding of Chd1 function, it may be possible to improve the potency of some stem cells, such as in the creation of iPSCs, and it may also be possible to better direct desired stem cell differentiation for potential clinical applications downstream.

References

Gaspar-Maia, A., Alajem, A., Polesso, F., Sridharan, R., Mason, M. J., Heidersbach, A., Ramalho-Santos, J., McManus, M. T., Plath, K., Meshorer, E., Ramalho-Santos, M. Chd1 regulates open chromatin and pluripotency of embryonic stem cells. Nature. 2009. 460: 863-868.
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Efroni, S., Duttagupta, R., Cheng, J., Dehghani, H., Hoeppner, D. J., Dash, C., Bazett-Jones, D. P., Grice, S. L., McKay, R. D. G., Buetow, K. H., Gingeras, T. R., Misteli, T., Meshorer, E. Global Transcription in Pluripotent Embryonic Stem Cells. Cell Stem Cell. 2008. 2(5): 437-447.
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Delmas, V., Stokes, D. G., Perry, R. P. A mammalian DNA-binding protein that contains a chromodomain and an SNF2/SWI2-like helicase domain. Proc. Natl. Acad. Sci. 1993. 90(6): 2414-2418.
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Image of “Chromatin” was taken from Wikipedia and redistributed freely as it is in the public domain.

The most recent and most publicized link between iPSCs and cancer is p53. p53, also known as protein 53 (53 referring to its molecular mass), is a well-studied protein whose normal function is important in preventing cancer. Though p53 has many different roles, they are quite related. In essence, the job of p53 is to make sure the cell does not accumulate DNA damage, or DNA mutations, which could eventually make the cell cancerous. When a cell has its DNA damaged, often from external stresses, p53 stops the normal cell cycle to fix the DNA damage. If the damage is too great to repair, p53 can prevent the cell from dividing, which would create more damaged cells; p53 initiates programmed cell death, or apoptosis. The potential tumor cell dies. Overall, p53 functions as a “tumor suppressor” to prevent abnormal cells from occurring and multiplying into a cancer (Vazquez et al., 2008). Consequently, it has been found that p53 is mutated in approximately 50% of all human tumors, and other tumors may have mutations in the pathway regulating p53 activity (Vazquez et al., 2008). p53 is therefore well-studied as an oncogene, or a gene that when not functioning normally can contribute to a normal cell becoming cancerous.

So what does p53 have to do with iPSCs? One recently discovered connection is with the generation of iPSCs. Recently, many research groups discovered that when p53 is deleted from, or damaged in, their cells, they could more easily become iPSCs (Hong et al., 2009; Kawamura et al., 2009; Utikal et al., 2009; Li et al., 2009; Zhao et al., 2008). As posted earlier, iPSCs are cells that were originally from adult tissues, but have been “reprogrammed” to be pluripotent stem cells, or stem cells able to become all the adult cells of the body, looking and functioning nearly identical to human embryonic stem cells (hESCs) (Takahashi et al., 2007; Yu et al., 2007).

To create iPSCs, adult cells are exposed to “reprogramming factors,” or transcription factors, thought to be important for pluripotency. Researchers have tested different groups of reprogramming factors, and originally four different factors were found to work best. However, the process is rather inefficient; only a very small percentage of cells exposed to the set of factors actually becomes reprogrammed. Shinya Yamanaka’s group, one of the two that first created human iPSCs, found that iPSC generation increased by up to 20% in cells without p53 (Hong et al., 2009). Other groups have reported similar results; cells with non-functional p53 mutants, or mutations in the p53 pathway, had increased reprogramming efficiencies (Utikal et al., 2009; Li et al., 2009), some even with fewer reprogramming factors than are usually needed (Kawamura et al., 2009). Some researchers speculate that p53 is acting to protect the cells from the DNA damage that the reprogramming factors can cause, as p53 is turned on immediately after the factors are introduced (Kawamura et al., 2009). For a more detailed review of these papers, check out (Dolgin, 2009). Overall, these many separate recent reports clearly show the importance of p53 in creating iPSCs. As a side note, a very interesting remaining question is how exactly these cells become pluripotent after bypassing p53 activity.

What are the implications of the fact that decreasing p53 activity greatly increases iPSC derivation? It could imply that just about any cell in the human body has a greatly increased potential to initiate a cancer by losing p53 activity. Interestingly, this actually runs counter to the cancer stem cell hypothesis, which theorizes that not just any cell, but specifically a rare stem cell, may gain mutations over time and give rise to some cancers (see Figure)(Kawamura et al., 2009). This is a somewhat frightening prospect, suggesting that a far greater number of cells have tumorigenic potential than believed by the stem cell hypothesis, though it may help scientists better understand how cancer develops and consequently how it can be successfully fought.

The cancer stem cell hypothesis (top) theorizes that a (rare) mutated stem cell may gain mutations over time and give rise to cancer. Running counter to this (bottom), some evidence suggests that a mutation in p53, causing it to no longer function, in any given cell (including, but not only, stem cells) may greatly increase the cell’s potential to initiate a cancer.

p53 is not the only cancer-related factor important for the creation of iPSCs; most of the reprogramming factors have actually been suggested to be oncogenes and implicated in the generation of different cancers. C-Myc is a widely-used iPSC reprogramming factor (Takahashi et al., 2008) and is also responsible for the regulation of a very large number of different genes within the cell, including controlling cell proliferation. When c-Myc is over-expressed, or expressed at higher levels than normal, it can cause cancer, and high levels of c-Myc have been detected in many different tumor types (Hermeking, 2003). Other studies have shown that all the other reprogramming factors may also be oncogenes, with one exception (Lin28, which has been shown to not even be required for iPSC generation) (Liu, 2008A). But is this really that surprising? The reprogramming factors are genes expressed in embryonic, pluripotent tissues that are thought to be involved in making the tissue pluripotent. The cells that create cancerous tumors are most likely multipotent, being able to become multiple different cell types, because tumors are heterogenous tissues. Consequently, it is not that surprising that researchers have found connections between these embryonic reprogramming factors, and other embryonic-specific genes, and the creation of tumors and cancer stem cells (Wong et al., 2008; Gunaratne, 2009).

While such reports have caused some researchers to label iPSCs as “man-made cancer stem cells” (Liu, 2008B), it is important to keep in mind the distinct differences between iPSCs and cancer. iPSCs indeed, by definition, can create teratoma tumors when injected into animals and express many embryonic proteins which allow them to differentiate into multiple cell types just like tumors can. However, unlike cancer, iPSCs are grown in laboratories under controlled settings and it is only when they are undifferentiated that they have these tumorigenic potentials. To be used in therapies all cells must be carefully differentiated to the desired, adult cell type. iPSCs must lose their multipotency and consequently their tumorigenic potential. Researchers are currently working on many ways to make iPSCs safer for therapeutic use: using p53 to select for iPSCs that have no introduced DNA damage (Kawamura et al., 2009), optimizing the purification of differentiated populations, improving transient expression of the reprogramming genes, and more. Aside from therapies and regenerative medicine, iPSCs have great potential for creating cellular disease models, creating cell lines from reprogrammed diseased tissue to allow for greater study in laboratories; cancer cells cannot do this in the same fashion. Despite their similarities, iPSCs have the ability to offer scientists many important research opportunities that studying cancer by itself does not.

Understanding the connections between iPSCs and cancer has great potential for improving our treatment of cancer. Because iPSCs are reprogrammed adult cells, it may be possible to think of tumors as reprogrammed adult cells as well. Since iPSCs can be differentiated into specific, desired cell types, some researchers think it may also be possible to differentiate tumors into non-malignant cell types. However, the human body is a much more complicated environment than cells in a controlled laboratory setting (Blelloch et al., 2004; Yang et al., 2008). To potentially improve our abilities to treat cancer, it will take a great open-mindedness and understanding of not only the behavior of these cells in the laboratory, but also of their possible similarities to cancer initiation as it occurs in an organism.

References

Blelloch, R. B., Hochedlinger, K., Yamada, Y., Brennan, C., Kim, M., Mintz, B., Chin, L., and Jaenisch, R. Nuclear cloning of embryonal carcinoma cells. PNAS. 2004. 101(39): 13985-13990.
View Article

Dolgin, E. Immortality improves cell reprogramming: Knocking out genes with a role in cancer prevention helps produce stem cells. Nature News. 2009.
View Article

Gunaratne, P. H. Embryonic Stem Cell MicroRNAs: Defining Factors in Induced Pluripotent (iPS) and Cancer (CSC) Stem Cells? Curr Stem Cell Res Ther. 2009.
View Article

Hermeking, H. The MYC Oncogene as a Cancer Drug Target. Current Cancer Drug Targets. 2003. 3(3): 163-175.
View Article

Hong, H., Takahashi, K., Ichisaka, T., Aoi, T., Kanagawa, O., Nakagawa, M., Okita, K., and Yamanaka, S. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 460, 1132-1135 (27 August 2009).
View Article

Kawamura, T., Suzuki, J., Wang, Y. V., Menendez, S., Morera, L. B., Raya, A., Wahl, G. M., and Izpisúa Belmonte, J. C. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature. 2009. 460: 1140-1144.
View Article

Li, H., Collado, M., Villasante, A., Strati, K., Ortega, S., Cañamero, M., Blasco, M. A., and Serrano, M. The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature. 2009. 460: 1136-1139.
View Article

Liu, S. V. iPS Cells: A More Critical Review. Stem Cells and Development. 2008A. 17: 391-397.
View Article

Liu, S. V. IPS Cells are Man-Made Cancer Cells. Logical Biology. 2008B. 8(1): 16-18.
View Article

Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts. Cell. 2007. 131: 1-12.
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Utikal, J., Polo, J. M., Stadtfeld, M., Maherali, N., Kulalert, W., Walsh, R. M., Khalil, A., Rheinwald, J. G., and Hochedlinger, K. Immortalization eliminates a roadblock during cellular reprogramming into iPS cells. Nature. 2009. 460: 1145-1148.
View Article

Vazquez, A., Bond, E. E., Levine, A. J., and Bond, G. L. The genetics of the p53 pathway, apoptosis and cancer therapy. Nature Reviews Drug Discovery. 2008. 7: 979-987.
View Article

Wong, D. J., Liu, H., Ridky, T. W., Cassarino, D., Segal, E., and Chang, H. Y. Module Map of Stem Cell Genes Guides Creation of Epithelial Cancer Stem Cells. Cell Stem Cell. 2008. 2(4): 333-344.
View Article

Yang, Y., Zhang, L., Wei, Y., Wang, H., Fukuma, M., Xu, H., Xiong, W., and Zheng, J.
Neural differentiation arrest in embryonal carcinoma cells with forced expression of EWS-FLI1. Journal of Neuro-Oncology. 2008. 90(2): 141-150.
View Article

Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I., and Thomson, J. A. Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. Science. 2007. 318(5858): 1917-1920.
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Zhao, Y., Yin, X., Qin, H., Zhu, F., Liu, H., Yang, W., Zhang, Q., Xiang, C., Hou, P., Song, Z., Liu, Y., Yong, J., Zhang, P., Cai, J., Liu, M., Li, H., Li, Y., Qu, X., Cui, K., Zhang, W., Xiang, T., Wu, Y., Zhao, Y., Liu, C., Yu, C., Yuan, K., Lou, J., Ding, M., and Deng, H. Two supporting factors greatly improve the efficiency of human iPSC generation. Cell Stem Cell. 2008. 3(5): 475-9.
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Applying Somatic Cell Nuclear Transfer in the Creation of Dolly the Cloned Sheep. Dolly the sheep was cloned through somatic cell nuclear transfer (SCNT). An adult cell from the mammary gland of a Finn-Dorset ewe acted as the nuclear donor; it was fused with an enucleated egg from a Scottish Blackface ewe, which acted as the cytoplasmic (or egg) donor. An electrical pulse acted to fuse the cells and activate the oocyte after injection into the surrogate mother ewe. A successfully implanted oocyte developed into the lamb Dolly, a clone of the nuclear donor, the Finn-Dorset ewe.

The idea of reprogramming a cell from adult tissue into an embryonic-like, pluripotent cell existed long before the creation of iPSCs. In 1938, Hans Spemann showed that a nucleus from a fertilized salamander egg that had already undergone cell division several times could be implanted into a cell from a newly fertilized salamander egg that is enucleated (has had its nucleus removed) and create an entire adult salamander (Spemann, 1938). Consequently, Spemann’s work suggests that an embryonic nucleus remains totipotent, or is able to develop into any cell type of the adult body, even after several cell divisions. Due to technical difficulties, it was several years before researchers could repeat these experiments using older nuclei to see how long the nucleus retains its pluripotency. In the early 1950s, Robert Briggs and Thomas King repeated Spemann’s experiments using a species of leopard frog, Rana pipiens, first with a nucleus from young embryos (Briggs and King, 1952) then from older embryos (King and Briggs, 1954); both the younger and older implanted nuclei could still be reprogrammed by the enucleated host cell. However, they also observed that the older the donor nucleus was, the more difficult it was to reprogram it to a totipotent state. For years it was unclear whether the nucleus from a fully differentiated, adult cell could be completely reprogrammed, as conflicting results were published by different groups (Briggs and King, 1957; Fishberg et al., 1958; Gurdon and Byrne, 2003).

Although the studies done by Spemann, Briggs, and King used nuclei from embryos, their results are the basis for somatic cell nuclear transfer (SCNT). SCNT is a technique wherein the nucleus from a somatic cell (an adult cell that is not a sperm or egg, i.e. not the gametes) is implanted into an enucleated egg cell which can then be implanted into, and develop in, a surrogate mother, and potentially become an adult organism. The resultant organism is a clone of the animal that donated the nucleus. The first widely-accepted successful use of SCNT came with the creation of the sheep Dolly in 1997, the first cloned animal from an adult cell and the first cloned mammal (Wilmut et al., 1997). Since then, several other animals have been successfully cloned, though many problems still remain and there are low success rates (Wilmut et al., 1997; Wakayama et al., 1998; Solter, 1998; McKinnell and Di Bernardino, 1999; Gurdon and Byrne, 2003).

With the living evidence of Dolly and other animals cloned from adult cells, the idea that an adult somatic cell could become a reprogrammed embryonic-like cell regained a spotlight in the scientific community. The creation of iPSCs began by studying proteins not only uniquely expressed in embryonic stem cells, but proteins known to be functionally important in creating the unique properties of these cells. In 2006, Shinya Yamanaka’s group made the first iPSCs by applying this knowledge in mouse cells (Takahashi and Yamanaka, 2006). They made adult fibroblastic mouse cells become essentially mouse embryonic stem cells, in appearance and function, by forcing the fibroblasts to express four key embryonic stem cell factors: Oct-4, Sox2, Klf4, and c-Myc. The induced expression of these factors was accomplished through transducing the fibroblasts with, or making the fibroblasts uptake, a retrovirus vector that produced the DNA for these four proteins. The DNA was then incorporated into the genome of the fibroblasts and translated into protein by the host cell.

The same principles applied to the creation of human iPSCs, which was reported only a year later concurrently, though independently, by the laboratories of Yamanaka and James Thomson (Yu et al., 2007; Takahashi et al., 2007). Yamanaka’s group used human adult dermal fibroblasts and induced them to become iPSCs, appearing and functioning like hESCs, by having them express the same proteins as he used with mouse cells: Oct-4, Sox2, Klf4, and c-Myc (Takahashi et al., 2007). Thomson’s group also created human iPSCs, but used fetal fibroblasts and foreskin fibroblasts and a different set of proteins; while both groups used Oct-4 and Sox2, Thomson’s group used Nanog and Lin28 instead of Klf4 and c-Myc (Yu et al., 2007). Even though different cell types were used as the initial starting materials, and they were made to produce different sets of proteins, both groups were able to identify and isolate hESC-like cell colonies only 20 to 30 days after transduction. Both groups reported that some factors were more important than others in inducing the adult cells to become embryonic; Oct-4 and Sox2 appear to be essential.

Though iPSCs and hESCs are both pluripotent and have a virtually infinite supply, there are distinct advantages and disadvantages associated with each cell type. Being created from adult cells, iPSCs overcome some ethical concerns associated with hESCs and can potentially be patient-specific, but may also have shorter life spans than hESCs due to the donor cell age. Additionally, iPSCs originally generated contain DNA randomly inserted into the genome from the retroviral vectors. However, iPSC technology has been making great leaps and bounds in the three years since their creation; researchers have found ways of making iPSCs with non-integrating vectors (Yu et al., 2009) and, more recently, have created iPSCs without directly altering the adult cell genome at all but instead delivered the key reprogramming proteins to the cells (Zhou et al., 2009). Researchers are quickly overcoming the hurdles to using iPSCs in human clinical trials, though some issues still remain to be addressed.

References:

Briggs, R. and King, T. J. Proc. Natl. Acad. Sci. 1952. 38: 455-463.

Briggs, R. and King, T. J. J. Embryol. Exp. Morphol. 1957. 100: 269-312.

Fishberg, M., Gurdon, J. B., and Elsdale, T. R. Nature. 1958. 181: 424.

Gurdon, J. B. and Byrne, J. A. The First Half-Century of Nuclear Transplantation.
PNAS. 2003. 100(14): 8048-8052.
View Article

King, T. J. and Briggs, R. Transplantation of Living Nuclei of Late Gastrulae into Enucleated Eggs of Rana pipiens. J. Embryol. Exp. Morphol. 1954. 2: 73-80.

McKinnell, R. G., and Di Bernardino, M. A. The Biology of Cloning: History and Rationale. BioScience. 1999. 49(11): 875-885.
View Article

Solter, D. Dolly is a Clone – and no Longer Alone. Nature. 1998. 394: 315-316.
View Article

Spemann, H. Embryonic Development and Induction. Yale University Press, New Haven. 1938.

Takahashi, K. and Yamanaka, S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell. 2006. 126: 663–676.
View Article

Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts. Cell. 2007. 131: 1-12.
View Article

Wakayama, T., Perry, A. C. F., Zuccotti, M., Johnson, K. R., and Yanagimachi, R. Full-
Term Development of Mice from Enucleated Oocytes Injected with Cumulus Cell Nuclei. Nature. 1998. 394: 369-374.
View Article

Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J., and Campbell, K. H. S. Viable Offspring Derived from Fetal and Adult Mammalian Cells. Cloning and Stem Cells. 1997. 9(1): 3-7.
View Article

Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I., and Thomson, J. A. Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. Science. 2007. 318(5858): 1917-1920.
View Article

Yu, J., Hu, K., Smuga-Otto, K., Tian, S., Stewart, R., Slukvin, I., and Thomson, J. A. Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences. Science. 2009. 324(5928): 797-801.
View Article

Zhou, H., Wu, S., Joo, J., Zhu, S., Han, D. W., Lin, T., Trauger, S., Bien, G., Yao, S., Zhu, Y., Siuzdak, G., Schöler, H. R., Duan, L., and Ding, S. Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins. Cell Stem Cell. 2009. 4(5): 381-384.
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Image of “Somatic Cell Nuclear Transfer to Create Dolly” was taken from Wikipedia and redistributed freely as it is in the public domain.