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

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

In brief, in most mammals the trophoblast is the part of the early embryo that later significantly contributes to the placenta of the fetus. The embryo and mother work together to create the placenta; while the trophoblast of the embryo becomes the chorion part of the placenta, the maternal uterine cells and surrounding blood vessels form the maternal placental components (Gilbert, 2003).

The placenta is the organ in mammals that connects the uterine wall to the developing fetus, bringing the two blood systems close together. The placenta allows the fetus to safely receive essential gases, such as oxygen, and nutrients from the mother. At the same time, it also lets the fetus expel waste through the mother’s kidneys. Additionally, the placenta releases essential pregnancy-related hormones and growth factors that, for example, let the uterus hold the fetus. Lastly, the placenta secretes immune response regulators to give the fetus immune protection against the mother (so that the fetus is not rejected by the mother’s immune system, as a tissue graft or organ transplant would be) (Rossant and Cross, 2001; Gilbert, 2003). Overall, the placenta plays a key role in early development; even small abnormalities in the placenta can lead to death of the fetus (Rossant and Cross, 2001).

Figure 1: The blastocyst is a hollow sphere made of approximately 150 cells and contains three distinct areas: the trophoblast, which is the surrounding outer layer that contains the trophoblast stem cells and later becomes the placenta, the blastocoel, which is a fluid-filled cavity within the blastocyst, and the inner cell mass, also known as the embryoblast, which can become the embryo proper, or fetus, and is where human embryonic stem cells are isolated from. When the late blastocyst is implanted in the uterine wall, at day 7 or 8 in human development, the trophoblast stem cells (in the trophoblast) quickly differentiate to form cells required for a firm implantation and, later, for the placenta.

While TSCs give rise to the placenta, these stem cells establish their identity long before the placenta develops; their fate is determined during the early embryo. Soon after the egg and sperm join during fertilization, the resultant zygote (fertilized egg cell) starts undergoing cell division. The resulting cells continue to undergo synchronous cell division. When the embryo is at the 16-cell stage (called a morula), it is a solid sphere of cells and already the precursors of the trophoblast cells are defined; the external, relatively larger cells mostly become the trophoblast cells. By the 64-cell stage, these cells’ fates are set; while the trophoblast will become the placenta, the other cells in the embryo can become the fetus. In mammalian development, this is the first differentiation event (Rossant and Cross, 2001; Gilbert, 2003).

A few cell divisions later, the trophoblast contributes to significant cellular rearrangements in the embryo which make it enter the blastocyst stage (see Figure 1). The blastocyst, which contains approximately 150 cells, is made up of three main parts: the blastocoel (an internal, fluid-filled cavity), the inner cell mass (ICM), and the trophoblast. When the embryo was a morula, the surrounding trophoblast precursors caused fluid to be secreted into the morula (utilizing sodium pumps in the trophoblast cell membranes); this secretion created the blastocoel cavity. The ICM is a cluster of cells inside the blastocyst that will later become the adult organism; human embryonic stem cells can be derived from the ICM, as was previously discussed. Lastly, the trophoblast is a monolayer of cells, specifically polarized epithelial cells, which surround the blastocoel and ICM, similar to their future role of surrounding the fetus as its placenta (Rossant and Cross, 2001; Gilbert, 2003).

A great deal of cell signaling occurs right before implantation of the late blastocyst into the uterine wall in order to ready the trophoblast for the next developmental stage, gastrulation. Prior to implantation, the ICM and neighboring trophoblast cells secrete proteins that make the trophoblast become highly proliferative and take on different, distinct trophoblast characteristics. Upon uterine implantation, which occurs around day 7 or 8 in human development, the trophoblast differentiates into a variety of extraembryonic (outside of the embryo proper) structures to assist with implantation and placenta development. To firmly implant the embryo, trophoblast cells ingress into the uterus wall.

During gastrulation, the blastula undergoes distinct cellular reorganizations to reach the next developmental stage, which is called a gastrula. (The hallmark of gastrulation is the creation of three distinct tissue, or germ, layers, in the embryo which will make up all the tissues of the future adult organism; these layers are the ectoderm, the mesoderm, and the endoderm [see Figure 2].)

Figure 2: After the blastocyst is implanted in the uterine wall, it rapidly and significantly undergoes cellular reorganizations to become a gastrula. The gastrula is distinct for having differentiated, or developed, into the three germ layers of the fetus, which are labeled: the endoderm, the ectoderm, and the mesoderm. While these three layers are the predecessors of the adult organism’s cells, the stem cells of the trophoblast differentiate into cells that support the developing fetus; trophoblast cells ingress into the uterine wall (top of the gastrula image, in green) to firmly attach the embryo and trophoblast cells also surround the entire embryo (green), significantly contributing to the formation of the placenta.

While the internal components of the developing gastrula undergo significant rearrangements, during gastrulation the extraembryonic portions are also rapidly changing, anchoring the embryo on the uterine wall and establishing an exchange of nutrients and gases with the mother. At this time, the TSCs give rise to cytotrophoblast stem cells, which are precursors of all trophoblast cells. The cytotrophoblast makes up the inner single cell layer of the trophoblast, surrounding the gastrula and anchoring the chorion to the maternal endometrium. Some cytotrophoblasts will later become stem cells in the chorionic villi, which provides more surface area on the chorion to maximize contact to the maternal blood supply (Rossant and Cross, 2001; Gilbert, 2003).

TSCs can ultimately become one of many different trophoblast cell types, making them multipotent stem cells. The TSCs on the ICM proliferate and become the extraembryonic ectoderm and the ectoplacental cone (Oda et al., 2006). The extraembryonic ectoderm includes cells that can become syncytiotrophoblasts, which make up the outer trophoblast layer. This outer layer becomes thick, multinucleate, and is exterior to the gastrula; these are the cells that ingress, or enter, into the uterine tissue (invading the maternal extracellular matrix, which is a collection of proteins surrounding cells that help the cells adhere and communicate with each other) (Rossant and Cross, 2001; Gilbert, 2003). To become motile and ingress, the trophoblasts undergo an epithelial-mesenchymal transition, becoming moving mesenchymal cells, but after they have reached their destination and implantation is complete, they undergo a mesenchymal-epithelial transition, becoming epithelial and much less mobile (Abell et al., 2009). The syncytiotrophoblast also secretes progesterone and other hormones to sustain pregnancy.

The ectoplacental cone contains a variety of cells, including ones involved in implantation as well as the creation of the placenta. Some cells of the ectoplacental cone become spongiotrophoblasts, which form the middle layer of the placenta. TSCs on the side of the embryo opposite of the ICM become the trophoblast giant cells, which are non-proliferative cells required for implantation that are made up of several subtypes (Rossant and Cross, 2001; Moore et al., 2008; Liu et al., 2009; He et al., 2008). The ICM is thought to signal nearby TSCs to remain in a proliferative, stem cell state, while further away TSCs differentiate (Rossant and Cross, 2001).

By understanding the early embryo, researchers have learned how to better culture these TSCs, and at the same time TSCs have taught scientists much about this unique developmental environment. TSCs are thought to exist during the blastocyst to early gastrula stages, including after implantation. Like most stem cells, TSCs lose their potency as development progresses, although some rare TSCs have been found in later-stage extraembryonic and chorionic layers (Oda et al., 2006). Fibroblast growth factor 4 (FGF4) is known to activate TSC self-renewal and proliferation, and it is a necessary supplement for growing TSCs in culture (Abell et al., 2009; Himeno et al., 2008). Interestingly, TSCs have recently been found to also require an environment free of male hormones, which was suspected (Epple-Farmer et al., 2009). However, TSCs have not yet been successfully isolated from humans, though they have been isolated from mice, which is what most of the data presented here is based on, as well as monkeys. TSCs are very likely present in humans as well (Douglas et al., 2009).

TSCs have potential for use in regenerative medicine as well as developmental models. Their great appeal for use in regenerative medicine is due to their being immune privileged, as this is one of their roles during development of the embryo. TSCs have been found to also allow immunoprotection to other cell types they are co-transplanted with. However, there is some concern over this use of TSCs because they are such highly proliferative and invasive cells (Epple-Farmer et al., 2009). Additionally, TSCs can serve as a model for placental development. Although human embryonic stem cells can become trophoblast-like cells in culture, these cells are not very proliferative; TSCs are thought to be much better models (Moore et al., 2008).

References

Abell, A. N., Granger, D. A., Johnson, N. L., Vincent-Jordan, N., Dibble, C. F., and Johnson, G. L. Trophoblast Stem Cell Maintenance by Fibroblast Growth Factor 4 Requires MEKK4 Activation of Jun N-Terminal Kinase. Molecular and Cellular Biology. 2009. 29(10): 2748-2761.
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He, S., Pant, D., Schiffmacher, A., Meece, A., Keefer, C. L. Lymphoid enhancer factor 1-mediated Wnt signaling promotes the initiation of trophoblast lineage differentiation in mouse embryonic stem cells. Stem Cells. 2008. 26(4): 842-849.
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Himeno, E., Tanaka, S., and Kunath, T. Isolation and manipulation of mouse trophoblast stem cells. Curr. Protoc. Stem Cell Biol. 2008. Unit 1E.4.
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Liu, J., Xu, W., Sun, T., Wang, F., Puscheck, E., Brigstock, D., Wang, Q. T., Davis, R., and Rappolee, D. A. Hyperosmolar Stress Induces Global mRNA Responses in Placental Trophoblast Stem Cells that Emulate Early Post-implantation Differentiation. Placenta. 2008. 30(1): 66-73.
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Moore, H., Udayashankar, R., and Aflatoonian, B. Stem cells for reproductive medicine. Molecular and Cellular Endocrinology. 2008. 288(1-2): 104-110.
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Oda, M., Shiota, K., and Tanaka, S. Trophoblast stem cells. Methods in Enzymology. 2006. 419: 387-400.
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Original “The Blastocyst” image modified from the Wikimedia Commons and original “The Three Germ Layers” image from the Wikimedia Commons. Both are redistributed freely as they are in the public domain.

The most recent article, “Likely Suspects in Cancer Growth,” is on cancer stem cells — it is a modified version of the “All Things Stem Cell” post “Cancer Stem Cells: A Possible Path to a Cure” to fit a more lay public audience.

Tune in to “Biology Bytes” for weekly stories on a wide array of fascinating biology topics, including more accessible explanations of stem cell biology.

In the first reported instance of using stem cells to bioengineer a functional human organ, Paolo Macchiarini and his research group used a patient’s own stem cells to generate an airway, specifically a bronchus, and successfully grafted it into the patient to replace her damaged bronchus (See Figure 1). Macchiarini’s group bypassed the problem of immune rejection by using the patient’s own stem cells. Additionally, by combining a variety of bioengineering efforts, no synthetic parts were involved in the creation of the organ; it was made entirely of cadaveric and patient-derived tissues (Macchiarini et al., 2008; Hollander et al., 2009).

Figure 1. In order to create a patient-compatible replacement bronchus, Macchiarini’s group removed and decellularized a trachea from a cadaveric donor, grew cells removed from the patient on the trachea in a bioreactor, and then transplanted the bioengineered airway into the patient, successfully replacing their defective bronchus (Macchiarini et al., 2008).

The relatively unique and tragic situation of the patient led Macchiarini’s group to test this novel organ transplant on her, which had previously been tried in mouse and pig models. Due to a severe tuberculosis infection, the 30-year-old female patient’s left bronchus had become near-collapse; breathing was so impaired that the patient could no longer carry out simple domestic chores. After several other approaches did not succeed in fixing the bronchus, it was decided that the best option was to remove and replace the bronchus. Normally replacement of large airway pieces and other organs is a significant problem because the patient must be on immunosuppressant medications for life to prevent rejection of the new tissue, and this can shorten the patient’s lifespan by 10 years on average; using the patient’s own stem cells got around rejection (Macchiarini et al., 2008; Hollander et al., 2009).

To create the replacement bronchus, a cadaveric donor airway was obtained and decellularized, or treated so that all donor cells would be removed. A segment of trachea was removed from a cadaveric donor and all connected tissues carefully detached. To prevent immune rejection by the patient, which can be caused by the presence of foreign cells and different major histocompatibility complexes (MHC), all cells and parts of cells had to be removed from the donor trachea. To ensure complete removal of all donor cellular components, the trachea underwent an extensive, previously established decellularization procedure over a period of 6 weeks, which involved the trachea being incubated with detergents and deoxyribonucleases (enzymes that degrade DNA) for 25 cycles (Macchiarini et al., 2008; Conconi et al., 2005). The researchers confirmed that donor cells, including MHC-positive cells, were absent, leaving only the cartilage of the trachea intact (Macchiarini et al., 2008).

The decellularized trachea acted as a scaffold for the patient’s cells to be grown on; the stripped airway was incubated in a novel bioreactor with two different kinds of cells from the patient. Epithelial cells were removed from the mucosa, or moist tissue lining, of the patient’s right bronchus. These cells were taken and cultured, or grown, inside the donor trachea. The second type of cell used was chondrocytes. To create chondrocytes the researchers removed bone marrow from the patient and isolated out a population of mesenchymal stem cells (MSCs). The MSCs were induced to differentiate into, or become, chondrocytes using a standard protocol (i.e. specific factors were added to the growth media) for three days. These chondrocytes were seeded on the outside of the trachea. The cells were grown in different media used inside and outside of the bioreactor, media specific to the epithelial cells or chondrocytes. The cells were cultured on the trachea in the bioreactor for four days, at which point the researchers had bioengineered a human airway lacking any synthetic parts (Macchiarini et al., 2008).

The portion of the patient’s left bronchus that was near-collapse was removed and successfully replaced by the bioengineered trachea, now acting as a segment of bronchus. After a month in the patient, the transplanted trachea was indistinguishable from a normal bronchus, as compared to the patient’s unaffected right bronchus and the surrounding bronchus tissue. The transplanted airway quickly also displayed completely normal function (Macchiarini et al., 2008). One year later, the graft and patient are still doing fine (Asnaghi et al., 2009).

While the case of this successfully bioengineered and transplanted organ is a breakthrough, improvements are needed to make such transplants feasible. Because Macchiarini’s group used a donor graft, the original cadaveric trachea segment, these transplants are limited by available donors. It is hoped that research efforts will lead to fully-tissue engineered organ transplants without the need of such donor grafts. If this is possible, the current shortage of donor tissue and organs can be dealt with and a large aging population can be much more effectively treated (Hollander et al., 2009).

Aside from Macchiarini’s report, several other research groups have made breakthroughs in bioengineering organs and tissues recently. One group reported creating skeletal muscle segments using a synthetic scaffold to shape and grow cells on (Bian and Bursac, 2009). Specifically, these researchers used a silicon-based polymer (polydimethylsiloxane, or PDMS) to create micromolds with pegs, or elongated posts, sticking up from the molds. Muscle cells in a gel solution were poured onto the mold and polymerized together. This created a porous skeletal muscle network that was densely packed, with uniformly aligned muscle fibers that spontaneous contracted at the macroscopic level. In the future this approach could create customized, functional skeletal muscle tissue for reconstructing damaged muscle (Bian and Bursac, 2009). Similarly, another group discusses potential in using stem cells to rescue damaged heart muscles (Shimizu et al., 2009). Researchers are also investigating the feasibility of using epithelial stem cells in bioengineered intestines, based on polymer scaffold experiments performed in rats (Day, 2006). Intestinal transplantation, often needed for short bowel syndrome caused by a variety of reasons, is a significant problem because of the extremely active immune system of the intestines (Day, 2006). Other researchers are focusing on the great potential of mesenchymal stem cells (such as were used in Macchiarini’s report) in general wound healing; these cells can differentiate into many different kinds of cells, be isolated in significant numbers, potentially migrate to areas they are needed in, and may be immunosuppressive (Fu and Li, 2009). The use of nanomaterials, which can mimic proteins on the surface of cells and tissues, also hold much potential for future scaffold designs in regenerative medicine (Zhang and Webster, 2008).

While Macchiarini’s patient represents a significant breakthrough, it is still a single success that must be repeated to be proven. The transition to the clinic of other stem cell-based regenerative therapies will also require extremely careful characterization of each individual procedure. There are still many obstacles to overcome before such therapies can become common practice. Those interested in receiving stem cell therapies should be aware of the possible risks involved; the Department of Health’s Gene Therapy Advisory Committee lists such potential hazards associated with undergoing stem cell therapies.

References

Asnaghi, M. A., Jungebluth, P., Raimondi, M. T., Dickinson, S. C., Rees, L. E. N., Go, T., Cogan, T. A., Dodson, A., Parnigotto, P. P., Hollander, A. P., Birchall, M. A., Conconi, M. T., Macchiarini, P., and Mantero, S. A double-chamber rotating bioreactor for the development of tissue-engineered hollow organs: From concept to clinical trials. Biomaterials. 2009. 30(29): 5260-5269.
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Bian, W. and Bursac, N. Engineered skeletal muscle tissue networks with controllable architecture. Biomaterials. 2009. 30(7): 1401-1412.
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Conconi , M. T., De Coppi, P., Di Liddo, R., Vigolo, S., Zanon, G. F., Parnigotto, P. P., and Nussdorfer, G. G. Tracheal matrices, obtained by a detergent-enzymatic method, support in vitro the adhesion of chondrocytes and tracheal epithelial cells. Transpl. Internat. 2005. 18(6): 727-734.
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Day, R. M. Epithelial stem cells and tissue engineered intestine. Curr. Stem Cell Res. Ther. 2006. 1(1): 113-120.
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Fu, X. and Li, H. Mesenchymal stem cells and skin wound repair and regeneration: possibilities and questions. Cell and Tiss. Res. 2009. 335(2): 317-321.
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Hollander, A., Macchiarini, P., Gordijn, B., and Birchall, M. The first stem cell-based tissue-engineered organ replacement: implications for regenerative medicine and society. Regen. Med. 2009. 4(2): 147-148.
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Macchiarini, P., Jungebluth, P., Go, T., Asnaghi, M. A., Rees, L. E., Cogan, T. A., Ddson, A., Martorell, J., Bellini, S., Parnigotto, P. P., Dickinson, S. C., Hollander, A. P., Mantero, S., Conconi, M. R., Birchall, M. A. Clinical transplantation of a tissue-engineered airway. The Lancent. 2008. 372(9655): 2023-2030.
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Shimizu,T., Sekine, H., Yamato, M., Okano, T. Cell Sheet-Based Myocardial Tissue Engineering: New Hope for Damaged Heart Rescue. Curr. Pharm. Design. 2009. 15(24): 2807-2814.
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Zhang, L., and Webster, T. J. Nanotechnology and nanomaterials: Promises for improved tissue regeneration. Nanotoday. 2009. 4(1): 66-80.
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Image of “Macchiarini’s Bioengineered Bronchus Replacement” was modified 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.
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Dolgin, E. Immortality improves cell reprogramming: Knocking out genes with a role in cancer prevention helps produce stem cells. Nature News. 2009.
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Gunaratne, P. H. Embryonic Stem Cell MicroRNAs: Defining Factors in Induced Pluripotent (iPS) and Cancer (CSC) Stem Cells? Curr Stem Cell Res Ther. 2009.
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Hermeking, H. The MYC Oncogene as a Cancer Drug Target. Current Cancer Drug Targets. 2003. 3(3): 163-175.
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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).
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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.
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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.
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Liu, S. V. iPS Cells: A More Critical Review. Stem Cells and Development. 2008A. 17: 391-397.
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Liu, S. V. IPS Cells are Man-Made Cancer Cells. Logical Biology. 2008B. 8(1): 16-18.
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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.
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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.
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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.
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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.
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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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In salamanders, when a limb is severed the resultant limb bud undergoes a distinct process to regenerate the lost limb. The epithelial layer quickly spreads across the amputation site, closing the wound within 24 hours (Mescher, 1996). This epithelial layer thickens and becomes what is referred to as the wound epithelium (WE). As the immune system responds to the injury, macrophages and neutrophils arrive to clean up the wound site beneath the WE. The existing injured tissues and cells are broken down as well as the extracellular matrix, which is made up of proteins that surround cells to hold them together and stimulate normal cellular functions. It was thought that at this time in the regenerative process other resident cells below the WE become multipotent mesenchymal stem cells (MSCs) (see Figure). These eventually form a mass of MSCs called a blastema (Mescher, 1996; Brockes and Kumar, 2005). The blastema was thought to contain a homogenous group of pluripotent stem cells that had “dedifferentiated” or “redifferentiated,” meaning they had reverted back from their committed fates to function as very potent stem cells in order to recreate the limb. The WE stimulates the cells in the blastema to proliferate, making new cells and extracellular matrix, though more than is required for simple repair; the WE signals the blastema cells to regenerate the entire lost limb (Mescher, 1996; Kragl et al., 2009).

Limb regeneration in the salamander after limb amputation (time course going from the top down). Shortly after the limb is amputated, the epithelium layer covers the exposed limb bud, forming the wound epithelium (WE). A group of stem cells collects below this layer, forming the blastema (at the tip of the bud). The WE signals the stem cells below it to rebuild the limb, recreating the limb from the point of injury out towards the hand. The final regenerated limb is indistinguishable from the original.

Recently, Elly Tanaka’s group showed that multiple different groups of stem cells with relatively limited fates, being only multipotent or unipotent, may actually regenerate the salamander limb, in contrast to the previously held belief that one homogenous group of pluripotent stem cells was responsible (Kragl et al., 2009). The group labeled the major tissue types in the limb (using green fluorescent protein [GFP]) to track the tissue types during regeneration. While the blastema does appear histologically homogenous, they found it may be made up of all the different tissue types found in the complete limb; all the cells in the blastema may be types of progenitor cells that can become only one or two specific adult tissue types. In short, many different cell types may coordinate to recreate the limb (Kragl et al., 2009).

Only one tissue cell type in the blastema was able to differentiate into a cell of a different tissue layer in Tanaka’s experiments. Specifically, Tanaka’s group reported that precursor cells for muscle, epidermis, cartilage, or Schwann cells (neural cells) could only create muscle, epidermis, cartilage, or Schwann cells, respectively; each of these cell types was limited to become its own cell type. The dermis tissue layer was the only cell type found to be able to become more than one fate; the dermis could become both dermis and skeleton/cartilage (but not muscle or Schwann cells). Dermis and cartilage have a common developmental origin in the mesoderm, which helps explain why the dermis layer cells could become both of these cell types. Additionally, the group investigated whether these progenitors know their proper final position in the limb along the proximal-distal (i.e. shoulder-hand) axis. The researchers found that cartilage precursors do not have such positioning abilities, while the Schwann cells do. This indicates that the positional identity is tissue specific (Kragl et al., 2009).

While this research was conducted using salamanders, it may be quite relevant for future regenerative medicine research in humans; it shows that we may not need a pluripotent state for complex tissue regeneration, but instead could use multiple different stem cells with much more restricted fates. However, this does not necessarily make the process more feasible technically, but it may give researchers a more focused direction for future studies. To read other coverage of this ground-breaking work by Tanaka’s group, reported just last month, take a look at (Baker, 2009) or (Johnson, 2009).

References

Baker, M. Regenerating limb tissue may not dedifferentiate. Nat. Rep. Stem Cells. 2009.
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Brockes, J. P. and Kumar, A. Appendage Regeneration in Adult Vertebrates and Implications for Regenerative Medicine. Science. 2005. 310(5756): 1919-1923.
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Johnson, S. L. Memory of Fate and Position, Colorized. Dev. Cell. 2009. 17(1): 5-6.
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Kragl, M., Knapp, D., Nacu, E., Khattak, S., Maden, M., Epperlein, H. H., Tanaka, E. M. Cells keep a memory of their tissue of origin during axolotl limb regeneration. Nature. 2009. 460: 60–65.
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Mescher, A. L. The cellular basis of limb regeneration in urodeles. Int. J. Dev. Biol. 1996. 40: 785-795.
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Original “Salamander Limb Regeneration” image modified from the Wikimedia Commons and redistributed freely as it is under GNU Free Documentation License.

Although the theory of cancer stem cells has been around since the 1970s (Hamburger and Salmon, 1977), recently it has gained a spotlight in the scientific community. The first functional identification of CSCs was in 1997 in acute myeloid leukemia (Bonnet and Dick, 1997). Researchers found that although there are many different populations of cells within a tumor, only one population has the ability to generate the tumor. This was determined by separating the populations from each other and engrafting them into an immuno-compromised (NOD/SCID) mouse; the population identified as CSCs was able to recreate the original tumor, including morphology and the specific differentiated cell types observed within the tumor (Vermeulen et al., 2008).

The different populations within a tumor can be separated and identified according to the proteins expressed (or produced) on the surface of a particular cell; cells expressing the same set of proteins are grouped into one population. Because such proteins are commonly used to identify and categorize cells, they are called cell markers. CSCs from the same tumor type usually have the same set of markers expressed, although the markers expressed can vary much more between CSCs from different tissues (Vermeulen et al., 2008). For example, breast cancer CSCs have been found to express a marker called CD44, but are distinct for also not expressing the marker CD24 (making this CSC population be labeled CD44⁺/CD24^-) (Al-Hajj et al., 2002). In comparison, pancreatic cancer CSCs express CD44, but also express CD24 (Li et al., 2007). Although there are differences like this in marker expression between CSCs from different tumor types, some markers are present in CSCs from many different types of tumors, such as CD44. CSCs from ovarian tumors (Zhang et al., 2008) and head and neck squamous cell carcinomas (Prince et al., 2006) have also been found to express CD44. Another major marker protein expressed in CSCs across tissue types is CD133; it is expressed by CSCs found in brain (Singh et al., 2003), prostate (Lang et al., 2008), colon (O’Brien et al., 2007), lung (Eramo et al., 2007), and hepatic (Suetsugu et al., 2006) tumors. For a more detailed summary of marker expression of CSCs from the different tumors they have been discovered in, see Table 1.

Table 1. Cancer Stem Cell Populations Detected in Different Cancerous Tumors (CSC Markers and Percent of the Total Tumor)

The markers expressed by cancer stem cells are also standard markers for many different stem cells from non-cancerous tissues. CD133⁺ is associated with many different kinds of stem cells: neural (Hill, 2006), hematopoietic (Suetsugu et al., 2006), endothelial, epithelial, and other stem cell types (Eramo et al., 2007). CD44 is expressed in hematopoietic (Morrison et al., 1995) and mesenchymal (Pittenger et al., 1999; Mitchell et al., 2006) stem cells. As becomes apparent when comparing these associations to the CSC markers reported in Table 1, often CSCs share the same markers as stem cells found in normal tissue in the tumor origin, but surprisingly sometimes the CSCs express very different markers from the stem cells normally present in the same healthy tissue (Vermeulen et al., 2008). It is still unclear why this is; these CSCs may be migrating from another tissue to the tumor site where they thrive due to components of the new cellular environment (Vermeulen et al., 2008), or the CSCs may be expressing these markers for other, unknown reasons. Like other stem cells, CSCs can differentiate and change expression of their markers; CSCs most likely have a marker expression profile very different from their progenitor cells (Eramo et al., 2007; Vermeulen et al., 2008).

Another cancer stem cell attribute of note is that CSCs account for only a small percentage of the total number of cells in the tumor. Shown in Table 1, the percentage of CSCs in a tumor can vary from as little as 0.002% to around 30%, depending on the type of tumor, but appears to most often be less than 10% (Singh et al., 2003; O’Brien et al., 2007; Eramo et al., 2007; Zhang et al., 2008; Li et al., 2007; Prince et al., 2006). Additionally, it has been reported that only some of the cells in the CSC population, as identified by their markers, can actually form tumors (Hill, 2006; O’Brien et al., 2007). Consequently, some researchers say that selecting for CSCs on the basis of their markers is only enriching for the true, functional CSC population, but it is not isolating them from cells that cannot form tumors (Hill, 2006). Additionally, there may be cells in the tumors other than CSCs that are capable of creating tumors (Hill, 2006), although they would most likely not be as able to form tumors as the identified CSC populations.

In order to comprehend how cancer stem cells are created it is important to understand the theories behind the creation of cancerous tumors and how this applies to CSCs. Cancer can occur when mutations have accumulated in genes related to controlling cell growth and differentiation. Specifically, such key genes are referred to as oncogenes and tumor suppressor genes. If one of the first mutations affects the regulation of cell growth, this can result in the expansion of an already mutated, potentially cancerous stem cell population. This population can gain mutations that upregulate, or increase, their ability to self-renew, further increasing the population size. The Wnt and BMI1 signaling pathways, which normally regulate cell proliferation and self-renewal, are often mutated in CSCs (Vermeulen et al., 2008). When enough mutations accumulate, a cell can overcome the normal cell growth restrictions and grow out of control, becoming cancerous (Vermeulen et al., 2008).

As more evidence is reported pointing at the key role of cancer stem cells in the creation of cancerous tumors, it becomes more crucial for researchers to have a thorough understanding of these stem cells. Although the CSC populations can be identified by different protein markers and their ability to create tumors in a mouse model, there is still much about them that is not well understood: how they are created, how their origins are related to non-cancerous stem cells, whether they are present in all cancerous tumors, and how they are affected by their cellular environment (Vermeulen et al., 2008; Hill, 2006). As more answers come to light, we will be able to answer the most important question: how can we use our knowledge of CSCs to most effectively combat them?

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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.

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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.
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