So please be sure to get out the word on stem cells this October 3! For more information on International Stem Cell Awareness Day (and free wallpapers and downloadable stem cell images!), visit StemCellsOfferHope.com, which is affiliated with the Sue & Bill Gross Stem Cell Research Center at the University of California, Irvine. Read on for a summary of stem cell history and recent research breakthroughs and highlights.

With all of the breaking news stories that come out on cutting-edge stem cell findings all the time, it can be easy to lose sight of the bigger picture. Yes, the stem cell family, which includes all of the varieties of stem cells that have been discovered so far, is very large, and growing larger with new children, cousins, uncles, and aunts being discovered or created all the time. But a key feature they all share is their potential to improve our lives.

Our understanding of these cells and their incredible potential for treating diseases, fight cancers, heal wounds, and, in essence, saving lives, has grown hugely since we first unknowingly used them in World War II. However, the more we learn about them the more we realize we have yet to understand. This blog has strived to explore the different stem cell types in detail, including their biology, history, potential, clinical applications, and numerous remaining questions. However, the ways in which the different types of stem cells came to be accepted into the stem cell family is itself an interesting story, and one that can help paint a useful bigger picture, and that is why this story will be the focus for this blog post to celebrate International Stem Cell Awareness Day.

The first time stem cells were successfully used to treat patients was during World War II. However, at the time, people did not know they were using stem cells. During World War II, people exposed to lethal doses of radiation were given bone marrow transplants that, somehow, could cure them. Much later it was discovered that the responsible agents in the bone marrow were hematopoietic stem cells (HSCs) (which have been discussed in several blog posts). Because HSCs are rapidly growing cells, they’re particularly damaged by exposure to radiation. Consequently, a radiation victim may need a transplant of healthy HSCs to replace their own. As this history makes apparent, HSCs reside in bone marrow (as well as other tissues), making bone marrow a good HSC transplantation source.

But what do these HSCs do exactly? They’re quite important. They can turn into all the different types of blood cells in the body. This is why transplants of HSCs (from bone marrow) are also used to treat cancers of the hematopoietic (blood) system, such as leukemia or lymphoma. Today HSCs are one of the few adult stem cells that are widely used clinically.

While bone marrow donor centers were being established in the 1980s, another stem cell family tree branch was developing that would draw much attention: Nearly 30 years ago, embryonic stem cells were isolated from early-stage mouse embryos. It was not until 1998 that the same feat was accomplished with human embryos, by James Thomson, who holds a faculty appointment at the University of Wisconsin and the University of California at Santa Barbara.

These human embryonic stem cells (hESCs), which have been discussed in numerous blog posts, are isolated from early stage embryos. Technically called blastocysts, these are embryos that have not yet implanted in the uterus. They have existed for only five days after fertilization and contain only about 150 cells. One of the most promising qualities of hESCs is their ability to become virtually any cell type; this potential means they are “pluripotent.” As we’ll see later on, this has already contributed to their receiving FDA-approval for use in a clinical trial.

Around the same time that researchers were figuring out how to isolate embryonic stem cells from humans, yet another large group of stem cells was finally admitted into the stem cell family. Although researchers reported the existence of mesenchymal stem cells (MSCs) as early as the 1960s and 1970s, as discussed in a previous blog post, they were excluded from the stem cell family for decades. Why the prejudice? MSCs had somewhat questionable origins; they’re most commonly harvested from adipose tissue (fat) or bone marrow. Researchers already knew bone marrow was home to HSCs and it was difficult to accept that they shared their home with yet another group of stem cells. But by the late 1990s, MSCs were firmly established and allowed into the family.

MSCs hold great potential for the field of regenerative medicine, as they can become many different types of cells (they’re “multipotent”), most typically bone, cartilage, and fat cells. In 2000, a new member was added to the mesenchymal stem cell branch, when researchers discovered that teeth are home to dental pulp stem cells, which were explored in a previous blog post.

During the last decade, a number of new stem cell types have joined the every-growing family, through discovery or creation. In 2007, stem cells were found to reside in menstrual blood, as covered previously. It’s unclear exactly what branch these “endometrial regenerative cells” belong to in the stem cell family; they have similarities with both ESCs and MSCs. Nonetheless, because they can be obtained in a non-invasive manner, and in large quantities, they offer much potential for cellular therapies.

2007 also saw one of the most game-changing developments in the stem cell field; researchers learned how to create cells like embryonic stem cells, but instead of coming from an embryo these cells are created from adult cells, potentially cells from any tissue in the human body. These cells, called induced pluripotent stem cells (iPSCs), which were discussed in several previous blog posts, are created by forcing adult cells to produce proteins that are specific to ESC functions, causing the adult cells to look and act like ESCs. iPSCs have significantly altered the field ethically, as they have the utility of embryonic stem cells but do not require harvesting cells from a blastocyst. They also alter it clinically: It’s now possible, in theory, to grow patient-specific pluripotent cells.

But the idea of changing a mature cell’s identity had already been around for a few decades. Since the late 1980s, researchers had used “direct reprogramming” to, for example, make different types of adult cells turn into muscle cells. They did this by making the adult cells produce proteins essential to the identity of muscle cells, which was discussed in a previous blog post. It was found that other cells could have their “established” identities altered in similar ways, with a group in 2008 reporting that some pancreas cells (exocrine cells) could be turned into other, insulin-producing pancreas cells (beta-cells), which may hold promise for treating diabetes. In 2010, another group found that fibroblast cells (cells active in connective tissue) taken from mouse tails could be made into nerve cells, or neurons.

Although many stem cell therapies are still in their infancy, in the last few years there have been several important publications on the successful use of novel stem cell treatments in patients.

In 2008, Claudia Castillo had her near-collapsed bronchus replaced by a trachea from a cadaver that had her own cells grown on it, which was discussed in detail in another blog post. These cells included chondrocytes (cartilage cells) that were made from MSCs that had been taken from her bone marrow; turning these MSCs into chondrocytes only took researchers three days. Since 2008, this technique has been improved upon and successfully used in other patients.

Although HSCs have been used since World War II to treat victims of radiation, a potential, significant new application was reported in 2009: HSCs were used to successfully treat a patient with HIV, which was discussed previously, although the procedure is currently risky and much additional research is necessary for it to be widely accepted and used.

2009 also saw the first FDA-approval of the use of hESCs in a clinical trial, with the first patients receiving treatment in 2010. Hans Keirstead and colleagues developed the spinal cord therapy used in the clinical trial; they made hESCs become oligodendrocytes and then showed that these cells could help cure spinal cord injuries in animals. StemCells Inc. and collaborators at the Sue & Bill Gross Stem Cell Research Center announced at a meeting in September, 2012, that some patients with spinal cord injuries in a clinical trial regained some capacity to feel heat and touch. These trials hold much promise not only for those with spinal cord injuries, but for other hESC-based therapies that may now have a better chance at receiving FDA-approval for clinical trials.

Stem cells have also helped us better understand cancer, through investigation of the emerging idea of the cancer stem cell, which has been discussed multiple times before, and the many similarities cancer shares with different members of the stem cell family. Such studies may even help researchers develop better cancer vaccines, also as previously discussed.

And that’s not all that stem cells have done. Here are a few more recent stem cell research highlights:

• Studying stem cells may have provided us with a key to understanding male pattern baldness, as discussed in a previous blog post.
• Stem cells are used to help us understand the toxicity of different compounds on different tissues, as outlined in this article, and as recently published using ESCs.
• Just one recent example of the many diseases that stem cells are being used to treat: A patient with tumors caused by the human papillomavirus (HPV), specifically recurrent respiratory papillomatosis with tumor invasion, had normal and tumorous cells removed and reprogrammed to create cell cultures. The researchers found that the HPV genome was normal in the normal cells, but much larger in the tumorous cells, due to the presence of multiple oncogene regions. Findings such as this may help us better treat complications caused by HPV, and other viral, infections.
• Another stem cell member may be tentatively added to the family with the characterization of cells that generate esophageal tissue.

As our understanding of this complex and constantly growing family continues to grow, so too should our understanding of how the medical field can best use the different members to improve our lives. So, again, on this October 3 please be sure to spread the word on the amazing research being done with stem cells! For more information on International Stem Cell Awareness Day and free stem cell images, visit StemCellsOfferHope.com.

References:

California Institute for Regenerative Medicine (CIRM). CIRM grantees show preliminary signs of success in spinal cord injury trial. September 4, 2012. View Article

Chi, K. R. Stemming the Toxic Tide: How to Screen for Toxicity Using Stem Cells. The Scientist. September 2, 2012. View Article

Kushner, J. A. Esophageal Stem Cells, Where Art Thou? Development. August 31, 2012. View Article

Rowland, T. J. A Night with Dr. Hans Keirstead. Santa Barbara Independent: Biology Bytes. June 4, 2010. View Article

Rowland, T. J. The Stem Cell Family. Santa Barbara Independent: Biology Bytes. June 11, 2010. View Article

Seiler, A. E. M., and Spielmann, H. The validated embryonic stem cell test to predict embryotoxicity in vitro. Nature Protocols. June 16, 2011. View Article

StemCellsOfferHope.com. Sue & Bill Gross Stem Cell Research Center, University of California, Irvine.

Yuan, H., Myers, S., Wang, J., et al. Use of Reprogrammed Cells to Identify Therapy for Respiratory Papillomatosis. The New England Journal of Medicine. September 27, 2012. View Article

The science presented in the film is well-explained and even though the focus of the film is on medical breakthroughs accomplished using stem cells, the scientists interviewed do not try to over-hype current stem cell applications. Most helpful in making the technical information accessible are several short, accurate, and intriguing animations (made by Cameron Duguid). During a segment on Yamanaka’s research, one of these animations is particularly useful in explaining how chromatin regulation of gene expression is different in different types of tissues. However, it is repeatedly jarring when the interviews with down-to-earth stem cell scientists, who mostly do not over-hype their research, are bookended by interviews with Margaret Atwood (a writer who is confusingly repeatedly interviewed in a laboratory setting). She makes repeated references to The Fountain of Youth – at odds with the scientists’ messages. Similarly, repeatedly interspersed videos of a topless man doing what looked to be the Brazilian martial art of Capoeira seemed out of place.

Perhaps the only shortcoming of the film, if a bit minor, is that it shies away from getting into some of the nitty-gritty of why iPSCs may be better than hESCs or vice versa, but instead falls back upon the standard argument that hESCs are surrounded by ethical concerns. For a 71-minute-long film, it only makes sense that some issues be simplified, but additional details may have helped viewers better understand this important and hotly-debated topic. Specifically, a lot of the ethical arguments against hESCs are outdated or ill-founded. Probably most importantly, in 2006, Irina Klimanskaya and colleagues found how to isolate hESCs while leaving the donor embryo intact and potentially able to develop normally, weakening the argument against the generation of hESC lines on the grounds that they require the destruction of a potential embryo. Additionally, many researchers use blastocysts that would have been discarded by the in vitro fertilization clinic because the embryos were damaged in some way and would never develop properly. However, a significant strike against using hESCs in treatments, which the film does not touch upon, is the potential for immune rejection. Human iPSCs, on the other hand, are very appealing because they potentially may not have immune rejection problems in treatments, as mentioned in the film. However, human iPSCs are much newer to the stem cell scene and have similarities with cancer cells that researchers should probably better understand before iPSCs are widely used clinically. It is also a little surprising that Jamie Thomson is not mentioned in the human iPSC segment, as his group independently created human iPSCs at the same time as Yamanaka’s group.

The researchers interviewed in the film emphasize the importance of striking a balance between regulation and progress, but then the film seems to not take its own advice and gets bogged down in the regulation of stem cells in the very last segment of the film, when it may have been more useful to focus on the near-future applications of these cells. There’s a surprising focus on the hypothetical ethical arguments that would arise should human iPSCs be made into function eggs and sperm (which has not been done yet, and may not even be possible). However, it may be more useful to first focus on whether human iPSCs can even be successfully used in the clinic before diverting attention to this hypothetical ethical argument, which is much further down the road. It would also have been nice to see a mention of direct reprogramming, the latest stem cell technology that may one day make even iPSCs obsolete.

While there are amazing advances being made with stem cell technology, the film rightly cautions viewers about the dangers of going to a stem cell clinic abroad. A great resource for those considering stem cell treatments abroad is A Closer Look at Stem Cell Treatments, a website made by the reputable International Society for Stem Cell Research.

Overall, “STEM CELL REVOLUTIONS” is a great film for anyone wanting to learn more about the history of stem cells, hear legendary researchers talk about their ground-breaking work and patients talk about how stem cell therapies have changed their lives, and still get a down-to-earth idea of what is realistically being accomplished with these cells.

A Hair’s Life-Cycle: In order to understand AGA and the newly discovered key role of these progenitor cells in it, it’s helpful to first review the normal life of a hair. In the skin, every hair sits inside a hair follicle, a little cavity that goes down through the dermis layer and has connected sebaceous glands (which lubricate the hair by secreting an oily substance) an arrector pili (a small bundle of muscles that can make the hair stand on end) (see Hair Follicle figure). Each hair carries out its own life-cycle. The first lifecycle phase is called anagen, a growing period that about 85 percent of the hairs on a person’s head are in at any given time. During anagen, which can last two to six years for one hair, the hair grows at the rate of about five inches a year. After anagen, the hair enters catagen, a transitional one- to two week-long stage when the hair follicle and root both shrink. The hair then enters the last stage, telogen, which is a resting phase that lasts about five to six weeks, during which time the old hair does not grow. At the end of telogen the hair follicle re-enters anagen, the growth phase, and often a new hair will push the old one out, starting the growth cycle over again (Furdon & Clark, 2003; Garza et al., 2011).

Every hair sits inside a hair follicle, which goes down through the epidermis and dermis of the skin. Connected to the follicle are sebaceous glands, which release oils onto the hair, and arrector pili muscles, which can cause hairs to stand on end. The bulge is where the majority of the hair follicle stem cells reside, and these can give rise to multipotent progenitor cells.

Androgenetic Alopecia: Normally, the new hair will grow similar to how the last one did. However, with AGA this isn’t the case. In AGA, hair follicles get smaller over time, and consequently make smaller and smaller, eventually microscopic, hairs. How is this caused? It’s not that well understood; it’s known that testosterone is necessary for this miniaturization (as inhibiting testosterone conversion to its active form can delay AGA progression), but not much else is known about what causes AGA (Garza et al., 2011).

But even if it’s not known what happens to cause AGA, researchers have done a lot of work to figure out what stem cells are normally active in the hair follicle. Within a hair follicle, there are stem cells that reside in an area called the hair follicle “bulge,” which is a small compartment located where the outer root sheath meets the arrector pili muscle (see Hair Follicle figure). The stem cells in the bulge are multipotent epithelial stem cells, and can become, or differentiate into, all the epithelial cell types in the follicle (including hair follicles, epidermis, and sebaceous glands) (Oshima et al., 2001). They’re intimately involved in the hair follicle lifecycle. Given this, it shouldn’t come as a surprise that if these stem cells are destroyed, so is the hair follicle (Ohyama et al., 2006; Ohyama 2007).

Are bulge stem cells involved in AGA? In the recent study, the researchers first set out to determine whether the stem cells in the hair follicle bulge play a key role in AGA. To do this, they looked at the expression of keratin 15 (KRT15), which is highly expressed by the stem cells in the bulge (which are also keratinocyte stem cells), but not by the cells in the surrounding hair follicle area (Lyle et al., 1998). These bulge stem cells, as identified by high expression of KRT15, have been shown to be small cells that are primarily quiescent (mostly inactive) in vivo, only springing into action when a new hair follicle starts to grow.

To investigate whether the bulge stem cells play a key role in AGA, Garza et al. isolated epithelial cells from the heads of people with AGA and compared the expression of KRT15 in cells from areas that were bald to areas that were haired (non-bald). Using flow cytometry, the researchers specifically looked at cells expressing the highest amount of KRT15 (the top 5 percent), a characteristic of the bulge stem cells, as well as the most integrin alpha 6 (ITGA6) (a protein in epithelial basal cells). The researchers found that, surprisingly, the percentage of cells expressing high amounts of KRT15 and ITGA6 were the same in the bald and haired samples. Because this reveals that bald scalp can form even in the presence of bulge stem cells, this indicates that other, unidentified cellular players may be primarily involved in causing AGA.

A novel progenitor population involved in AGA: By using other cellular “markers” in addition to KRT15, the researchers found that there was indeed a different population of cells that was present in the haired, but not the bald, epithelial cell samples. (Markers are proteins usually made by distinct cell populations.) Specifically, a population of cells that expressed high levels of CD200 and ITGA6 had relatively very decreased numbers in bald samples. Why did they look at CD200? CD200 is a membrane glycoprotein that has increased expression on the surface of cells in the bulge, and is consequently used to identify cells in the bulge, but it is also expressed in nearby cell layers (Ohyama et al., 2006). Additionally, while the cells it is expressed by greatly overlaps with those that express KRT15, the overlap is incomplete. Garza et al. found that the population of cells expressing high levels of CD200 and ITGA6 did not express the high levels of KRT15 found in the primary bulge stem cell population, and that the CD200/ITGA6-expressing cell population contained only 10% of all of the local cells that express CD200. Consequently, these cells represent a distinct cell population that has not been investigated previously.

Additionally, the newly identified population had hallmark signs of being progenitor cells: Garza et al. showed that the cells were slightly larger in size (about 130%) and less quiescent (with slightly fewer cells in the G0 quiescent stage) than the KRT15-highly expressing bulge stem cells. Because small size is often associated with being a stem cell, these cells’ relatively larger size further reinforces the idea that they have a more activated (less quiescent) identity than the bulge stem cells. Based on this evidence, the authors hypothesized that these novel cells most likely represent a progenitor cell population.

Enter CD34: Yet more progenitors involved: After discovering the presence of this novel cell progenitor population, the researchers decided to see if there were any other progenitor populations hiding around the bulge that had not been associated with AGA before. They did indeed find another progenitor population, this time with the help of a marker called CD34. A population of cells that lives just below the bulge, in the outer root sheath, was already known to highly express CD34 during the anagen (growing) phase, and then undergo apoptosis when anagen ends (Inoue et al., 2009). Additionally, these cells were already suspected to be a progenitor population that originated from the bulge, although some have argued that CD34 does not truly label a progenitor population (Gutierrez-Rivera et al., 2010).

However, in the recent Garza et al. study, researchers again presented evidence supporting the idea that the CD34-expressing population is made up of progenitors (based on relatively low KRT15 production and larger cell size compared to the highly KRT15-expressing cells). The researchers also, for the first time, showed evidence supporting this population’s potential role in AGA; the CD34-positive cells were present in bald cell samples at one tenth the level they were found in haired scalp samples.

Where exactly are these novel progenitor populations? Going back to their novel cell progenitor population, although the researchers could tell that it expressed certain markers (CD200 and ITGA6) and had other distinguishing characteristics (a larger cell size and lower levels of KRT15 compared to the bulge stem cells), they weren’t sure exactly where it called home. They used another marker (follistatin, or FST) to find that their novel population had members located outside of the bulge (FST is only in the bulge, but 15% of the novel progenitors did not express it).

They also determined that some of the progenitors resided in an area home to the “secondary germ cells.” The researchers discovered this by using two different markers: the epithelial cell adhesion molecule (EPCAM), which stains secondary germ cells, labeled 16% of the progenitors, and the leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), which labels bulge and secondary germ progenitors, was significantly elevated in the progenitors (and had decreased expression in bald scalp relative to haired samples). Consequently, the novel progenitor population was thought to reside in both the bulge and the secondary germ.

This isn’t that surprising though; other progenitors derived from the bulge are known to reside in the secondary germ cells (Ito et al., 2004). During catagen, the bulge produces progenitor cells which make their way down to the hair follicle base. There they are responsible for generating a new hair (mainly the hair shaft and root sheath) at the beginning of anagen. Some of these progenitors can also, amazingly, “dedifferentiate” into bulge cells if a bulge cells are lost when a hair is removed from the follicle (Ito et al., 2004). But none of this had been tied to AGA previously.

Multipotent progenitors: Lastly, the researchers showed that their novel progenitors were multipotent and could successfully create hair follicles. To determine their potency, researchers injected the progenitors into immunodeficient mice and, when combined with neonatal dermis, this resulted in the production of all hair follicle lineages, complete with outer and inner root sheaths and sebaceous glands.

Concluding reflections and next steps: The findings from the Garza et al., 2011 paper not only help us better understand how baldness in AGA may be caused, but also how AGA may be treated. The authors hypothesize that in AGA bulge stem cells that have somehow become inactivated may result in a loss of CD200/ITGA6-expressing progenitor cells. Because the CD200/ITGA6-expressing progenitors have been shown in this study to be multipotent, generating a number of important hair follicle lineages, this may lead to the miniaturization of the hair follicle, a hallmark sign of AGA. But because there are still stem cells present in all of the hair follicles of patients with AGA, it suggests that the AGA condition may be reversible. The next steps will be to figure out what signals are necessary for the creation of this vital progenitor population, and how stem cells that are still alive in the hair follicles of bald AGA scalp can be used to regenerate the lost progenitors.

References:

Furdon, S. A., Clark, D. A. Scalp hair characteristics in the newborn infant. Advances in Neonatal Care. 2003. 3(6): 286-296.
View Article

Garza, L. A., Yang, C., Zhao, T., Blatt, H. B., et al. Bald scalp in men with androgenetic alopecia retains hair follicle stem cells but lacks CD200-rich and CD34-positive hair follicle progenitor cells. The Journal of Clinical Investigation. 2011. 121(2): 613-622.
View Article

Gutierrez-Rivera, A., Pavon-Rodriguez, A., Jimenez-Acosta, F., Poblet, E., et al. Functional characterization of highly adherent CD34+ keratinocytes isolated from human skin. Experimental Dermatology. 2010. 19(7): 685-688.
View Article

Inoue, K., Aoi, N., Sato, T., Yamauchi, Y., et al. Differential expression of stem-cell-associated markers in human hair follicle epithelial cells. Laboratory Investigation. 2009. 89: 844-856.
View Article

Ito, M., Kizawa, K., Hamada, K., Cotsarelis, G. Hair follicle stem cells in the lower bulge form the secondary germ, a biochemically distinct but functionally equivalent progenitor cell population, at the termination of catagen. Differentiation. 2004. 72(9-10): 548-57.
View Article

Lyle, S., Christofidou-Solomidou, M., Liu, Y., Elder, D. E., et al. The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells. The Journal of Cell Science. 1998. 111(21): 3179-88.
View Article

Ohyama, M. Advances in the Study of Stem-Cell-Enriched Hair Follicle Bulge Cells: A Review Featuring Characterization and Isolation of Human Bulge Cells. Dermatology. 2007. 214(4): 342-351.
View Article

Ohyama, M., Terunuma, A., Tock, C. L., Radonovich, M. F., et al. Characterization and isolation of stem cell-enriched human hair follicle bulge cells. The Journal of Clinical Investigation. 2006. 116(1): 249-260.
View Article

Oshima, H., Rochat, A., Kedzia, C., Kobayashi, K., Barrandon, Y. Morphogenesis and Renewal of Hair Follicles from Adult Multipotent Stem Cells. Cell. 2001. 104(2): 233-245.
View Article

Image of “Hair Follicle” was created by the author based off of images from Wikimedia Commons and other medical resources.

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:

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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.

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

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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

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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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Douglas, G. C., VandeVoort, C. A., Kumar, P., Chang, T., Golos, T. G. Trophoblast Stem Cells: Models for Investigating Trophectoderm Differentiation and Placental Development. Endocrine Reviews. 2009. 30(3): 228-240.
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Epple-Farmer, J., Debeb, B. G., Smithies, O., Binas, B. Gender-Dependent Survival of Allogeneic Trophoblast Stem Cells in Liver. Cell Transplantation. 2009. 18(7): 769-776.
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Gilbert, S. F. Developmental Biology. Seventh edition. Sinauer Associates, 2003.

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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Rossant, J. and Cross, J. C. Placental development: Lessons from mouse mutants. Nature Reviews Genetics. 2001. 2: 538-548.
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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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