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Introduction
For decades, diabetes researchers have been searching for ways to replace the insulin-producing cells of the pancreas that are destroyed by a patient’s own immune system. Now it appears that this may be possible. Each year, diabetes affects more people and causes more deaths than breast cancer and AIDS combined. Diabetes is the seventh leading cause of death in the United States today, with nearly 200,000 deaths reported each year. The American Diabetes Association estimates that nearly 16 million people, or 5.9 percent of the United States population, currently have diabetes.
Diabetes is actually a group of diseases characterized by abnormally high levels of the sugar glucose in the bloodstream. This excess glucose is responsible for most of the complications of diabetes, which include blindness, kidney failure, heart disease, stroke, neuropathy, and amputations. Type 1 diabetes, also known as juvenile-onset diabetes, typically affects children and young adults. Diabetes develops when the body’s immune system sees its own cells as foreign and attacks and destroys them. As a result, the islet cells of the pancreas, which normally produce insulin, are destroyed. In the absence of insulin, glucose cannot enter the cell and glucose accumulates in the blood. Type 2 diabetes, also called adult-onset diabetes, tends to affect older, sedentary, and overweight individuals with a family history of diabetes. Type 2 diabetes occurs when the body cannot use insulin effectively. This is called insulin resistance and the result is the same as with type 1 diabetes—a build up of glucose in the blood.
There is currently no cure for diabetes. People with type 1 diabetes must take insulin several times a day and test their blood glucose concentration three to four times a day throughout their entire lives. Frequent monitoring is important because patients who keep their blood glucose concentrations as close to normal as possible can significantly reduce many of the complications of diabetes, such as retinopathy (a disease of the small blood vessels of the eye which can lead to blindness) and heart disease, that tend to develop over time. People with type 2 diabetes can often control their blood glucose concentrations through a combination of diet, exercise, and oral medication. Type 2 diabetes often progresses to the point where only insulin therapy will control blood glucose concentrations.
Each year, approximately 1,300 people with type 1 diabetes receive whole-organ pancreas transplants. After a year, 83 percent of these patients, on average, have no symptoms of diabetes and do not have to take insulin to maintain normal glucose concentrations in the blood. However, the demand for transplantable pancreases outweighs their availability. To prevent the body from rejecting the transplanted pancreas, patients must take powerful drugs that suppress the immune system for their entire lives, a regimen that makes them susceptible to a host of other diseases. Many hospitals will not perform a pancreas transplant unless the patient also needs a kidney transplant. That is because the risk of infection due to immunosuppressant therapy can be a greater health threat than the diabetes itself. But if a patient is also receiving a new kidney and will require immunosuppressant drugs anyway, many hospitals will perform the pancreas transplant.
Over the past several years, doctors have attempted to cure diabetes by injecting patients with pancreatic islet cells—the cells of the pancreas that secrete insulin and other hormones. However, the requirement for steroid immunosuppressant therapy to prevent rejection of the cells increases the metabolic demand on insulin-producing cells and eventually they may exhaust their capacity to produce insulin. The deleterious effect of steroids is greater for islet cell transplants than for whole-organ transplants. As a result, less than 8 percent of islet cell transplants performed before last year had been successful.
More recently, James Shapiro and his colleagues in Edmonton, Alberta, Canada, have developed an experimental protocol for transplanting islet cells that involves using a much larger amount of islet cells and a different type of immunosuppressant therapy. In a recent study, they report that [17], seven of seven patients who received islet cell transplants no longer needed to take insulin, and their blood glucose concentrations were normal a year after surgery. The success of the Edmonton protocol is now being tested at 10 centers around the world.
If the success of the Edmonton protocol can be duplicated, many hurdles still remain in using this approach on a wide scale to treat diabetes. First, donor tissue is not readily available. Islet cells used in transplants are obtained from cadavers, and the procedure requires at least two cadavers per transplant. The islet cells must be immunologically compatible, and the tissue must be freshly obtained—within eight hours of death. Because of the shortage of organ donors, these requirements are difficult to meet and the waiting list is expected to far exceed available tissue, especially if the procedure becomes widely accepted and available. Further, islet cell transplant recipients face a lifetime of immunosuppressant therapy, which makes them susceptible to other serious infections and diseases.
Development of the Pancreas
Before discussing cell-based therapies for diabetes, it is important to understand how the pancreas develops. In mammals, the pancreas contains three classes of cell types: the ductal cells, the acinar cells, and the endocrine cells. The endocrine cells produce the hormones glucagon, somatostatin, pancreatic polypeptide (PP), and insulin, which are secreted into the blood stream and help the body regulate sugar metabolism. The acinar cells are part of the exocrine system, which manufactures digestive enzymes, and ductal cells from the pancreatic ducts, which connect the acinar cells to digestive organs.
In humans, the pancreas develops as an outgrowth of the duodenum, a part of the small intestine. The cells of both the exocrine system—the acinar cells—and of the endocrine system—the islet cells—seem to originate from the ductal cells during development. During development these endocrine cells emerge from the pancreatic ducts and form aggregates that eventually form what is known as Islets of Langerhans. In humans, there are four types of islet cells: the insulin-producing beta cells; the alpha cells, which produce glucagon; the delta cells, which secrete somatostatin; and the PP-cells, which produce pancreatic polypeptide. The hormones released from each type of islet cell have a role in regulating hormones released from other islet cells. In the human pancreas, 65 to 90 percent of islet cells are beta cells, 15 to 20 percent are alpha-cells, 3 to 10 percent are delta cells, and one percent is PP cells. Acinar cells form small lobules contiguous with the ducts (see Figure 7.1. Insulin Production in the Human Pancreas). The resulting pancreas is a combination of a lobulated, branched acinar gland that forms the exocrine pancreas, and, embedded in the acinar gland, the Islets of Langerhans, which constitute the endocrine pancreas.

Figure 7.1. Insulin Production in the Human Pancreas. The pancreas is located in the abdomen, adjacent to the duodenum (the first portion of the small intestine). A cross-section of the pancreas shows the islet of Langerhans which is the functional unit of the endocrine pancreas. Encircled is the beta cell that synthesizes and secretes insulin. Beta cells are located adjacent to blood vessels and can easily respond to changes in blood glucose concentration by adjusting insulin production. Insulin facilitates uptake of glucose, the main fuel source, into cells of tissues such as muscle.
(© 2001 Terese Winslow, Lydia Kibiuk)
During fetal development, new endocrine cells appear to arise from progenitor cells in the pancreatic ducts. Many researchers maintain that some sort of islet stem cell can be found intermingled with ductal cells during fetal development and that these stem cells give rise to new endocrine cells as the fetus develops. Ductal cells can be distinguished from endocrine cells by their structure and by the genes they express. For example, ductal cells typically express a gene known as cytokeratin-9 (CK-9), which encodes a structural protein. Beta islet cells, on the other hand, express a gene called PDX-1, which encodes a protein that initiates transcription from the insulin gene. These genes, called cell markers, are useful in identifying particular cell types.
Following birth and into adulthood, the source of new islet cells is not clear, and some controversy exists over whether adult stem cells exist in the pancreas. Some researchers believe that islet stem cell-like cells can be found in the pancreatic ducts and even in the islets themselves. Others maintain that the ductal cells can differentiate into islet precursor cells, while others hold that new islet cells arise from stem cells in the blood. Researchers are using several approaches for isolating and cultivating stem cells or islet precursor cells from fetal and adult pancreatic tissue. In addition, several new promising studies indicate that insulin-producing cells can be cultivated from embryonic stem cell lines.
Development of Cell-Based Therapies for DiabeteS
In developing a potential therapy for patients with diabetes, researchers hope to develop a system that meets several criteria. Ideally, stem cells should be able to multiply in culture and reproduce themselves exactly. That is, the cells should be self-renewing. Stem cells should also be able to differentiate in vivo to produce the desired kind of cell. For diabetes therapy, it is not clear whether it will be desirable to produce only beta cells—the islet cells that manufacture insulin—or whether other types of pancreatic islet cells are also necessary. Studies by Bernat Soria and colleagues, for example, indicate that isolated beta cells—those cultured in the absence of the other types of islet cells—are less responsive to changes in glucose concentration than intact islet clusters made up of all islet cell types. Islet cell clusters typically respond to higher-than-normal concentrations of glucose by releasing insulin in two phases: a quick release of high concentrations of insulin and a slower release of lower concentrations of insulin. In this manner the beta cells can fine-tune their response to glucose. Extremely high concentrations of glucose may require that more insulin be released quickly, while intermediate concentrations of glucose can be handled by a balance of quickly and slowly released insulin.
Isolated beta cells, as well as islet clusters with lower-than-normal amounts of non-beta cells, do not release insulin in this biphasic manner. Instead insulin is released in an all-or-nothing manner, with no fine-tuning for intermediate concentrations of glucose in the blood [5, 18]. Therefore, many researchers believe that it will be preferable to develop a system in which stem or precursor cell types can be cultured to produce all the cells of the islet cluster in order to generate a population of cells that will be able to coordinate the release of the appropriate amount of insulin to the physiologically relevant concentrations of glucose in the blood.
Fetal Tissue as Source for Islet Cells
Several groups of researchers are investigating the use of fetal tissue as a potential source of islet progenitor cells. For example, using mice, researchers have compared the insulin content of implants from several sources of stem cells—fresh human fetal pancreatic tissue, purified human islets, and cultured islet tissue [2]. They found that insulin content was initially higher in the fresh tissue and purified islets. However, with time, insulin concentration decreased in the whole tissue grafts, while it remained the same in the purified islet grafts. When cultured islets were implanted, however, their insulin content increased over the course of three months. The researchers concluded that precursor cells within the cultured islets were able to proliferate (continue to replicate) and differentiate (specialize) into functioning islet tissue, but that the purified islet cells (already differentiated) could not further proliferate when grafted. Importantly, the researchers found, however, that it was also difficult to expand cultures of fetal islet progenitor cells in culture [7].
Adult Tissue as Source for Islet Cells
Many researchers have focused on culturing islet cells from human adult cadavers for use in developing transplantable material. Although differentiated beta cells are difficult to proliferate and culture, some researchers have had success in engineering such cells to do this. For example, Fred Levine and his colleagues at the University of California, San Diego, have engineered islet cells isolated from human cadavers by adding to the cells’ DNA special genes that stimulate cell proliferation. However, because once such cell lines that can proliferate in culture are established, they no longer produce insulin. The cell lines are further engineered to express the beta islet cell gene, PDX-1, which stimulates the expression of the insulin gene. Such cell lines have been shown to propagate in culture and can be induced to differentiate to cells, which produce insulin. When transplanted into immune-deficient mice, the cells secrete insulin in response to glucose. The researchers are currently investigating whether these cells will reverse diabetes in an experimental diabetes model in mice [6, 8].
These investigators report that these cells do not produce as much insulin as normal islets, but it is within an order of magnitude. The major problem in dealing with these cells is maintaining the delicate balance between growth and differentiation. Cells that proliferate well do not produce insulin efficiently, and those that do produce insulin do not proliferate well. According to the researchers, the major issue is developing the technology to be able to grow large numbers of these cells that will reproducibly produce normal amounts of insulin [9].
Another promising source of islet progenitor cells lies in the cells that line the pancreatic ducts. Some researchers believe that multipotent (capable of forming cells from more than one germ layer) stem cells are intermingled with mature, differentiated duct cells, while others believe that the duct cells themselves can undergo a differentiation, or a reversal to a less mature type of cell, which can then differentiate into an insulin-producing islet cell.
Susan Bonner-Weir and her colleagues reported last year that when ductal cells isolated from adult human pancreatic tissue were cultured, they could be induced to differentiate into clusters that contained both ductal and endocrine cells. Over the course of three to four weeks in culture, the cells secreted low amounts of insulin when exposed to low concentrations of glucose, and higher amounts of insulin when exposed to higher glucose concentrations. The researchers have determined by immunochemistry and ultrastructural analysis that these clusters contain all of the endocrine cells of the islet [4].
Bonner-Weir and her colleagues are working with primary cell cultures from duct cells and have not established cells lines that can grow indefinitely. However the cells can be expanded. According to the researchers, it might be possible in principle to do a biopsy and remove duct cells from a patient and then proliferate the cells in culture and give the patient back his or her own islets. This would work with patients who have type 1 diabetes and who lack functioning beta cells, but their duct cells remain intact. However, the autoimmune destruction would still be a problem and potentially lead to destruction of these transplanted cells [3]. Type 2 diabetes patients might benefit from the transplantation of cells expanded from their own duct cells since they would not need any immunosuppression. However, many researchers believe that if there is a genetic component to the death of beta cells, then beta cells derived from ductal cells of the same individual would also be susceptible to autoimmune attack.
Some researchers question whether the ductal cells are indeed undergoing a dedifferentiation or whether a subset of stem-like or islet progenitors populate the pancreatic ducts and may be co-cultured along with the ductal cells. If ductal cells die off but islet precursors proliferate, it is possible that the islet precursor cells may overtake the ductal cells in culture and make it appear that the ductal cells are dedifferentiating into stem cells. According to Bonner-Weir, both dedifferentiated ductal cells and islet progenitor cells may occur in pancreatic ducts.
Ammon Peck of the University of Florida, Vijayakumar Ramiya of Ixion Biotechnology in Alachua, FL, and their colleagues [13, 14] have also cultured cells from the pancreatic ducts from both humans and mice. Last year, they reported that pancreatic ductal epithelial cells from adult mice could be cultured to yield islet-like structures similar to the cluster of cells found by Bonner-Weir. Using a host of islet-cell markers they identified cells that produced insulin, glucagon, somatostatin, and pancreatic polypeptide. When the cells were implanted into diabetic mice, the diabetes was reversed.
Joel Habener has also looked for islet-like stem cells from adult pancreatic tissue. He and his colleagues have discovered a population of stem-like cells within both the adult pancreas islets and pancreatic ducts. These cells do not express the marker typical of ductal cells, so they are unlikely to be ductal cells, according to Habener. Instead, they express a marker called nestin, which is typically found in developing neural cells. The nestin-positive cells do not express markers typically found in mature islet cells. However, depending upon the growth factors added, the cells can differentiate into different types of cells, including liver, neural, exocrine pancreas, and endocrine pancreas, judged by the markers they express, and can be maintained in culture for up to eight months [20].
Embryonic Stem Cells
The discovery of methods to isolate and grow human embryonic stem cells in 1998 renewed the hopes of doctors, researchers, and diabetes patients and their families that a cure for type 1 diabetes, and perhaps type 2 diabetes as well, may be within striking distance. In theory, embryonic stem cells could be cultivated and coaxed into developing into the insulin-producing islet cells of the pancreas. With a ready supply of cultured stem cells at hand, the theory is that a line of embryonic stem cells could be grown up as needed for anyone requiring a transplant. The cells could be engineered to avoid immune rejection. Before transplantation, they could be placed into nonimmunogenic material so that they would not be rejected and the patient would avoid the devastating effects of immunosuppressant drugs. There is also some evidence that differentiated cells derived from embryonic stem cells might be less likely to cause immune rejection (see Chapter 10. Assessing Human Stem Cell Safety). Although having a replenishable supply of insulin-producing cells for transplant into humans may be a long way off, researchers have been making remarkable progress in their quest for it. While some researchers have pursued the research on embryonic stem cells, other researchers have focused on insulin-producing precursor cells that occur naturally in adult and fetal tissues.
Since their discovery three years ago, several teams of researchers have been investigating the possibility that human embryonic stem cells could be developed as a therapy for treating diabetes. Recent studies in mice show that embryonic stem cells can be coaxed into differentiating into insulin-producing beta cells, and new reports indicate that this strategy may be possible using human embryonic cells as well.
Last year, researchers in Spain reported using mouse embryonic stem cells that were engineered to allow researchers to select for cells that were differentiating into insulin-producing cells [19]. Bernat Soria and his colleagues at the Universidad Miguel Hernandez in San Juan, Alicante, Spain, added DNA containing part of the insulin gene to embryonic cells from mice. The insulin gene was linked to another gene that rendered the mice resistant to an antibiotic drug. By growing the cells in the presence of an antibiotic, only those cells that were activating the insulin promoter were able to survive. The cells were cloned and then cultured under varying conditions. Cells cultured in the presence of low concentrations of glucose differentiated and were able to respond to changes in glucose concentration by increasing insulin secretion nearly sevenfold. The researchers then implanted the cells into the spleens of diabetic mice and found that symptoms of diabetes were reversed.
Manfred Ruediger of Cardion, Inc., in Erkrath, Germany, is using the approach developed by Soria and his colleagues to develop insulin-producing human cells derived from embryonic stem cells. By using this method, the non-insulin-producing cells will be killed off and only insulin-producing cells should survive. This is important in ensuring that undifferentiated cells are not implanted that could give rise to tumors [15]. However, some researchers believe that it will be important to engineer systems in which all the components of a functioning pancreatic islet are allowed to develop.
Recently Ron McKay and his colleagues described a series of experiments in which they induced mouse embryonic cells to differentiate into insulin-secreting structures that resembled pancreatic islets [10]. McKay and his colleagues started with embryonic stem cells and let them form embryoid bodies—an aggregate of cells containing all three embryonic germ layers. They then selected a population of cells from the embryoid bodies that expressed the neural marker nestin (see Appendix B. Mouse Embryonic Stem Cells). Using a sophisticated five-stage culturing technique, the researchers were able to induce the cells to form islet-like clusters that resembled those found in native pancreatic islets. The cells responded to normal glucose concentrations by secreting insulin, although insulin amounts were lower than those secreted by normal islet cells (see Figure 7.2. Development of Insulin-Secreting Pancreatic-Like Cells From Mouse Embryonic Stem Cells). When the cells were injected into diabetic mice, they survived, although they did not reverse the symptoms of diabetes.

Figure 7.2. Development of Insulin-Secreting Pancreatic-Like Cells From Mouse Embryonic Stem Cells. Mouse embryonic stem cells were derived from the inner cell mass of the early embryo (blastocyst) and cultured under specific conditions. The embryonic stem cells (in blue) were then expanded and differentiated. Cells with markers consistent with islet cells were selected for further differentiation and characterization. When these cells (in purple) were grown in culture, they spontaneously formed three-dimentional clusters similar in structure to normal pancreatic islets. The cells produced and secreted insulin. As depicted in the chart, the pancreatic islet-like cells showed an increase in release of insulin as the glucose concentration of the culture media was increased. When the pancreatic islet-like cells were implanted in the shoulder of diabetic mice, the cells became vascularized, synthesized insulin, and maintained physical characteristics similar to pancreatic islets.
(© 2001 Terese Winslow, Caitlin Duckwall)
According to McKay, this system is unique in that the embryonic cells form a functioning pancreatic islet, complete with all the major cell types. The cells assemble into islet-like structures that contain another layer, which contains neurons and is similar to intact islets from the pancreas [11]. Several research groups are trying to apply McKay’s results with mice to induce human embryonic stem cells to differentiate into insulin-producing islets.
Recent research has also provided more evidence that human embryonic cells can develop into cells that can and do produce insulin. Last year, Melton, Nissim Benvinisty of the Hebrew University in Jerusalem, and Josef Itskovitz-Eldor of the Technion in Haifa, Israel, reported that human embryonic stem cells could be manipulated in culture to express the PDX-1 gene, a gene that controls insulin transcription [16]. In these experiments, researchers cultured human embryonic stem cells and allowed them to spontaneously form embryoid bodies (clumps of embryonic stem cells composed of many types of cells from all three germ layers). The embryoid bodies were then treated with various growth factors, including nerve growth factor. The researchers found that both untreated embryoid bodies and those treated with nerve growth factor expressed PDX-1. Embryonic stem cells prior to formation of the aggregated embryoid bodies did not express PDX-1. Because expression of the PDX-1 gene is associated with the formation of beta islet cells, these results suggest that beta islet cells may be one of the cell types that spontaneously differentiate in the embryoid bodies. The researchers now think that nerve growth factor may be one of the key signals for inducing the differentiation of beta islet cells and can be exploited to direct differentiation in the laboratory. Complementing these findings is work done by Jon Odorico of the University of Wisconsin in Madison using human embryonic cells of the same source. In preliminary findings, he has shown that human embryonic stem cells can differentiate and express the insulin gene [12].
More recently, Itskovitz-Eldor and his Technion colleagues further characterized insulin-producing cells in embryoid bodies [1]. The researchers found that embryonic stem cells that were allowed to spontaneously form embryoid bodies contained a significant percentage of cells that express insulin. Based on the binding of antibodies to the insulin protein, Itskovitz-Eldor estimates that 1 to 3 percent of the cells in embryoid bodies are insulin-producing beta-islet cells. The researchers also found that cells in the embryoid bodies express glut-2 and islet-specific glucokinase, genes important for beta cell function and insulin secretion. Although the researchers did not measure a time-dependent response to glucose, they did find that cells cultured in the presence of glucose secrete insulin into the culture medium. The researchers concluded that embryoid bodies contain a subset of cells that appear to function as beta cells and that the refining of culture conditions may soon yield a viable method for inducing the differentiation of beta cells and, possibly, pancreatic islets.
Taken together, these results indicate that the development of a human embryonic stem cell system that can be coaxed into differentiating into functioning insulin-producing islets may soon be possible.
Future Directions
Ultimately, type 1 diabetes may prove to be especially difficult to cure, because the cells are destroyed when the body’s own immune system attacks and destroys them. This autoimmunity must be overcome if researchers hope to use transplanted cells to replace the damaged ones. Many researchers believe that at least initially, immunosuppressive therapy similar to that used in the Edmonton protocol will be beneficial. A potential advantage of embryonic cells is that, in theory, they could be engineered to express the appropriate genes that would allow them to escape or reduce detection by the immune system. Others have suggested that a technology should be developed to encapsulate or embed islet cells derived from islet stem or progenitor cells in a material that would allow small molecules such as insulin to pass through freely, but would not allow interactions between the islet cells and cells of the immune system. Such encapsulated cells could secrete insulin into the blood stream, but remain inaccessible to the immune system.
Before any cell-based therapy to treat diabetes makes it to the clinic, many safety issues must be addressed (see Chapter 10. Assessing Human Stem Cell Safety). A major consideration is whether any precursor or stem-like cells transplanted into the body might revert to a more pluripotent state and induce the formation of tumors. These risks would seemingly be lessened if fully differentiated cells are used in transplantation.
But before any kind of human islet-precursor cells can be used therapeutically, a renewable source of human stem cells must be developed. Although many progenitor cells have been identified in adult tissue, few of these cells can be cultured for multiple generations. Embryonic stem cells show the greatest promise for generating cell lines that will be free of contaminants and that can self renew. However, most researchers agree that until a therapeutically useful source of human islet cells is developed, all avenues of research should be exhaustively investigated, including both adult and embryonic sources of tissue.
References
1. Assady, S., Maor, G., Amit, M., Itskovitz-Eldor, J., Skorecki, K.L., and Tzukerman, M. (2001). Insulin production by human embryonic stem cells. Diabetes. 50. http://www.diabetes.org/Diabetes_Rapids/Suheir_Assady_ 06282001.pdf
2. Beattie, G.M., Otonkoski, T., Lopez, A.D., and Hayek, A. (1997). Functional beta-cell mass after transplantation of human fetal pancreatic cells: differentiation or proliferation? Diabetes. 46, 244–248.
3. Bonner-Weir, S., personal communication.
4. Bonner-Weir, S., Taneja, M., Weir, G.C., Tatarkiewicz, K., Song, K.H., Sharma, A., and O’Neil, J.J. (2000). In vitro cultivation of human islets from expanded ductal tissue. Proc. Natl. Acad. Sci. U. S. A. 97, 7999–8004.
5. Bosco, D. and Meda, P. (1997). Reconstructing islet function in vitro. Adv. Exp. Med. Biol. 426, 285–298.
6. Dufayet de la Tour, D., Halvorsen, T., Demeterco, C., Tyrberg, B., Itkin-Ansari, P., Loy, M., Yoo, S.J., Hao, S., Bossie, S., and Levine, F. (2001). B-cell differentiation from a human pancreatic cell line in vitro and in vivo. Mol. Endocrinol. 15, 476–483.
7. Hayek, A., personal communication.
8. Itkin-Ansari, P., Demeterco, C., Bossie, S., Dufayet de la Tour, D., Beattie, G.M., Movassat, J., Mally, M.I., Hayek, A., and Levine, F. (2001). PDX-1 and cell-cell contact act in synergy to promote d-cell development in a human pancreatic endocrine precursor cell line. Mol. Endocrinol. 14, 814–822.
9. Levine, F., personal communication.
10. Lumelsky, N., Blondel, O., Laeng, P., Velasco, I., Ravin, R., and McKay, R. (2001). Differentiation of Embryonic Stem Cells to Insulin-Secreting Structures Similiar to Pancreatic Islets. Science. 292, 1389–1394.
11. McKay, R., personal communication
12. Odorico, J. S., personal communication.
13. Peck, A., personal communication.
14. Ramiya, V. K., personal communication.
15. Ruediger, M., personal communication.
16. Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D., and Benvenisty, N. (2000). Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 97, 11307–11312.
17. Shapiro, J., Lakey, J.R.T., Ryan, E.A., Korbutt, G.S., Toth, E., Warnock, G.L., Kneteman, N.M., and Rajotte, R.V. (2000). Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N. Engl. J. Med. 343, 230–238.
18. Soria, B., Martin, F., Andreu, E., Sanchez-Andrés, J.V., Nacher, V., and Montana, E. (1996). Diminished fraction of blockable ATP-sensitive K+ channels in islets transplanted into diabetic mice. Diabetes. 45, 1755–1760.
19. Soria, B., Roche, E., Berná, G., Leon-Quinto, T., Reig, J.A., and Martin, F. (2000). Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocininduced diabetic mice. Diabetes. 49, 157–162.
20. Zulewski, H., Abraham, E.J., Gerlach, M.J., Daniel, P.B., Moritz, W., Muller, B., Vallejo, M., Thomas, M.K., and Habener, J.F. (2001). Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes. 50, 521–533.

Introduction
The body’s main line of defense against invasion by infectious organisms is the immune system. To succeed, an immune system must distinguish the many cellular components of its own body (self) from the cells or components of invading organisms (nonself). “Nonself” should be attacked while “self” should not. Therefore, two general types of errors can be made by the immune system. If the immune system fails to quickly detect and destroy an invading organism, an infection will result. However, if the immune system fails to recognize self cells or components and mistakenly attacks them, the result is known as an autoimmune disease. Common autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosis (lupus), type 1 diabetes, multiple sclerosis, Sjogren’s syndrome and inflammatory bowel disease. Although each of these diseases has different symptoms, they share the unfortunate reality that, for some reason, the body’s immune system has turned against itself (see Box 6.1. Immune System Components: Common Terms and Definitions).
How Does the Immune System Normally Keep Us Healthy?
The “soldiers” of the immune system are white blood cells, including T and B lymphocytes, which originate in the bone marrow from hematopoietic stem cells. Every day the body comes into contact with many organisms such as bacteria, viruses, and parasites. Unopposed, these organisms have the potential to cause serious infections, such as pneumonia or AIDS. When a healthy individual is infected, the body responds by activating a variety of immune cells. Initially, invading bacteria or viruses are engulfed by an antigen presenting cell (APC), and their component proteins (antigens) are cut into pieces and displayed on the cell’s surface. Pieces of the foreign protein (antigen) bind to the major histocompatibility complex (MHC) proteins, also known as human leukocyte antigen (HLA) molecules, on the surface of the APCs (see Figure 6.1 Immune Response to Self or Foreign Antigens). This complex, formed by a foreign protein and an MHC protein, then binds to a T cell receptor on the surface of another type of immune cell, the CD4 helper T cell. They are so named because they “help” immune responses proceed and have a protein called CD4 on their surface. This complex enables these T cells to focus the immune response to a specific invading organism. The antigen-specific CD4 helper T cells divide and multiply while secreting substances called cytokines, which cause inflammation and help activate other immune cells. The particular cytokines secreted by the CD4 helper T cells act on cells known as the CD8 “cytotoxic” T cells (because they can kill the cells that are infected by the invading organism and have the CD8 protein on their surface). The helper T cells can also activate antigen-specific B cells to produce antibodies, which can neutralize and help eliminate bacteria and viruses from the body. Some of the antigen-specific T and B cells that are activated to rid the body of infectious organisms become long-lived “memory” cells. Memory cells have the capacity to act quickly when confronted with the same infectious organism at later times. It is the memory cells that cause us to become “immune” from later reinfections with the same organism.

Figure 6.1. Immune Response to Self or Foreign Antigens.
(© 2001 Terese Winslow)
How Do the Immune Cells of the Body Know What to Attack and What Not To?
All immune and blood cells develop from multipotent hematopoietic stem cells that originate in the bone marrow. Upon their departure from the bone marrow, immature T cells undergo a final maturation process in the thymus, a small organ located in the upper chest, before being dispersed to the body with the rest of the immune cells (e.g., B cells). Within the thymus, T cells undergo an important process that “educates” them to distinguish between self (the proteins of their own body) and nonself (the invading organism’s) antigens. Here, the T cells are selected for their ability to bind to the particular MHC proteins expressed by the individual. The particular array of MHCs varies slightly between individuals, and this variation is the basis of the immune response when a transplanted organ is rejected. MHCs and other less easily characterized molecules called minor histocompatibility antigens are genetically determined and this is the reason why donor organs from relatives of the recipient are preferred over unrelated donors.
Box 6.1. Immune System Components:
Common Terms and Definitions
Antibody—A Y-shaped protein secreted by B cells in response to an antigen. An antibody binds specifically to the antigen that induced its production. Antibodies directed against antigens on the surface of infectious organisms help eliminate those organisms from the body.
Antigen—A substance (often a protein) that induces the formation of an antibody. Antigens are commonly found on the surface of infectious organisms, transfused blood cells, and organ transplants.
Antigen presenting cells (APC)—One of a variety of cells within the body that can process antigens and display them on their surface in a form recognizable by T cells.
Autoantibody—An antibody that reacts with antigens found on the cells and tissues of an individual’s own body. Autoantibodies can cause autoimmune diseases.
Autoimmune disease—A condition that results from the formation of antibodies that attack the cells or tissues of an individual’s own body.
B cells—Also known as B lymphocytes. Each B cell is capable of making one specific antibody. When stimulated by antigen and helper T cells, B cells mature into plasma cells that secrete large amounts of their specific antibody.
Bone marrow—The soft, living tissue that fills most bone cavities and contains hematopoietic stem cells, from which all red and white blood cells evolve. The bone marrow also contains mesenchymal stem cells that a number of cell types come from, including chondrocytes, which produce cartilage.
Cytokines—A generic term for a large variety of regulatory proteins produced and secreted by cells and used to communicate with other cells. One class of cytokines is the interleukins, which act as intercellular mediators during the generation of an immune response.
Immune system cells—White blood cells or leukocytes that originate from the bone marrow. They include antigen presenting cells, such as dendritic cells, T and B lymphocytes, and neutrophils, among many others.
Lymphatic system—A network of lymph vessels and nodes that drain and filter antigens from tissue fluids before returning lymphocytes to the blood.
Memory cells—A subset of antigen-specific T or B cells that “recall” prior exposure to an antigen and respond quickly without the need to be activated again by CD4 helper T cells.
Major histocompatibility complex (MHC)—A group of genes that code for cell-surface histocompatibility antigens. These antigens are the primary reason why organ and tissue transplants from incompatible donors fail.
T cells—Also known as T lymphocytes. There are two primary subsets of T cells. CD4 helper T cells (identified by the presence of the CD4 protein on their surfaces) are instrumental in initiating an immune response by supplying special cytokines. CD8 cytotoxic (killer) T cells (identified by the presence of the CD8 protein on their surfaces), after being activated by the CD4 helper cells, are capable of killing infected cells in the body. CD4 helper T cells are destroyed by the HIV virus in AIDS patients, resulting in an ineffective immune system.
Thymus—A lymphoid organ located in the upper chest cavity. Maturing T cells leave the bone marrow and go directly to the thymus, where they are educated to discriminate between self and nonself proteins. (See tolerance.)
Tolerance—A state of specific immunologic unresponsiveness. Individuals should normally be tolerant of the cells and tissues that make up our own bodies. Should tolerance fail, an autoimmune disease may result.
In the bone marrow, a highly diverse and random array of T cells is produced. Collectively, these T cells are capable of recognizing an almost unlimited number of antigens. Because the process of generating a T cell’s antigen specificity is a random one, many immature T cells have the potential to react with the body’s own (self) proteins. To avoid this potential disaster, the thymus provides an environment where T cells that recognize self-antigens (autoreactive or self-reactive T cells) are deleted or inactivated in a process called tolerance induction.
Tolerance usually ensures that T cells do not attack the “autoantigens” (self-proteins) of the body. Given the importance of this task, it is not surprising that there are multiple checkpoints for destroying or inactivating T cells that might react to auto-antigens.
Autoimmune diseases arise when this intricate system for the induction and maintenance of immune tolerance fails. These diseases result in cell and tissue destruction by antigen-specific CD8 cytotoxic T cells or autoantibodies (antibodies to self-proteins) and the accompanying inflammatory process. These mechanisms can lead to the destruction of the joints in rheumatoid arthritis, the destruction of the insulinproducing beta cells of the pancreas in type 1 diabetes, or damage to the kidneys in lupus. The reasons for the failure to induce or maintain tolerance are enigmatic. However, genetic factors, along with environmental and hormonal influences and certain infections, may contribute to tolerance and the development of autoimmune disease [4, 7].
Hematopoietic Stem Cell Therapy for Autoimmune Diseases
The current treatments for many autoimmune diseases include the systemic use of anti-inflammatory drugs and potent immunosuppressive and immunomodulatory agents (i.e., steroids and inhibitor proteins that block the action of inflammatory cytokines). However, despite their profound effect on immune responses, these therapies are unable to induce clinically significant remissions in certain patients. In recent years, researchers have contemplated the use of stem cells to treat autoimmune disorders. Discussed here is some of the rationale for this approach, with a focus on experimental stem cell therapies for lupus, rheumatoid arthritis, and type 1 diabetes.
The immune-mediated injury in autoimmune diseases can be organ-specific, such as type 1 diabetes which is the consequence of the destruction of the pancreatic beta islet cells or multiple sclerosis which results from the breakdown of the myelin covering of nerves. These autoimmune diseases are amenable to treatments involving the repair or replacement of damaged or destroyed cells or tissue (see Chapter 7. Stem Cells and Diabetes and Chapter 11. Use of Genetically Modified Stem Cells in Experimental Gene Therapies). In contrast, non-organ-specific autoimmune diseases, such as lupus, are characterized by widespread injury due to immune reactions against many different organs and tissues.
One approach is being evaluated in early clinical trials of patients with poorly responsive, life-threatening lupus. This is a severe disease affecting multiple organs in the body including muscles, skin, joints, and kidneys as well as the brain and nerves. Over 239,000 Americans, of which more than 90 percent are women, suffer from lupus. In addition, lupus disproportionately afflicts African-American and Hispanic women [11]. A major obstacle in the treatment of non-organ-specific autoimmune diseases such as lupus is the lack of a single specific target for the application of therapy.
The objective of hematopoietic stem cell therapy for lupus is to destroy the mature, long-lived, and auto-reactive immune cells and to generate a new, properly functioning immune system. In most of these trials, the patient’s own stem cells have been used in a procedure known as autologous (from “one’s self”) hematopoietic stem cell transplantation. First, patients receive injections of a growth factor, which coaxes large numbers of hematopoietic stem cells to be released from the bone marrow into the blood stream. These cells are harvested from the blood, purified away from mature immune cells, and stored. After sufficient quantities of these cells are obtained, the patient undergoes a regimen of cytotoxic (cell-killing) drug and/or radiation therapy, which eliminates the mature immune cells. Then, the hematopoietic stem cells are returned to the patient via a blood transfusion into the circulation where they migrate to the bone marrow and begin to differentiate to become mature immune cells. The body’s immune system is then restored. Nonetheless, the recovery phase, until the immune system is reconstituted represents a period of dramatically increased susceptibility to bacterial, fungal, and viral infection, making this a high-risk therapy.
Recent reports suggest that this replacement therapy may fundamentally alter the patient’s immune system. Richard Burt and his colleagues [18] conducted a long-term follow-up (one to three years) of seven lupus patients who underwent this procedure and found that they remained free from active lupus and improved continuously after transplantation, without the need for immunosuppressive medications. One of the hallmarks of lupus is that during the natural progression of disease, the normally diverse repertoire of T cells become limited in the number of different antigens they recognize, suggesting that an increasing proportion of the patient’s T cells are autoreactive. Burt and colleagues found that following hematopoietic stem cell transplantation, levels of T cell diversity were restored to those of healthy individuals. This finding provides evidence that stem cell replacement may be beneficial in reestablishing tolerance in T cells, thereby decreasing the likelihood of disease reoccurrence.
Development of Hematopoietic Stem Cell Lines for Transplantation
The ability to generate and propagate unlimited numbers of hematopoietic stem cells outside the body—whether from adult, umbilical cord blood, fetal, or embryonic sources—would have a major impact on the safety, cost, and availability of stem cells for transplantation. The current approach of isolating hematopoietic stem cells from a patient’s own peripheral blood places the patient at risk for a flare-up of their autoimmune disease. This is a potential consequence of repeated administration of the stem cell growth factors needed to mobilize hematopoietic stem cells from the bone marrow to the blood stream in numbers sufficient for transplantation. In addition, contamination of the purified hematopoietic stem cells with the patient’s mature autoreactive T and B cells could affect the success of the treatment in some patients. Propagation of pure cell lines in the laboratory would avoid these potential drawbacks and increase the numbers of stem cells available to each patient, thus shortening the at-risk interval before full immune reconstitution.
Whether embryonic stem cells will provide advantages over stem cells derived from cord blood or adult bone marrow hematopoietic stem cells remains to be determined. However, hematopoietic stem cells, whether from umbilical cord blood or bone marrow, have a more limited potential for self-renewal than do pluripotent embryonic stem cells. Although new information will be needed to direct the differentiation of embryonic stem cells into hematopoietic stem cells, hematopoietic cells are present in differentiated cultures from human embryonic stem cells [9] and from human fetal-derived embryonic germ stem cells [17].
One potential advantage of using hematopoietic stem cell lines for transplantation in patients with autoimmune diseases is that these cells could be generated from unaffected individuals or, as predisposing genetic factors are defined, from embryonic stem cells lacking these genetic influences. In addition, use of genetically selected or genetically engineered cell types may further limit the possibility of disease progression or reemergence.
One risk of using nonself hematopoietic stem cells is of immune rejection of the transplanted cells. Immune rejection is caused by MHC protein differences between the donor and the patient (recipient). In this scenario, the transplanted hematopoietic stem cells and their progeny are rejected by the patient’s own T cells, which are originating from the patient’s surviving bone marrow hematopoietic stem cells. In this regard, embryonic stem cell-derived hematopoietic stem cells may offer distinct advantages over cord blood and bone marrow hematopoietic stem cell lines in avoiding rejection of the transplant. Theoretically, banks of embryonic stem cells expressing various combinations of the three most critical MHC proteins could be generated to allow close matching to the recipient’s MHC composition.
Additionally, there is evidence that embryonic stem cells are considerably more receptive to genetic manipulation than are hematopoietic stem cells (see Chapter 11. Use of Genetically Modified Stem Cells in Experimental Gene Therapies).
This characteristic means that embryonic stem cells could be useful in strategies that could prevent their recognition by the patient’s surviving immune cells. For example, it may be possible to introduce the recipient’s MHC proteins into embryonic stem cells through targeted gene transfer. Alternatively, it is theoretically possible to generate a universal donor embryonic stem cell line by genetic alteration or removal of the MHC proteins. Researchers have accomplished this by genetically altering a mouse so that it has little or no surface expression of MHC molecules on any of the cells or tissues. There is no rejection of pancreatic beta islet cells from these genetically altered mice when the cells are transplanted into completely MHC-mismatched mice [13]. Additional research will be needed to determine the feasibility of these alternative strategies for prevention of graft rejection in humans [6].
Jon Odorico and colleagues have shown that expression of MHC proteins on mouse embryonic stem cells and differentiated embryonic stem cell progeny is either absent or greatly decreased compared with MHC expression on adult cells [8]. These preliminary findings raise the intriguing possibility that lines derived from embryonic stem cells may be inherently less susceptible to rejection by the recipient’s immune system than lines derived from adult cells. This could have important implications for the transplantation of cells other than hematopoietic stem cells.
Another potential advantage of using pure populations of donor hematopoietic stem cells achieved through stem cell technologies would be a lower incidence and severity of graft-versus-host disease, a potentially fatal complication of bone marrow transplantation. Graft-versus-host disease results from the immune-mediated injury to recipient tissues that occurs when mature organ-donor T cells remain within the organ at the time of transplant. Such mature donor alloreactive T cells would be absent from pure populations of multipotent hematopoietic stem cells, and under ideal conditions of immune tolerance induction in the recipient’s thymus, the donor-derived mature T cell population would be tolerant to the host.
Gene Therapy and Stem Cell Approaches for the Treatment of Autoimmune Diseases
Gene therapy is the genetic modification of cells to produce a therapeutic effect (see Chapter 11. Use of Genetically Modified Stem Cells in Experimental Gene Therapies). In most investigational protocols, DNA containing the therapeutic gene is transferred into cultured cells, and these cells are subsequently administered to the animal or patient. DNA can also be injected directly, entering cells at the site of the injection or in the circulation. Under ideal conditions, cells take up the DNA and produce the therapeutic protein encoded by the gene.
Currently, there is an extensive amount of gene therapy research being conducted in animal models of autoimmune disease. The goal is to modify the aberrant, inflammatory immune response that is characteristic of autoimmune diseases [15, 19]. Researchers most often use one of two general strategies to modulate the immune system. The first strategy is to block the actions of an inflammatory cytokine (secreted by certain activated immune cells and inflamed tissues) by transferring a gene into cells that encodes a “decoy” receptor for that cytokine. Alternatively, a gene is transferred that encodes an anti-inflammatory cytokine, redirecting the auto-inflammatory immune response to a more “tolerant” state. In many animal studies, promising results have been achieved by using these approaches, and the studies have advanced understanding of the disease processes and the particular inflammatory cytokines involved in disease progression [15, 19].
Serious obstacles to the development of effective gene therapies for humans remain, however. Foremost among these are the difficulty of reliably transferring genetic material into adult and slowly dividing cells (including hematopoietic stem cells) and of producing long-lasting expression of the intended protein at levels that can be tightly controlled in response to disease activity. Importantly, embryonic stem cells are substantially more permissive to gene transfer compared with adult cells, and embryonic cells sustain protein expression during extensive self-renewal. Whether adult-derived stem cells, other than hematopoietic stem cells, are similarly amenable to gene transfer has not yet been determined.
Ultimately, stem cell gene therapy should allow the development of novel methods for immune modulation in autoimmune diseases. One example is the genetic modification of hematopoietic stem cells or differentiated tissue cells with a “decoy” receptor for the inflammatory cytokine interferon gamma to treat lupus. For example, in a lupus mouse model, gene transfer of the decoy receptor, via DNA injection, arrested disease progression [12]. Other investigators have used a related but distinct approach in a mouse model of type 1 diabetes. Interleukin-12 (IL-12), an inflammatory cytokine, plays a prominent role in the development of diabetes in these mice. The investigators transferred the gene for a modified form of IL-12, which blocks the activity of the natural IL-12, into pancreatic beta islet cells (the target of autoimmune injury in type 1 diabetes). The islet cell gene therapy prevented the onset of diabetes in these mice [20]. Theoretically, embryonic stem cells or adult stem cells could be genetically modified before or during differentiation into pancreatic beta islet cells to be used for transplantation. The resulting immune-modulating islet cells might diminish the occurrence of ongoing autoimmunity, increase the likelihood of long-term function of the transplanted cells, and eliminate the need for immunosuppressive therapy following transplantation.
Researchers are exploring similar genetic approaches to prevent progressive joint destruction and loss of cartilage and to repair damaged joints in animal models of rheumatoid arthritis. Rheumatoid arthritis is a debilitating autoimmune disease characterized by acute and chronic inflammation, in which the immune system primarily attacks the joints of the body. In a recent study, investigators genetically transferred an anti-inflammatory cytokine, interleukin-4 (IL-4), into a specialized, highly efficient antigen-presenting cell called a dendritic cell, and then injected these IL-4-secreting cells into mice that can be induced to develop a form of arthritis similar to rheumatoid arthritis in humans. These IL-4-secreting dendritic cells are presumed to act on the CD4 helper T cells to reintroduce tolerance to self-proteins. Treated mice showed complete suppression of their disease and, in addition to its immune-modulatory properties, IL-4 blocked bone resorption (a serious complication of rheumatoid arthritis), making it a particularly attractive cytokine for this therapy [10]. However, one obstacle to this approach is that human dendritic cells are difficult to isolate in large numbers.
Investigators have also directed the differentiation of dendritic cells from mouse embryonic stem cells, indicating that a stem cell-based approach might work in patients with rheumatoid arthritis [5]. Longer-term follow-up and further characterization will be needed in animal models before researchers proceed with the development of such an approach in humans. In similar studies, using other inhibitors of inflammatory cytokines such as a decoy receptor for tumor necrosis factor-? (a prominent inflammatory cytokine in inflamed joints), an inhibitor of nuclear factor-?B (a protein within cells that turns on the production of many inflammatory cytokines), and interleukin-13 (an anti-inflammatory cytokine), researchers have shown promising results in animal models of rheumatoid arthritis [19]. Because of the complexity and redundancy of immune system signaling networks, it is likely that a multifaceted approach involving inhibitors of several different inflammatory cytokines will be successful, whereas approaches targeting single cytokines might fail or produce only short-lived responses. In addition, other cell types may prove to be even better vehicles for the delivery of gene therapy in this disease.
Chondrocytes, cells that build cartilage in joints, may provide another avenue for stem cell-based treatment of rheumatoid arthritis. These cells have been derived from human bone marrow stromal stem cells derived from human bone marrow [14]. Little is known about the intermediate cells that ultimately differentiate into chondrocytes. In addition to adult bone marrow as a source for stromal stem cells, human embryonic stem cells can differentiate into precursor cells believed to lead ultimately to the stromal stem cells [16]. However, extensive research is needed to reliably achieve the directed derivation of the stromal stem cells from embryonic stem cells and, subsequently, the differentiation of chondrocytes from these stromal stem cells.
The ideal cell for optimum cartilage repair may be a more primitive cell than the chondrocyte, such as the stromal cell, or an intermediate cell in the pathway (e.g., a connective tissue precursor) leading to the chondrocyte. Stromal stem cells can generate new chondrocytes and facilitate cartilage repair in a rabbit model [3]. Such cells may also prove to be ideal targets for the delivery of immune-modulatory gene therapy. Like hematopoietic stem cells, stromal stem cells have been used in animal models for delivery of gene therapy [1]. For example, a recent study demonstrated that genetically engineered chondrocytes, expressing a growth factor, can enhance the function of transplanted chondrocytes [2].
Two obstacles to the use of adult stromal stem cells or chondrocytes are the limited numbers of these cells that can be harvested and the difficulties in propagating them in the laboratory. Embryonic stem cells, genetically modified and expanded before directed differentiation to a connective tissue stem cell, may be an attractive alternative.
Collectively, these results illustrate the tremendous potential these cells may offer for the treatment of rheumatoid arthritis and other autoimmune diseases.
Conclusion
Stem cell-based therapies offer many exciting possibilities for the development of novel treatments, and perhaps even cures, for autoimmune diseases. A challenging research effort remains to fully realize this potential and to address the many remaining questions, which include how best to direct the differentiation of specific cell types and determine which particular type of stem cell will be optimum for each therapeutic approach. Gene therapy with cytokines or their inhibitors is still in its infancy, but stem cells or their progeny may provide one of the better avenues for future delivery of immune-based therapies. Ultimately, the potential to alleviate these devastating chronic diseases with the use of stem cell-based technologies is enormous.
References
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Despite the vast experience with hematopoietic stem cells, scientists face major roadblocks in expanding their use beyond the replacement of blood and immune cells. First, hematopoietic stem cells are unable to proliferate (replicate themselves) and differentiate (become specialized to other cell types) in vitro (in the test tube or culture dish). Second, scientists do not yet have an accurate method to distinguish stem cells from other cells recovered from the blood or bone marrow. Until scientists overcome these technical barriers, they believe it is unlikely that hematopoietic stem cells will be applied as cell replacement therapy in diseases such as diabetes, Parkinson’s Disease, spinal cord injury, and many others.

Introduction

Blood cells are responsible for constant maintenance and immune protection of every cell type of the body. This relentless and brutal work requires that blood cells, along with skin cells, have the greatest powers of self-renewal of any adult tissue.
The stem cells that form blood and immune cells are known as hematopoietic stem cells (HSCs). They are ultimately responsible for the constant renewal of blood—the production of billions of new blood cells each day. Physicians and basic researchers have known and capitalized on this fact for more than 50 years in treating many diseases. The first evidence and definition of blood-forming stem cells came from studies of people exposed to lethal doses of radiation in 1945.
Basic research soon followed. After duplicating radiation sickness in mice, scientists found they could rescue the mice from death with bone marrow transplants from healthy donor animals. In the early 1960s, Till and McCulloch began analyzing the bone marrow to find out which components were responsible for regenerating blood [56]. They defined what remain the two hallmarks of an HSC: it can renew itself and it can produce cells that give rise to all the different types of blood cells (see Chapter 4. The Adult Stem Cell).
What Is a Hematopoietic Stem Cell?
A hematopoietic stem cell is a cell isolated from the blood or bone marrow that can renew itself, can differentiate to a variety of specialized cells, can mobilize out of the bone marrow into circulating blood, and can undergo programmed cell death, called apoptosis—a process by which cells that are detrimental or unneeded self-destruct.
A major thrust of basic HSC research since the 1960s has been identifying and characterizing these stem cells. Because HSCs look and behave in culture like ordinary white blood cells, this has been a difficult challenge and this makes them difficult to identify by morphology (size and shape). Even today, scientists must rely on cell surface proteins, which serve, only roughly, as markers of white blood cells.
Identifying and characterizing properties of HSCs began with studies in mice, which laid the groundwork for human studies. The challenge is formidable as about 1 in every 10,000 to 15,000 bone marrow cells is thought to be a stem cell. In the blood stream the proportion falls to 1 in 100,000 blood cells. To this end, scientists began to develop tests for proving the self-renewal and the plasticity of HSCs.
The “gold standard” for proving that a cell derived from mouse bone marrow is indeed an HSC is still based on the same proof described above and used in mice many years ago. That is, the cells are injected into a mouse that has received a dose of irradiation sufficient to kill its own blood-producing cells. If the mouse recovers and all types of blood cells reappear (bearing a genetic marker from the donor animal), the transplanted cells are deemed to have included stem cells.
These studies have revealed that there appear to be two kinds of HSCs. If bone marrow cells from the transplanted mouse can, in turn, be transplanted to another lethally irradiated mouse and restore its hematopoietic system over some months, they are considered to be long-term stem cells that are capable of self-renewal. Other cells from bone marrow can immediately regenerate all the different types of blood cells, but under normal circumstances cannot renew themselves over the long term, and these are referred to as short-term progenitor or precursor cells. Progenitor or precursor cells are relatively immature cells that are precursors to a fully differentiated cell of the same tissue type. They are capable of proliferating, but they have a limited capacity to differentiate into more than one cell type as HSCs do. For example, a blood progenitor cell may only be able to make a red blood cell (see Figure 5.1. Hematopoietic and Stromal Stem Cell Differentiation).

Figure 5.1. Hematopoietic and Stromal Stem Cell Differentiation.
(© 2001 Terese Winslow, Lydia Kibiuk)
Harrison et al. write that short-term blood-progenitor cells in a mouse may restore hematopoiesis for three to four months [36]. The longevity of short-term stem cells for humans is not firmly established. A true stem cell, capable of self-renewal, must be able to renew itself for the entire lifespan of an organism. It is these long-term replicating HSCs that are most important for developing HSC-based cell therapies. Unfortunately, to date, researchers cannot distinguish the long-term from the short-term cells when they are removed from the bloodstream or bone marrow.
The central problem of the assays used to identify long-term stem cells and short-term progenitor cells is that they are difficult, expensive, and time-consuming and cannot be done in humans. A few assays are now available that test cells in culture for their ability to form primitive and long-lasting colonies of cells, but these tests are not accepted as proof that a cell is a long-term stem cell. Some genetically altered mice can receive transplanted human HSCs to test the cells’ self-renewal and hematopoietic capabilities during the life of a mouse, but the relevance of this test for the cells in humans—who may live for decades—is open to question.
The difficulty of HSC assays has contributed to two mutually confounding research problems: definitively identifying the HSC and getting it to proliferate, or increase its numbers, in a culture dish. More rapid research progress on characterizing and using HSCs would be possible if they could be readily grown in the laboratory. Conversely, progress in identifying growth conditions suitable for HSCs and getting the cells to multiply would move more quickly if scientists could reliably and readily identify true HSCs.
Can Cell Markers Be Used to Identify Hematopoietic Stem Cells?
HSCs have an identity problem. First, the ones with long-term replicating ability are rare. Second, there are multiple types of stem cells. And, third, the stem cells look like many other blood or bone marrow cells. So how do researchers find the desired cell populations? The most common approach is through markers that appear on the surface of cells. (For a more detailed discussion, see Appendix E.i. Markers: How Do Researchers Use Them to Identify Stem Cells?) These are useful, but not perfect tools for the research laboratory.
In 1988, in an effort to develop a reliable means of identifying these cells, Irving Weissman and his collaborators focused attention on a set of protein markers on the surface of mouse blood cells that were associated with increased likelihood that the cell was a long-term HSC [50]. Four years later, the laboratory proposed a comparable set of markers for the human stem cell [3]. Weissman proposes the markers shown in Table 5.1 as the closest markers for mouse and human HSCs [62].

Table 5.1. Proposed cell-surface markers of undifferentiated hematopoietic stem cells.
Listed here are cell surface markers found on mouse and human hematopoietic stem cells as they exist in their undifferentiated state in vivo and in vitro. As these cells begin to develop as distinct cell lineages the cell surface markers are no longer identified.
Mouse Human
CD34low/- CD 34+
SCA-1+ CD59+*
Thy1+/low Thy1+
CD38+ CD38low/-
C-kit+ C-kit-/low
lin-* lin-**
* Only one of a family of CD59 markers has thus far been evaluated.
** Lin- cells lack 13 to 14 different mature blood-lineage markers.
Such cell markers can be tagged with monoclonal antibodies bearing a fluorescent label and culled out of bone marrow with fluorescence-activated cell sorting (FACS).
The groups of cells thus sorted by surface markers are heterogeneous and include some cells that are true, long-term self-renewing stem cells, some shorter-term progenitors, and some non-stem cells. Weissman’s group showed that as few as five genetically tagged cells, injected along with larger doses of stem cells into lethally irradiated mice, could establish themselves and produce marked donor cells in all blood cell lineages for the lifetime of the mouse. A single tagged cell could produce all lineages for as many as seven weeks, and 30 purified cells were sufficient to rescue mice and fully repopulate the bone marrow without extra doses of backup cells to rescue the mice [49]. Despite these efforts, researchers remain divided on the most consistently expressed set of HSC markers [27, 32]. Connie Eaves of the University of British Columbia says none of the markers are tied to unique stem cell functions or truly define the stem cell [14]. “Almost every marker I am aware of has been shown to be fickle,” she says.
More recently, Diane Krause and her colleagues at Yale University, New York University, and Johns Hopkins University, used a new technique to home in on a single cell capable of reconstituting all blood cell lineages of an irradiated mouse [27]. After marking bone marrow cells from donor male mice with a nontoxic dye, they injected the cells into female recipient mice that had been given a lethal dose of radiation. Over the next two days, some of the injected cells migrated, or homed, to the bone marrow of the recipients and did not divide; when transplanted into a second set of irradiated female mice, they eventually proved to be a concentrated pool of self-renewing stem cells. The cells also reconstituted blood production. The scientists estimate that their technique concentrated the long-term stem cells 500 to 1,000- fold compared with bone marrow.
What Are the Sources of Hematopoietic Stem Cells?
Bone Marrow
The classic source of hematopoietic stem cells (HSCs) is bone marrow. For more than 40 years, doctors performed bone marrow transplants by anesthetizing the stem cell donor, puncturing a bone—typically a hipbone—and drawing out the bone marrow cells with a syringe. About 1 in every 100,000 cells in the marrow is a long-term, blood-forming stem cell; other cells present include stromal cells, stromal stem cells, blood progenitor cells, and mature and maturing white and red blood cells.
Peripheral Blood
As a source of HSCs for medical treatments, bone marrow retrieval directly from bone is quickly fading into history. For clinical transplantation of human HSCs, doctors now prefer to harvest donor cells from peripheral, circulating blood. It has been known for decades that a small number of stem and progenitor cells circulate in the bloodstream, but in the past 10 years, researchers have found that they can coax the cells to migrate from marrow to blood in greater numbers by injecting the donor with a cytokine, such as granulocyte-colony stimulating factor (GCSF). The donor is injected with GCSF a few days before the cell harvest. To collect the cells, doctors insert an intravenous tube into the donor’s vein and pass his blood through a filtering system that pulls out CD34+ white blood cells and returns the red blood cells to the donor. Of the cells collected, just 5 to 20 percent will be true HSCs. Thus, when medical researchers commonly refer to peripherally harvested “stem cells,” this is something of a misnomer. As is true for bone marrow, the CD34+ cells are a mixture of stem cells, progenitors, and white blood cells of various degrees of maturity.
In the past three years, the majority of autologous (where the donor and recipient are the same person) and allogeneic (where the donor and recipient are different individuals) “bone marrow” transplants have actually been white blood cells drawn from peripheral circulation, not bone marrow. Richard Childs, an intramural investigator at the NIH, says peripheral harvest of cells is easier on the donor—with minimal pain, no anesthesia, and no hospital stay—but also yields better cells for transplants [6]. Childs points to evidence that patients receiving peripherally harvested cells have higher survival rates than bone marrow recipients do. The peripherally harvested cells contain twice as many HSCs as stem cells taken from bone marrow and engraft more quickly. This means patients may recover white blood cells, platelets, and their immune and clotting protection several days faster than they would with a bone marrow graft. Scientists at Stanford report that highly purified, mobilized peripheral cells that have CD34+ and Thy-1+ surface markers engraft swiftly and without complication in breast cancer patients receiving an autologous transplant of the cells after intensive chemotherapy [41].
Umbilical Cord Blood
In the late 1980s and early 1990s, physicians began to recognize that blood from the human umbilical cord and placenta was a rich source of HSCs. This tissue supports the developing fetus during pregnancy, is delivered along with the baby, and, is usually discarded. Since the first successful umbilical cord blood transplants in children with Fanconi anemia, the collection and therapeutic use of these cells has grown quickly. The New York Blood Center’s Placental Blood Program, supported by NIH, is the largest U.S. public umbilical cord blood bank and now has 13,000 donations available for transplantation into small patients who need HSCs. Since it began collecting umbilical cord blood in 1992, the center has provided thousands of cord blood units to patients. Umbilical cord blood recipients—typically children—have now lived in excess of eight years, relying on the HSCs from an umbilical cord blood transplant [31, 57].
There is a substantial amount of research being conducted on umbilical cord blood to search for ways to expand the number of HSCs and compare and contrast the biological properties of cord blood with adult bone marrow stem cells. There have been suggestions that umbilical cord blood contains stem cells that have the capability of developing cells of multiple germ layers (multipotent) or even all germ layers, e.g., endoderm, ectoderm, and mesoderm (pluripotent). To date, there is no published scientific evidence to support this claim. While umbilical cord blood represents a valuable resource for HSCs, research data have not conclusively shown qualitative differences in the differentiated cells produced between this source of HSCs and peripheral blood and bone marrow.
Fetal Hematopoietic System
An important source of HSCs in research, but not in clinical use, is the developing blood-producing tissues of fetal animals. Hematopoietic cells appear early in the development of all vertebrates. Most extensively studied in the mouse, HSC production sweeps through the developing embryo and fetus in waves. Beginning at about day 7 in the life of the mouse embryo, the earliest hematopoietic activity is indicated by the appearance of blood islands in the yolk sac (see Appendix A. Early Development). The point is disputed, but some scientists contend that yolk sac blood production is transient and will generate some blood cells for the embryo, but probably not the bulk of the HSCs for the adult animal [12, 26, 44]. According to this proposed scenario, most stem cells that will be found in the adult bone marrow and circulation are derived from cells that appear slightly later and in a different location. This other wave of hematopoietic stem cell production occurs in the AGM—the region where the aorta, gonads, and fetal kidney (mesonephros) begin to develop. The cells that give rise to the HSCs in the AGM may also give rise to endothelial cells that line blood vessels. [13]. These HSCs arise at around days 10 to 11 in the mouse embryo (weeks 4 to 6 in human gestation), divide, and within a couple of days, migrate to the liver [11]. The HSCs in the liver continue to divide and migrate, spreading to the spleen, thymus, and—near the time of birth—to the bone marrow.
Whereas an increasing body of fetal HSC research is emerging from mice and other animals, there is much less information about human fetal and embryonic HSCs. Scientists in Europe, including Coulombel, Peault, and colleagues, first described hematopoietic precursors in human embryos only a few years ago [20, 53]. Most recently, Gallacher and others reported finding HSCs circulating in the blood of 12- to 18-week aborted human fetuses [16, 28, 54] that was rich in HSCs. These circulating cells had different markers than did cells from fetal liver, fetal bone marrow, or umbilical cord blood.
Embryonic Stem Cells and Embryonic Germ Cells
In 1985, it was shown that it is possible to obtain precursors to many different blood cells from mouse embryonic stem cells [9]. Perkins was able to obtain all the major lineages of progenitor cells from mouse embryoid bodies, even without adding hematopoietic growth factors [45].
Mouse embryonic stem cells in culture, given the right growth factors, can generate most, if not all, the different blood cell types [19], but no one has yet achieved the “gold standard” of proof that they can produce long-term HSCs from these sources—namely by obtaining cells that can be transplanted into lethally irradiated mice to reconstitute long-term hematopoiesis [32].
The picture for human embryonic stem and germ cells is even less clear. Scientists from James Thomson’s laboratory reported in 1999 that they were able to direct human embryonic stem cells—which can now be cultured in the lab—to produce blood progenitor cells [23]. Israeli scientists reported that they had induced human ES cells to produce hematopoietic cells, as evidenced by their production of a blood protein, gamma-globin [21]. Cell lines derived from human embryonic germ cells (cultured cells derived originally from cells in the embryo that would ultimately give rise to eggs or sperm) that are cultured under certain conditions will produce CD34+ cells [47]. The blood-producing cells derived from human ES and embryonic germ (EG) cells have not been rigorously tested for long-term self-renewal or the ability to give rise to all the different blood cells.
The Stem Cell Database
http://stemcell.princeton.edu
Ihor Lemischka and colleagues at Princeton University and the Computational Biology and Informatics Laboratory at the University of Pennsylvania are collaborating to record all the findings about hematopoietic stem cell (HSC) genes and markers in the Stem Cell Database.
The collaborators started the database five years ago. Its goal is listing and annotating all the genes that are differentially expressed in mouse liver HSCs and their cellular progeny. The database is growing to include human HSCs from different blood sources, and a related database, constructed in collaboration with Kateri A. Moore, also at Princeton University, will document all genes active in stromal cells, which provide the microenvironment in which stem cells are maintained. The combined power of the two databases, along with new tools and methods for studying molecular biology, will help researchers put together a complete portrait of the hematopoietic stem cell and how it works. The databases will continue to grow and take advantage of other efforts, such as those to complete the gene sequences of mammals. Data will be publicly available to researchers around the world.
As sketchy as data may be on the hematopoietic powers of human ES and EG cells, blood experts are intrigued by their clinical potential and their potential to answer basic questions on renewal and differentiation of HSCs [19]. Connie Eaves, who has made comparisons of HSCs from fetal liver, cord blood, and adult bone marrow, expects cells derived from embryonic tissues to have some interesting traits. She says actively dividing blood-producing cells from ES cell culture—if they are like other dividing cells—will not themselves engraft or rescue hematopoiesis in an animal whose bone marrow has been destroyed. However, they may play a critical role in developing an abundant supply of HSCs grown in the lab. Indications are that the dividing cells will also more readily lend themselves to gene manipulations than do adult HSCs. Eaves anticipates that HSCs derived from early embryo sources will be developmentally more “plastic” than later HSCs, and more capable of self-renewal [14].
How Do HSCs from Varying Sources Differ?
Scientists in the laboratory and clinic are beginning to measure the differences among HSCs from different sources. In general, they find that HSCs taken from tissues at earlier developmental stages have a greater ability to self-replicate, show different homing and surface characteristics, and are less likely to be rejected by the immune system—making them potentially more useful for therapeutic transplantation.
Stem cell populations of the bone marrow
When do HSCs move from the early locations in the developing fetus to their adult “home” in the bone marrow? European scientists have found that the relative number of CD34+ cells in the collections of cord blood declined with gestational age, but expression of cell-adhesion molecules on these cells increased.
The authors believe these changes reflect preparations for the cells to relocate—from homing in fetal liver to homing in bone marrow [52].
The point is controversial, but a paper by Chen et al. provides evidence that at least in some strains of mice, HSCs from old mice are less able to repopulate bone marrow after transplantation than are cells from young adult mice [5]. Cells from fetal mice were 50 to 100 percent better at repopulating marrow than were cells from young adult mice were. The specific potential for repopulating marrow appears to be strain-specific, but the scientists found this potential declined with age for both strains. Other scientists find no decreases or sometimes increases in numbers of HSCs with age [51]. Because of the difficulty in identifying a long-term stem cell, it remains difficult to quantify changes in numbers of HSCs as a person ages.
Effectiveness of Transplants of Adult versus Umbilical Cord Blood Stem Cells
A practical and important difference between HSCs collected from adult human donors and from umbilical cord blood is simply quantitative. Doctors are rarely able to extract more than a few million HSCs from a placenta and umbilical cord—too few to use in a transplant for an adult, who would ideally get 7 to 10 million CD34+ cells per kilogram body weight, but often adequate for a transplant for a child [33, 48].
Leonard Zon says that HSCs from cord blood are less likely to cause a transplantation complication called graft-versus-host disease, in which white blood cells from a donor attack tissues of the recipient [65]. In a recent review of umbilical cord blood transplantation, Laughlin cites evidence that cord blood causes less graft-versus-host disease [31]. Laughlin writes that it is yet to be determined whether umbilical cord blood HSCs are, in fact, longer lived in a transplant recipient.
In lab and mouse-model tests comparing CD34+ cells from human cord with CD34+ cells derived from adult bone marrow, researchers found cord blood had greater proliferation capacity [24]. White blood cells from cord blood engrafted better in a mouse model, which was genetically altered to tolerate the human cells, than did their adult counterparts.
Effectiveness in Transplants of Peripheral Versus Bone Marrow Stem Cells
In addition to being far easier to collect, peripherally harvested white blood cells have other advantages over bone marrow. Cutler and Antin’s review says that peripherally harvested cells engraft more quickly, but are more likely to cause graft-versus-host disease [8]. Prospecting for the most receptive HSCs for gene therapy, Orlic and colleagues found that mouse HSCs mobilized with cytokines were more likely to take up genes from a viral vector than were non-mobilized bone marrow HSCs [43].
What Do Hematopoietic Stem Cells Do and What Factors Are Involved in These Activities?
As stated earlier, an HSC in the bone marrow has four actions in its repertoire: 1) it can renew itself, 2) it can differentiate, 3) it can mobilize out of the bone marrow into circulation (or the reverse), or 4) it can undergo programmed cell death, or apoptosis. Understanding the how, when, where, which, and why of this simple repertoire will allow researchers to manipulate and use HSCs for tissue and organ repair.
Self-renewal of Hematopoietic Stem Cells
Scientists have had a tough time trying to grow—or even maintain—true stem cells in culture. This is an important goal because cultures of HSCs that could maintain their characteristic properties of self-renewal and lack of differentiation could provide an unlimited source of cells for therapeutic transplantation and study. When bone marrow or blood cells are observed in culture, one often observes large increases in the number of cells. This usually reflects an increase in differentiation of cells to progenitor cells that can give rise to different lineages of blood cells but cannot renew themselves. True stem cells divide and replace themselves slowly in adult bone marrow.
New tools for gene-expression analysis will now allow scientists to study developmental changes in telomerase activity and telomeres. Telomeres are regions of DNA found at the end of chromosomes that are extended by the enzyme telomerase. Telomerase activity is necessary for cells to proliferate and activity decreases with age leading to shortened telomeres. Scientists hypothesize that declines in stem cell renewal will be associated with declines in telomere length and telomerase activity. Telomerase activity in hematopoietic cells is associated with self-renewal potential [40].
Because self-renewal divisions are rare, hard to induce in culture, and difficult to prove, scientists do not have a definitive answer to the burning question: what puts—or perhaps keeps—HSCs in a self-renewal division mode? HSCs injected into an anemic patient or mouse—or one whose HSCs have otherwise been suppressed or killed—will home to the bone marrow and undergo active division to both replenish all the different types of blood cells and yield additional self-renewing HSCs. But exactly how this happens remains a mystery that scientists are struggling to solve by manipulating cultures of HSCs in the laboratory.
Two recent examples of progress in the culturing studies of mouse HSCs are by Ema and coworkers and Audet and colleagues [2, 15]. Ema et al. found that two cytokines—stem cell factor and thrombo-poietin—efficiently induced an unequal first cell division in which one daughter cell gave rise to repopulating cells with self-renewal potential. Audet et al. found that activation of the signaling molecule gp130 is critical to survival and proliferation of mouse HSCs in culture.
Work with specific cytokines and signaling molecules builds on several earlier studies demonstrating modest increases in the numbers of stem cells that could be induced briefly in culture. For example, Van Zant and colleagues used continuous-perfusion culture and bioreactors in an attempt to boost human HSC numbers in single cord blood samples incubated for one to two weeks [58]. They obtained a 20-fold increase in “long-term culture initiating cells.”
More clues on how to increase numbers of stem cells may come from looking at other animals and various developmental stages. During early developmental stages—in the fetal liver, for example—HSCs may undergo more active cell division to increase their numbers, but later in life, they divide far less often [30, 42]. Culturing HSCs from 10- and 11-day-old mouse embryos, Elaine Dzierzak at Erasmus University in the Netherlands finds she can get a 15-fold increase in HSCs within the first 2 or 3 days after she removes the AGM from the embryos [38]. Dzierzak recognizes that this is dramatically different from anything seen with adult stem cells and suggests it is a difference with practical importance. She suspects that the increase is not so much a response to what is going on in the culture but rather, it represents the developmental momentum of this specific embryonic tissue. That is, it is the inevitable consequence of divisions that were cued by that specific embryonic microenvironment. After five days, the number of HSCs plateaus and can be maintained for up to a month. Dzierzak says that the key to understanding how adult-derived HSCs can be expanded and manipulated for clinical purposes may very well be found by defining the cellular composition and complex molecular signals in the AGM region during development [13].
In another approach, Lemischka and coworkers have been able to maintain mouse HSCs for four to seven weeks when they are grown on a clonal line of cells (AFT024) derived from the stroma, the other major cellular constituent of bone marrow [39]. No one knows which specific factors secreted by the stromal cells maintain the stem cells. He says ongoing gene cloning is rapidly zeroing in on novel molecules from the stromal cells that may “talk” to the stem cells and persuade them to remain stem cells—that is, continue to divide and not differentiate.
If stromal factors provide the key to stem cell self-renewal, research on maintaining stromal cells may be an important prerequisite. In 1999, researchers at Osiris Therapeutics and Johns Hopkins University reported culturing and expanding the numbers of mesenchymal stem cells, which produce the stromal environment [46]. Whereas cultured HSCs rush to differentiate and fail to retain primitive, self-renewing cells, the mesenchymal stem cells could be increased in numbers and still retained their powers to generate the full repertoire of descendant lineages.
Differentiation of HSCs into Components of the Blood and Immune System
Producing differentiated white and red blood cells is the real work of HSCs and progenitor cells. M.C. MacKey calculates that in the course of producing a mature, circulating blood cell, the original hematopoietic stem cell will undergo between 17 and 19.5 divisions, “giving a net amplification of between ~170,000 and ~720,000″ [35].
Through a series of careful studies of cultured cells—often cells with mutations found in leukemia patients or cells that have been genetically altered—investigators have discovered many key growth factors and cytokines that induce progenitor cells to make different types of blood cells. These factors interact with one another in complex ways to create a system of exquisite genetic control and coordination of blood cell production.
Migration of Hematopoietic Stem Cells Into and Out of Marrow and Tissues
Scientists know that much of the time, HSCs live in intimate connection with the stroma of bone marrow in adults (see Chapter 4. The Adult Stem Cell). But HSCs may also be found in the spleen, in peripheral blood circulation, and other tissues. Connection to the interstices of bone marrow is important to both the engraftment of transplanted cells and to the maintenance of stem cells as a self-renewing population. Connection to stroma is also important to the orderly proliferation, differentiation, and maturation of blood cells [63].
Weissman says HSCs appear to make brief forays out of the marrow into tissues, then duck back into marrow [62]. At this time, scientists do not understand why or how HSCs leave bone marrow or return to it [59]. Scientists find that HSCs that have been mobilized into peripheral circulation are mostly non-dividing cells [64]. They report that adhesion molecules on the stroma, play a role in mobilization, in attachment to the stroma, and in transmitting signals that regulate HSC self-renewal and progenitor differentiation [61].
Apoptosis and Regulation of Hematopoietic Stem Cell Populations
The number of blood cells in the bone marrow and blood is regulated by genetic and molecular mechanisms. How do hematopoietic stem cells know when to stop proliferating? Apoptosis is the process of programmed cell death that leads cells to self-destruct when they are unneeded or detrimental. If there are too few HSCs in the body, more cells divide and boost the numbers. If excess stem cells were injected into an animal, they simply wouldn’t divide or would undergo apoptosis and be eliminated [62]. Excess numbers of stem cells in an HSC transplant actually seem to improve the likelihood and speed of engraftment, though there seems to be no rigorous identification of a mechanism for this empirical observation.
The particular signals that trigger apoptosis in HSCs are as yet unknown. One possible signal for apoptosis might be the absence of life-sustaining signals from bone marrow stroma. Michael Wang and others found that when they used antibodies to disrupt the adhesion of HSCs to the stroma via VLA-4/VCAM-1, the cells were predisposed to apoptosis [61].
Understanding the forces at play in HSC apoptosis is important to maintaining or increasing their numbers in culture. For example, without growth factors, supplied in the medium or through serum or other feeder layers of cells, HSCs undergo apoptosis. Domen and Weissman found that stem cells need to get two growth factor signals to continue life and avoid apoptosis: one via a protein called BCL-2, the other from steel factor, which, by itself, induces HSCs to produce progenitor cells but not to self-renew [10].
What Are the Clinical Uses of Hematopoietic Stem Cells?
Leukemia and Lymphoma
Among the first clinical uses of HSCs were the treatment of cancers of the blood—leukemia and lymphoma, which result from the uncontrolled proliferation of white blood cells. In these applications, the patient’s own cancerous hematopoietic cells were destroyed via radiation or chemotherapy, then replaced with a bone marrow transplant, or, as is done now, with a transplant of HSCs collected from the peripheral circulation of a matched donor. A matched donor is typically a sister or brother of the patient who has inherited similar human leukocyte antigens (HLAs) on the surface of their cells. Cancers of the blood include acute lymphoblastic leukemia, acute myeloblastic leukemia, chronic myelogenous leukemia (CML), Hodgkin’s disease, multiple myeloma, and non-Hodgkin’s lymphoma.
Thomas and Clift describe the history of treatment for chronic myeloid leukemia as it moved from largely ineffective chemotherapy to modestly successful use of a cytokine, interferon, to bone marrow trans-plants—first in identical twins, then in HLA-matched siblings [55]. Although there was significant risk of patient death soon after the transplant either from infection or from graft-versus-host disease, for the first time, many patients survived this immediate challenge and had survival times measured in years or even decades, rather than months. The authors write, “In the space of 20 years, marrow transplantation has contributed to the transformation of [chronic myelogenous leukemia] CML from a fatal disease to one that is frequently curable. At the same time, experience acquired in this setting has improved our understanding of many transplant-related problems. It is now clear that morbidity and mortality are not inevitable consequences of allogeneic transplantation, [and] that an allogeneic effect can add to the anti-leukemic power of conditioning regimens…”
In a recent development, CML researchers have taken their knowledge of hematopoietic regulation one step farther. On May 10, 2001, the Food and Drug Administration approved Gleevec™ (imatinib mesylate), a new, rationally designed oral drug for treatment of CML. The new drug specifically targets a mutant protein, produced in CML cancer cells, that sabotages the cell signals controlling orderly division of progenitor cells. By silencing this protein, the new drug turns off cancerous overproduction of white blood cells, so doctors do not have to resort to bone marrow transplantation. At this time, it is unknown whether the new drug will provide sustained remission or will prolong life for CML patients.
Inherited Blood Disorders
Another use of allogeneic bone marrow transplants is in the treatment of hereditary blood disorders, such as different types of inherited anemia (failure to produce blood cells), and inborn errors of metabolism (genetic disorders characterized by defects in key enzymes need to produce essential body components or degrade chemical byproducts). The blood disorders include aplastic anemia, beta-thalassemia, Blackfan-Diamond syndrome, globoid cell leukodystrophy, sickle-cell anemia, severe combined immunodeficiency, X-linked lymphoproliferative syndrome, and Wiskott-Aldrich syndrome. Inborn errors of metabolism that are treated with bone marrow transplants include: Hunter’s syndrome, Hurler’s syndrome, Lesch Nyhan syndrome, and osteopetrosis. Because bone marrow transplantation has carried a significant risk of death, this is usually a treatment of last resort for otherwise fatal diseases.
Hematopoietic Stem Cell Rescue in Cancer Chemotherapy
Chemotherapy aimed at rapidly dividing cancer cells inevitably hits another target—rapidly dividing hematopoietic cells. Doctors may give cancer patients an autologous stem cell transplant to replace the cells destroyed by chemotherapy. They do this by mobilizing HSCs and collecting them from peripheral blood. The cells are stored while the patient undergoes intensive chemotherapy or radiotherapy to destroy the cancer cells. Once the drugs have washed out of a patient’s body, the patient receives a transfusion of his or her stored HSCs. Because patients get their own cells back, there is no chance of immune mismatch or graft-versus-host disease. One problem with the use of autologous HSC transplants in cancer therapy has been that cancer cells are sometimes inadvertently collected and reinfused back into the patient along with the stem cells. One team of investigators finds that they can prevent reintroducing cancer cells by purifying the cells and preserving only the cells that are CD34+, Thy-1+[41].
The National Marrow Donor Program
http://www.marrow.org
Launched in 1987, the National Marrow Donor Program (NMDP) was created to connect patients who need blood-forming stem cells or bone marrow with potential nonrelated donors. About 70 percent of patients who need a life-saving HSC transplant cannot find a match in their own family.
The NMDP is made up of an international network of centers and banks that collect cord blood, bone marrow, and peripherally harvested stem cells and that recruit potential donors. As of February 28, 2001, the NMDP listed 4,291,434 potential donors. Since its start, the Minneapolis-based group has facilitated almost 12,000 transplants—75 percent of them for leukemia. Major recruiting efforts have led to substantial increases in the number of donations from minorities, but the chance that African Americans, Native Americans, Asian/Pacific Islanders, or Hispanics will find a match is still lower than it is for Caucasians.
Graft-Versus-Tumor Treatment of Cancer
One of the most exciting new uses of HSC transplantation puts the cells to work attacking otherwise untreatable tumors. A group of researchers in NIH’s intramural research program recently described this approach to treating metastatic kidney cancer [7]. Just under half of the 38 patients treated so far have had their tumors reduced. The research protocol is now expanding to treatment of other solid tumors that resist standard therapy, including cancer of the lung, prostate, ovary, colon, esophagus, liver, and pancreas.
This experimental treatment relies on an allogeneic stem cell transplant from an HLA-matched sibling whose HSCs are collected peripherally. The patient’s own immune system is suppressed, but not totally destroyed. The donor’s cells are transfused into the patient, and for the next three months, doctors closely monitor the patient’s immune cells, using DNA fingerprinting to follow the engraftment of the donor’s cells and regrowth of the patient’s own blood cells. They must also judiciously suppress the patient’s immune system as needed to deter his/her T cells from attacking the graft and to reduce graft-versus-host disease.
A study by Joshi et al. shows that umbilical cord blood and peripherally harvested human HSCs show antitumor activity in the test tube against leukemia cells and breast cancer cells [22]. Grafted into a mouse model that tolerates human cells, HSCs attack human leukemia and breast cancer cells. Although untreated cord blood lacks natural killer (NK) lymphocytes capable of killing tumor cells, researchers have found that at least in the test tube and in mice, they can greatly enhance the activity and numbers of these cells with cytokines IL-15 [22, 34].
Other Applications of Hematopoietic Stem Cells
Substantial basic and limited clinical research exploring the experimental uses of HSCs for other diseases is underway. Among the primary applications are autoimmune diseases, such as diabetes, rheumatoid arthritis, and system lupus erythematosis. Here, the body’s immune system turns to destroying body tissues. Experimental approaches similar to those applied above for cancer therapies are being conducted to see if the immune system can be reconstituted or reprogrammed. More detailed discussion on this application is provided in Chapter 6. Autoimmune Diseases and the Promise of Stem Cell-Based Therapies. The use of HSCs as a means to deliver genes to repair damaged cells is another application being explored. The use of HSCs for gene therapies is discussed in detail in Chapter 11. Use of Genetically Modified Stem Cells in Experimental Gene Therapies.
Plasticity of Hematopoietic Stem Cells
A few recent reports indicate that scientists have been able to induce bone marrow or HSCs to differentiate into other types of tissue, such as brain, muscle, and liver cells. These concepts and the experimental evidence supporting this concept are discussed in Chapter 4. The Adult Stem Cell.
Research in a mouse model indicates that cells from grafts of bone marrow or selected HSCs may home to damaged skeletal and cardiac muscle or liver and regenerate those tissues [4, 29]. One recent advance has been in the study of muscular dystrophy, a genetic disease that occurs in young people and leads to progressive weakness of the skeletal muscles. Bittner and colleagues used mdx mice, a genetically modified mouse with muscle cell defects similar to those in human muscular dystrophy. Bone marrow from non-mdx male mice was transplanted into female mdx mice with chronic muscle damage; after 70 days, researchers found that nuclei from the males had taken up residence in skeletal and cardiac muscle cells.
Lagasse and colleagues’ demonstration of liver repair by purified HSCs is a similarly encouraging sign that HSCs may have the potential to integrate into and grow in some non-blood tissues. These scientists lethally irradiated female mice that had an unusual genetic liver disease that could be halted with a drug. The mice were given transplants of genetically marked, purified HSCs from male mice that did not have the liver disease. The transplants were given a chance to engraft for a couple of months while the mice were on the liver-protective drug. The drug was then removed, launching deterioration of the liver—and a test to see whether cells from the transplant would be recruited and rescue the liver. The scientists found that transplants of as few as 50 cells led to abundant growth of marked, donor-derived liver cells in the female mice.
Recently, Krause has shown in mice that a single selected donor hematopoietic stem cell could do more than just repopulate the marrow and hematopoietic system of the recipient [27]. These investigators also found epithelial cells derived from the donors in the lungs, gut, and skin of the recipient mice. This suggests that HSCs may have grown in the other tissues in response to infection or damage from the irradiation the mice received.
In humans, observations of male liver cells in female patients who have received bone marrow grafts from males, and in male patients who have received liver transplants from female donors, also suggest the possibility that some cells in bone marrow have the capacity to integrate into the liver and form hepatocytes [1].
What Are the Barriers to the Development of New and Improved Treatments Using Hematopoietic Stem Cells?
Boosting the Numbers of Hematopoietic Stem Cells
Clinical investigators share the same fundamental problem as basic investigators—limited ability to grow and expand the numbers of human HSCs. Clinicians repeatedly see that larger numbers of cells in stem cell grafts have a better chance of survival in a patient than do smaller numbers of cells. The limited number of cells available from a placenta and umbilical cord blood transplant currently means that cord blood banks are useful to pediatric but not adult patients. Investigators believe that the main cause of failure of HSCs to engraft is host-versus-graft disease, and larger grafts permit at least some donor cells to escape initial waves of attack from a patient’s residual or suppressed immune system [6]. Ability to expand numbers of human HSCs in vivo or in vitro would clearly be an enormous boost to all current and future medical uses of HSC transplantation.
Once stem cells and their progeny can be multiplied in culture, gene therapists and blood experts could combine their talents to grow limitless quantities of “universal donor” stem cells, as well as progenitors and specific types of red and white blood cells. If the cells were engineered to be free of markers that provoke rejection, these could be transfused to any recipient to treat any of the diseases that are now addressed with marrow, peripheral, cord, or other transfused blood. If gene therapy and studies of the plasticity of HSCs succeed, the cells could also be grown to repair other tissues and treat non-blood-related disorders [32].
Several research groups in the United States, Canada, and abroad have been striving to find the key factor or factors for boosting HSC production. Typical approaches include comparing genes expressed in primitive HSCs versus progenitor cells; comparing genes in actively dividing fetal HSCs versus adult HSCs; genetic screening of hematopoietically mutated zebrafish; studying dysregulated genes in cancerous hematopoietic cells; analyzing stromal or feeder-layer factors that appear to boost HSC division; and analyzing factors promoting homing and attachment to the stroma. Promising candidate factors have been tried singly and in combination, and researchers claim they can now increase the number of long-term stem cells 20-fold, albeit briefly, in culture.
The specific assays researchers use to prove that their expanded cells are stem cells vary, which makes it difficult to compare the claims of different research groups. To date, there is only a modest ability to expand true, long-term, self-renewing human HSCs. Numbers of progenitor cells are, however, more readily increased. Kobari et al., for example, can increase progenitor cells for granulocytes and macrophages 278-fold in culture [25].
Some investigators are now evaluating whether these comparatively modest increases in HSCs are clinically useful. At this time, the increases in cell numbers are not sustainable over periods beyond a few months, and the yield is far too low for mass production. In addition, the cells produced are often not rigorously characterized. A host of other questions remain—from how well the multiplied cells can be altered for gene therapy to their potential longevity, immunogenicity, ability to home correctly, and susceptibility to cancerous transformation. Glimm et al. [17] highlight some of these problems, for example, with their confirmation that human stem cells lose their ability to repopulate the bone marrow as they enter and progress through the cell cycleælike mouse stem cells that have been stimulated to divide lose their transplantability [18]. Observations on the inverse relationship between progenitor cell division rate and longevity in strains of mice raise an additional concern that culture tricks or selection of cells that expand rapidly may doom the cells to a short life.
Pragmatically, some scientists say it may not be necessary to be able to induce the true, long-term HSC to divide in the lab. If they can manipulate progenitors and coax them into division on command, gene uptake, and differentiation into key blood cells and other tissues, that may be sufficient to accomplish clinical goals. It might be sufficient to boost HSCs or subpopulations of hematopoietic cells within the body by chemically prodding the bone marrow to supply the as-yet-elusive factors to rejuvenate cell division.
Outfoxing the Immune System in Host, Graft, and Pathogen Attacks
Currently, the risks of bone marrow transplants—graft rejection, host-versus-graft disease, and infection during the period before HSCs have engrafted and resumed full blood cell production—restrict their use to patients with serious or fatal illnesses. Allogeneic grafts must come from donors with a close HLA match to the patient (see Chapter 6. Autoimmune Diseases and the Promise of Stem Cell-Based Therapies). If doctors could precisely manipulate immune reactions and protect patients from pathogens before their transplants begin to function, HSC transplants could be extended to less ill patients and patients for whom the HLA match was not as close as it must now be. Physicians might use transplants with greater impunity in gene therapy, autoimmune disease, HIV/AIDS treatment, and the preconditioning of patients to accept a major organ transplant.
Scientists are zeroing in on subpopulations of T cells that may cause or suppress potentially lethal host-versus-graft rejection and graft-versus-host disease in allogeneic-transplant recipients. T cells in a graft are a two-edged sword. They fight infections and help the graft become established, but they also can cause graft-versus-host disease. Identifying subpopulations of T cells responsible for deleterious and beneficial effects—in the graft, but also in residual cells surviving or returning in the host—could allow clinicians to make grafts safer and to ratchet up graft-versus-tumor effects [48]. Understanding the presentation of antigens to the immune system and the immune system’s healthy and unhealthy responses to these antigens and maturation and programmed cell death of T cells is crucial.
The approach taken by investigators at Stanford—purifying peripheral blood—may also help eliminate the cells causing graft-versus-host disease. Transplants in mouse models support the idea that purified HSCs, cleansed of mature lymphocytes, engraft readily and avoid graft-versus-host disease [60].
Knowledge of the key cellular actors in autoimmune disease, immune grafting, and graft rejection could also permit scientists to design gentler “minitransplants.” Rather than obliterating and replacing the patient’s entire hematopoietic system, they could replace just the faulty components with a selection of cells custom tailored to the patient’s needs. Clinicians are currently experimenting with deletion of T cells from transplants in some diseases, for example, thereby reducing graft-versus-host disease.
Researchers are also experimenting with the possibility of knocking down the patient’s immune system—but not knocking it out. A blow that is sublethal to the patient’s hematopoietic cells given before an allogeneic transplant can be enough to give the graft a chance to take up residence in the bone marrow. The cells replace some or all of the patient’s original stem cells, often making their blood a mix of donor and original cells. For some patients, this mix of cells will be enough to accomplish treatment objectives but without subjecting them to the vicious side effects and infection hazards of the most powerful treatments used for total destruction of their hematopoietic systems [37].
Understanding the Differentiating Environment and Developmental Plasticity
At some point in embryonic development, all cells are plastic, or developmentally flexible enough to grow into a variety of different tissues. Exactly what is it about the cell or the embryonic environment that instructs cells to grow into one organ and not another?
Could there be embryological underpinnings to the apparent plasticity of adult cells? Researchers have suggested that a lot of the tissues that are showing plasticity are adjacent to one another after gastrulation in the sheet of mesodermal tissue that will go on to form blood—muscle, blood vessels, kidney, mesenchyme, and notochord. Plasticity may reflect derivation from the mesoderm, rather than being a fixed trait of hematopoietic cells. One lab is now studying the adjacency of embryonic cells and how the developing embryo makes the decision to make one tissue instead of another—and whether the decision is reversible [65].
In vivo studies of the plasticity of bone marrow or purified stem cells injected into mice are in their infancy. Even if follow-up studies confirm and more precisely characterize and quantify plasticity potential of HSCs in mice, there is no guarantee that it will occur or can be induced in humans.
Summary
Grounded in half a century of research, the study of hematopoietic stem cells is one of the most exciting and rapidly advancing disciplines in biomedicine today. Breakthrough discoveries in both the laboratory and clinic have sharply expanded the use and supply of life-saving stem cells. Yet even more promising applications are on the horizon and scientists’ current inability to grow HSCs outside the body could delay or thwart progress with these new therapies. New treatments include graft-versus-tumor therapy for currently incurable cancers, autologous transplants for autoimmune diseases, and gene therapy and tissue repair for a host of other problems. The techniques, cells, and knowledge that researchers have now are inadequate to realize the full promise of HSC-based therapy.
Key issues for tapping the potential of hematopoietic stem cells will be finding ways to safely and efficiently expand the numbers of transplantable human HSCs in vitro or in vivo. It will also be important to gain a better understanding of the fundamentals of how immune cells work—in fighting infections, in causing transplant rejection, and in graft-versus-host disease as well as master the basics of HSC differentiation. Concomitant advances in gene therapy techniques and the understanding of cellular plasticity could make HSCs one of the most powerful tools for healing.
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