Promises and Challenges of Stem Cell Research for Regenerative Medicine

Ideas and Opinions Annals of Internal Medicine Promises and Challenges of Stem Cell Research for Regenerative Medicine

by Carl Power, PhD, and John E.J. Rasko, MBBS, PhD

In recent years, stem cells have generated increasing excitement, with frequent claims that they are revolutionizing medicine. For those not directly involved in stem cell research, however, it can be difficult to separate fact from fiction or realistic expectation from wishful thinking. This article aims to provide internists with a clear and concise introduction to the field. While recounting some sci- entific and medical milestones, the authors discuss the 3 main varieties of stem cells—adult, embryonic, and induced pluripotent—

Stem cells promise great advances for medicine. Clinical and preclinical studies suggest that stem cells could be used to regenerate a range of damaged tissues, treat a vari- ety of medical conditions, and assist in the development of new drugs. This article provides a concise introduction to the 3 main kinds of stem cells—adult, embryonic, and induced pluripotent—highlighting clinical applications of each and the many challenges that must be addressed.

Stem cells are undifferentiated cells that have the ca- pacity to self-renew and differentiate into more than one specialized cell type. It is largely due to their presence that our body is able to grow, develop, and repair itself. Har- nessing their therapeutic potential, safely and with careful regulatory oversight, is a central concern of regenerative medicine.

Different kinds of cells can be classified according to their degree of developmental potential. At the top end of the scale is totipotency, the capacity of a fertilized egg and its progeny, for several cell divisions, to form the entire embryo and the extra-embryonic tissues necessary for its development (1). To date, totipotent cell lines have not been grown long term in vitro.

The cultured cell lines that come closest to totipotency are known as pluripotent stem cells (2). They can generate all of the cell types (more than 200) in the human body, but they generally cannot produce extra-embryonic tissues. Embryonic stem (ES) cells are pluripotent, and cell lines derived from them can be maintained in culture for long periods—perhaps indefinitely. Human ES cells are most often harvested from blastocyst-stage embryos that have been created in vitro in fertility clinics and donated for research.

comparing their advantages and disadvantages for clinical medicine. The authors have sought to avoid the moral and political debates surrounding stem cell research, focusing instead on scientific and medical issues.

Ann Intern Med. 2011;155:706-713. www.annals.org For author affiliations, see end of text.

As an embryo develops, nearly all of its cells differ- entiate into specialized cell types. However, some undif- ferentiated cells remain in specific niches or circulate throughout the body. Ordinarily quiescent, they divide infrequently to help maintain homeostasis within tissues but can mobilize to assist repair in response to injury. These are “somatic” (bodily) stem cells, often referred to as “adult” stem cells, although the latter term is somewhat misleading because they also exist in embryos. Adult stem cells have been found in many organs; they can be culti- vated in vitro for limited periods and, being multipotent, can generate 2 or more mature lineages, usually belonging to their tissue of origin.

Medical students were traditionally taught that, from the fertilized egg to the adult organism, differentiation is an irreversible process that commits cells to ever more re- stricted lineages. It is now known that this is not a natural law, fixed and irrevocable, but a general rule that admits important exceptions and can be circumvented altogether by new techniques.

Efforts to “reverse engineer” the mechanisms of differ- entiation have, in recent years, given rise to the science of cell reprogramming. It is now relatively easy to dedifferen- tiate ordinary body cells, such as fibroblasts from a skin sample, pushing them back up the developmental slope so that they enter an ES cell–like state. These “induced plu- ripotent stem” (iPS) cells are very similar to ES cells in that they self-renew indefinitely in vitro and have the potential to generate any cell type in the organism (3). New research demonstrates that reprogramming can also achieve trans- differentiation. This involves the direct conversion of one differentiated cell type into another without its passing through a pluripotent state (4).

To a considerable extent, advances in regenerative medicine depend on our ability to isolate, cultivate, and manipulate stem cells. Each kind of stem cell—adult, em- bryonic, and induced pluripotent—has unique characteris- tics that make it suitable for certain therapeutic uses (Fig- ure 1). Each presents its own set of medical opportunities and drawbacks, which will be examined in turn.

See also:

Print

KeySummaryPoints …………………..707

Web-Only

Conversion of graphics into slides

706 © 2011 American College of Physicians

ADULT STEM CELLS

Although organ regeneration has long been imagined (5), the stem cells responsible for such phenomena are a recent discovery. The study of blood first brought to light the existence of adult stem cells. The idea that the cell types that compose blood all have a common cellular origin— hematopoietic stem cells (HSCs)—was proposed in the late 19th century, but was not experimentally confirmed until the early 1960s (6). Remarkably, HSCs entered the clinic at about the same time. Human bone marrow transplanta- tion was first attempted in the late 1950s, with the earliest successes involving syngeneic grafts between identical twins. It took another decade and a much better grasp of histocompatibility before allogeneic marrow grafts—where the donor is genetically different from the recipient—first succeeded (7). This laid the foundations of regenerative medicine.

Hematopoietic stem cells can be isolated from many sources, including bone marrow, peripheral blood, and umbilical cord blood, and are commonly used to rescue bone marrow function in patients who have received che- motherapy for hematologic cancer, such as leukemia, lym- phoma, and multiple myeloma. In some circumstances, a patient’s own (autologous) HSCs are removed after cycles of induction chemotherapy, stored frozen, and returned to the patient after high-dose chemotherapy (8). The infused HSCs “home” to the bone marrow where they create new blood cells and restore immune function.

The great benefit of autologous cell transplants is that they avoid risks for rejection and graft-versus-host disease. One drawback, however, is that autologous grafts may be contaminated with malignant cells (9, 10). For some types of leukemia, this danger is great enough to make allogeneic transplants preferable, despite the difficulty of finding a close HLA match, the risk for graft-versus-host disease, and problems associated with the use of immunosuppressive medications. Another advantage of allogeneic HSCs is the graft-versus-leukemia effect, mediated by lymphocytes, which can target and destroy residual malignant cells in the patient’s body, thus reducing the chances of relapse (11).

Mesenchymal stem cells (MSCs)—also called mesen- chymal or multipotent stromal cells—are becoming in- creasingly important for regenerative medicine. They can be found in diverse sources, such as bone marrow, blood, fat, and Wharton’s jelly of the umbilical cord, and can differentiate most readily into connective tissue cells. At present, more than 120 clinical trials (mostly phase 1 and 2) worldwide are testing the use of MSCs for a wide range of conditions, including diseases of the bone, cartilage, heart, liver, gastrointestinal tract, and even the nervous system (12).

Mesenchymal stem cells are known to home to injured tissue where they can assist repair; however, the mechanism involved is often unclear. Growing evidence suggests that transplanted MSCs can contribute to regeneration not by re-

placing damaged cells, but indirectly, through paracrine effects (13). Mesenchymal stem cells secrete factors that reduce in- flammation, suppress the host’s immune response, and per- haps stimulate the stem cells already present in injured tissue.

Several studies have shown that MSC infusions not only enhance an organ’s own regenerative capacities but improve engraftment of other transplanted cells— especially HSCs. Of note is an MSC-based product currently in phase 2 and 3 trials as a treatment for graft-versus-host disease; acute myo- cardial infarction; and autoimmune disorders, such as type 1 diabetes and Crohn disease. Although allogeneic MSCs are used in these trials, they do not evoke a classical immune response and can in fact turn down T-cell proliferation. Re- sults so far have been mixed (14); however, the basic idea of harnessing the paracrine effects of stem cells remains a power- ful one (15, 16).

Although there are too many adult stem cell types to survey here, one more type deserves mention: neural stem cells (NSCs). They are currently being tested in clinical trials for patients with spinal injury; stroke; and neurode- generative conditions, such as Parkinson disease and amyo- trophic lateral sclerosis. In most of these trials, NSCs are derived from fetal brain tissues—a controversial practice— but they can also be obtained from patients themselves by means of brain biopsies (17) and even nasal biopsies (18). Although some successes have been reported in the use of

Stem Cell Research and Regenerative Medicine

Ideas and Opinions

Key Summary Points

Stem cells are undifferentiated cells that have the capacity to self-renew and, given the appropriate cues, to differen- tiate into more than one cell type.

Embryonic stem (ES) cells are typically derived from the inner cell mass of blastocyst-stage embryos; these cells can self-renew indefinitely in culture and can develop into any cell type of the body.

Adult (or somatic) stem cells can be derived from most organs and typically differentiate into cell types specific to that organ; they are currently used to treat diverse condi- tions and are being evaluated in numerous clinical trials.

In vitro techniques enable fully differentiated adult cells to be reprogrammed so that they enter an ES cell–like state. Although these “induced pluripotent stem” (iPS) cells are similar to ES cells in terms of morphology, self-renewal capacity, and pluripotentiality, early reports suggest possi- ble dissimilarities, including genetic and epigenetic differences.

Embryonic stem cells and iPS cells have the potential to supply an unlimited number of diverse cell types for trans- plantation; however, their use in cell therapies involves significant obstacles that must be overcome. Clinical trials of ES cells have only recently begun.

www.annals.org

15 November 2011 Annals of Internal Medicine Volume 155 • Number 10 707

Ideas and Opinions Stem Cell Research and Regenerative Medicine
Figure 1. Derivation and differentiation of human stem cells for cell-based therapies.

ES embryonic stem; iPS induced pluripotent stem; SC stem cell. A. ES cells are usually derived from the inner cell mass of a blastocyst. B. iPS cells are produced by in vitro reprogramming of adult cells so that they enter an ES cell–like state. Both ES and iPS cells are pluripotent—they can be differentiated into a wide range of specialized cell types via multipotent intermediate cells. C. Adult SCs can be collected from a variety of sources, including bone marrow, peripheral blood, adipose tissue, and neural tissue. They are generally believed to be multipotent, or able to generate specialized cell types belonging to the organ from which they were derived. ES embryonic stem; iPS induced pluripotent stem; SC stem cell.

NSCs, results have not been uniform, and more research is needed to assess their clinical value.

Adult stem cells hold great promise for the treatment of injury and disease; however, substantial hurdles must be overcome before this promise can be fulfilled. Some hur- dles are common to all kinds of stem cells, such as those associated with tissue engineering (see Box) (19–23). An- other general concern is that culturing stem cells can mod- ify their genetic and epigenetic characteristics in potentially harmful ways. For instance, when cultured long term in vitro, MSCs exhibit genomic instability (24, 25). However, this is not an insurmountable problem; techniques have been developed to maintain karyotypically normal MSCs, and many clinical laboratories could do so according to “good manufacturing practice.”

The widespread use in the culture process of animal products, such as mouse fibroblasts and bovine serum, also raises concern. Although the risk for transmission of infec- tious agents seems to be low, nonhuman proteins may be more likely to trigger an adverse immune response in graft recipients (26). Because the long-term effects of animal proteins and pathogens remain unknown, their use in the manufacture of stem cell therapeutics must include rigor- ous safety testing.

Perhaps the main obstacle to the study and clinical use of adult stem cells is their limited capacity to proliferate

outside their bodily “niche.” Unlike ES cells, which can divide indefinitely ex vivo, adult stem cells undergo prolif- erative senescence and have a finite life span (27). Human MSCs, for example, usually cease replicating before 20 population doublings and progressively lose their ability to differentiate (28).

To secure an adequate supply of adult stem cells for the laboratory and clinic, it is vital to improve methods for increasing numbers while maintaining their phenotype outside the body. This is not only true for rare stem cells, such as those in the liver, but even for those that are rela- tively easy to collect, such as HSCs (19, 29).

EMBRYONIC STEM CELLS

Because they are pluripotent, ES cells promise to over- come the chief obstacle facing adult stem cells—securing an adequate supply of transplantable cells. The modern understanding of pluripotency grew out of the study of teratocarcinomas (malignant tumors that develop from germ cells of the testis and ovary). In 1964, it was shown that mouse teratocarcinomas include cells that self-renew indefinitely in vitro and can differentiate into lineages across all 3 germ layers (2, 30). These were the first pluri- potent cells ever cultivated, and they serve to remind us that all pluripotent cells have the potential to form tumors.

708 15 November 2011 Annals of Internal Medicine Volume 155 • Number 10

www.annals.org

Embryonic stem cells were first derived from mouse blastocysts in 1981 (31, 32). The same feat with human blastocysts proved much more difficult and was achieved only in 1998 (33). Today, more than 1000 “immortal” human ES cell lines exist, stored or registered in cell banks worldwide.

Robust protocols have been developed to differentiate human ES cells into a wide variety of cell types, including hepatocytes, neurons, retinal cells, blood cells, pancreatic -cells, and cardiomyocytes. Proof-of-principle animal ex- periments provide an indication of their therapeutic use and potential toxicities. For instance, human ES cell– derived pancreatic cells implanted into immunodeficient mice are known to protect them from an induced form of diabetes (34).

Of particular note are ongoing experiments with hu- man ES cell–derived oligodendrocyte progenitors, which produce the myelin sheaths that insulate axons in the ner- vous system. Transplanted into rodents with spinal cord injuries, these have been found to promote repair of dam- aged tissue and improve motor ability (35, 36). These encouraging results have led to the first testing of a similar procedure in humans. In October 2010, a phase 1 study commenced to assess the safety and tolerability of ES cell–derived oligodendrocyte progenitors in patients with “complete” spinal cord injuries (37). More recently, ES cell–derived retinal cells have become the subject of 2 more clinical trials (phase 1/2) in patients with macular degeneration (38, 39).

Embryonic stem cells have opened up a whole new field of biomedical research because they provide diverse opportunities to examine the development and function of both normal and diseased tissues in vitro (40). It may be possible, using ES cells, to model the effects of a range of factors on embryonic development and to identify those that predispose to disease in later life.

Modeling human diseases “in a dish” is especially use- ful for conditions that are otherwise difficult to study. The causes of acute childhood leukemia, for instance, resist analysis with patient samples (because these diseases may already manifest in utero) and with mouse models (because the latter may not recapitulate important aspects of the disease). Moreover, if a disease can be modeled in vitro, then high-throughput screens for potential treatments can be designed.

Embryonic stem cell lines may have an even broader application for drug discovery. When a new drug is devel- oped, it must undergo extensive preclinical evaluation. Most commonly, it is screened for heart and liver toxicity. Embryonic stem cell lines offer an unlimited supply of normal human heart and liver cells for routine early testing of new drugs.

Many obstacles must be faced before ES cells can gain widespread clinical application. The most publicized is the ethical controversy surrounding ES cell research, which has resulted in major restrictions in federal funding in the

Box. Tissue Engineering

Stem Cell Research and Regenerative Medicine

Ideas and Opinions

Creating tissues and organs in the laboratory poses many challenges. Bioreactors that mimic developmental condi- tions within the body are needed to culture stem cells and differentiate them into a sufficient number of specialized cells. Tissue scaffolds may be used to support stem cell niches and to organize cells into complex structures (19,
20). Because cells that are more than 1 or 2 mm from the graft’s surface will perish because of an inadequate blood supply, some engineered tissues may need to be vascularized by, for instance, including blood vessel precursors in the initial cell culture.

Despite the many difficulties associated with tissue engi- neering, some clinical milestones have been achieved.
Since the 1980s, severe burns have been treated with skin grafts cultured from a patient’s own epidermal stem cells (21). A similar approach is now being used to restore vi- sion to patients who have had chemical burns and other in- juries to the cornea. Limbal stem cells taken from a pa- tient’s healthy eye can be expanded in culture on a fibrin support and then grafted into the injured eye (22). Recent successes in tissue engineering include the creation of arti- ficial tracheas for patients with tuberculosis and cancer. A donor trachea can be decellularized and then seeded with the patient’s own epithelial cells and mesenchymal stem cell− derived chondrocytes, thereby avoiding the need for antirejection medications (23).

United States. From a purely medical perspective, however, safety issues are the main concern.

It is vital that transplants derived from ES cells (or any pluripotent cells) contain, as far as possible, only enriched populations of differentiated cells. Even a small number of pluripotent cells can pose a risk for teratoma formation, although just how great a risk is an open question (41, 42). For this reason, improved methods are needed to direct differentiation into the desired cell types and to minimize the presence of undifferentiated cells.

Perhaps the most obvious drawback of ES cells is that they do not allow for patient-specific (autologous) cell therapies. Their use places the recipient at risk for alloge- neic graft rejection. There are a number of ways to deal with this problem. One is to develop treatments involving sites in the body that are relatively protected against an inflammatory immune response, such as the brain, spinal cord, and eyes. This is the strategy adopted by the current clinical trials of ES cell products.

Another strategy is to establish large ES cell banks from which histocompatible matches could be selected (43, 44). But even with “close” matches, transplant recipients may need to use immunosuppressive drugs for the rest of their lives, which brings additional risks.

www.annals.org

15 November 2011 Annals of Internal Medicine Volume 155 • Number 10 709

Ideas and Opinions Stem Cell Research and Regenerative Medicine

A more radical strategy is to produce patient-specific ES cells through somatic cell nuclear transfer—that is, therapeutic cloning. By taking a body cell (or just the nucleus) from a patient and fusing it with an ovum whose nucleus has been removed, an early embryo can be created and, from this, ES cells can be extracted. For many reasons—scientific, legal, and ethical—this idea has been extremely difficult to put into practice. Nuclear transfer has produced ES cell lines from mice and monkeys but not yet from humans, although cloned human blastocysts have been created (45, 46).

INDUCED PLURIPOTENT STEM CELLS

Cloning was the earliest method devised to dedifferen- tiate a mature body cell. In 2006, Takahashi and Ya- manaka from Kyoto University, Japan, invented a much easier method. Using retroviruses, they inserted 4 genes into mouse fibroblasts, which caused them to enter a plu- ripotent state (3). The same in vitro techniques were ap- plied to human skin cells, and in 2007, researchers in Ja- pan and the United States created the first human iPS cells (47– 49).

The obvious advantage of iPS cells over ES cells is that they make the derivation of cell lines from almost anyone possible. In the past few years, human iPS cells have been differentiated into many functional cell types, including insulin-secreting pancreatic cells, cardiac muscle cells, neu- rons, and retinal cells. If such cells prove safe for clinical

Figure 2. Modeling diseases with iPS cells.

use, a wide range of autologous cell replacement therapies may be developed.

This does not mean that, before long, everyone could have their own stem cell lines. The time, effort, and cost of producing personalized iPS cell therapies are likely to limit their clinical application (50). A cheaper and more practi- cal alternative would be to use ready-made, immune- matched cells from a public cell bank. Reprogramming may make it much easier to develop a bank of compatible pluripotent stem cell lines (51). Instead of depending on randomly donated embryos to cover the most common haplotypes of a given population, individuals with those haplotypes could be selected as cell donors.

Cell reprogramming also makes it easier to model dis- eases in vitro (Figure 2). Dozens of laboratories around the world have derived iPS cells from patients with such dis- eases as amyotrophic lateral sclerosis, Parkinson disease, type 1 diabetes, and schizophrenia. So far, genetic condi- tions (especially those linked to a single gene malfunction) have been the main focus of this approach, but reprogram- ming could also be used to study the contribution of non- genetic factors.

Modeling a disease often involves differentiating a disease-specific iPS cell line into an affected cell type. Not only can such models provide valuable information about the disease, they can be used to test potential therapies. To give just one example, skin cells taken from patients with Fanconi anemia were genetically corrected using gene ther-

Cells can be obtained from a patient with almost any disease, reprogrammed to a pluripotent state, expanded in vitro, and differentiated into a cell type that expresses features of the disease. Such disease models “in a dish” can be used to test new drugs and gene therapies. Induced pluripotent stem cells derived from healthy donors can also assist in the development of new drugs by offering an unlimited supply of heart, liver, and neural cells for toxicology testing. iPS induced pluripotent stem.

710 15 November 2011 Annals of Internal Medicine Volume 155 • Number 10 www.annals.org

apy and reprogrammed into iPS cells. When they were subsequently differentiated into hematopoietic cells, they displayed a disease-free phenotype in vitro (52).

Shepherding iPS cells from the laboratory to the clinic raises many of the same problems associated with ES cells. Strict methods of cell differentiation are needed so that, as far as possible, grafts are free of undifferentiated cells and the associated risk for teratoma formation.

Other dangers are specific to iPS cells. Of the 4 genes first used to induce pluripotency, at least 2 are oncogenic. Also, the viral vectors initially used to transport these genes can insert within the cell’s genome and cause harmful mu- tations, cellular dysfunction, and tumorigenesis (53). To address these dangers, reprogramming methods are being developed that avoid oncogenes and that make use of non- integrative or excisable vectors. Some of the most promis- ing new techniques introduce no genetic elements or viral vectors into cells (54–56).

Although iPS cells are similar to ES cells in morphol- ogy, self-renewal capacity, and pluripotentiality, important dissimilarities seem to be emerging. Unfortunately, iPS cells often suffer in this comparison. According to recent studies, iPS cells are prone to higher levels of genetic and epigenetic abnormalities than ES cells (57–59). Also disap- pointing are reports that, compared with ES cells, iPS cells differentiate into functional cell types with less efficiency and fidelity (60) and that the differentiated cells they pro- duce have less ability to proliferate (61).

The task of comparing ES and iPS cells is rendered more complex by the differences between iPS cells them- selves. There is evidence that iPS cells retain an “epigenetic memory” of their tissue of origin; this makes them easier to differentiate along that same tissue’s lineages but may limit their broader therapeutic utility (62). Another source of variation is the diversity of cell reprogramming techniques. Different methods are likely to affect the behavior of iPS cells and introduce different risks.

It should be emphasized that reprogramming is in its infancy. Given the huge inherent heterogeneity in human cell lines generally and the limited number of iPS lines that have been systematically examined, the true statistical sig- nificance of the differences between iPS and ES cells re- mains uncertain.

BALANCING ASSETS AND LIABILITIES

Stem cells promise great advances in the treatment of injury and disease, but many problems must be overcome before their clinical potential can be realized. Each kind of stem cell presents unique opportunities and challenges, as- sets, and liabilities. To determine the medical value of each, we must compare their ever-changing balance sheets.

We have avoided most ethical issues and have barely mentioned the stem cell controversy. This controversy has prompted claims that ES cells can be replaced, without

www.annals.org

significant loss, by iPS cells, a topic we recently addressed (63).

Here, we merely wish to point out that the validity of such claims—made in the heat of public debate— can only be assessed scientifically. To know whether one stem cell can replace another in the clinic, it is necessary to identify similarities and differences from a range of perspectives. These include the source and availability of each kind of stem cell; its proliferative potential; the types of cells it can generate; the diseased and injured tissues it might be used to treat; the optimum scalable methods for its collection, culture, identification, puri- fication, potency testing, and delivery; and its short- and long-term risks. These are complex issues that will take many years to resolve.

From the Centenary Institute, University of Sydney, and the Royal Prince Alfred Hospital, Sydney, Australia.

Acknowledgment: The authors thank Cure The Future (Cell and Gene Trust) and the Brocher Foundation for support. The figures were pro- duced using Servier Medical Art (www.servier.com).

Potential Conflicts of Interest: Disclosures can be viewed at www .acponline.org/authors/icmje/ConflictOfInterestForms.do?msNum M11 -1370.

Requests for Single Reprints: John E.J. Rasko, MBBS, PhD, Centenary Institute, Locked Bag 6, Newtown, New South Wales 2042, Australia; e-mail, j.rasko@cenint.org.

Current author addresses and author contributions are available at www .annals.org.

References

1. Seydoux G, Braun RE. Pathway to totipotency: lessons from germ cells. Cell. 2006;127:891-904. [PMID: 17129777]
2. Yu J, Thomson JA. Pluripotent stem cell lines. Genes Dev. 2008;22:1987-97. [PMID: 18676805]

3. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663- 76. [PMID: 16904174]
4. Szabo E, Rampalli S, Risuen ̃o RM, Schnerch A, Mitchell R, Fiebig-Comyn A, et al. Direct conversion of human fibroblasts to multilineage blood progeni- tors. Nature. 2010;468:521-6. [PMID: 21057492]

5. Power C, Rasko JE. Whither Prometheus’ liver? Greek myth and the science of regeneration. Ann Intern Med. 2008;149:421-6. [PMID: 18794562]
6. Becker AJ, McCulloch EA, Till JE. Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature. 1963;197:452-4. [PMID: 13970094]

7. Thomas ED. Bone marrow transplantation from the personal viewpoint. Int J Hematol. 2005;81:89-93. [PMID: 15765774]
8. Vaughan W, Seshadri T, Bridges M, Keating A. The principles and overview of autologous hematopoietic stem cell transplantation. Cancer Treat Res. 2009; 144:23-45. [PMID: 19779877]

9. Brenner MK, Rill DR, Moen RC, Krance RA, Mirro J Jr, Anderson WF, et al. Gene-marking to trace origin of relapse after autologous bone-marrow transplantation. Lancet. 1993;341:85-6. [PMID: 8093407]
10. Brenner MK, Rill DR, Moen RC, Krance RA, Heslop HE, Mirro J Jr, et al. Gene marking and autologous bone marrow transplantation. Ann N Y Acad Sci. 1994;716:204-14. [PMID: 8024195]

11. Horowitz MM, Gale RP, Sondel PM, Goldman JM, Kersey J, Kolb HJ, 15 November 2011 Annals of Internal Medicine Volume 155 • Number 10 711

Stem Cell Research and Regenerative Medicine Ideas and Opinions

Ideas and Opinions Stem Cell Research and Regenerative Medicine

et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood. 1990;75:555-62. [PMID: 2297567]
12. Trounson A, Thakar RG, Lomax G, Gibbons D. Clinical trials for stem cell therapies. BMC Med. 2011;9:52. [PMID: 21569277]

13. Prockop DJ. Repair of tissues by adult stem/progenitor cells (MSCs): con- troversies, myths, and changing paradigms. Mol Ther. 2009;17:939-46. [PMID: 19337235]
14. Allison M. Genzyme backs Osiris, despite Prochymal flop. Nat Biotechnol. 2009;27:966-7. [PMID: 19898434]

15. Bernardo ME, Locatelli F, Fibbe WE. Mesenchymal stromal cells. Ann N Y Acad Sci. 2009;1176:101-17. [PMID: 19796238]
16. Tolar J, Le Blanc K, Keating A, Blazar BR. Concise review: hitting the right spot with mesenchymal stromal cells. Stem Cells. 2010;28:1446-55. [PMID: 20597105]

17. Levesque ME, Neuman T, Rezak M. Therapeutic microinjection of autol- ogous adult human neural stem cells and differentiated neurons for Parkinson’s disease: five-year post-operative outcome. Open Stem Cell J. 2009;1:20-9.
18. Mackay-Sim A, Fe ́ron F, Cochrane J, Bassingthwaighte L, Bayliss C, Davies W, et al. Autologous olfactory ensheathing cell transplantation in human para- plegia: a 3-year clinical trial. Brain. 2008;131:2376-86. [PMID: 18689435]

19. Holst J, Watson S, Lord MS, Eamegdool SS, Bax DV, Nivison-Smith LB, et al. Substrate elasticity provides mechanical signals for the expansion of hemo- poietic stem and progenitor cells. Nat Biotechnol. 2010;28:1123-8. [PMID: 20890282]

20. Grayson WL, Martens TP, Eng GM, Radisic M, Vunjak-Novakovic G. Biomimetic approach to tissue engineering. Semin Cell Dev Biol. 2009;20:665- 73. [PMID: 19146967]
21. Green H. The birth of therapy with cultured cells. Bioessays. 2008;30:897- 903. [PMID: 18693268]

22. Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G. Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med. 2010;363:147-55. [PMID: 20573916]
23. Macchiarini P, Jungebluth P, Go T, Asnaghi MA, Rees LE, Cogan TA, et al. Clinical transplantation of a tissue-engineered airway. Lancet. 2008;372: 2023-30. [PMID: 19022496]

24. Li H, Fan X, Kovi RC, Jo Y, Moquin B, Konz R, et al. Spontaneous expression of embryonic factors and p53 point mutations in aged mesenchymal stem cells: a model of age-related tumorigenesis in mice. Cancer Res. 2007;67: 10889-98. [PMID: 18006834]

25. Bork S, Pfister S, Witt H, Horn P, Korn B, Ho AD, et al. DNA methyl- ation pattern changes upon long-term culture and aging of human mesenchymal stromal cells. Aging Cell. 2010;9:54-63. [PMID: 19895632]
26. Horwitz EM, Gordon PL, Koo WK, Marx JC, Neel MD, McNall RY, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci U S A. 2002;99:8932-7. [PMID: 12084934]

27. Sieburg HB, Rezner BD, Muller-Sieburg CE. Predicting clonal self-renewal and extinction of hematopoietic stem cells. Proc Natl Acad Sci U S A. 2011;108: 4370-5. [PMID: 21368169]
28. Digirolamo CM, Stokes D, Colter D, Phinney DG, Class R, Prockop DJ. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol. 1999;107:275-81. [PMID: 10583212]

29. Delaney C, Heimfeld S, Brashem-Stein C, Voorhies H, Manger RL, Bern- stein ID. Notch-mediated expansion of human cord blood progenitor cells capa- ble of rapid myeloid reconstitution. Nat Med. 2010;16:232-6. [PMID: 20081862]

30. Kleinsmith LJ, Pierce GB Jr. Multipotentiality of single embryonal carci- noma cells. Cancer Res. 1964;24:1544-51. [PMID: 14234000]
31. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981;292:154-6. [PMID: 7242681]

32. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A. 1981;78:7634-8. [PMID: 6950406]
33. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145-7. [PMID: 9804556]

34. Kroon E, Martinson LA, Kadoya K, Bang AG, Kelly OG, Eliazer S, et al. Pancreatic endoderm derived from human embryonic stem cells generates

712 15 November 2011 Annals of Internal Medicine Volume 155 • Number 10

glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol. 2008;26:443- 52. [PMID: 18288110]
35. Keirstead HS, Nistor G, Bernal G, Totoiu M, Cloutier F, Sharp K, et al. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci. 2005;25: 4694-705. [PMID: 15888645]

36. Sharp J, Frame J, Siegenthaler M, Nistor G, Keirstead HS. Human embry- onic stem cell-derived oligodendrocyte progenitor cell transplants improve recov- ery after cervical spinal cord injury. Stem Cells. 2010;28:152-63. [PMID: 19877167]

37. A Phase 1 Safety Study of GRNOPC1 in Patients With Neurologically Complete, Subacute, Spinal Cord Injury [clinical trial]. ClinicalTrials.gov regis- tration number: NCT01217008. Accessed at http://clinicaltrials.gov/ct2/show /NCT01217008 on 11 June 2011.

38. A Phase I/II, Open-Label, Multi-Center, Prospective Study to Determine the Safety and Tolerability of Sub-retinal Transplantation of Human Embry- onic Stem Cell Derived Retinal Pigmented Epithelial (MA09-hRPE) Cells in Patients With Stargardt’s Macular Dystrophy (SMD) [clinical trial]. Clinical Trials.gov registration number: NCT01345006. Accessed at http://clinicaltrials .gov/ct2/show/NCT01345006 on 11 June 2011.

39. A Phase I/II, Open-Label, Multi-Center, Prospective Study to Deter- mine the Safety and Tolerability of Sub-retinal Transplantation of Human Embryonic Stem Cell Derived Retinal Pigmented Epithelial (MA09-hRPE) Cells in Patients With Advanced Dry AMD [clinical trial]. ClinicalTrials.gov registration number: NCT01344993. Accessed at www.clinicaltrials.gov/ct2 /show/NCT01344993 on 11 June 2011.

40. Menendez P, Bueno C, Wang L. Human embryonic stem cells: a journey beyond cell replacement therapies. Cytotherapy. 2006;8:530-41. [PMID: 17148029]
41. Li JY, Christophersen NS, Hall V, Soulet D, Brundin P. Critical issues of clinical human embryonic stem cell therapy for brain repair. Trends Neurosci. 2008;31:146-53. [PMID: 18255164]

42. Miura K, Okada Y, Aoi T, Okada A, Takahashi K, Okita K, et al. Variation in the safety of induced pluripotent stem cell lines. Nat Biotechnol. 2009;27: 743-5. [PMID: 19590502]
43. Taylor CJ, Bolton EM, Pocock S, Sharples LD, Pedersen RA, Brad- ley JA. Banking on human embryonic stem cells: estimating the number of donor cell lines needed for HLA matching. Lancet. 2005;366:2019-25. [PMID: 16338451]

44. Nakajima F, Tokunaga K, Nakatsuji N. Human leukocyte antigen matching estimations in a hypothetical bank of human embryonic stem cell lines in the Japanese population for use in cell transplantation therapy. Stem Cells. 2007;25: 983-5. [PMID: 17185611]

45. French AJ, Adams CA, Anderson LS, Kitchen JR, Hughes MR, Wood SH. Development of human cloned blastocysts following somatic cell nuclear transfer with adult fibroblasts. Stem Cells. 2008;26:485-93. [PMID: 18202077]
46. Li J, Liu X, Wang H, Zhang S, Liu F, Wang X, et al. Human embryos derived by somatic cell nuclear transfer using an alternative enucleation approach. Cloning Stem Cells. 2009;11:39-50. [PMID: 19196043]

47. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861-72. [PMID: 18035408]
48. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318:1917-20. [PMID: 18029452]

49. Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA, et al. Repro- gramming of human somatic cells to pluripotency with defined factors. Nature. 2008;451:141-6. [PMID: 18157115]
50. Cyranoski D. Stem cells: 5 things to know before jumping on the iPS bandwagon. Nature. 2008;452:406-8. [PMID: 18368095]

51. Nakatsuji N, Nakajima F, Tokunaga K. HLA-haplotype banking and iPS cells [Letter]. Nat Biotechnol. 2008;26:739-40. [PMID: 18612291]
52. Raya A, Rodr ́ıguez-Piza` I, Guenechea G, Vassena R, Navarro S, Barrero MJ, et al. Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells. Nature. 2009;460:53-9. [PMID: 19483674] 53. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulf- fraat N, Leboulch P, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003;302:415-9. [PMID: 14564000]

54. Zhou H, Wu S, Joo JY, Zhu S, Han DW, Lin T, et al. Generation of www.annals.org

induced pluripotent stem cells using recombinant proteins. Cell Stem Cell. 2009; 4:381-4. [PMID: 19398399]
55. Kim D, Kim CH, Moon JI, Chung YG, Chang MY, Han BS, et al. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell. 2009;4:472-6. [PMID: 19481515]

56. Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell. 2010;7:618-30. [PMID: 20888316]

57. Gore A, Li Z, Fung HL, Young JE, Agarwal S, Antosiewicz-Bourget J, et al. Somatic coding mutations in human induced pluripotent stem cells. Nature. 2011;471:63-7. [PMID: 21368825]
58. Hussein SM, Batada NN, Vuoristo S, Ching RW, Autio R, Na ̈rva ̈ E, et al. Copy number variation and selection during reprogramming to pluripotency. Nature. 2011;471:58-62. [PMID: 21368824]

Ode to an Everyman

I hold you in my hands,
an embryo
no bigger than a dust mote

within a droplet— one of many nurtured in the dark

of an incubator this day. Conjured from a sperm and egg, each a life to be fulfilled—

ascendance, then decay. What might you become once I place you there—

a cutpurse or a Liszt, or die a little death within the womb?

Richard Bronson, MD

Stony Brook University Medical Center Stony Brook, NY 11794-8091

Stem Cell Research and Regenerative Medicine Ideas and Opinions

59. Lister R, Pelizzola M, Kida YS, Hawkins RD, Nery JR, Hon G, et al. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature. 2011;471:68-73. [PMID: 21289626]
60. Hu BY, Weick JP, Yu J, Ma LX, Zhang XQ, Thomson JA, et al. Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency. Proc Natl Acad Sci U S A. 2010;107:4335- 40. [PMID: 20160098]

61. Feng Q, Lu SJ, Klimanskaya I, Gomes I, Kim D, Chung Y, et al. He- mangioblastic derivatives from human induced pluripotent stem cells exhibit lim- ited expansion and early senescence. Stem Cells. 2010;28:704-12. [PMID: 20155819]

62. Kim K, Doi A, Wen B, Ng K, Zhao R, Cahan P, et al. Epigenetic memory in induced pluripotent stem cells. Nature. 2010;467:285-90. [PMID: 20644535] 63. Power C, Rasko JE. Will cell reprogramming resolve the embryonic stem cell controversy? A narrative review. Ann Intern Med. 2011;155:114-21. [PMID: 21768585]

Ad Libitum

www.annals.org

15 November 2011 Annals of Internal Medicine Volume 155 • Number 10 713

Current Author Address: Richard Bronson, MD, Department of Obstetrics & Gynecology, Stony Brook University Medical Center, HSC T9-080, Stony Brook, NY 11794-8091.

© 2011 American College of Physicians

Annals of Internal Medicine

Current Author Addresses: Drs. Power and Rasko: Centenary Institute, Locked Bag 6, Newtown, New South Wales 2042, Australia.

Author Contributions: Conception and design: C. Power, J.E.J. Rasko. Drafting of the article: C. Power, J.E.J. Rasko.
Critical revision of the article for important intellectual content: C. Power, J.E.J. Rasko.

Final approval of the article: C. Power, J.E.J. Rasko. Obtaining of funding: J.E.J. Rasko.
Collection and assembly of data: C. Power, J.E.J. Rasko.