Monitoring Stem Cell Research

Table of Contents

Recent Developments in Stem Cell Research and Therapy

Research using human and animal stem cells is an extremely active area of current biomedical inquiry. It is contributing new knowledge about the pathways of normal and abnormal cell differentiation and organismal development. It is opening vistas of new cell transplantation therapies for human diseases. Although the availability of a variety of human stem cells is relatively recent-the isolation of human embryonic stem cellsⁱ was first reported only in 1998-much is happening in both publicly funded and privately funded research centers around the world. It is difficult for anyone to stay abreast of all the results now rapidly accumulating.

To help us fulfill our mandate to "monitor stem cell research," the President's Council on Bioethics asked several experts to survey the recent published scientific literature and to contribute articles on various areas of stem cell research to this report (see articles by Drs. Gearhart,¹ Ludwig and Thomson,² Verfaillie,³ Prentice,⁴ Itescu,^{5, 6} and Jaenisch⁷ in the Appendices). These reviews and the present chapter emphasize peer-reviewed, published work with human stem cells through July 2003. Interested readers should also consult the wide variety of other review articles that have appeared.⁸

This chapter should be read in conjunction with the commissioned review articles cited above. It draws on their findings, as well as on the Council's own monitoring activities, but it makes no attempt to summarize all the complexity of stem cell research or the vast array of results. Rather we offer here some general observations and specific examples that might help non-scientist readers understand the overall state of present human stem cell research, its therapeutic promise, and some of the problems that need to be solved if the research is to yield sound knowledge and clinical benefit. To that end, we highlight the importance of well-characterized, stable preparations of stem cells for obtaining reproducible experimental results, and we identify several problems that must be solved before these requirements can be fully met. This chapter then describes, by way of illustration and example, some of the better-characterized adult and embryonic stem cells. It also indicates some of the specific investigations that are being conducted with their aid. Finally, it considers how human stem cells are being used to explore their potential for treating disease, using experiments in animal models of Type-1 diabetes as an example, and it points out some of the difficulties that must be overcome before stem cell-based remedies may be available to treat human diseases.

We confine our attention here to newly identified types of human stem cells and their potential use in research and future medical treatment. Accordingly, we do not consider those stem cell types that are already well established in medical practice and research. Specifically, we will not examine those preparations of bone marrow cells that have been clinically used for some years to treat various forms of anemia and cancer.⁹ Neither will we deal with hematopoietic (blood-forming) stem cells that have been isolated and purified from bone marrow and are now being intensively studied.¹⁰ Although these developments lie beyond the scope of this report, the demonstrated usefulness of these cells for research and therapy encourages many researchers to expect similar benefits from the newer stem cells that we shall consider here.

I. Stem Cells and Their Derivatives

The adult human body, and all its differentiated cells, tissues, and organs, arise from a small group of cells contained within the early embryo at the blastocyst stage of its development. During in vivo embryonic development, these cells, constituting the inner cell mass (ICM), will divide and differentiate in concert with each other and with the whole of which they are a part, eventually producing the specialized and integrated tissues and organs of the body. But when embryos are grown [using in vitro fertilization (IVF)] in a laboratory setting, these ICM cells may be removed and isolated, and under appropriate conditions some will proliferate in vitro and become embryonic stem cell lines.

These embryonic stem cells are capable of becoming many different types of differentiated cells if stimulated to do so in vitro [see endnote 2 for references]. However, it is not yet clear that the cells that survive the in vitro selection process to become embryonic stem cells have all of the same biological properties and potentials as the ICM cells of the blastocyst.⁷ In particular, it is not known for certain that human embryonic stem cells in vitro can give rise to all the different cell types of the adult body.ⁱⁱ

As noted in the Introduction to this report, stem cells are a diverse class of cells, which can now be isolated from a variety of embryonic, fetal, and adult tissues. Stem cells share two characteristic properties: (1) unlimited or prolonged self-renewal (that is, the capacity to maintain a pool of stem cells like themselves), and (2) potency for differentiation, the potential to produce more differentiated cell types-usually more than one and, in some cases, many.ⁱⁱⁱ

When stem cells head down the pathway toward differentiation, they usually proceed by first giving rise to a more specialized kind of stem cell (sometimes called "precursor cells" or "progenitor cells"), which can in turn either proliferate through se

lf-renewal or produce fully specialized or differentiated cells (see Figure 1).

Figure 1. Schematic Diagram of Some Stages in Cell Differentiation

At the top of the figure is an undifferentiated stem cell; in the central box are more "specialized" stem cells (or "precursor cells" or "progenitor cells"); at the bottom are various differentiated cells that are derived from the specialized stem cells. Dashed arrows indicate symmetrical (in the sense that both the daughter cells are stem cells) cell divisions thatproduce more stem cells (self-renewal). Solid arrows indicate asymmetric cell divisions that produce moredifferentiated daughter cells. (There may also be self-renewal with asymmetric division-not shown here-in which one daughter cell initiates a differentiation pathway while the other remains a stem cell.) Differentiation signals can be supplied by both soluble proteins and by specific, cell-surface binding sites. Some of the specialized stem cells inside the dashed box, for example, mesenchymal stem cells, can be isolated from tissues after birth and correspond to adult stem cells. Scientists are currently investigating whether, at least in some cases, the process can be reversed, that is, whether specialized cells may, on appropriate signals, dedifferentiate to become precursor or even fully undifferentiated stem cells.

The terminology used to describe different stem cell types can be confusing. As used in this chapter, stem cells are self-renewing, cultured cells, grown and preserved in vitro, that are capable-upon exposure to appropriate signals-of differentiating themselves into (usually more than one) specialized cell types. Stem cells may be classified either according to their origins or according to their developmental potential.

Stem cells may be obtained from various sources: from embryos, from fetal tissues, from umbilical cord blood, and from tissues of adults (or children). Thus, depending on their origin, stem cell preparations may be called adult stem cells,^iv embryonic stem cells, embryonic germ cells, or fetal stem cells. Adult stem cells [see (4)] are cells derived from various tissues or organs in humans or animals that have the two characteristic properties of stem cells (self-renewal and potency for differentiation). Embryonic stem cells (ESCs) [see (2)] are derived from cells isolated from the inner cell mass of early embryos. Embryonic germ cells (EGCs) [see (1)] are stem cells derived from the primordial germ cells of a fetus. Fetal stem cells (not further discussed in this chapter, but included for the sake of completeness) are derived from the developing tissues and organs of fetuses; because they come (unlike EGCs) from already differentiated tissues, they are (like adult stem cells) "non-embryonic," and may be expected to behave as such.

Depending on their developmental potential, cells may be called pluripotent, multipotent, or unipotent. Cells that can produce all the cell types of the developing body, such as the ICM cells of the blastocyst, are said to be pluripotent. The somewhat more specialized stem cells, of the sort found in the developed organs or tissues of the body, are said to be multipotent if they produce more than one differentiated tissue cell type, and unipotent if they produce only one differentiated tissue cell type.

We introduce in this chapter an additional term: stem cell preparation. A stem cell preparation is a population of stem cells, prepared, grown, and preserved under certain conditions. Because different laboratories (or even the same one) can have different preparations of the same type of stem cell, it is important to recognize the potential differences between particular preparations of embryonic stem cells.^v It will sometimes be important to call attention to this fact, by speaking of a "preparation of ES cells" (or a preparation of adult stem cells) rather than of "ES cells," pure and simple. We will use the term "stem cell preparations" when we are speaking of a diverse group of stem cell cultures, when we are speaking of stem cell cultures that contain an admixture of other types of cells, or when the developmental homogeneity of the stem cells in the population has not been defined.

Adult and embryonic stem cell populations have also been called "stem cell lines." In the past, the term "cell line" denoted a cell population (usually of cancer cells containing abnormal chromosome numbers or structure, or both) that could grow "indefinitely" in vitro. Embryonic and some adult stem cell preparations are capable of prolonged growth beyond 50 population doublings in vitro while retaining their characteristic stem cell properties and initially with no change in the chromosome numbers and structure. It is not yet known whether any preparation of human ES cells (generally believed to be much longer-lived than adult stem cells) will continue to grow "indefinitely," without breaking down.

Under the influence of various cell-differentiation signals, embryonic stem cells differentiate into numerous distinct types of more specialized cells. Some of these are specialized stem cells that can also self-renew, while retaining their ability also to differentiate into multiple cell types. Recent research has led to the isolation of an increasing number of adult (non-embryonic) stem cells (dashed box area of Figure 1) from such tissues as bone marrow (for example, hematopoietic and mesenchymal stem cells), brain (for example, neural stem cells) and other tissues [see (4)]. Although these stem cell preparations differ from one another in their future fates, they tend to be grouped together (especially in the public policy debates) under the name "adult stem cells," even though they may have been obtained from children or even from umbilical cord blood obtained at the time of childbirth.

Subsequent exposure to additional differentiation signals can cause these specialized stem cells to differentiate further, so that they finally give rise to the variety of differentiated cells that make up the adult body (labeled A-D in Figure 1). At each stage of the differentiation process, specific sets of genes are expressed (or "turned on") and other sets are repressed (or "turned off"), to produce the specific proteins that give each cell its distinctive properties. At each stage along the way, proteins called transcription factors play key roles in determining which sets of genes are expressed and repressed, and therefore what sort of a cell the newly differentiated cell will become.

II. Reproducible Results Using Stem Cell Preparations and Their Derivatives

A major goal of scientific research is the acquisition of reliable knowledge based on experiments that yield reproducible results. Reproducible results are possible only if the materials used in experiments remain constant and stable. To obtain reproducible results in experiments using stem cells, it is essential to produce, preserve, characterize, and continually re-characterize preparations of stem cells in ways that increase the likelihood that the cells used to repeat experiments will remain unchanged-a technically challenging task. The tendency of stem cells in vitro to differentiate spontaneously into more specialized cells makes the task of obtaining homogeneous and stable stem cell preparations especially challenging, and much basic research is needed to learn how to control the fate of these cells. Failure to control the cells may yield experimental results that are difficult or impossible to reproduce. The following more specific observations make clear the dimensions of this difficulty.

Isolation of adult stem cells from source tissues such as bone marrow, brain, or muscle initially yields a heterogeneous cell preparation. The initial preparation contains the several cell types found in the source tissue, and it may also include red blood cells, white blood cells, and (possibly) circulating stem cells, owing to the presence of blood flowing through the tissue in question. Initial mixtures of cells may then be treated in various ways to remove unwanted contaminating cells, thereby increasing the proportion of stem cells in the preparation. But seldom, if ever, does one produce an adult stem cell preparation that is 100 percent stem cells, unless the adult stem cell preparation has been "single-cell cloned" in vitro (see below).

The way in which human embryonic stem cells have been produced from ICM cells also raises a question about the "species homogeneity" of the initial cell preparations. In the past, human embryonic stem cells were isolated and maintained by in vitro growth on top of irradiated (so that they no longer divide) "feeder layers" of mouse cells. It is thought that the feeder cells secrete factor(s) that enable the stem cells to divide while maintaining a relatively undifferentiated state. Although the mouse cells have been treated to prevent their cell division, should any of them happen to survive, human embryonic stem cells prepared in this way may contain some viable mouse cells.^vi More recently, several groups have shown that it is possible to grow ESCs on feeder layers of human cells, including fibroblasts obtained from skin biopsies, or without any feeder cell layer at all.¹¹ One way to be certain that human embryonic stem cell preparations do not contain any mouse feeder cells is through "single cell cloning" (see below).

Some preparations of stem cells growing in vitro have been "single cell cloned," that is, grown as a population derived from a single stem cell. By placing a cylinder over a single cell located with a microscope, scientists are able to isolate within the cylinder all the progeny produced by subsequent cell divisions beginning from this single cell. The result is a stem cell preparation in which all the cells are descended from the original single cell. The cells within the cylinder are then harvested and grown to greater numbers in vitro, and the resulting stem cell preparation is said to be "single cell cloned." The stem cells within a "single cell cloned" population are, at least to begin with, genetically homogeneous because they are all derived from the same original cell. Some of the ESC preparations produced prior to August 9, 2001 have been "single cell cloned."¹²

C. Expansion in Vitro, Preservation, and Storage

Reproducible results require that preparations of stem cells, even if genetically homogenous when first isolated, remain stable over time and during preservation. This, too, is not a simple matter with stem cells, despite the fact that the self-renewal characteristic of human embryonic and adult stem cells enables them-unlike differentiated cells from many human tissues-to be grown in large numbers in vitro while maintaining their essential stem cell characteristics. After such expansion, many, presumably identical, vials of the cells can be frozen and preserved at very low temperatures. Frozen stem cell preparations can later be thawed and grown again in vitro to produce larger numbers of cells.

As with all dividing cells, stem cells are subject to a very small but definite chance of mutation during DNA replication; thus, prolonged growth in vitro could introduce genetic heterogeneity into an originally homogeneous population. During this process of repeated expansion and preservation, subtle changes in the growth conditions or other variables may give rise to "selective pressures" that can increase the heterogeneity in a stem cell preparation by favoring the multiplication of advantaged cell variants in the population. It is not known at present how many of the 78 human ESC preparations, designated as eligible for federal funding under the current policy, have developed genetic variants that may make them unsuitable for further research.

Whether several cycles of freezing and thawing change the phenotypic characteristics of stem cell preparations needs detailed study. However, the practical advantages of preserving stem cell preparations by freezing are too large to ignore. Such preservation makes it possible to repeat an experiment many times with a very similar stem cell preparation. It would also make it possible, should stem cell based therapies be developed in the future, to treat multiple patients with a common, well-characterized cell preparation derived from a single initial stem cell sample.

D. Chromosome Changes

In addition to the possible loss of homogeneity in stem cell preparations owing to variability in growth conditions or to freezing and thawing, there is the possibility of variation being introduced during the processes of growth and cell division. Normal human stem cells (like all human somatic cells) have 46 chromosomes. During the copying of chromosomal DNA and the separation of daughter chromosomes at cell division, rare mistakes occur that lead to the formation of abnormal chromosomes or maldistribution of normal ones. Cells with abnormal chromosomes or chromosome numbers can progress to malignancy, so retention of the normal human chromosome number and structure is an essential characteristic of useful human stem cell preparations. The most studied preparations of human stem cells generally have normal human chromosome numbers and structure.^{3 13 vii} Nevertheless, vigilance is needed, for even a small number of chromosomally abnormal cells could end up causing cancer in future clinical trials of stem cell based therapies.

The in vitro growth conditions and the presence of specific chemicals or proteins, or both, in the culture medium can influence the differentiation pathway taken by stem cells as they start to differentiate. Thus, even initially homogeneous, "single cell cloned" stem cell preparations may become developmentally heterogeneous over time, with respect to the percentage of cells in the preparation that are in one or another differentiated state. For example, a stem cell preparation after growth in vitro under specific conditions might contain 75 percent fully differentiated (insulin-producing) cells and 25 percent partially differentiated cells. The biological properties of the fully differentiated cells and the partially differentiated cells are likely to be different. If such a cell preparation is used in research, or transplanted into an animal model of human disease and a biological effect is observed, one must do additional experiments to determine whether the effect was due to the fully differentiated cells or to the partially differentiated cells (or perhaps to both acting together) in the now mixed preparation.

F. Microbial Contamination

Stem cell preparations originally isolated from humans and expanded in vitro may also be variably contaminated with human viruses, bacteria, fungi and mycoplasma. ESC preparations isolated using mouse feeder cell layers might also be contaminated with mouse viruses. Specific tests need to be performed on the source tissue and periodically on the resulting stem cell preparations to rule out the presence of these contaminants. Some of these contaminants can also multiply when stem cells are grown in vitro, and their presence can influence the results obtained when stem cell preparations are used in subsequent experiments. The presence of such contaminants can also potentially affect the reproducibility of the results of experiments in which stem cell preparations are studied in vivo in experimental animals.

In summary, there are numerous challenges to obtaining and preserving the uniform and stable preparations of stem cells necessary for reliable research and, eventually, for safe and effective possible therapies. Researchers must address multiple factors in order to maximize the probability of obtaining reproducible results with human stem cell preparations. Human stem cell preparations that are

• "single cell cloned," with a normal chromosome structure and number, and

• stored as multiple samples that are preserved at very low temperature, and

• compared in experiments where cells from the same lot of frozen material are used, and

• well-characterized as to the absence of cellular, viral, bacterial, fungal, and mycoplasma contaminants, and

• tested to determine the proportion of stem cells and various differentiated cells in the cell preparation used in the experiments,

III. Major Examples of Human Stem Cells

In this section we discuss major examples of human stem cells that meet many of the criteria listed above. Among human adult stem cells, we focus on mesenchymal stem cells (MSCs),⁴ multipotent adult progenitor cells (MAPCs),³ and neural stem cells, and among human embryonic stem cells, on ESC² and EGC¹ cells. For information on the wide variety of other human stem cell preparations isolated from adult tissues, see reference (4) (Appendix K).

Further research on some of these other adult stem cell preparations may demonstrate that they can also be "single cell cloned," expanded considerably by growth in vitro with retention of normal chromosome structure and number, and preserved by freezing and storage at low temperatures. At that point, it would be very important to compare the properties of these other adult stem cells, and the more differentiated cells that can be derived from them, with the already characterized human embryonic and adult stem cell preparations.

A. Human Adult Stem Cells

1. Human Mesenchymal Stem Cells.

Bone marrow contains at least two major kinds of stem cells, hematopoietic stem cells¹⁰ that give rise to the red cells and white cells of the blood, and mesenchymal stem cells^viii that can be reproducibly isolated and expanded in vitro, and that can differentiate in vitro into cells with properties of cartilage, bone, adipose (fat), and muscle cells.¹⁴

The characteristics (morphology, expressed proteins, and biological properties) of these cells have been somewhat difficult to specify, because they appear to vary depending upon the in vitro culture conditions and the specific cell preparation.¹⁵ However, there is a recent report indicating that MSCs, if isolated using three somewhat different methods, give rise to stem cell preparations whose properties are very similar to one another.¹⁶ Using dual antibody staining and fluorescence-activated cell sorting, Gronthos and colleagues¹⁷ isolated human MSCs in almost pure form and expanded them substantially in vitro. Thus, human MSC preparations isolated in different laboratories by different methods may have similar but not identical properties.

A molecular analysis of genes expressed in a single-cell-derived colony of MSCs provided evidence for the activity of genes also turned on in bone, cartilage, adipose, muscle, hematopoiesis-supporting stromal, endothelial, and neuronal cells.¹⁵ These results are surprising in that MSCs derived from a single cell appear to be expressing genes associated with multiple major cell lineages. It is possible that different cells within the colony had already entered into distinct differentiation pathways, resulting in a developmentally heterogeneous population composed of several different cell types.

Mesenchymal stem cells are important for research and therapy for several reasons. First, because they can be differentiated in vitro into multiple cell types, they make possible detailed research on the molecular events underlying differentiation into bone,¹⁸ cartilage, and fat cell lineages. Second, they have recently been shown to support the in vitro growth of human embryonic stem cells.¹⁹ Thus, they could replace the mouse feeder cells used previously, obviating the need to satisfy FDA requirements for xenotransplantation, should the ESCs or their derivatives ever be used in human clinical research or transplantation therapy. Third, clinical studies are already underway in which MSCs are co-transplanted with autologous hematopoietic stem cells into cancer patients to replace their blood cell-forming system, destroyed by radiation or high dose chemotherapy.²⁰ It is believed that the MSCs will support the repopulation of the bone marrow by the injected hematopoietic stem cells.

In addition, injecting allogeneic MSCs (MSCs from a genetically different human donor) may also prove valuable in modulating the immune system to make it more accepting of foreign tissue grafts [see Itescu review, reference (5)]. Finally, MSCs have the potential for cell-replacement therapies in injuries involving bone, tendon, or cartilage and possibly other diseases. They are, in fact, already being tested as experimental therapies for osteogenesis imperfecta,²¹ metachromatic leukodystrophy, and Hurler syndrome.²² These last two studies are of great interest, since allogeneic MSCs were used and no serious adverse immune reactions were noted.

2. Multipotent Adult Progenitor Cells (MAPCs).

Verfaillie and coworkers recently described the isolation of MAPCs from rat, mouse, and human bone marrow [see (3) and references cited therein]. Like MSCs, MAPCs can also be differentiated in vitro into cells with the properties of cartilage, bone, adipose, and muscle cells. In addition, there is evidence for the in vitro differentiation of human MAPCs into functional, hepatocyte-like cells,²³ a potential that has not so far been shown for MSCs. There is increasing interest in MAPCs, both as potential precursors of multiple differentiated tissues and, ultimately, for possible autologous transplantation therapy.

The relationship between human MSCs and the human MAPCs described by Verfaillie and coworkers [see (3)] needs to be clarified by further research. Both kinds of cells are isolated from bone marrow aspirates as cells that adhere to plastic. Each can be differentiated in vitro into cells with cartilage, bone, and fat cell properties. They express several of the same cell antigens, but are reported to differ in a few others.³ MAPCs have to be maintained at specific, low cell densities when grown in vitro, otherwise they tend to differentiate into MSCs.³ It remains important that the isolation and properties of MAPCs be reproduced in additional laboratories.

3. Human Neural Stem Cells.

The nervous system is made up of three major types of cells; neurons or nerve cells proper, and two kinds of supporting or glial cells (oligodendrocyte, astrocyte). Stem cells capable of differentiating into one or more of these neural cell lineages can be isolated from brain tissue (particularly the olfactory bulb and lining of the ventricles)^24,25 and grown in vitro. In the presence of purified growth-factor proteins, the population of cells can be expanded by growth in vitro as round clumps of cells called neurospheres. However, many neurospheres grown in culture are developmentally heterogeneous in that they contain more than one neural cell type, and the number of self-renewing cells is frequently low (less than five percent).²⁶

Although neural stem cells are still insufficiently understood, they are already proving valuable in basic research on neural development. The ability to grow reproducible neural stem cells in vitro has facilitated identification of important neural stem cell growth factors and their cellular receptors. For example, human neural stem cells from the developing human brain cortex, expanded in culture in the presence of leukemia inhibitory factor (LIF), allowed growth of a self-renewing neural stem cell preparation for up to 110 population doublings. Withdrawal of LIF led to decreased expression of about 200 genes,²⁷ which were specifically identified through use of "gene chips" manufactured by Affymetrix. These genes are presumably involved in promoting or preserving the stem cell's capacity for self-renewal in the undifferentiated state. The number and specificity of the molecular changes characterized in these experiments powerfully illustrate the usefulness of neural and other stem cell preparations in basic biomedical research.

Human neural stem cells are also being injected into animals to test their effects on animal models of human neurological disease. To track the fate of the introduced human cells, they must first be modified or "marked" in ways that permit their specific detection.^ix Marked human neural stem cells are easily tracked after they are injected into experimental animals, making it possible to determine whether they survive and migrate following injection. Studies of this type have provided evidence that human neural cells can migrate extensively in the brain after injection.²⁸ In addition, such cells can be injected into animal models of human diseases such as intracerebral hemorrhage and Parkinson Disease (PD) to study their effect on the progression of the disease.²⁹ Although human neural stem cells may not yet be as well characterized as MSCs or ESCs, they are being actively studied with the hope that they can be used in future treatments for devastating neurological diseases such as Alzheimer Disease and PD.

4. Adult Stem Cells from Other Sources.

Prentice [see (4)] has summarized a large amount of recent information on preparations of stem cells isolated from amniotic fluid, peripheral blood, umbilical cord blood, umbilical cord, brain tissue, muscle, liver, pancreas, cornea, salivary gland, skin, tendon, heart, cartilage, thymus, dental pulp, and adipose tissue. Studies of many of the stem cell preparations from these sources are just getting started, and further work is needed to determine their biological properties and their relatedness to other stem cell types. In some cases, the long-term expandability in vitro of these stem cells has not been demonstrated. Yet, the demonstration that they can be isolated from such tissue compartments in animals should spur the search for similar human stem cell types.

As Prentice also reports,⁴ many attempts have already been made using various preparations of adult stem cells to influence or alter the course of diseases in animal models. Despite the fact that the stem cell preparations used are not well characterized, and reproducible results have yet to be obtained, preliminary findings are sometimes encouraging. It is of course not yet clear whether the injected cells are functioning as stem cells, fusing with existing host cells, or stimulating the influx of the host's own stem cells into the target tissue.^x But, if reproduced, these preliminary findings may point the way to future therapies, even in the absence of precise knowledge of the mechanism(s) of cellular action.

B. Human Embryonic Stem Cells

1. Human Embryonic Stem Cells (ESCs).

Human embryonic stem cells have been isolated from the inner cell masses of blastocyst-stage human embryos in multiple laboratories around the world.^xi There is great interest in understanding the properties of these cells because they hold out the promise of being able to be differentiated into a large number of different cell types for possible cell therapies, as contrasted with the more limited number of cell types available by differentiation of specific adult stem cell preparations. As of July 2003, 12 ESC preparations (up from 2 such preparations a year earlier) out of a total of 78 "eligible" preparations of human ESCs were available for shipment to recipients of U.S. federal research grants.^xii The review by Ludwig and Thomson² lists more than 40 peer-reviewed human ESC primary research papers that have been published since the initial publication in 1998.

Although isolated from different blastocyst-stage human embryos in laboratories in different parts of the world, ESCs have a number of properties in common. These include the presence of common cell surface antigens (recognized by binding of specific antibodies), expression of the enzymes alkaline phosphatase and telomerase, and production of a common gene-regulating transcription factor known as Oct-4. At least 12 different preparations of ESCs have been expanded by growth in vitro, frozen and stored at low temperature, and at least partially characterized.¹³ Some of these ESC preparations have been "single-cell cloned."

Human ESCs have been differentiated in vitro into neural (neurons, astrocytes, and oligodendrocytes), cardiac (synchronously contracting cardiomyocytes), endothelial (blood vessels), hematopoietic (multiple blood cell lineages), hepatocyte (liver cell), and trophoblast (placenta) lineages.2 In the case of neural and cardiac lineages, similar results have been obtained in different laboratories using different preparations of ESCs, thus fulfilling the "reproducible results" criterion described above. For other lineages, the results described have not yet been reproduced in another laboratory.

2. Embryonic Germ Cells.

Human embryonic germ cells are isolated from the primordial germ tissues of aborted fetuses. Gearhart¹ has summarized the results of recent research with human and mouse EG cells. One study focused on regulation of imprinted genes in EG cells: it showed "that general dysregulation of imprinted genes will not be a barrier to their (EG cell) use in transplantation studies."^{30 xiii}In addition, Kerr and coworkers³¹ showed that cells derived from human EG cells, when introduced into the cerebrospinal fluid of rats, became extensively distributed over the length of the spinal cord and expressed markers of various nerve cell types. Rats paralyzed by virus-induced nerve-cell loss recovered partial motor function after transplantation with the human cells. The authors suggested that this could be due to the secretion of transforming growth factor-&alpha and brain-derived growth factor by the transplanted cells and subsequent enhancement of rat neuron survival and function.

Until recently, work with human EG cells came primarily from one laboratory. Recently the isolation and properties of human EG cells have been independently confirmed.³² Because human EG cells share many (but not all) properties with ESCs, these cells offer another important avenue of inquiry.

3. Embryonic Stem Cells from Cloned Embryos (Cloned ESCs).

Although it has yet to be accomplished in practice, somatic cell nuclear transfer (SCNT) could create cloned human embryos from which embryonic stem cells could be isolated that would be genetically virtually identical to the person who donated the nucleus for SCNT: hence cloned ESCs [see (7)]. In theory, using such cloned embryonic stem cells from individual patients might provide a way around possible immune rejection (see below), though in practice this could require individual cloned embryos for each prospective patient-a daunting task. And clinical uses might require a separate FDA approval for every single cloned stem cell line or its derivatives.

The ability to produce cloned mouse stem cells and genetically modify them in vitro has made possible an experiment demonstrating the potential of cloned human embryonic stem cells in the possible future treatment of human genetic diseases. Rideout et al.³³ used a mutant mouse strain that was deficient in immune system function. They produced a cloned mouse embryonic stem cell line carrying the mutation, and then specifically repaired that gene mutation in vitro. The repaired cloned stem cell preparation was then differentiated in vitro into bone marrow precursor cells. When these precursor cells were injected back into the genetically mutant mice, they produced partial restoration of immune system function.

Production of cloned human embryonic stem cell preparations remains technically very difficult and ethically controversial. Recently however, Chen and coworkers³⁴ have reported that fusion of human fibroblasts with enucleated rabbit oocytes in vitro leads to the development of embryo-like structures from which cell preparations with properties similar to human embryonic stem cells can be isolated. This work needs to be confirmed by repetition in other laboratories.

In addition, further work is needed to decisively settle the question of whether rabbit (or human egg donor) mitochondrial DNA and rabbit (or human egg donor) mitochondrial proteins persist in the embryonic stem cell preparations. Persistence of these foreign mitochondrial proteins in these human ESC-like preparations could possibly increase the probability of immune rejection of the cloned cells, thus limiting their clinical application, although the immune reaction might not be as severe as that to foreign proteins produced under the direction of chromosomal genes.  The presence of foreign or aberrant mitochondria also carries the risk of transmitting mitochondrial disease (caused by defects in mitochondrial DNA) that could be detrimental to the cells and to the recipient into whom they might eventually be transplanted.

IV. Basic Research Using Human Stem Cells

Human stem cells are proving useful in basic research in several ways. They are useful in unraveling the complex molecular pathways governing human differentiation. For example, because ESCs can be stimulated in vitro to produce more differentiated cells, this transition can be studied in greater detail and under better-controlled conditions than it can be in vivo. In the best circumstances, these differentiated cells can be grown as largely homogeneous cell populations, and their gene expression profiles can be compared in detail.

Also, stem cell preparations can be used to produce populations of specialized cells that are not easily obtained in other ways. In one case, for example, this approach has provided large quantities of human trophoblast-like cells that have not been previously available.³⁵ In addition, cultures of differentiated cells derived from stem cells could be used to test new drugs and chemical compounds for toxicity and mutagenicity.³⁶ As experience with these differentiated derivatives of human ESCs grows, it may become possible to reduce or eliminate the use of live animals in such testing protocols.

In the near future, the differentiated state of various human cell types will be characterized not just by a few biological markers, but by the pattern and levels of expression of hundreds or thousands of genes. Integration of this knowledge with the catalog of all human genes produced during the Human Genome Project will gradually give us knowledge of which genes are key regulators of human development and which genes are central to maintaining the stem cell state.³⁷ Increased understanding of the molecular pathways of human cell differentiation should eventually lead to the ability to direct in vitro differentiation along pathways that yield cells useful in medical treatment. In addition, when the normal range of gene expression patterns is known, researchers can then determine which genes are expressed abnormally in various diseases, thus increasing our understanding of and ability to treat these diseases.

A group of stem cell researchers has recently outlined a set of important research questions, which, once answered will greatly enhance our understanding of human embryonic stem cells and their potential fates and possible uses.³⁸ They include the following:

• What is the most effective way to isolate and grow ESCs?
• How is the self-renewal of ESCs regulated?
• Are all ESC lines the same?
• How can ESCs be genetically altered?
• What controls the processes of ESC differentiation?
• What new tools are needed to measure ESC differentiation in vitro and in vivo?

V. Human Stem Cells and the Treatment of Disease

A major goal of stem cell research is to provide healthy differentiated cells that, once transplanted, could repair or replace a patient's diseased or destroyed tissues. In pursuit of this goal, one likely approach would start by isolating  stem cells that could be expanded substantially in vitro. A large number of the cultivated stem cells could then be stored in the frozen state, extensively tested for safety and efficacy as outlined above, and used as reproducible starting material from which to prepare differentiated cell preparations that will express the needed beneficial properties when they are transplanted into patients with specific diseases or deficiencies.

To make more concrete both the potential of this approach and the obstacles it faces, we will summarize, as a case study example, some current information on the properties of cells derived from human stem cell populations that have been used in an animal model of Type-1 diabetes. But before doing so, we discuss an obstacle to any successful program of stem cell-based transplantation therapy: the problem of immune rejection of the transplanted cells.

Much of the impetus for human stem cell research comes from the hope that stem cells (or more likely, differentiated cells derived from them) will one day prove useful in cell transplantation therapies for a variety of human diseases. Such cell transplantation would augment the current practice of whole organ transplantation. To the extent that the healing process works with in vitro derived cells, the need for organ donors and long waiting lists for organ donation might be reduced or even eliminated.

Will the recipient (patient) accept or reject the transplanted human cells? In principle, the problem might seem avoidable altogether: adult stem cells could be obtained from each individual patient needing treatment. They could then be grown or modified to produce the desired (autologous and hence rejection-proof) transplantable cells. But the logistical difficulties in processing separate and unique materials for each patient suggest that this approach may not be practical. The cost and time required to produce sufficient numbers of well-characterized cells suitable for therapy suggest that it will be cells derived from one or another unique stem cell line that will be used to treat many (genetically different) individual patients (allogeneic cell transplantation).

When allogeneic organ or tissue transplantation is currently done using, for example, bone marrow, kidney, or heart, powerful immunosuppressive drugs-carrying undesirable side effects-must be used to prevent immunological rejection of the transplanted tissue.⁵ Without such immunosuppression, the patient's T-lymphocytes and natural killer (NK) cells recognize surface molecules on the transplanted cells as "foreign" and attack and destroy the cells. Also, in whole organ transplantation donor T-lymphocytes and NK cells, entering the recipient with the transplanted organ, can also destroy the tissues of the transplant recipient (called "graft versus host" disease).

Are the differentiated derivatives of human stem cells as likely to incite immune rejection, when transplanted, as are solid organs? Do their surfaces carry those protein antigens that will be recognized as "foreign"? Experiments have been done to examine human ESC and MSC preparations growing in vitro for the expression of surface molecules known to play important roles in the immune rejection process. Drukker and coworkers³⁹ showed that embryonic stem cells in vitro express very low levels of the immunologically crucial major histocompatibility complex class I (MHC-I) proteins on their cell surface. The presence of MHC-I proteins increased moderately when the ESCs became differentiated, whether in vitro or in vivo. A more pronounced increase in MHC-I antigen expression was observed when the ESCs were exposed to gamma-interferon, a protein produced in the body during immune reactions. Thus, under some circumstances, human ESC-derived cells can express cell surface molecules that could lead to immune rejection upon allogeneic transplantation.

Similarly, Majumdar and colleagues showed that human mesenchymal stem cells in vitro express multiple proteins on their cell surfaces that would enable them to bind to, and interact with, T-lymphocytes. They also observed that gamma-interferon increased expression of both human leukocyte antigen (HLA) class I and class II molecules on the surface of these MSCs.⁴⁰ These results indicate that it will probably not be possible to predict, solely on the basis of in vitro experiments, the likelihood that transplanted allogeneic MSCs would trigger immune rejection processes in vivo.

Many further studies in this area are badly needed. At this time there is insufficient information to determine which, if any, of the approaches to get around the rejection problem will eventually prove successful.

1. The Disease and Its Causes.

The human body converts the sugar glucose into cell energy for heart and brain functioning, and indeed, for all bodily and mental activities. Glucose is derived from dietary carbohydrates, is stored as glycogen in the liver, and is released again when needed into the bloodstream. A protein hormone called insulin, produced by the beta cells in the islets of the pancreas, facilitates the entrance of glucose from the bloodstream into the cells, where it is then metabolized. Insulin is critical for regulating the body's use of glucose and the glucose concentration in the circulating blood.

The body's failure to produce sufficient amounts of insulin results in diabetes, an extremely common metabolic disease affecting over 10 million Americans, often with widespread and devastating consequences. In some five to ten percent of cases, known as Type-1 diabetes (or "juvenile diabetes"), the disease is caused by "autoimmunity," a process in which the body's immune system attacks "self." ^xiv T-lymphocytes attack the patient's own insulin-producing beta cells in the pancreas. Eventually, this results in destruction of ninety percent or so of the beta cells, resulting in the diabetic state.

With a deficiency or absence of insulin the blood glucose becomes elevated and may lead to diabetic coma, a fatal condition if untreated. Chronic diabetes, both Type-1 and the much more common Type-2 diabetes (which is not autoimmune, but largely genetic), causes late complications in the retina, kidneys, nerves, and blood vessels. It is the leading cause of blindness, kidney failure, and amputations in the U.S. and a major cause of strokes and heart attacks.

Type-1 diabetes is a devastating, lifelong condition that currently affects an estimated 550,000-1,100,000 Americans,⁴¹ including many children. It imposes a significant burden on the U.S. healthcare system and the economy as a whole, over and above the disabilities and impairments borne by individual sufferers. Recent estimates suggest that treatment of all forms of diabetes costs Americans a total of $132 billion per year.⁴² At 5-10 percent of all diabetes cases, the costs of Type-1 diabetes can be estimated as $6.5-13 billion per year.

2. Current Therapy Choices and Outcomes.

The current treatment of Type-1 diabetes consists of insulin injections, given several times a day in response to repeatedly measured blood glucose levels. Although this treatment is life-prolonging, the procedures are painful and burdensome, and in many cases they do not adequately control blood glucose concentrations. Whole pancreas transplants can essentially cure Type-1 diabetes, but less than 2,000 donor pancreases become available for transplantation in the U.S. each year, and they are primarily used to treat patients who also need a kidney transplant. Like all recipients of donated organs, pancreas transplant recipients must continuously take powerful drugs to suppress the immunological rejection of the transplanted pancreas.

In addition to treatment with whole pancreas transplantation, small numbers of Type-1 diabetes patients have been treated by transplantation of donor pancreatic islets into the liver of the patient coupled with a less intensive immunosuppressive treatment (the Edmonton protocol).⁴³ Expanded clinical trials of this procedure are currently underway. Scientists are also evaluating methods of slowing the original autoimmune destruction of pancreatic beta cells that produces the disease in the first place.

Whole pancreas and islet cell transplants ameliorate Type-1 diabetes, but there is nowhere near enough of these materials to treat all in need. To overcome this shortage, people hope that human stem cells can be induced-at will and in bulk-to differentiate in vitro into functional pancreatic beta cells, available for transplantation. Of course, it would still be crucial to prevent immunological destruction of the newly transplanted stem cell-derived beta cells.

3. Stem Cell Therapy for Type-1 Diabetes?

Initial experiments in mice suggested that insulin-producing cells could be obtained from mouse embryonic stem cells following in vitro differentiation.⁴⁴ Can this approach be extended to human stem cells? A number of attempts have been made, with promising initial findings, yet they are not easily evaluated, partly because the criteria for characterizing the cells are not standardized. In a recent paper, Lechner and Habener provided a list of six criteria to define the characteristics of pancreas-derived "beta-like" cells that could be potentially useful in treatment of Type-1 diabetes.⁴⁵

We have used those criteria to facilitate assessment of the current state of progress toward development of functional "beta-like" cells that might eventually be tested in Type-1 diabetes patients. Table 1 summarizes and compares the properties of human cell preparations recently produced in research seeking this objective by Abraham et al.,⁴⁶Zulewski et al.,⁴⁷ Assady et al.,⁴⁸ Zhao et al.,⁴⁹ and Zalzman et al.,⁵⁰ and tested in mouse models of human diabetes.

Table 1: Comparison of Insulin-Producing Cells Derived from Human Stem Cells

Beta-cell-specific markers: PDX-1: (a.k.a IPF-1), a regulatory gene important for beta-cell function; Glucokinase (GK), an enzyme that detects high levels of glucose and modulates insulin release; GLUT-2, a protein associated with glucose-responsive insulin secretion. CK-19 is a marker for pancreatic duct cells. Insulin production criteria: synthesis of messenger RNA for insulin or preproinsulin; tests for the presence of insulin protein; and ultrastructural studies (electron microscopy) to determine the presence of typical insulin secretory granules. In addition, the glucose-responsiveness of insulin production and release, an essential characteristic of normal beta-cell function, was assessed in a number of the studies described above. Both mouse models of Type-1 diabetes used mice that had a condition known as Severe Combined Immunodeficiency (SCID) and were treated with streptozotocin (STZ), a drug that induces selective destruction of the insulin-producing cells. The mice in the Zalzman study were also born with a form of mouse diabetes, and are called Non-Obese Diabetic (NOD) mice.

As the results described in Table 1 indicate, cells derived from some human stem cells transplanted into specific strains of mice mimicking major aspects of Type-1 human diabetes⁵¹ were able to reverse high blood glucose concentrations. Although these results are encouraging, the transplant rejection question remains unanswered because the likely immune rejection of the transplanted human cells was prevented in these experiments by using special strains of immunodeficient mice that lack the capacity to recognize and attack foreign cells.

No tumors were observed in the transplanted mice, but the experiments were terminated after about three months, an insufficient time for much tumor development to occur. Because many Type-1 diabetes patients are children and because a largely effective therapy (insulin injection) is currently available, the introduction of islet cell transplant therapy will need a high degree of certainty that the introduced cells or their derivatives will not become malignant over the course of the patient's life. Stringent tests of the cancer-causing potential of candidate cell preparations will be required, including multi-year studies in animals that live longer than mice or rats. Long-term follow-up of children and adult patients who had received bone marrow transplants many years ago has revealed an increased risk of severe neurologic complications⁵² and a variety of types of cancer.⁵³

Before stem cell based therapies are used to treat human diseases, they will have to gain approval through the Food and Drug Administration (FDA) regulatory process. The first step in this process is filing an Investigational New Drug (IND) application. As of July 2003, four IND applications have been filed for clinical applications of mesenchymal stem cells. The disease indications include: (1) providing MSC support for peripheral blood stem cell transplantation in cancer treatment, (2) providing MSC support for cord blood transplantation in cancer treatment, (3) using MSCs to stimulate regeneration of cardiac tissue after acute myocardial infarction (heart attack), and (4) using MSCs to stimulate regeneration of cardiac tissue in cases of congestive heart failure. The first two applications are currently in Phase II of the regulatory process, with pivotal Phase III trials scheduled to begin in 2004.⁵⁴

VI. Private Sector Activity

In the United States, much of the basic research on animal stem cells and human adult stem cells has been publicly funded. Yet before 2001, research in the U.S., using human ESCs could only be done in the private sector (the locus also of much research on animal and human adult stem cells). The current state of knowledge about human ESCs (and also about human MSCs) reflects pioneering and on-going stem cell research funded by the private sector in the U.S.^54,55 For example, the work that led to the 1998 reports of the first isolation of both ESCs and EGCs, was funded by Geron Corporation. Embryonic and adult stem cell research is today vigorously pursued by many companies and supported by several private philanthropic foundations,⁵⁶ and the results of some of this research have been published in peer-reviewed journals.⁵⁷ Private sector organizations have pursued and been awarded patents on the stem cells themselves and methods for producing and using them to treat disease. As noted above, at least one company (Osiris Therapeutics) has protocols under review at the FDA for clinical trials with MSCs. It seems likely that private sector companies will continue to play large roles in the future development of stem cell based therapies.

VII. Preliminary Conclusions

While it might be argued that it is too soon to attempt to draw any conclusions about the state of a field that is changing as rapidly as stem cell research, we draw the following preliminary conclusions regarding the current state of the field.

Human stem cells can be reproducibly isolated from a variety of embryonic, fetal, and adult tissue sources. Some human stem cell preparations (for example, human ESCs, EGCs, MSCs, and MAPCs) can be reproducibly expanded to substantially larger cell numbers in vitro, the cells can be stored frozen and recovered, and they can be characterized and compared by a variety of techniques. These cells are receiving a large share of the attention regarding possible future (non-hematopoietic) stem cell transplantation therapies.

Preparations of ESCs, EGCs, MSCs, and MAPCs can be induced to differentiate in vitro into a variety of cells with properties similar to those found in differentiated tissues.

Research using these human stem cell preparations holds promise for: (a) increased understanding of the basic molecular process underlying cell differentiation, (b) increased understanding of the early stages of genetic diseases (and possibly cancer), and (c) future cell transplantation therapies for human diseases.

The case study of developing stem cell-based therapies for Type-1 diabetes illustrates that, although insulin-producing cells have been derived from human stem cell preparations, we could still have a long way to go before stem cell-based therapies can be developed and made available for this disease. This appears to be true irrespective of whether one starts from human embryonic stem cells or from human adult stem cells. The transplant rejection problem remains a major obstacle, but only one among many.

Human mesenchymal stem cells are currently being evaluated in pre-clinical studies and clinical trials for several specific human diseases.

Much basic and applied research remains to be done if human stem cells are to achieve their promise in regenerative medicine.⁵⁸ This research is expensive and technically challenging, and requires scientists willing to take a long perspective in order to discover, through painstaking research, which combinations of techniques could turn out to be successful.  Strong financial support, public and private, will be indispensable to achieving success.

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FOOTNOTES

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ENDNOTES

• The stem or progenitor cell should be clonally isolated or marked; "enrichment" of a certain cell type alone is not sufficient.
• In vitro differentiation to a fully functional beta cell should be unequivocally established. Insulin expression per se does not make a particular cell a beta cell. The expression of other markers of beta cells (e.g. Pdx1/Ipf1, GLUT2, and glucokinase) or other endocrine islet cells should be demonstrated.
• Ultrastructural studies should confirm the formation of mature endocrine cells by identification of characteristic insulin secretory granules.
• The in vitro function of endocrine cells, differentiated from stem cells, should be reminiscent of the natural counterparts. For beta cells, this would imply a significant glucose-responsive insulin secretion, adequate responses to incretin hormones and secretagogues, and the expected electrophysiological properties.
• In vivo studies in diabetic animals should demonstrate a reproducible and durable effect of the stem/progenitor-derived tissue on the attenuation of the diabetic phenotype. It should also be demonstrated that removal of the stem cell-derived graft after a certain period of time leads to reappearance of the diabetes.
• For future clinical use, the tumorigenicity of stem/progenitor tissue should be determined.
• Additionally, immune responses toward the transplanted cells should be examined.

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