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 cells,10 which give rise to the red cells and white cells of the blood, and mesenchymal stem cells,viii which 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.14The 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).26Although 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-a 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.