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.
A. Initial Stem Cell Preparations Can Contain Multiple Cell Types
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).
B. Genetically Homogenous Stem Cells through Single Cell Cloning
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.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.
E. Developmental Heterogeneity of Stem Cell Preparations
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, are most likely to yield experimental results that will be reproducible. Preparations with these properties will be the most useful both in basic research and in investigations of possible clinical applications.
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