Adult stem cells.

• Author: Prentice, David A.

Within just a few years, the possibility that the human body contains cells that can repair and regenerate damaged and diseased tissue has gone from an unlikely proposition to a virtual certainty. Adult stem cells have been isolated from numerous adult tissues, umbilical cord, and other non-embryonic sources, and have demonstrated a surprising ability for transformation into other tissue and cell types and for repair of damaged tissues. This paper will examine the published literature regarding the identity of adult stem cells and possible mechanisms for their observed differentiation into tissue types other than their tissue of origin. Reported data from both human and animal studies will be presented on the various tissue sources of adult stem cells and the differentiation and repair abilities for each source, especially with regards to current and potential therapeutic treatments.

Adult stem cells have received intense scrutiny over the past few years due to surprising discoveries regarding heretofore unknown abilities to form multiple cell and tissue types, as well as the discovery of such cells in an increasing number of tissues. The term "adult stem cell" is somewhat of a misnomer, because the cells are present even in infants and similar cells exist in umbilical cord and placenta. More accurate terms have been proposed, such as tissue stem cells, somatic stem cells, or post-natal stem cells. However, because of common usage this review will continue to use the term adult stem cell.

This paper will review the literature related to adult stem cells, including current and potential clinical applications (with apologies to the many who are not cited, due to the exponential increase in papers regarding adult stem cells and the limitations of this review.) The focus will be on human adult stem cells, but will also include results from animal studies which bear on the potential of adult stem cells to be used therapeutically for patients.

This paper will not attempt to review the literature related to hematopoietic stem cells, i.e., the bone marrow stem cell that is the immediate precursor for blood cells, and the formation of typical blood cells. Nor will this paper review the substantial literature regarding clinical use of bone marrow or bone marrow stein cell transplants for hematopoietic conditions such as various cancers and anemias, nor the striking clinical results seen for conditions such as scleromyxedema, multiple sclerosis, systemic lupus, arthritis, Crohn's disease, etc. (1) In these instances, the stem cells are used primarily to replace the hematopoietic system of the patient, after ablation of the patient's own bone marrow hematopoietic system. Finally, multipotent adult progenitor cells (MAPC'S), a bone marrow stem cell that has shown significant abilities at proliferation in culture and differentiation into other body tissues, (2) have been reviewed by Dr. Catherine Verfaillie in a separate paper for the President's Council on Bioethics, and the reader is directed to that review for more information.

Key questions regarding adult stem cells are: (1) their identity, (2) their tissue source of origin, (3) their ability to form other cell or tissue types, and (4) the mechanisms behind such changes in differentiation and effects on tissues and organs. Historically only a few stem cells were recognized in humans, such as the hematopoietic stem cell which produces all of the blood cell types, the gastrointestinal stem cell associated with regeneration of the gastrointestinal lining, the stem cell responsible for the epidermal layer of skin, and germ cell precursors (in the adult human, the spermatogonial stem cell.) These stem cells were considered to have very limited repertoires, related to replenishment of cells within their tissue of origin. These limitations were considered to be a normal part of the developmental paradigm in which cells become more and more restricted in their lineage capabilities, leading to defined and specific differentiated cells in body tissues. Thus, discovery of stem cells in other tissues, or with the ability to cross typical lineage boundaries, is both exciting and confusing because such evidence challenges the canonical developmental paradigm.

Stem Cell Markers

Identification of cells typically relies on use of cell surface markers--cellular differentiation (CD) antigens--that denote the expression of particular proteins associated with genomic activity related to a particular differentiation state of the cell. Identification also has relied on morphological and molecular indications of function, such as expression of specific enzymes. Since stem cells by definition have not yet taken on a specific differentiated function, their identification has relied primarily on use of cell surface markers, and only secondarily on production of differentiated products in various tissues. One stated goal has been to isolate a single putative adult stem cell, characterized fully by specific markers and molecular characteristics, and then to follow the differentiation of this single cell (and/or its progeny) to show that it indeed has multipotent or pluripotent capabilities (clonogenic ability). For bone marrow stem cells, selection of putative adult stem cells has usually excluded typical markers for hematopoietic lineages (lin (-)), CD45, CD38, with inclusion or exclusion of the hematopoietic marker CD34 and inclusion of the marker c-kit (CD117). Other proposed markers for adult stem cells are AC133-2 (CD133), which is found on many stem cell populations, (3) and C1q[R.sub.p], the receptor for complement molecule Clq, (4) found on a subset of CD3[4.sup.[+ or -]] human stem cells from bone marrow and umbilical cord blood. When transplanted into immunodeficient mice, C1q[R.sub.p] -pOsitive human stem cells formed not only hematopoietic cells but also human hepatocytes. Other methods of isolation and identification include the ability of putative stem cells to exclude fluorescent dyes (rhodamine 123, Hoechst 33342), allowing isolation by fluorescence-activated cell sorter (FACS) of a "side population" of cells within a tissue that have stem cell characteristics. Expression of the Bcrp1 gene (ABCG2 gene in humans) is apparently responsible for this dye exclusion, and could provide a common molecular expression marker for stem cells (50. A study of expressed genes from a single cell-derived colony of human mesenchymal stem cells identified transcripts from numerous cell lineages, (6) and a similar attempt at profiling the gene expression of human neural stem cell in culture with leukemia inhibitory factor (LIF) has been done, (7) perhaps providing an expressed molecular milieu which could identify candidate stem cells. Attempts to determine the complete molecular signature of gene expression common to human and mouse stem cells have shown over 200 common genes between hematopoietic and neural stem cells, with some considerable overlap with mouse embryonic stem cells as well. (8) The function of many of these genes is as yet unknown, but may provide distinctive markers for identification of adult stem cells in different tissues.

However, dependence on particular markers for prospective identification and isolation of adult stem cells seems unreliable. In particular, the use of specific hematopoietic markers such as the presence or absence of CD34, has yielded mixed results in terms of the identification of putative stem cells. There is evidence that the expression of CD34 and CD133 can actually change over time, and its expression may be part of a cycling phenomenon among human hematopoietic and mesenchymal stem cells in the bone marrow and peripheral blood, and perhaps in other tissues, (9) i.e., an isolated CD34 (+) cell may become CD34 (-), and then reacquire CD34 expression. Likewise, a systematic analysis of the cell surface markers and differentiation potential of supposedly distinct isolated populations of human bone marrow stem cells revealed no differences in practice between the cell populations. (10) Moreover, an analysis of genetic and ultrastructural characteristics of human mesenchymal stem cells undergoing differentiation and dedifferentiation has revealed reversibility in the characteristics studied. (11)

Thus, any attempt to isolate a single type of adult stem cell for study may not actually capture the intended cell, or may, by using a particular set of isolation or growth conditions, alter its gene expression. This idea has been elaborated by Thiese and Krause, (120 who note that this "uncertainty principle" means any attempt to isolate and characterize a cell necessarily alters its environment, and thereby potentially its gene expression, identity, and potential ability to differentiate along various lineages. Likewise, the stochastic nature of cell differentiation in such dynamic and interacting systems means that attempts to delineate differentiation pathways must include descriptions of each parameter associated with the conditions used, and still may lead only to a probabilistic outcome for differentiation of a stem cell into a particular tissue. Blau et al. (13) have raised the question of whether there may be a "universal" adult stem cell, residing in multiple tissues and activated dependent on cellular signals, e.g., tissue injury. When recruited to a tissue, the stem cell would take its cues from the local tissue milieu in which it finds itself (including the soluble growth factors, extracellular matrix, and cell-cell contacts.) Examples of such environmental influences on fate choice have been noted previously. (14) Thus, it may not be surprising to see examples of cells isolated using the same marker set showing disparate differentiative potentials, (15, 16, 17, 18) based on the context of the isolation or experimental conditions, or to see cells with different marker sets showing similar differentiation. In the final analysis, description...