HEMATOPOIETIC STEM CELLS

CHAPTER44 HEMATOPOIETIC STEMCELLS A n n e Kessinger and G r a h a m Sharp INTRODUCTION Avll of the mature functional cells of the hematopoietic and immune systems originate from a ery small number of undifferentiated cells called hematopoietic stem cells (HSCs), the most primitive of which constitute about 1 in 105 of nucleated hematopoietic cells in bone marrow. HSCs also circulate in blood at a frequency of approximately 1 log less than their counterparts in the marrow. These cells, by a process of sequential clonal amplification and differentiation of their progeny, populate the hematopoietic and immune systems. Manipulation of the total mass of hematopoietic and immune (lymphoid) tissues presents special challenges because these interrelated cellular systems are arranged both as discrete masses of tissue as well as diffuse distributions throughout most other organs of the body. Migration is an essential component of their function (Sharp, 1993). Hematopoietic precursor cells in blood can establish endothelial cells in culture. In the adult, whether HSC and endothelial precursors are separate and distinct populations or whether they are closely related, perhaps even daughter cells of the same rare primitive stem cell, is not clear. In the embryo, HSCs and vascular stem cells probably are closely related and share a common precursor (Kluppel et al., 1997; Shalaby et al., 1997). Whether distinct precursors to these two cell types are established and their common stem cell is lost or whether a rare common stem cell persists but is dwarfed in frequency by slightly more differentiated lineage-restricted progeny of hematopoietic versus endothelial cells in the adult is as yet unknown. A similar puzzle exists in adult bone marrow that clearly contains HSCs, mesenchymal stem cells (Vogel, 1999), and cells that appear to be able to give rise to muscle (Ferrari et al., 1998). Are these stem cell populations entirely distinct or are they related? If they are related, when during development do they diverge? Is the divergence complete or do rare stem cells persist that are a common precursor? In other words, exactly how much plasticity exists in the most primitive HSC of adults? Neural stem cells can give rise to blood cells (Bjornson et al., 1999). Is the converse true? Some reviews of this topic imply this might be so (Bjorklund and Svendsen, 1999), but for now these important questions remain unanswered. The answers will have profound implications for tissue engineering of the HSC, because HSC collection is not controversial and considerable experience in transplanting them exists. Might HSCs be a noncontroversial alternative source to embryonic stem cells (O'Shea, 1999; Solter and Gearhart, 1999; Vogel, 1999) for a variety of tissues? In the embryo, hematopoietic stem cells originate in a region associated with the dorsal aorta, the lateral plate mesenchyme, and the yolk sac. These initial HSCs migrate via the embryonic circulation to the liver (which then becomes the primary hematopoietic organ of the fetus), to the spleen, and, before birth, to the developing bone marrow spaces to establish the primary hematopoietic organ of postnatal life (Lansdorp, 1995). The process of HSC migration to the bone marrow involves the expression of the chemokine receptor CXCR4 and interactions with its ligand, stromal-derived factor 1 (SDF1). CXCR4 knockout mice exhibit a defect of bone marrow hematopoiesis (Aiuti et al., 1997; Ma et al., 1998; Nagasawa et al., 1996); SDF1 is also implicated in the successful engraftment of human HSCs in immunodeficient N O D / S C I D mice (Peled Principles of Tissue Engineering Second Edition

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Kessinger and Sharp et al., 1999). The possibility that a small reserve of HSCs remains in the circulation has been postulated but remains unproved. In the adult, the active red marrow retracts largely to the axial skeleton, leaving the long bones populated with yellow (fatty) marrow (Tavassoli, 1989). The progeny of HSCs differentiate, migrate to establish the lympho-hematopoietic component of other organs (thymus, spleen, lymph node, Peyer's patches), and circulate throughout the tissues of the body. They express essential properties of hematopoietic and immune cell effector functions, cytokine secretion, and provision of key enzymes required by tissues for detoxification of harmful materials. Consequently, congenital abnormalities of HSCs can lead to hematopoietic, immune, and systemic diseases. Tissue engineering by way of replacement (transplantation) or manipulation of hematopoietic stem cells offers an approach to the therapy of these diseases. Because bone marrow and all of the lymphoid components of the body cannot be removed surgically, systemic therapies are required for manipulations of these tissues. These therapies may be either cytotoxic (radiation, chemotherapy, immunotherapy) or regulatory (recombinantly engineered cytokines), and have potential effects and toxicities for uninvolved tissues. Conversely, therapies targeted at nonhematopoietic or nonimmune tissues can damage the hematopoietic and immune systems. Such iatrogenic events can require cellular or tissue engineering to restore hematopoietic and immune function. The diffuse nature of hematopoietic and immune tissues presents challenges, as well as opportunities. The most primitive HSCs and other bone marrow or lymphoid cells can be harvested from the body, manipulated, and reinfused. The reinfused cells home to appropriate microenvironments and resume their original in viva functions or, if altered in function, begin their new activity. These properties can be exploited to introduce genes into the body to correct congenitally deficient genes, to add entirely new genes, or to alter genes expressed by the infused cells. Although genetic engineering of HSCs has succeeded in principle (Brenner and Rill, 1994), many practical issues remain to be solved before this approach will become an effective therapy. For example, the proportion of primitive HSCs in the cell cycle is a few percent at best (Goodell et al., 1996). Consequently, HSCs are difficult to transduce with vectors that require a proliferating target (AgrawaU et al., 1996; Kohn, 1996). The use of cytokines to stimulate proliferation can promote terminal differentiation and cytokine-exposed cells may not engraft well (Kittler et al., 1997). Even if transduction is successful, clonal succession of primitive HSCs can complicate the in vivo pattern of gene expression (Lansdorp, 1995). Systemic cytoreductive therapy may or may not be needed to "create space" for the infused manipulated cells. Quesenberry and colleagues have followed up on original observations by Brecher et al. (1982) that HSCs (bone marrow) infused into unmanipulated recipient mice competitively establish a significant degree of chimerism (Quesenberry et al., 1994). The key to this process appears to involve daily infusion of donor cells over a few days, rather than a single infusion. If purified primitive HSCs are infused, their competitive repopulating capacity is substantial. Additionally, such cells do divide following transplantation, which might lead to integration of genetic vectors present in the cells (Bradford et al., 1997). Clearly, the field of tissue engineering using genetically altered HSCs presents many interesting and exciting challenges to overcome before achieving the status of a routine clinical procedure. In this review, an overview of hematopoiesis and its regulation together with the practical aspects of harvesting and processing of HSCs is presented as a basis for the discussion of the tissue engineering of HSCs.

OVERVIEW OF HEMATOPOIESIS- PROPERTIES A N D REGULATION OF HEMATOPOIETIC STEM CELLS RELEVANT TO TISSUE ENGINEERING The hematopoietic stem cells compartment comprises a differentiation hierarchy in which relatively rare and uncommitted peripotential "primitive" stem cells give rise to multiple lineages of mature cells by a linear branching differentiation process (Sharp, 1993). Within each lineage, the cells are increasingly committed stochastically to the type of mature cells they produce (Ogawa, 1994). The existence of several "levels" of differentiation within this compartment potentially leads to confusion over the use of the terms "stem" cell, "progenitor" cell, "precursor" cell, etc. As a simplification, here all of these cell types are considered stem cells. Hematopoiesis occurs in specific stromal microenvironments (Tavassoli and Takahashi, 1982), which vary in location during development and aging, and in pathologic situations. Stro-

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mal cells in these microenvironments have multiple interactions with developing hematopoietic cells (Dexter, 1982; Lambertsen, 1984). These include the provision of extracellular matrix molecules (Anklesaria et al., 1991; Siczkowski et al., 1992; Yoder and Williams, 1995; Zipori et al., 1985), soluble and membrane-bound growth and cell survival factors (Du and Williams, 1994; Whetton and Spooncer, 1998), and adhesion molecules (Conget and Minguell, 1995; Verfaillie et al., 1994), as well as the regulation of the release of cells to the circulation at an appropriate stage of differentiation (Weiss and Geduldig, 1991). The potentials of marrow stromal cells have been reviewed (Owen, 1988). Stromal cell lines have been developed that duplicate many of these functions (Zipori, 1989a). A mesenchymal stem cell from bone marrow has been identified (Pittenger et al., 1999). The use of such cells combined with HSCs might be necessary to solve some of the tissue engineering challenges presented by HSCs. Some of the progeny of primitive stem cells must pass through, or otherwise be influenced by, additional microenvironments, for example, the thymus, for key differentiation events to occur. Collectively, these processes produce the multiple mature cell types needed to sustain the hematologic and immunologic needs of the organism. Attempts to engineer these tissues, therefore, require a duplication of their key components, for example, provision of an appropriate population of stromal cells. The functional status of these stromal cells, i.e., whether they are promoting quiescence versus active replication of their associated HSC, also may be important. Maybe more than one stromal cell type will be necessary to provide a microenvironment "compleat" (Walton, 1653) for HSCs?

ORGANIZATION OF THE STEM CELL COMPARTMENT Do the most primitive hematopoietic stem cells comprise a "cell renewal" system to provide replacement of any cells lost from this compartment by cell division, or are a fixed number of stem cells available in an organism large enough to provide vast numbers of progeny without becoming significantly depleted in a lifetime (Harrison et al., 1987; Lansdorp, 1995; Micldem et al., 1983)? These two hypotheses of stem cell compartment organization are not necessarily mutually exclusive. Both receive some experimental support, and experiments that test unequivocally which of these alternatives, if either, is correct have been difficult to devise (Van Zant et al., 1997). The answer underlies the likely success or failure of attempts to expand or genetically manipulate most primitive stem cells ex vivo for the purposes of tissue engineering (Lansdorp, 1995). The consequences of telomere shortening in this circumstance are a concern (Engelhardt et al., 1997; Lansdorp et al., 1997). The best available evidence suggests that, in humans, HSCs, as identified by the CD34 antigen, self-renew in fetal liver to establish the physiologically necessary stem cell compartment. In the adult, few, if any, CD34-positive cells self-renew under normal circumstances (Landsdorp et al., 1993). Fetal hematopoietic stem cells may differ significantly in their properties and perhaps in their regulation from stem cells in the adult. Although controversial for clinical use, fetal tissue might offer some advantages in engineering new or replacement tissues (Harrison et al., 1997). Cord blood is an additional and potentially very useful source of stem cells for tissue engineering (Wagner et al., 1995). In the mouse, the most primitive HSCs are CD34-negative cells, and CD34positive cells are their progeny (Osawa et al., 1996). CD34-negative stem cells likely exist in humans, but their proportion and place in the stem cell hierarchy remain to be defined (Goodell et aL, 1997).

STROMAL MICROENVIRONMENTS Just as hematopoiesis in vivo is limited to specific sites during development, hematopoietic cells demand stringent conditions for their maintenance in culture. For many years, the data suggested that mixed stromal cell populations, including large "blanket" cells, were necessary to maintain hematopoietic stem cells in culture (Dexter, 1982; Tavassoli and Takahashi, 1982). Stromal cell lines that support hematopoiesis have now been developed (Itoh et al., 1989; Zipori, 1989a). Although an intimate association of stem cells with stromal cells is probably essential, contact may not be needed for their survival and differentiation (Verfaillie, 1992). Currently, all of the interactive properties of stromal cells with stem cells cannot be completely duplicated with known recombinant survival, growth, and differentiation factors. Consequently, the mechanisms by which cloned stromal cells support hematopoietic stem cells from several species (mouse, swine, and humans) in culture are incompletely defined. Potentially, these stromal cells either produce soluble

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growth factor(s) or present a membrane-bound growth factor(s), or both, which regulate stem cell survival and production or inhibition of apoptosis and self-renewal versus differentiation (Whetton and Spooncer, 1998; Williams, 1994). Stromal cells or other cell types may also be a source of inhibitors of stem cell differentiation (Waegell et al., 1994; Zipori, 1989b), and the presence and possible adherence of more differentiated hematopoietic cells (VerfaiUie et al., 1994) to stromal cells may be a feedback signal necessary to maintain inhibitor levels. If these differentiated cells or the inhibitors are removed, the inhibition of stem cell differentiation ceases and the stem cells may differentiate out of existence. Primitive HSCs likely have several signal transduction pathways that can be triggered by different signals (or combinations of signals). Stromal cells might be involved in regulating asymmetric versus symmetric division, which potentially involves a role for the notch gene and its ligands (Morrison et al., 1997; Whetton and Spooncer, 1998). Different signaling pathways may be used for stem cell survival (vs. apoptosis), self-renewal, proliferation, and lineagespecific differentiation. Indeed, regulation of cell production by modification of the rate of apoptosis of hematopoietic cells might be a critical component of the process. Methods to alter (suppress) apoptosis have received little attention in tissue engineering, although this may be one of the mechanisms by which microenvironmental stromal cells act. In myelodysplastic syndromes, increased apoptosis appears to be a critical problem (Mundle et al., 1994). Other than in very early embryogenesis, the majority of stromal cell precursors are likely distinct from primitive hematopoietic stem cells (Dexter and Allen, 1992; Huang and Terstappen, 1992, 1994). Consequently, in any engineered system, using adult cells might require both hematopoietic stem cells and stromal cells, but in a temporal order, i.e., stromal cells first followed by HSCs might be necessary to establish appropriate stromal niches (Allen, 1981). AssAYs oF STrM CELLS Assays to enumerate stem cells at specific stages of differentiation, especially assays that require the production of mature progeny, must be designed to meet the physiologic demands of the cell types assayed. For example, the most primitive stem cells demand either an in vivo environment or a culture system that permits an intimate association with stromal cells. Assays that meet this criterion include the long-term repopulating ability (LTRA) assay using marked cells, e.g., the Y chromosome of male cells transplanted into female recipients (Watt and Visser, 1992), or, secondarily, the assay of long-term culture initiating cells (LTC-ICs) (Sutherland et al., 1989). If the stem cells are grown on stromal lines and not harvested for assay of secondary colonies, but instead are assessed by hematopoietic cobblestone areas in the primary cultures, the assayed cells are designated as long-term culture cobblestone-area-forming cells (LTC-CAFCs) (Ploemacher et al., 1991; Neben et al., 1993). The frequency of CD34 § cells in human bone marrow is at least 100 times greater than the frequency of LTC-ICs (Watt and Visser, 1992). Because the frequency of LTC-ICs correlates most closely with the LTRA in the mouse (the only species in which LTRA frequency has been accurately determined), this implies that in humans only a small fraction of CD34 § cells actually belongs to the most primitive category of stem cells. The frequency of LTCICs in the blood of normal individuals is very low, 3/ml or 1/2 • 106 nucleated cells, or approximately 100 times lower than in bone marrow (Udomsakdi et al., 1992). More differentiated stem cells will grow in a semisolid medium, e.g., agar or methylcellulose, provided their physiologic regulator(s) of differentiation, i.e., colony-stimulating factors (CSFs) and interleukins (ILs), are provided (Watt and Visser, 1992). Sudden removal of CSFs or ILs can trigger apoptosis of the target cells, indicating the critical importance of these regulators (Williams et a/., 1990). Because the differentiation hierarchy from primitive stem cell to the most differentiated progenitor cell is a linear branching system, the mature functional progenies of the more differentiated members of this hierarchy become evident much faster in colony-forming cell (CFC) assays, e.g., mature cells from committed granulocyte-monocyte CFCs (GM-CFC) at 7 days. The mature progenies of intermediately differentiated, high-proliferative potential colony-forming cells (HPP-CFCs) are evident after 14-28 days of culture compared to LTC-ICs, which require 4 - 8 weeks for assay (Ploemacher et al., 1991). The more differentiated committed progenitors, GM-CFCs, require a single cytokine, GM-CSF, whereas HPP-CFCs need multiple CSFs or ILs and LTC-ICs must be grown on irradiated bone marrow stromal cells or carefully screened cloned stromal cell lines (Watt and Visser, 1992; Sutherland et al., 1989; Ploemacher et al., 1991).

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HUMAN H S C HARVESTING AND PROCESSING The posterior crests of the pelvic bone provide the only readily accessible site From which marrow HSCs in amounts sufficient to provide a successful hematopoietic graft can be extracted. Marrow cells are collected by aspiration through specially designed needles. Approximately 2 • 108 nucleated cells/kg recipient weight (approximately 11 ml/kg) are sufficient. The aspiration is usually performed while the donor is under the influence of general anesthesia. Collection of HSCs From the circulation currently requires apheresis, because the numbers of HSCs per milliliter of blood are quite small. The HSC donor can be treated with cytokines or, in the case of autologous donors, with myelosuppressive chemotherapy plus cytokines to increase the number of circulating CD34 § cells (Brugger et al., 1992). When sufficient numbers of these cells are mobilized into the circulation, an adequate graft product can sometimes be generated with a single apheresis procedure (Pettengell et al., 1993). However, autologous collections From patients who have already received substantial amounts of antitumor cy~otoxic therapy often require several apheresis procedures to collect a useful graft product. Not all normal donors mobilize successfully. About 10-20% of normal donors are poor mobilizers, which suggests that Factors other than prior cytotoxic therapy are implicated in poor mobilization. An undefined circulating inhibitor has been implicated (Kessinger and Sharp, 1998). Harvested marrow cells or peripheral HSCs are cryopreserved and stored in either a liquid nitrogen freezer or in temperatures at or below -80~ unless they are to be infused within 24-48 hr. If ex vivo manipulations of these cells are performed, such procedures generally occur prior to cryopreservation. At the time of infusion, the cells are thawed by placing their plastic storage bags in a warm water bath. The thawed cells are immediately infused into the circulation of an individual whose marrow function has been ablated with cytotoxic therapy. From there, the cells make their way to the appropriate niches in the marrow stroma to grow and restore hematopoietic function. Approximately 7 days are required after infusion before mature functional cells begin to appear in the circulation. In the meanwhile, patients are supported with red cell and platelet transfusions. Therefore, the first evidence that marrow function is returning after transplantation is the reappearance of circulating white blood cells. Replacement of congenitally defective H S C Patients with congenitally defective stem cells present with a myriad of clinical manifestations, depending on the specific defect. The resultant diseases include severe combined immune deftciency (SCID), a disorder of the lymphoid stem cell (Wiscott-Aldrich syndrome), a disorder of the lymphoid and hematopoietic stem cells (thalassemia, or erythroid disorder), Fanconi anemia, and osteopetrosis (a disorder of osteoclasts), among others. Destruction of the defective marrow and replacement with normal marrow from a compatible donor will reverse these otherwise fatal diseases. For patients who lack donors, genetically engineered stem cell grafts are an alternative (Kohn, 1996; Liu et al., 1997). In addition, patients with generalized congenital enzyme deficiencies such as osteopetrosis and Gaucher's disease can benefit from allogeneic hematopoietic stem cell transplant because the transplanted normal marrow cells provide progenies with sufficient amounts of enzyme to control the symptomatology of these disorders. Alternatively, they require engineered autologous grafts (Xu et al., 1994; Dunbar and Kohn, 1996). Patients with acquired stem cell defects can also be treated with allogeneic stem cell transplants. Disorders such as aplastic anemia, leukemia, myeloma, and dysmyelopoietic syndromes have been cured when the diseased marrow was destroyed and replaced with normal cells. Certain diseases (e.g., aplastic anemia subsequent to radiation or administration of chemotherapeutic agents) might arise from a microenvironmental defect (Testa et al., 1985; Mauch et al., 1995). Such disorders may not be amenable to cure by infusion of stem cells alone but could also require replacement of the microenvironment. Potentially; such defects would be primary targets for therapy with engineered bone marrow (Naughton et al., 1994). Unfortunately, observations in patients undergoing genetically engineered autologous transplants confirm data from primates and to some extent mice that, using current techniques, the transfection frequency of HSCs with retroviral vectors is low. The quiescent status of the majority of primitive hematopoietic stem cells is believed to be the cause of this problem. Forced cycling

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Kessinger and Sharp of the HSCs by cytokines increases the transfection frequency (Luskey et al., 1992). A concern with this approach, especially in adults, is that if the HSCs that are forced to cycle also differentiate, they may lose some of their "stemness" (Peters et al., 1995). The survival time of such transfected cells on transplantation will be reduced and the loss of transfected gene expression or perhaps even complete loss of the grafted cells might occur. Potentially, the application of an engineered system that more closely duplicates in vivo hematopoiesis and in particular provides stromal cells will lead to improved gene transduction successes (Xu et al., 1995; Naughton et al., 1992). This is the attraction of some of the newly developed perfusion systems. Artificial bone m a r r o w and perfused hematopoietic culture systems Historical

The evidence suggesting the need for a supporting matrix to maintain hematopoiesis has been reviewed by Fliedner and Cairo (1978). Knospe and colleagues, following the lead of Seki (1973), employed cellulose ester membranes that were coated with various cell types then implanted in vivo (Knospe, 1989). Long-term bone marrow cultures (LTBMCs) (Dexter et al., 1977) have been acknowledged to represent the most physiological ex vivo model of hematopoiesis. Hematopoietic colony-forming cells can be detected in human LTBMCs for about 6 months and much longer in cultures from subhuman primates and mice (Dexter et al., 1984). The demonstration that hematopoietic stem cells could be maintained in culture paved the way to the engineering of artificial bone marrow and perfused systems to support and grow stem cells. The application in modern times of tissue engineering to hematopoietic stem cells in bone marrow can be traced to pioneering work with artificial capillary cultures described by Knazek et aL (1972; Gullino and Knazek, 1979), who grew mouse fibroblasts and human choriocarcinoma cells, and to studies by Chick et al. (1975), who attempted to create an artificial pancreas. Based on this work, starting in the mid-1970s attempts to grow normal hematopoietic cells in artificial capillary cultures were made. The original intent was to grow bone marrow stroma or stromal cell lines in the extracapillary space and circulate hematopoietic cells suspended in medium through the stromal cell-surrounded capillaries. The technical challenges of this approach became quickly apparent. There was a very high cell death rate of the circulating hematopoietic cells. At that time damage by shear forces was thought to be primarily responsible. In retrospect, normal cell death due to physiological apoptosis was also a likely significant contributor. As an alternative, an attempt was made to grow an "artificial marrow" in the extracapillary spaces, which were perfused with medium flowing through the capillaries. Although this was somewhat more successful, the system was far from ideal because the extracapillary volume was relatively large compared to the volume of cells, perhaps because an inadequate matrix was available for normal cell growth. Additionally, the long-term (weeks to months) maintenance of sterility was difficult (Anderson and Sharp, 1981), emphasizing the need for relatively sophisticated engineering solutions to these problems. Consequently, partnerships with companies have become an essential element of tissue engineering of hematopoietic stem cells. In the remainder of this review reference is made to several company products; however, the references are not exhaustive and no endorsement of company products is implied. In order to obtain a better overview of available resources, a search of the Worldwide Web is recommended. The company names mentioned can serve as a measure of the effectiveness of the search engine employed. Naughton and colleagues have employed nylon mesh templates as a matrix on which to grow rodent hematopoietic cells. Mature cells and late-stage precursors were observed for 39 weeks (Naughton et al., 1987). Subsequently, they described the role of stromal cells in this system using rat, nonhuman primate, and human cells (Naughton and Naughton, 1989). The threedimensional nylon mesh system supports human bone marrow stem cell differentiation into multiple lineages (Naughton et al., 1990) and can be used to evaluate the effects of drugs on hematopoiesis (Naughton et al., 1991) and for gene transfer studies (Naughton et al., 1992). Naughton et aL (1994) evaluated the effects of surgical implantation of bioengineered bone marrow tissue in rats. Active hematopoiesis was observed at sites of implantation for up to 110 days without the addition of exogenous growth factors. A company, Cytomatrix, has also evaluated a porous biocompatible matrix to support hematopoiesis (Rosenzweig, 1999). The problem of shear stress has been

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addressed by investigators at the National Aeronautics and Space Administration (NASA), who have devised microgravity bioreactors that are distributed by Synthecon (Goodwin et al., 1993). Although this latter approach appears successful, it is unlikely to become a routine laboratory procedure. Bone marrow organoids

In an attempt to circumvent the high loss of normal cells, established long-term bone marrow culture-derived cells were employed as starting material to establish bone marrow organoids. The majority of differentiated normal hematopoietic cells are lost from long-term cultures during the first few weeks of culture, which permits harvesting ofhematopoietic stem cells associated with the adherent layer. The supernatant is discarded from the culture and the remaining cells are scraped from the flask. The cells can be concentrated to a slurry by gently pelleting in a conical centrifuge tube. When cultured for a subsequent period, these cells form bone marrow organoids. A micropipette is used to deposit a volume of this cell slurry onto a polycarbonate filter floating on a "raft" in medium. The hematopoietic reconstituting potential of these organoids can be demonstrated by transplanting them to lethally irradiated syngeneic recipients (Sharp et al., 1985). If these organoids were transplanted under the kidney capsule of intact adult mice, then a fat organ resembling yellow bone marrow was formed. In contrast, in irradiated recipients not only did the organoid show hematopoietic differentiation, but spleen colonies were formed presumably by hematopoietic stem cells that migrated from the organoid (Sharp et al., 1985). Such organoids are also a potential long-term source of hematopoietic regulatory factors (Brockbank and van Peer, 1983). The full scope of the potential of these hematopoietic organoids has yet to be explored. Because there are reports that graft-versus-host disease (GVHD) does not occur with long-term cultured cells (Spooncer and Dexter, 1983), whether organoids or artificial bone marrow alter the chimeric status and extent of GVHD in allogeneic recipients should be examined. Much more information has been gathered following infusion of bone marrow or blood as a cell suspension, which produces regeneration in the context of recipient stroma rather than the organoids, which possess donor stroma. P e r f u s e d systems

Engineering solutions to the problems of shear stress, nutrition, oxygenation, etc. have been applied and this has led to the development of successful perfusion systems in which hematopoieric stem cell numbers can be amplified. Schwartz et al. (1991) demonstrated that increased longterm bone marrow perfusion was superior to the traditional approach. Establishment of more optimal perfusion conditions supported the continuous stable generation of human progenitor cells for over 5 months in culture (Schwartz et al., 1991). This information was employed to design and construct a perfusion culture system employing bioreactors, which provides at least 1 log expansion over 14 days of total cells and of various progenitor cell types and almost 1 log expansion of the more primitive LTC-ICs (Koller et al., 1993). The original system required establishment of a stromal layer and represents an automated media exchange system rather than a fully perfused culture system. Even so, an automated version of this system marketed by Aastrom permits from 10to 15-fold marrow cell expansion under good manufacturing practice (GMP) conditions with minimal operator intervention (Bachier et aL, 1999). Stem and progenitor cells expanded using this system are now being evaluated in clinical trials (Koller et al., 1993; Palsson, 1994). Bioreactots for stem cell expansion have also been described by Wang and Wu (1992), providing a severalfold increase in cell output over an 8-week period (Wang et al., 1995). Several other groups are working on perfusion systems (Kompala and Highfill, 1994; Papoutsakis et al., 1994). Ex vivo expanded progenitor cells have been shown to contribute to reconstitution of transplanted patients (Brugger et al., 1995). Newer technologies are approaching the goal of fully perfused culture systems. This approach is being pursued by Cytometrix. Acordis Research has also developed a fully perfused, oxygenated, waste-exchanging, culture perfusion unit (Glockner et aL, 1999), which should permit duplication of the environment experienced by blood stem cells. This system also accommodates the establishment of a stromal matrix that might more closely duplicate bone marrow microenvironment. Such systems may provide the environment for controlled proliferation of primitive stem cells without their terminal differentiation, thus duplicating events during the first 2 weeks post-

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transplantation in vivo. Controlled proliferation of primitive stem cells might significantly increase the frequency of gene transduction, which should greatly amplify the potential applications of tissue-engineered hematopoietic stem cells. In addition to the application of gene-marked stem cells (Brenner and Rill, 1994), HSCs engineered for therapy have been employed in the treatment of adenosine deaminase SCID (ADA-SCID) (Kohn, 1996), Gaucher's disease (Dunbar and Kohn, 1996), and Fanconi anemia (Liu et al., 1997), and HSCs engineered to be resistant to the human immunodeficiency virus (HIV) by Systemix are currently in a phase I/II trial (Dutton, 1999). Additionally, work has begun on the development of an artificial thymus (Chen, 1994). After about 25 years in development, tissue-engineered hematopoietic stem cells have finally been administered to humans. The outcome of transplantation of even more primitive stem cells grown and manipulated ex vivo is currently awaited (Kessinger, 1995), and the full potential of engineered stem cells has become an exciting topic of both research and contemplation.

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