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Advances in hematopoietic stem cell culture
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Advances in hematopoietic stem cell culture Julie Audet*, Peter W Zandstra*, Connie J Eavest and James M Piret* Recent advances in our understanding of the earliest stages of hematopoietic cell differentiation, and how these may be manipulated under defined conditions in vitro, have set the stage for the development of robust bioprocess technology applicable to hematopoietic cells. Sensitive and specific assays now exist for measuring the frequency of hematopoietic stem cells with long-term in vivo repopulating activity from human as well as murine sources. The production of natural or engineered ligands through recombinant DNA and/or combinatorial chemistry strategies is providing new reagents for enhancing the productivity of hematopoietic cell cultures. Multifactorial and dose-response analyses have yielded new insight into the different types and concentrations of factors required to optimize the rate and the extent of amplification of specific subpopulations of primitive hematopoietic cells. In addition, the rate of cytokine depletion from the medium has also been found to be dependent on the types of cell present. The discovery of these cell-type-specific parameters affecting cytokine concentrations and responses has introduced a new level of complexity into the design of optimized hematopoietic bioprocess systems.
Addresses *Biotechnology Laboratory and Department of Chemical and Bio-Resource Engineering, and tDepartment of Medical Genetics, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada :.tTerryFox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, V5Z 1L3, Canada Current Opinion in Biotechnology 1998, 9:146-151 htt p://biomednet.com/elecref/0958166900900146 © Current Biology Ltd ISSN 0958-1669
Abbreviations CFCs colony-forming cells Epo erythropoietin FL flt3-1igand IL interleukin LTC-lC long-termculture-initiating cells SF Steel factor slL-6R solubleversion of IL-6 receptor Tpo thrombopoietin
Introduction Hematopoietic stem cells capable of producing very large numbers of all blood cell types persist in the marrow throughout adult life. Current evidence indicates that the process by which these stem cells ultimately generate mature blood ceils in vivo normally spans many cell generations and is unidirectional. These concepts underlie the description of the hematopoietic system as a hierarchy of progenitors in which the most primitive stem cells
are uniquely characterized by their dual capacity for multi-lineage differentiation and self-renewal. Because different types of primitive hematopoietic progenitors are not morphologically distinguishable, their identification and characterization has had to rely on the development of other procedures. The most sensitive and discriminating of these are functional assays that measure the ability of primitive progenitor cells to give rise to specific numbers and types of mature blood cells either in vitro or in fifo. In vitro, such assays can detect a variety of apparently lineage-restricted and muhi-potent progenitors. These cells generate a corresponding spectrum of colony types when stimulated by particular growth factors in semi-solid media. The progenitors of these colonies are therefore called colony-forming cells (CFCs). An even more primitive compartment of hematopoietic cells, referred to as long-term culture-initiating cells (LTC-IC) has also been defined. LTC-IC continue to differentiate into CFC after five or more weeks of coculture with stromal fibroblasts [1]. To date, most hematopoietic cell bioreactor and bioprocess optimization studies have focused on improving CFC and LTC-IC yields as surrogate indicators of stem cell amplification. Recently, methodology for quantitating transplantable murine stem cells with long-term in vivo repopulating activity by limiting dilution analysis has been successfully adapted to human hematopoietic stem cells using immunocompromised mice as recipients [2°']. In addition, it has been shown that the number of human stem cells thus identified [2",3"*], like their murine counterparts [4"°], can be expanded in vitro without restriction of their transplantable stem cell properties. A number of biochemical engineering reviews have compared the outputs of various types of more differentiated hematopoietic cells in different types of bioreactor systems and have also examined the relative importance of some of the bioprocess parameters (e.g. 0 2 tension and perfusion rate) that affect these endpoints as well as summarizing initial clinical results obtained with transplants of in vitro expanded cells [5-7]. Here we focus on what has been learned recently about parameters that may limit the ability of various cytokines to support the expansion in vitro of the most primitive hematopoietic cell types. Increased production and availability of these primitive cells should advance the whole spectrum of hematopoietic stem cell culture applications. Cytokine requirements The unique properties of believed to be dictated by subset of genes, changes in
o f h e m a t o p o i e t i c cells hematopoietic stem cells are their expression of a specific which presumably herald and
Advances in hematopoietic stem cell culture Audet et aL
bring about the irreversible exit of cells from the stem cell compartment. T h e genes involved in such changes are just beginning to be identified and almost nothing is known about how their activation or repression is regulated. Nevertheless, it is clear that hematopoietic stem cells interact with many molecules in their extracellular milieu via transmembrane receptors (or receptor complexes) to maintain their viability, and to effect changes in their cell cycle progression and differentiated state. A key feature of any hematopoietic culture system, therefore, is the combination of cytokines it delivers to the microenvironment of the cells to be stimulated and how the concentrations of these cytokines are maintained over time, as both of these aspects of cytokine delivery will influence the cumulative response obtained. The most studied cytokine receptors expressed on hematopoietic cells belong to three general families, each family being defined by the structural similarities of its members: the tyrosine kinase receptors; the so-called hematopoietic growth factor receptors; and the gpl30 receptor family. Flk-2/flt3, the receptor for flt3-1igand (FL) [8] and c-kit, the receptor for Steel factor (SF) [9], are two examples of tyrosine kinase receptors. Both are expressed on very primitive hematopoietic cells and both are implicated in the regulation of early stages of hematopoiesis. In fact, the possibility that FL could play an important role in the stimulation of hematopoietic stem cells in vitro was first suggested by the restricted expression of its receptor on primitive hematopoietic cells [10,11] and later reinforced by the results of receptor antisense experiments which demonstrated an inhibition of CFC production in human long-term cultures [12]. Soon thereafter, potent effects of FL on both human and murine LTC-IC and repopulating cells, either in the presence or absence of stroma, were reported, particularly when certain other cytokines such as SF are present [2°',4"°,13,14°°,15]. T h e hematopoietic cytokine receptors do not possess an intrinsic tyrosine kinase domain but some share a number of other common features with the gpl30 family of receptors [16], such as an immunoglobulin-like region and some amino acid residue motifs. T h e ligands for two of these, interleukin (IL)-3 and thrombopoietin (Tpo; or c-mpl ligand), are of particular interest here. IL-3 is required (together with FL and SF) to maximize the expansion of human LTC-IC from CD34+CD38 - cells isolated from normal adult marrow [14"°]. If IL-3 is added at inappropriately high levels, however, it can also have a negative impact [17 °° ] similar to what has been described for primitive murine hematopoietic cells [15,18]. T p o can also act on very primitive cells and, in the mouse, T p o directly stimulates the proliferation and differentiation in vitro of long-term repopulating stem cells; however, the latter effect is dependent on the simultaneous exposure of the cells to other cytokines (SF, IL-3, erythropoietin [Epo]) [19]. T p o in concert with FL, SF and IL-3 has been found to be a significant stimulator of erythroid
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CFC production in short term cultures of adult human marrow CD34+CD38 - cells (Zandstra et al., unpublished data) and, as a single factor, T p o was shown to stimulate within 10 days a small net increase of LTC-IC from human CD34+CD38 - marrow cells [14°°,20]. Interestingly, in both of these latter studies, a significantly increased proportion of the CFC produced from Tpo-amplified LTC-IC were erythroid suggesting an additional ability of T p o to alter uncommitted cell differentiative behavior several cell generations later. T h e gpl30 receptor family includes receptors for IL-6, IL-11, leukemia inhibitory factor and oncostatin M. All of these cytokines interact with receptor complexes that contain the signal transducer subunit, gpl30. IL-11 is produced by stromal cells and has been shown, in concert with SF and FL, to stimulate the amplification in vitro of adult marrow murine stem cells [4°°,15,18,21°°]. Gpl30 appears to be the sole mediator of both IL-6 and IL-11-stimulated responses [16]; however, the particular ligand by which this is achieved may vary according to the cell being stimulated and its level of expression of the IL-6 versus the IL-11 binding protein. These restrictions can, however, be bypassed by the addition of a soluble version of the IL-6 receptor (slL-6R) together with IL-6 at a concentration sufficient to saturate all gpl30 molecules [22"]. Recent progress in understanding the molecular composition and three-dimensional structure of cytokine receptors and their accessory molecules expressed on hematopoietic cells as well as their need for dimerization [23] to activate intracellular kinase reactions has allowed a number of novel agonists to be developed. Examples include a hybrid cytokine of human IL-6 and the slL-6R [24"], human IL-3 and granulocyte-macrophage colony-stimulating factor [25], human IL-3 and Epo [26] and a divalent Epo mimetic [27°]. Although very few of these molecules have been analyzed for their ability to stimulate the proliferation and self-renewal of stem cells, the use and further development of engineered ligands to elicit such responses can be expected in the near future. Multifactorial analysis has proven to be a powerful experimental tool for identifying which cytokines can play a role (either alone or in combination) in stimulating the rapid expansion of particular types of primitive hematopoietic cells [14",17"',28]. Thus far, FL has been identified as the most important cytokine with such activity, although achieving maximal stem cell (LTC-IC or repopulating cell) amplification has required the presence of other cytokines, SF and IL-3 (in the case of human adult marrow cells) and IL-11/IL-6 plus slL-6R (in the case of murine adult marrow cells [28] or human cord blood cells [Zandstra eta/., unpublished data]). Interestingly, maximal concomitant production of CFC has been found to require additional factors: either IL-6, granulocyte/colony-stimulating factor or 13-nerve growth factor in the case of adult human
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marrow, or FL plus SF or IL-6/slL-6R in the case of human cord blood.
Dose-related effects of cytokines on hematopoieUc cell populations Investigations of the effects of cytokine concentration (both relative and absolute in cases where multiple cytokines are added) on primitive hematopoictic cell responses have also been informative. Not surprisingly, stimulation of the proliferation and differentiation of these cells shows typical log dose-response dependencies; however, different responses of the same original cells do not necessarily show the same cytokine concentration dependence. For example, in 10 day cultures of adult human marrow CD34+CD38 - cells, maximal expansion (60-fold) of LTC-IC requires the presence of 30-fold higher cytokine concentrations than are necessary to stimulate near maximal (280-fold) expansions of CFC in the same cultures [17"]. Furthermore, from studies of thcse effects at the single cell level, it has been possible to demonstrate that variability in LTC-IC amplification is determined by the extraccllular concentration of cytokines to which the cells are exposed and occurs largely independently of effects on their viability or proliferation. These findings support an emerging model of cytokine signaling where the cellular context in which the cytokinc initiates an intracellular signal is as important to the final response outcome as is the initial competence of the cell to bind the ligand. This context ultimately determines the magnitude and persistence of the cytokine signal over critical periods of time. Hence, both temporal and spatial aspects of cytokine delivery may modulate the ultimate response of a target cell to a given cytokine [29] which may be additionally influenced by ligand-induccd receptor down-modulation and cross-talk between receptors for different ligands, or their downstream signaling intermediates. Such issues are likely to be crucial to the development of large scale systems suitable for the controlled manipulation of hematopoietic stem cells in vitro. In particular, they highlight the importance of being able to measure and regulate cytokine concentrations in the culture medium, especially as the numbers and types of hematopoietic cell populations change over time.
Bioprocess developments in hematopoietic cell culture A small (threefold) but significant net expansion in vitro of human adult bone marrow LTC-IC was first reported in 1993 by Koller et al. [30]. This expansion of LTC-IC was achieved in a flat-plate stroma-containing bioreactor culture perfused by medium supplemented with the recombinant cytokines IL-3, SF and IL-6. Subsequently, in a similar bioreactor, additional cytokines were used (i.e. granulocyte-macrophage colony-stimulating factor, Epo and SF) and a 10-fold expansion of LTC-IC was
reported [31,32]. Depletion of the added cytokines was also noted [32]. It is difficult, however, to estimate how the actual concentration of a cytokine in the vicinity of the target cells in such a system would compare to the measured concentration of that cytokine in the flowing medium, due to local cytokine concentration gradients and the ability of hematopoietic cells to interact directly with cytokincs on the extracellular matrix or on the surface of adjacent stromal cells. More recent investigations have shown that similar expansions of primitive hematopoietic cells can be obtained in the absence of any stroma if an appropriate mixture of cytokines is provided [33"]. These responses have been obtained in cultures of purified subpopulations of cells (to eliminate stromal cells in the input innoculum) suspended in serum-free or serum-containing medium supplemented with various combinations of cytokines. This approach greatly reduces the complexity of the extracellular environment, which allows more reliable predictions regarding the outcome of the cultures because conditions are more easily reproduced. Furthermore, it has been suggested that direct contact of primitive hematopoietic cells with stromal cells may decrease the proliferative activity of the primitive hematopoietic cells [34,35°,36]. A modified culture chamber with multiple micro-grooves has recently been developed to provide a surface where less strongly anchored stroma-free cells can be easily retained while medium continuously perfuses above and at right angles to the grooves [37°]. Scaled-up suspension cultures for use in clinical trials have utilized mainly gas-permeable culture bags or T-flasks (tissue culture flasks designed for cell growth in vitro) to allow the cells to be incubated for the period required. For example, Moore and Hoskins [38] have reported an 18-fold expansion of human cord blood LTC-IC in gas-permeable bags supplemented with IL-1 and IL-3, and Bhatia et aL [39] measured a fivefold expansion of LTC-IC when CD34+DR - bone marrow cells from patients with chronic myeloid leukemia were cultured for 14 days in bags. Studies of human cells maintained in 50 or 100mL flat-bottomed spinner flasks with constant stirring (40 rpm) have been described by Sardonini and Wu [40] and Zandstra et aL [41,42"]. In these, the greatest expansion of total cell numbers was obtained in cultures initiated at a relatively high concentration of light density human marrow cells (106 cells/mL), which creates a more enriched population of primitive cells, with frequent replenishment of the medium. Bi-weekly addition of 10 ng/mL of IL-3, IL-6 and IL-11, plus 50ng/mL of FL and SF allowed within two weeks a 66-fold expansion of CFC numbers and a ninefold expansion of LTC-IC [42*]. Nevertheless, this feeding schedule did not sustain concentrations of IL-3, SF and FL at input levels. In addition, it was observed that the rate of cytokine depletion was increased at higher initial concentrations of the cytokin¢ [42"]. Thus,
Advances in hematopoietic stem cell culture Audet et aL
designing a feeding schedule to maintain optimal cytokine levels is probably much more complex than originally anticipated. Studies of hematopoietic factor-dependent cell lines have been a useful tool in cell culture research. Exposure of factor-dependent cell lines to high cytokine concentrations in vitro has generally been found to lead to a decrease in the expression by the cells of the corresponding receptor due to ligand-induced internalization of ligand-receptor complexes. Physiologically, this mechanism could be responsible for a desensitization of the cells via an attenuation of intracellular receptor-initiated signaling. This attenuation is due to two related but distinct phenomena generated by endocytic trafficking. First, receptors are lost from the cell surface by the combination of endocytosis and degradation, with the result that reduced numbers of receptors are available to interact with free extracellular ligand (receptor downregulation). Second, ligand is depleted from the extracellular medium by the same combination of endocytosis and degradation. T h e extent of c-kit (SF receptor) internalization by hematopoietic cells has been shown to be proportional to the extracellular SF concentration [9,43]. Thus, receptor/ligand internalization is a common and important mechanism in regulating factor-dependent proliferative responses of hematopoietic cells. Interestingly, primitive human adult marrow cells (defined by their CD34+CD38 - phenotype) are characterized by a 35-fold higher cell-specific rate of cytokine consumption when compared to other more differentiated types of marrow cells [42"]. These results confirm and extend those of Koller et al. [32] who found that the consumption of SF by marrow cells in culture increases as the cell numbers expand. T a k e n together, these findings are consistent with the detection of higher numbers of cytokine receptors on more primitive cells [44,45]. Physiological differences between the rates of glucose metabolism in primitive and mature hematopoietic cells have also been suggested. For example, Collins et al. [46] recently reported a correlation between specific glucose uptake rates, specific lactate generation rates and the percentage of CFC present over a broad range of culture conditions, including cells maintained in spinner flasks or T-flasks, and in serum-containing or serum-free medium. Accordingly, these authors suggested that lactate production rates might form the basis of a real-time analysis method for predicting when maximum concentrations of CFC have been reached.
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systems. T h e ultimate goal of in vitro production of human hematopoietic stem cells with long-term repopulating ability poses new challenges because of their unique cytokine requirements and the low incidence of these cells among normally accessible primary sources of human hematopoietic cell populations. An increased appreciation of many of the relevant parameters has recently begun to emerge from systematic analyses of cellular responses to, and depletion of, different cytokine supplements. In particular, such studies have revealed the very high cytokine dose requirements to maximize the expansion of primitive hematopoietic cells [17"] and their associated ability to deplete extracellular cytokine concentration at faster rates than their more differentiated progeny [42°]. Further developments can be expected from the cloning or engineering of new cytokines. T h e design of new ligands with increased binding affinity for specific cytokine receptors is an approach that has been used in other fields to create new therapeutic agents; however, potency has been shown to be dependent upon ligand/receptor trafficking dynamics as well as ligand-receptor affinities [47",48]. Thus, another approach for the engineering of cytokines would be to alter their intracellular trafficking properties. In future clinical applications, the in vitro expansion of human hematopoietic stem cells could be useful in transplantation, gene and cellular therapies, and for tumor purging. Recent developments in mature blood cell culture include the production of platelets, dendritic cells and T-lymphocytes in addition to red cells and granulocytes. Induction of donor-specific tolerance by bone marrow transplantation to allow organ allografting and tumor immunotherapy is another promising new avenue that might benefit from the availability of hematopoietic stem cell expansion technology, since this approach requires the transplantation of high doses of hematopoietic stem cells to overcome host histo-incompatibility barriers [49]. T h e development of a technology for amplifying human hematopoietic stem cells for clinical purposes now appears a feasible objective. In this review, the importance of a stringent control of the cytokine concentration and the implications of this principle in the future design and operation of bioreactor culture systems for stem cell expansion have been summarized. Additional investigations to define the dose-related effects of improved cytokine combinations and delivery schedules should enable significant further optimization of hematopoietic stem cell expansion culture systems.
Conclusions and future perspectives Over the past decade, principles of biochemical engineering have contributed to the development of ex vivo hematopoietic cell production processes through the design and testing of different types of systems that might be suitable for clinical scale applications. These include perfused flat-plate, membrane-immobilized and stirred
Acknowledgements The work performed by the authors was supported by grants from StemCell Technologies and the British Columbia Science Council, Novartis Pharmaceuticals Canada, and the National Cancer Institute of Canada with funds from the Terry Fox Run. Julie Audet and Peter W Zandstra held studentships from the Natural Sciences and Engineering Research Council of Canada and Connie J Eaves is a Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada.
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Wagner JE, Collins D, Fuller S, Schain LR, Berson AE, AImici C, Hall MA, Chen KE, Okarma TB, Lebkowski iS: Isolation of small, primitive human hematopoietic stem cells: distribution of cell surface cytokine receptors and growth in SCID-hu mice. Blood 1995, 86:512-523.
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Collins PC, Nielsen LK, Wong C-K, Papoutsakis ET, Miller WM: Real-time method for determining the colony-forming cell content of human hematopoietic cell cultures. Biotechnol Bioeng 1997, 55:693-700.
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Verfaillie CM: Direct contact between human primitive hematopoietic progenitors and bone marrow stroma is not required for long-term in vitro hematopoiesis. Blood 1992, 79:2821-2826.
Sandstrom CE, Bender JG, Miller WM, Papoutsakis ET: Development of a novel perfusion chamber to retain nonadherent cells and its use for comparison of human mobilized peripheral blood mononuclear cell cultures with and without irradiated bone marrow stroma. Biotechno/Bioeng 1996, 50:493-504. A novel stroma-free perfusion bioreactor for hematopoietic cell culture was developed. This culture system includes a chamber with multiple micro-
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Reddy CC, Niyogi SK, Wells A, Wiley HS, Lauffenburger DA: Engineering epidermal growth factor for enhanced mitogenic potency. Nat Biotechno/1996, 14:1696-1699. An epidermal growth factor (EGF) analog with a significantly reduced receptot binding affinity was a more potent mitogen than wild type EGF, demonstrating that cellular trafficking processes controlling the fate of both the ligand and the receptor can be as important as their binding affinity. 48.
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Advances in hematopoietic stem cell culture Julie Audet*, Peter W Zandstra*, Connie J Eavest and James M Piret* Recent advances in our understanding of the earliest stages of hematopoietic cell differentiation, and how these may be manipulated under defined conditions in vitro, have set the stage for the development of robust bioprocess technology applicable to hematopoietic cells. Sensitive and specific assays now exist for measuring the frequency of hematopoietic stem cells with long-term in vivo repopulating activity from human as well as murine sources. The production of natural or engineered ligands through recombinant DNA and/or combinatorial chemistry strategies is providing new reagents for enhancing the productivity of hematopoietic cell cultures. Multifactorial and dose-response analyses have yielded new insight into the different types and concentrations of factors required to optimize the rate and the extent of amplification of specific subpopulations of primitive hematopoietic cells. In addition, the rate of cytokine depletion from the medium has also been found to be dependent on the types of cell present. The discovery of these cell-type-specific parameters affecting cytokine concentrations and responses has introduced a new level of complexity into the design of optimized hematopoietic bioprocess systems.
Addresses *Biotechnology Laboratory and Department of Chemical and Bio-Resource Engineering, and tDepartment of Medical Genetics, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada :.tTerryFox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, V5Z 1L3, Canada Current Opinion in Biotechnology 1998, 9:146-151 htt p://biomednet.com/elecref/0958166900900146 © Current Biology Ltd ISSN 0958-1669
Abbreviations CFCs colony-forming cells Epo erythropoietin FL flt3-1igand IL interleukin LTC-lC long-termculture-initiating cells SF Steel factor slL-6R solubleversion of IL-6 receptor Tpo thrombopoietin
Introduction Hematopoietic stem cells capable of producing very large numbers of all blood cell types persist in the marrow throughout adult life. Current evidence indicates that the process by which these stem cells ultimately generate mature blood ceils in vivo normally spans many cell generations and is unidirectional. These concepts underlie the description of the hematopoietic system as a hierarchy of progenitors in which the most primitive stem cells
are uniquely characterized by their dual capacity for multi-lineage differentiation and self-renewal. Because different types of primitive hematopoietic progenitors are not morphologically distinguishable, their identification and characterization has had to rely on the development of other procedures. The most sensitive and discriminating of these are functional assays that measure the ability of primitive progenitor cells to give rise to specific numbers and types of mature blood cells either in vitro or in fifo. In vitro, such assays can detect a variety of apparently lineage-restricted and muhi-potent progenitors. These cells generate a corresponding spectrum of colony types when stimulated by particular growth factors in semi-solid media. The progenitors of these colonies are therefore called colony-forming cells (CFCs). An even more primitive compartment of hematopoietic cells, referred to as long-term culture-initiating cells (LTC-IC) has also been defined. LTC-IC continue to differentiate into CFC after five or more weeks of coculture with stromal fibroblasts [1]. To date, most hematopoietic cell bioreactor and bioprocess optimization studies have focused on improving CFC and LTC-IC yields as surrogate indicators of stem cell amplification. Recently, methodology for quantitating transplantable murine stem cells with long-term in vivo repopulating activity by limiting dilution analysis has been successfully adapted to human hematopoietic stem cells using immunocompromised mice as recipients [2°']. In addition, it has been shown that the number of human stem cells thus identified [2",3"*], like their murine counterparts [4"°], can be expanded in vitro without restriction of their transplantable stem cell properties. A number of biochemical engineering reviews have compared the outputs of various types of more differentiated hematopoietic cells in different types of bioreactor systems and have also examined the relative importance of some of the bioprocess parameters (e.g. 0 2 tension and perfusion rate) that affect these endpoints as well as summarizing initial clinical results obtained with transplants of in vitro expanded cells [5-7]. Here we focus on what has been learned recently about parameters that may limit the ability of various cytokines to support the expansion in vitro of the most primitive hematopoietic cell types. Increased production and availability of these primitive cells should advance the whole spectrum of hematopoietic stem cell culture applications. Cytokine requirements The unique properties of believed to be dictated by subset of genes, changes in
o f h e m a t o p o i e t i c cells hematopoietic stem cells are their expression of a specific which presumably herald and
Advances in hematopoietic stem cell culture Audet et aL
bring about the irreversible exit of cells from the stem cell compartment. T h e genes involved in such changes are just beginning to be identified and almost nothing is known about how their activation or repression is regulated. Nevertheless, it is clear that hematopoietic stem cells interact with many molecules in their extracellular milieu via transmembrane receptors (or receptor complexes) to maintain their viability, and to effect changes in their cell cycle progression and differentiated state. A key feature of any hematopoietic culture system, therefore, is the combination of cytokines it delivers to the microenvironment of the cells to be stimulated and how the concentrations of these cytokines are maintained over time, as both of these aspects of cytokine delivery will influence the cumulative response obtained. The most studied cytokine receptors expressed on hematopoietic cells belong to three general families, each family being defined by the structural similarities of its members: the tyrosine kinase receptors; the so-called hematopoietic growth factor receptors; and the gpl30 receptor family. Flk-2/flt3, the receptor for flt3-1igand (FL) [8] and c-kit, the receptor for Steel factor (SF) [9], are two examples of tyrosine kinase receptors. Both are expressed on very primitive hematopoietic cells and both are implicated in the regulation of early stages of hematopoiesis. In fact, the possibility that FL could play an important role in the stimulation of hematopoietic stem cells in vitro was first suggested by the restricted expression of its receptor on primitive hematopoietic cells [10,11] and later reinforced by the results of receptor antisense experiments which demonstrated an inhibition of CFC production in human long-term cultures [12]. Soon thereafter, potent effects of FL on both human and murine LTC-IC and repopulating cells, either in the presence or absence of stroma, were reported, particularly when certain other cytokines such as SF are present [2°',4"°,13,14°°,15]. T h e hematopoietic cytokine receptors do not possess an intrinsic tyrosine kinase domain but some share a number of other common features with the gpl30 family of receptors [16], such as an immunoglobulin-like region and some amino acid residue motifs. T h e ligands for two of these, interleukin (IL)-3 and thrombopoietin (Tpo; or c-mpl ligand), are of particular interest here. IL-3 is required (together with FL and SF) to maximize the expansion of human LTC-IC from CD34+CD38 - cells isolated from normal adult marrow [14"°]. If IL-3 is added at inappropriately high levels, however, it can also have a negative impact [17 °° ] similar to what has been described for primitive murine hematopoietic cells [15,18]. T p o can also act on very primitive cells and, in the mouse, T p o directly stimulates the proliferation and differentiation in vitro of long-term repopulating stem cells; however, the latter effect is dependent on the simultaneous exposure of the cells to other cytokines (SF, IL-3, erythropoietin [Epo]) [19]. T p o in concert with FL, SF and IL-3 has been found to be a significant stimulator of erythroid
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CFC production in short term cultures of adult human marrow CD34+CD38 - cells (Zandstra et al., unpublished data) and, as a single factor, T p o was shown to stimulate within 10 days a small net increase of LTC-IC from human CD34+CD38 - marrow cells [14°°,20]. Interestingly, in both of these latter studies, a significantly increased proportion of the CFC produced from Tpo-amplified LTC-IC were erythroid suggesting an additional ability of T p o to alter uncommitted cell differentiative behavior several cell generations later. T h e gpl30 receptor family includes receptors for IL-6, IL-11, leukemia inhibitory factor and oncostatin M. All of these cytokines interact with receptor complexes that contain the signal transducer subunit, gpl30. IL-11 is produced by stromal cells and has been shown, in concert with SF and FL, to stimulate the amplification in vitro of adult marrow murine stem cells [4°°,15,18,21°°]. Gpl30 appears to be the sole mediator of both IL-6 and IL-11-stimulated responses [16]; however, the particular ligand by which this is achieved may vary according to the cell being stimulated and its level of expression of the IL-6 versus the IL-11 binding protein. These restrictions can, however, be bypassed by the addition of a soluble version of the IL-6 receptor (slL-6R) together with IL-6 at a concentration sufficient to saturate all gpl30 molecules [22"]. Recent progress in understanding the molecular composition and three-dimensional structure of cytokine receptors and their accessory molecules expressed on hematopoietic cells as well as their need for dimerization [23] to activate intracellular kinase reactions has allowed a number of novel agonists to be developed. Examples include a hybrid cytokine of human IL-6 and the slL-6R [24"], human IL-3 and granulocyte-macrophage colony-stimulating factor [25], human IL-3 and Epo [26] and a divalent Epo mimetic [27°]. Although very few of these molecules have been analyzed for their ability to stimulate the proliferation and self-renewal of stem cells, the use and further development of engineered ligands to elicit such responses can be expected in the near future. Multifactorial analysis has proven to be a powerful experimental tool for identifying which cytokines can play a role (either alone or in combination) in stimulating the rapid expansion of particular types of primitive hematopoietic cells [14",17"',28]. Thus far, FL has been identified as the most important cytokine with such activity, although achieving maximal stem cell (LTC-IC or repopulating cell) amplification has required the presence of other cytokines, SF and IL-3 (in the case of human adult marrow cells) and IL-11/IL-6 plus slL-6R (in the case of murine adult marrow cells [28] or human cord blood cells [Zandstra eta/., unpublished data]). Interestingly, maximal concomitant production of CFC has been found to require additional factors: either IL-6, granulocyte/colony-stimulating factor or 13-nerve growth factor in the case of adult human
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marrow, or FL plus SF or IL-6/slL-6R in the case of human cord blood.
Dose-related effects of cytokines on hematopoieUc cell populations Investigations of the effects of cytokine concentration (both relative and absolute in cases where multiple cytokines are added) on primitive hematopoictic cell responses have also been informative. Not surprisingly, stimulation of the proliferation and differentiation of these cells shows typical log dose-response dependencies; however, different responses of the same original cells do not necessarily show the same cytokine concentration dependence. For example, in 10 day cultures of adult human marrow CD34+CD38 - cells, maximal expansion (60-fold) of LTC-IC requires the presence of 30-fold higher cytokine concentrations than are necessary to stimulate near maximal (280-fold) expansions of CFC in the same cultures [17"]. Furthermore, from studies of thcse effects at the single cell level, it has been possible to demonstrate that variability in LTC-IC amplification is determined by the extraccllular concentration of cytokines to which the cells are exposed and occurs largely independently of effects on their viability or proliferation. These findings support an emerging model of cytokine signaling where the cellular context in which the cytokinc initiates an intracellular signal is as important to the final response outcome as is the initial competence of the cell to bind the ligand. This context ultimately determines the magnitude and persistence of the cytokine signal over critical periods of time. Hence, both temporal and spatial aspects of cytokine delivery may modulate the ultimate response of a target cell to a given cytokine [29] which may be additionally influenced by ligand-induccd receptor down-modulation and cross-talk between receptors for different ligands, or their downstream signaling intermediates. Such issues are likely to be crucial to the development of large scale systems suitable for the controlled manipulation of hematopoietic stem cells in vitro. In particular, they highlight the importance of being able to measure and regulate cytokine concentrations in the culture medium, especially as the numbers and types of hematopoietic cell populations change over time.
Bioprocess developments in hematopoietic cell culture A small (threefold) but significant net expansion in vitro of human adult bone marrow LTC-IC was first reported in 1993 by Koller et al. [30]. This expansion of LTC-IC was achieved in a flat-plate stroma-containing bioreactor culture perfused by medium supplemented with the recombinant cytokines IL-3, SF and IL-6. Subsequently, in a similar bioreactor, additional cytokines were used (i.e. granulocyte-macrophage colony-stimulating factor, Epo and SF) and a 10-fold expansion of LTC-IC was
reported [31,32]. Depletion of the added cytokines was also noted [32]. It is difficult, however, to estimate how the actual concentration of a cytokine in the vicinity of the target cells in such a system would compare to the measured concentration of that cytokine in the flowing medium, due to local cytokine concentration gradients and the ability of hematopoietic cells to interact directly with cytokincs on the extracellular matrix or on the surface of adjacent stromal cells. More recent investigations have shown that similar expansions of primitive hematopoietic cells can be obtained in the absence of any stroma if an appropriate mixture of cytokines is provided [33"]. These responses have been obtained in cultures of purified subpopulations of cells (to eliminate stromal cells in the input innoculum) suspended in serum-free or serum-containing medium supplemented with various combinations of cytokines. This approach greatly reduces the complexity of the extracellular environment, which allows more reliable predictions regarding the outcome of the cultures because conditions are more easily reproduced. Furthermore, it has been suggested that direct contact of primitive hematopoietic cells with stromal cells may decrease the proliferative activity of the primitive hematopoietic cells [34,35°,36]. A modified culture chamber with multiple micro-grooves has recently been developed to provide a surface where less strongly anchored stroma-free cells can be easily retained while medium continuously perfuses above and at right angles to the grooves [37°]. Scaled-up suspension cultures for use in clinical trials have utilized mainly gas-permeable culture bags or T-flasks (tissue culture flasks designed for cell growth in vitro) to allow the cells to be incubated for the period required. For example, Moore and Hoskins [38] have reported an 18-fold expansion of human cord blood LTC-IC in gas-permeable bags supplemented with IL-1 and IL-3, and Bhatia et aL [39] measured a fivefold expansion of LTC-IC when CD34+DR - bone marrow cells from patients with chronic myeloid leukemia were cultured for 14 days in bags. Studies of human cells maintained in 50 or 100mL flat-bottomed spinner flasks with constant stirring (40 rpm) have been described by Sardonini and Wu [40] and Zandstra et aL [41,42"]. In these, the greatest expansion of total cell numbers was obtained in cultures initiated at a relatively high concentration of light density human marrow cells (106 cells/mL), which creates a more enriched population of primitive cells, with frequent replenishment of the medium. Bi-weekly addition of 10 ng/mL of IL-3, IL-6 and IL-11, plus 50ng/mL of FL and SF allowed within two weeks a 66-fold expansion of CFC numbers and a ninefold expansion of LTC-IC [42*]. Nevertheless, this feeding schedule did not sustain concentrations of IL-3, SF and FL at input levels. In addition, it was observed that the rate of cytokine depletion was increased at higher initial concentrations of the cytokin¢ [42"]. Thus,
Advances in hematopoietic stem cell culture Audet et aL
designing a feeding schedule to maintain optimal cytokine levels is probably much more complex than originally anticipated. Studies of hematopoietic factor-dependent cell lines have been a useful tool in cell culture research. Exposure of factor-dependent cell lines to high cytokine concentrations in vitro has generally been found to lead to a decrease in the expression by the cells of the corresponding receptor due to ligand-induced internalization of ligand-receptor complexes. Physiologically, this mechanism could be responsible for a desensitization of the cells via an attenuation of intracellular receptor-initiated signaling. This attenuation is due to two related but distinct phenomena generated by endocytic trafficking. First, receptors are lost from the cell surface by the combination of endocytosis and degradation, with the result that reduced numbers of receptors are available to interact with free extracellular ligand (receptor downregulation). Second, ligand is depleted from the extracellular medium by the same combination of endocytosis and degradation. T h e extent of c-kit (SF receptor) internalization by hematopoietic cells has been shown to be proportional to the extracellular SF concentration [9,43]. Thus, receptor/ligand internalization is a common and important mechanism in regulating factor-dependent proliferative responses of hematopoietic cells. Interestingly, primitive human adult marrow cells (defined by their CD34+CD38 - phenotype) are characterized by a 35-fold higher cell-specific rate of cytokine consumption when compared to other more differentiated types of marrow cells [42"]. These results confirm and extend those of Koller et al. [32] who found that the consumption of SF by marrow cells in culture increases as the cell numbers expand. T a k e n together, these findings are consistent with the detection of higher numbers of cytokine receptors on more primitive cells [44,45]. Physiological differences between the rates of glucose metabolism in primitive and mature hematopoietic cells have also been suggested. For example, Collins et al. [46] recently reported a correlation between specific glucose uptake rates, specific lactate generation rates and the percentage of CFC present over a broad range of culture conditions, including cells maintained in spinner flasks or T-flasks, and in serum-containing or serum-free medium. Accordingly, these authors suggested that lactate production rates might form the basis of a real-time analysis method for predicting when maximum concentrations of CFC have been reached.
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systems. T h e ultimate goal of in vitro production of human hematopoietic stem cells with long-term repopulating ability poses new challenges because of their unique cytokine requirements and the low incidence of these cells among normally accessible primary sources of human hematopoietic cell populations. An increased appreciation of many of the relevant parameters has recently begun to emerge from systematic analyses of cellular responses to, and depletion of, different cytokine supplements. In particular, such studies have revealed the very high cytokine dose requirements to maximize the expansion of primitive hematopoietic cells [17"] and their associated ability to deplete extracellular cytokine concentration at faster rates than their more differentiated progeny [42°]. Further developments can be expected from the cloning or engineering of new cytokines. T h e design of new ligands with increased binding affinity for specific cytokine receptors is an approach that has been used in other fields to create new therapeutic agents; however, potency has been shown to be dependent upon ligand/receptor trafficking dynamics as well as ligand-receptor affinities [47",48]. Thus, another approach for the engineering of cytokines would be to alter their intracellular trafficking properties. In future clinical applications, the in vitro expansion of human hematopoietic stem cells could be useful in transplantation, gene and cellular therapies, and for tumor purging. Recent developments in mature blood cell culture include the production of platelets, dendritic cells and T-lymphocytes in addition to red cells and granulocytes. Induction of donor-specific tolerance by bone marrow transplantation to allow organ allografting and tumor immunotherapy is another promising new avenue that might benefit from the availability of hematopoietic stem cell expansion technology, since this approach requires the transplantation of high doses of hematopoietic stem cells to overcome host histo-incompatibility barriers [49]. T h e development of a technology for amplifying human hematopoietic stem cells for clinical purposes now appears a feasible objective. In this review, the importance of a stringent control of the cytokine concentration and the implications of this principle in the future design and operation of bioreactor culture systems for stem cell expansion have been summarized. Additional investigations to define the dose-related effects of improved cytokine combinations and delivery schedules should enable significant further optimization of hematopoietic stem cell expansion culture systems.
Conclusions and future perspectives Over the past decade, principles of biochemical engineering have contributed to the development of ex vivo hematopoietic cell production processes through the design and testing of different types of systems that might be suitable for clinical scale applications. These include perfused flat-plate, membrane-immobilized and stirred
Acknowledgements The work performed by the authors was supported by grants from StemCell Technologies and the British Columbia Science Council, Novartis Pharmaceuticals Canada, and the National Cancer Institute of Canada with funds from the Terry Fox Run. Julie Audet and Peter W Zandstra held studentships from the Natural Sciences and Engineering Research Council of Canada and Connie J Eaves is a Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada.
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