Ex vivo expansion of hematopoietic cells from umbilical cord blood for clinical transplantation

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1 Ex vivo expansion of hematopoietic cells from umbilical cord blood for clinical transplantation Patricia D. Conrad* and Stephen G. Emerson* *Department of Pediatrics; Divisions of Hematology & Oncology, Children s Hospital of Philadelphia; and Department of Medicine; Division of Hematology/Oncology, University of Pennsylvania Abstract: Stem cell transplantation (SCT) has achieved significant therapeutic success over the last 10 years, providing a viable treatment option for many previously incurable diseases. However, several inherent limitations of the procedure have restricted its widespread use. These include: lack of sufficient donors for all recipients, a period of bone marrow (BM) aplasia leading to severe, prolonged neutropenia and thrombocytopenia, and the potential for tumor contamination in autologous SCT. Umbilical cord blood (UCB) provides a unique, and potentially more successful, approach to alleviating these limitations. Ex vivo manipulation of hematopoietic stem (HSCs) and progenitor cells (HPCs) derived from UCB using a liquid culture system has revealed that the primitive HSCs from UCB are not identical to their BM counterparts. In fact, these cells may derive from a more primitive stem cell compartment. Ultimately, successful engraftment of UCB HSCs manipulated in an ex vivo environment may lead to a larger number of these lifesaving procedures being performed and the full potential of SCT realized. J. Leukoc. Biol. 64: ; Key Words: stem cell transplantation neutropenia thrombocytopenia INTRODUCTION Autologous and allogeneic stem cell transplantation (SCT) have arisen over the last decade as viable therapeutic options for treating those diseases for which modern medicine previously could offer little hope. These include malignancies such as relapsed leukemia and neuroblastoma; hematological disorders such as sickle cell disease and aplastic anemia; and inherited diseases such as Wiskott-Aldrich Syndrome or adrenoleukodystrophy. The success of SCT is due in large part to the ability to deliver very large doses of chemotherapy, which eliminate residual tumor burden (if present) and ablate the bone marrow (BM). Subsequent replacement of native marrow from an allogeneic donor or previously harvested autologous cells repletes the hematopoietic compartment. BM has traditionally been the source for donor stem and progenitor cells. More recently, umbilical cord blood (UCB) and peripheral blood (PB) have demonstrated adequate stem and progenitor pools and in addition are much easier to obtain. The early promise of SCT has been tempered by several limitations. First, the number of potential marrow recipients for allogeneic transplants greatly exceeds the number of available HLA-matched donors. In addition, the genetic diversity of the National Marrow Donor Program (NMDP) reflects a European dominance and minorities are vastly under-represented, making matching for these groups more difficult. This limitation has been alleviated somewhat by the development of the UCB banks, which have the potential to more accurately reflect the general population. A second limitation to SCT is the prolonged period of BM aplasia after chemotherapy, which results in transient but potentially life-threatening neutropenia and thrombocytopenia. Transfusion of supplemental blood products and administration of exogenous hematopoietic growth factors has been of limited benefit; the patients remain at risk until hematopoiesis is fully restored. A final limitation, particular to autologous SCT, is the potential for tumor contamination of the transfused hematopoietic cells. Although modern techniques can remove the vast majority of tumor or leukemia cells, the risk of relapse remains larger when the patient s own BM or PB is used, compared with allogeneic BM or PB. However, when PB or BM from patients with chronic myeloid leukemia (CML) is cultured ex vivo, a greater loss of malignant clones is observed compared to normal nonclonal hematopoietic elements, leading to a preponderance of normal hematopoietic cells over time [1 3]. UCB hematopoietic cells present an intriguing and promising approach to addressing current limitations in SCT. This untapped resource was identified in the 1980 s and has proven to be an excellent source of hematopoietic stem (HSC) and progenitor cells (HPC). In addition to more closely representing the gene pool, cord blood is currently a wasted resource that can be utilized when needed. Preliminary results of cord blood Abbreviations: SCT, stem cell transplantation; BM, bone marrow; UCB, umbilical cord blood; HSC, hematopoietic stem cell; HPC, hematopoietic progenitor cell; PB, peripheral blood; NMDP, National Marrow Donor Program; CML, chronic myeloid leukemia; CFU-GM, colony-forming unit-granulocyte/ macrophage; LTC-IC, long-term culture-initiating cell; SCF, stem cell factor; IL-6, interleukin-6; GM-CSF, granulocyte-macrophage colony-stimulating factor; TPO, thrombopoietin; EPO, erythropoietin. Correspondence: Dr. Patricia Conrad, Department of Pediatrics, Children s Hospital of Philadelphia, Philadelphia, PA Received April 14, 1998; accepted April 14, Journal of Leukocyte Biology Volume 64, August

2 transplants in children suggest that there may be less graftversus-host disease [4, 5], but this is not certain. However, collection is limited to one time only and it is unclear whether there are enough HSCs and HPCs to successfully engraft a large adult. Furthermore, time to engraftment after UCB transplantation is prolonged compared to BM or mobilized PB, possibly reflecting a more primitive stem cell compartment. In an effort to overcome the above-mentioned limitations, ex vivo manipulation of allogeneic or autologous HSCs and HPCs before infusion into the recipient has been attempted. In theory, if insufficient numbers of HSCs were collected (as in UCB collection), and one were able to expand these HSCs (maintaining their characteristic stem cell abilities), more patients would then be eligible for this potentially curative procedure. It is important to emphasize that expansion of these cells must include perpetuation of self-renewal capacity in addition to production of differentiated progeny. Primitive HSCs are defined as those cells capable of multilineage differentiation and the ability to self-renew and remain in a primitive, undifferentiated state. In transplantation experiments, these cells lead to long-term hematopoietic engraftment in both animal models and humans after myeloablative therapy. In contrast, HPCs are not able to provide long-term hematopoiesis but are able to produce multilineage progeny. If the most primitive stem cells were only to differentiate and not selfreplicate, these differentiated daughter cells would not be capable of long-term hematopoietic reconstitution and ultimately the patient would experience bone marrow failure. Thus expansion of primitive hematopoietic cells ex vivo would be of little use clinically if such expansion led only to the terminal differentiation of most if not all of the primitive cells. Therefore, to be clinically useful, ex vivo expansion strategies should allow for proliferation of the primitive self-renewing stem cell compartment as well as their differentiation into daughter cells. The successful manipulation and expansion of HSCs ex vivo would have many clinical applications (Table 1). Not all techniques claiming the ability to expand human progenitor cells demonstrate that these cells retain primitive self-renewal characteristics, however. This article will review recent progress in fundamental and clinical research in ex vivo expansion, focusing on the demonstration of both proliferation and retention of self-renewal capacity in liquid culture, with particular emphasis on cord blood as the source for these primitive cells. Ex vivo culture of hematopoietic cells The initial description of HSCs and HPCs was made in 1961 by Till and McCulloch, who utilized a mouse model to demonstrate that a cell of bone marrow origin was capable of regenerating hematopoietic cells on the spleens of sublethally irradiated mice [6]. This experiment ushered in the field of stem cell biology and research attempting to uncover the in vivo as well as ex vivo nature of these primitive cells has continued at an TABLE 1. Potential Uses of Expanded Hematopoietic Cells Hematologic support after myeloablative therapy Manipulation of selected stem cells (i.e., CD34 ) Tumor purging for autologous transplantation Gene therapy TABLE 2. Benefits of Umbilical Cord Blood Pro Genetic diversity reflecting general population Readily available resource, currently discarded Less graft-versus-host disease (theoretically) Possibly more primitive stem cells compared to bone marrow No waiting period before transplant Con One-time chance at collection Limited sample size Delayed engraftment Needs to be processed, frozen, and stored in large volume, possibly for long term ever-increasing pace. Understanding the nature of these primitive cells has particular clinical relevance because the basic defect of many diseases (such as leukemia) originates in a stem or progenitor cell. Since the original early work, assays have been developed in both liquid and semisolid medium as well as in vivo assays utilizing immunodeficient mice. In semisolid medium the formation of colony-forming unit-granulocyte/ macrophage (CFU-GM) colonies is a reliable assay for a progenitor cell capable of short-term engraftment in transplantation experiments. Long-term liquid culture lasting 5 8 weeks is able to detect the long-term culture-initiating cell (LTC-IC), a fairly primitive member of the stem cell compartment [7]. In vivo assays include the transplantation of primitive human HSCs into sublethally irradiated immunodeficient mice, with or without the addition of exogenous human cytokines [8 13]. However, these assays remain technically difficult and cumbersome, limiting their widespread application. Clinical experience is accumulating in the pediatric population regarding the successful use of UCB in allogeneic transplantation (Table 2). Originally used as the donor source to treat Fanconi s Anemia, UCB is now used to treat many different diseases. In addition, the establishment of several cord blood banks have led to ever increasing use of this resource. These banks collect their samples from large maternity wards and in general are able to reflect the population, including minorities, better than the NMDP. Other benefits include easy availability of the placenta at delivery and ease of collection of the UCB. Due to the limitation of volume at the time of collection, however, it is unclear whether a large adult would be sufficiently engrafted by one cord blood sample. This has led to the basis for attempts at expansion of UCB, with the goal being that a limited number of HSCs and HPCs could be expanded to a number sufficient to engraft an average-sized adult. Several studies have examined the efficacy of UCB expansion and compared it to the expansion of BM or PB [14 18]. These studies demonstrated the ability to expand the absolute number as well as CD34 cells in liquid culture to a level that should be sufficient to engraft an average-sized adult. In addition, as discussed below, UCB HSCs and HPCs were shown to be different phenotypically as well as functionally when compared to adult BM or PB counterparts. Thus, for its ease and availability, UCB could be an optimal source for primitive stem and progenitor cells. The fact that cord blood is largely a wasted resource may make it the preferred source for hematopoietic 148 Journal of Leukocyte Biology Volume 64, August 1998

3 progenitors for both experimental and clinical uses in the future. Optimal conditions for ex vivo expansion Dexter, Allen, and Lajtha, in a series of publications between 1974 and 1977, reported the first successful attempts at cultures of hematopoietic progenitors in liquid media [19 21]. Briefly, murine BM was seeded in liquid culture and an adherent stromal layer allowed to form. This feeder layer of bone marrow stromal cells would then presumably provide various growth and regulatory factors necessary for the maintenance of primitive hematopoietic cells. Subsequently, pluripotent and committed progenitors were able to be collected over various lengths of time and analyzed for their qualitative characteristics. Hence, these liquid culture systems are now frequently referred to as Dexter cultures. Since the original inception of Dexter cultures, various groups have attempted to improve on the first system by manipulating various factors. This includes the use of liquid culture with and without a stromal feeder layer; addition of exogenous cytokines when no stromal layer is present; low or ambient oxygen tension; animal serum, human serum, or no serum, and continuous perfusion culture. Since these promising studies, two different approaches have been utilized to successfully expand stem cells ex vivo. The first general method includes the use of bone marrow stroma; this procedure takes advantage of the closely linked stromal and hematopoietic elements, the former having been shown to play a key role in hematopoiesis [22]. The second method utilizes high-dose cytokine combinations without a stromal layer. Each method provides for distinctive patterns of expansion of hematopoietic cell compartments. Source for primitive hematopoietic cells To sustain hematopoiesis, primitive hematopoietic stem and progenitor cells must be placed into the culture system. Phenotypically, the most primitive hematopoietic cells are believed to be CD34 lineage (lin ) and are by definition able to induce long-term engraftment in transplantation experiments. In addition to possessing a lineage-negative phenotype, these cells also demonstrate low forward light scatter via flow cytometry [23]. HSCs and HPCs may be derived from BM, PB, or UCB, depending on availability and specific needs of the recipient. Accumulating evidence suggests that the stem and progenitor cells in each of these compartments are in fact not identical. Quantitatively, bone marrow generally contains the largest percentage of CD34 cells (nearly 2%) with cord blood and unmobilized peripheral blood containing less (approximately 1 and 0.2%, respectively). Depending on the mobilization regimen, mobilized PB may contain anywhere from 0.6 2% CD34 cells [24]. Approximately 3.5% of density separated cord blood is CD34 in premature infants [25]. This percentage can vary due to influences such as course of labor and stress during delivery [26]. Recent studies have attempted to resolve the important question regarding the phenotype and proliferative capacity of cord blood progenitor and stem cells. Lansdorp et al. have demonstrated that primitive hematopoietic cells from adult BM, UCB, and fetal liver demonstrate qualitative differences when cultured ex vivo depending on the age of the donor [27]. The phenotype of primitive hematopoietic cells from UCB appears to differ from their adult BM counterpart because cord blood progenitors capable of initiating long-term culture belong to the CD34 HLA-DR Rh123 dull compartment and the respective cell from adult BM appears to be phenotypically HLA-DR [28]. It has also been demonstrated that UCB contains a greater percentage (possibly up to four-times) of CD38 cells in the CD34 fraction compared to adult BM [14, 24]. When compared to phenotypically similar cells from adult BM or mobilized PB, these cells demonstrate greater expandability and sensitivity to cytokine stimulation [15, 16, 24]. Using a semisolid assay for HPCs, cord blood was shown to produce equivalent numbers of colony-forming unit-granulocyte/ erythroid/macrophage/monocyte (CFU-GEMM), twice as many CFU-GM, and three times as many burst-forming unit-erythroid (BFU-E) colonies compared with the same cell type from adult BM [14]. The functional significance of these differences is not fully apparent; however, the stem cell compartment in UCB may be more primitive than its counterpart in mobilized PB or adult BM. This is borne out by the clinical transplantation experience, which reveals a longer time to engraftment with UCB and potentially less graft-versus-host disease. In vivo experiments using immunodeficient mice have confirmed that cord blood HPCs are not identical to their bone marrow counterparts. In addition to several published reports [8 13], work done in our laboratory has demonstrated that cord blood progenitors proliferate in NOD/SCID mice in the absence of exogenous cytokine support, producing both myeloid and lymphoid cells (Fig. 1, Table 3). This contrasts with the results of the same experiment using adult BM, in which exogenous human cytokines must be added for successful engraftment to occur. Possible explanations for this observation include the following: (1) the possibility that stromal elements are contained in the UCB sample; (2) that UCB HSCs are sensitive to murine cytokines (unlike adult BM as mentioned above); or (3) that the UCB HSCs are capable of an autocrine growth function. At issue is whether ex vivo cultured HSCs retain sufficient self-renewal ability to engraft an immunodeficient mouse long term. Work done by Dick and others has attempted to address this important issue. These experiments demonstrate that it appears possible to culture HSCs before successful transplantation, however, length of culture was extremely short (i.e., less than 2 weeks) [29, 30]. In addition, the frequency of the scid-repopulating cell or SRC, the cell presumably responsible for engraftment in these experiments, was greatest in UCB compared with BM or mobilized PB [31]. Selection of CD34 cells versus unselected cell populations The question of which primitive cell is best expanded and the degree of manipulation to obtain such a cell has been examined by several groups. To obtain such a cell for culture, several manipulations, including an enrichment technique, are required. Koller et al. examined the efficiency of density separation and immunoaffinity selection on the recovery and expandability of CD34 progenitors derived from adult BM. They reported that, with increasing enrichment, an overall loss Conrad and Emerson Ex vivo expansion of hematopoietic cells from umbilical cord blood 149

4 Fig. 1. Proliferation of human UCB B cells in NODSCID mice. NODSCID mice were irradiated with 350 cgy of ionizing radiation at day 0 and subsequently injected with UCB mononuclear cells. Tissue (bone marrow and spleen) was extracted at days 30 and 95. The tissue was stained for FACS analysis using fluorescence-conjugated antibodies to CD19 and CD3. Percent positivity is seen in the right upper quadrant. in total cell numbers, HPCs and HSCs occurred. Whole unmanipulated BM resulted in the greatest expansion over a range of inoculum densities. This may be due to accessory cell effects that are removed with further selection of the initial inoculum. There was a 73% loss of LTC-IC in the CD34 sample at the same time that total cell number expanded 269-fold. These authors concluded that CD34 cells can be expanded regardless of the extent of enrichment [32]. In fact, Xiao et al. demonstrated that expansion from as little as one TABLE 3. Human CD19 B Cell Engraftment in NODSCID Mice Input Day 30 Day 95 Bone marrow a Spleen Total human B cells a NODSCID mice were sublethally irradiated with 350 cgy and injected with mononuclear cells derived from UCB. Bone marrow and spleen were collected at days 30 and 95. FACS analysis was performed using fluorescenceconjugated antibody to CD19. Above values based on representative percentage of total cell number. CD34 cell derived from UCB has been shown to occur with maintenance of self-renewal capacity for at least 2 weeks. However, there was greater expansion of progenitor cells when initial inoculum consisted of 5000 CD34 cells compared to a single cell, reiterating the potential importance of cell-cell contact requirements, accessory effects, or unknown production of growth factors [17]. Eaves et al. [23] reported the ability to maintain LTC-IC at 100% at 10 days and 25% at 4 weeks. At issue is whether these expanded CD34 cells genuinely retain their primitive selfrenewal capacity. Several studies have shown that differentiation does occur with proliferation. When the phenotype of input CD34 cells is examined over time, by day 6 of liquid culture, 50% have matured to the point of CD34 negativity. By day 14 the vast majority of cells, if not all, are positive for various lineage markers including CD71 and glycophorin A [33, 34]. Thus, when primitive stem cells are cultured in liquid longterm cultures, many of these cells differentiate into more mature cells, limiting their potential usefulness in transplantation. 150 Journal of Leukocyte Biology Volume 64, August 1998

5 The fundamental question of these culture systems remains whether the most primitive cells are driven into activity and differentiation or remain quiescent. If there are remaining quiescent cells, are they able to cycle in and out, contributing to hematopoiesis over a longer period of time? Or are they not capable of contributing to long-term hematopoiesis and essentially remain in a resting state indefinitely? One study examined the role of cytokine stimulation on primitive HSCs residing in G 0 from bone marrow. The authors concluded that a control mechanism may exist that maintains proliferation of HSCs and that exogenous cytokine exposure may disrupt this cycle [35]. In another study examining recruitment of quiescent BM progenitors, different individual stem cells tended to show variable self-renewal capacity as well as variable differentiative ability [36]. Clearly, these cells are responsive to cytokine activation and can be moved into the cycling compartment, however slowly. UCB stem cells apparently are more active and/or more responsive to cytokine stimulation as suggested by the above results. This quality may be exploited when attempting to expand these primitive cells in liquid culture. Use of stromal feeder layers versus soluble cytokines When an inoculum of human or murine BM is placed into long-term culture medium, an adherent stromal layer quickly develops in association with the nonadherent soluble layer. This adherent layer includes such cells as adipocytes, fibroblasts, macrophages, and endothelial cells. One function of this layer is to produce the necessary cytokines for successful hematopoiesis; however, other functions may include unknown regulatory processes mediated via cell/cell contact [37, 38]. It is now generally accepted that the more primitive hematopoietic elements exist in the adherent layer and subsequently migrate into the nonadherent layer as they mature and differentiate [8, 39]. Formation of a stromal layer has not been consistently demonstrated for UCB. Hows et al. examined the requirement of stromal feeder layers in expansion experiments using UCB. Despite similar input numbers of clonogenic precursor cells, UCB progenitors were not able to maintain hematopoiesis for much longer than 2 weeks without a pre-formed stromal layer. If such a pre-formed stromal layer is provided, successful hematopoiesis can be maintained with duration and amplitude being greater than that for BM. On the other hand, BM was able to maintain hematopoiesis for up to 3 months after successfully forming a stromal layer [40]. In contrast to the above results, Ye et al. demonstrated that UCB is able to generate a fibroblastoid stromal layer that produced stem cell factor (SCF) and interleukin-6 (IL-6) but not interleukin-3 (IL-3) or granulocytemacrophage colony-stimulating factor (GM-CSF). After recharging this stromal layer with fresh UCB, hematopoiesis was maintained for 4 months and normal stromal elements (including fibroblasts, endothelial cells, and macrophages) were demonstrated. Of note, this group used a specialized coverslip that may have contributed to the successful formation of the stromal layer [41]. Cytokines for UCB expansion In addition to work in our laboratory, many groups have attempted to determine which cytokine combinations afford the greatest expansion and maintenance of primitive hematopoietic cells. SCF, G-and GM-CSF, Flt3Ligand (FL), interleukin-1 (IL-1), IL-3, IL-6, IL-11, thrombopoietin (TPO), and erythropoietin (EPO), in varying degrees and combinations, have most commonly been examined for their proliferative and differentiative effects [17, 42 49]. At least 190-fold expansion of clonogenic cells may be seen with the combination of SCF, EPO, IL-1, IL-3, and IL-6 [47]. IL-3 and IL-6 appear to be a better combination with regard to self-renewal over differentiation than IL-1 and IL-3; hematopoiesis was maintained longer with the former combination [45]. Xiao et al. demonstrated that one CD34 cell could produce over 5000 progeny after 4 weeks in culture when grown in combination with IL-1, IL-3, and SCF, although primitive progenitors were only demonstrated up to 2 weeks [17]. IL-11 appears to stimulate the differentiation of more primitive stem cells into a less multipotent progenitor cell and ultimately decrease the size of the stem cell compartment in liquid culture [48]. The minimum essential cocktail of cytokines for optimal expansion ex vivo must contain an early-acting agent such as FL or SCF and IL-6. Other additional cytokines that may lead to further differentiation appear to be variably successful in terms of rate of differentiation versus perpetuation of self-renewing capacity. FL, like SCF, acts as a synergistic amplifier of other hematopoietic cytokines. Several reports have demonstrated its efficacy in the expansion of hematopoietic progenitors. Koller et al. demonstrated that FL had a greater effect on the expansion of LTC-IC compared to SCF. In addition, they saw more primitive colonies produced by FL as evidenced by larger colonies with greater replating potential [49]. Gabbianelli et al. have also examined the action of FL on stem cells from PB [50]. They confirmed the stimulatory effect of FL on primitive HPCs and, unlike SCF, FL alone was able to expand primitive LTC-IC. Takahira et al. were able to demonstrate the efficacy of FL, in combination with SCF, on expansion of UCB HSCs and HPCs [51]. In mice, TPO has been shown to synergize with either FL or SCF to support multilineage colony formation, thus suggesting an additional cytokine with potential usefulness in human liquid culture [52]. TPO alone or in combination with other cytokines has been shown in humans to successfully expand LTC-IC over 10 days in one study [53], and over 200,000-fold over 6 months in another [18]. This latter study demonstrated the usefulness of FL in combination with TPO in maximally expanding primitive cells while maintaining their self-renewal characteristics for up to 6 months. The authors concluded that a pool of quiescent primitive cells was mobilized using this combination, which led to the long-term production of differentiated cells as well as maintenance of the primitive compartment. In this study, neither cytokine alone was sufficient to enable expansion. These promising early results suggest the potential usefulness of potential cytokine combinations, possibly of TPO, FL, and SCF, in enlarging the stem cell pool found in UCB. Thus, it may be possible to engraft not only children, but larger adults Conrad and Emerson Ex vivo expansion of hematopoietic cells from umbilical cord blood 151

6 after successful ex vivo expansion before transplant. In addition, manipulation of the quiescent and active compartments of the stem cell pool may alleviate the period of bone marrow hypoplasia and concommitant risks associated with neutropenia and thrombocytopenia. Optimum conditions for media In the original experiments of Dexter et al. [19 22], cells were placed in long-term culture media and fed weekly via replacement of 50% of supernatant with fresh media. Presumably, this method removed toxic metabolites and replenished diminishing growth factors. Since these early experiments, several groups have attempted to improve on liquid culture conditions. Serum from cord blood, peripheral blood, or fetal calf was examined by Ruggieri et al. [34]. They found that without the addition of cytokines, plasma alone (of any type) was not able to maintain cell viability. However, after adding cytokines to the liquid culture, cord blood plasma demonstrated a greater ability to expand UCB progenitor cells compared to peripheral blood plasma or fetal calf serum. The use of a serum-free culture system was examined by Lebkowski et al. [54]. Using bone marrow progenitors that were negatively selected then followed by a positive selection for CD34 cells, they demonstrated a 3.8-fold increase in the absolute number of CD34 cells compared to the identical experiment with serum-containing media. This peak occurred at days 5 10 and subsequently declined. No peak was observed in the serum-containing condition with gradual decline in CD34 cell number throughout the test period. Given the recent experience with newer cytokines, which show promising ability to expand HSCs (i.e., FL and TPO), it would be interesting to see if the superiority of serum-free culture media would still hold true. Low-oxygen environment Under physiological conditions, the oxygen level of most human tissues ranges from 2 5% [55]. Several groups have demonstrated that ambient (20%) oxygen tension may lead to inhibition of cell growth in culture [56, 57]. This may be due to the formation of toxic oxygen radicals at ambient oxygen saturation because inhibition is directly related to increasing numbers of phagocytic cells. In addition, antioxidants in the liquid media can decrease the levels of oxygen radicals and improve progenitor survival [58]. In 1986, both Broxmeyer et al. and Maeda et al. simultaneously published somewhat conflicting results on the effect of oxygen tension on hematopoietic progenitors in culture [59, 60]. Using cord blood progenitor cells, Broxmeyer et al. demonstrated that only from weeks 0 to 4 did lowering oxygen tension to 5% show any benefit in progenitor growth. However, after 5 weeks, progenitor cell growth as well as plating efficiency of CFU-GEMM and BFU-E was greater when cells were cultured at 20 versus 5%. Also, CFU-GM was maintained for up to 12 weeks in liquid culture at the higher oxygen tension. In a slightly different experimental design, Maeda et al. demonstrated that in fact there may well be some benefit to lowering oxygen tension. Using human bone marrow progenitors, all colony types preferentially grew better in a lower oxygen environment at all time points and the change at week 5 was not documented in this experiment. In fact, colony-forming uniterythroid (CFU-E) demonstrated increased sensitivity to EPO in the culture media at 5 versus 20% oxygen. The authors hypothesized that the crossover at week 5 seen by Broxmeyer et al. may be due to artifact secondary to bringing the culture system to ambient oxygen tension with each weekly feed. They surmised that paying strict attention to oxygen level during refeedings could remove this effect [59]. Following this line of investigation, Koller et al. explored the survival of cord blood progenitors in liquid culture using preformed stroma. They demonstrated significantly more total cell numbers and progenitors under all conditions studied in a low-oxygen atmosphere. However, this beneficial effect of reducing oxygen tension disappeared when bone marrow stroma and cytokines were added to the culture system. In another condition, cells were cultured with cytokines but without stroma, and 5% oxygen produced greater numbers of progenitors from weeks 2 8 [44]. These findings support those of Maeda et al. using the same source for progenitors as Broxmeyer et al. A definitive answer to the issue of appropriate oxygen tension to optimally expand primitive hematopoietic cells is thus unclear at this time. However, experimental results strongly indict toxic radicals produced at higher oxygen levels as a likely culprit leading to the suppression of hematopoiesis in liquid culture. Continuous perfusion culture One of the limitations of the liquid culture system has been the consistent decline in input HSCs over time. This has been thought to be due to either terminal differentiation related to excessive stimulation, accumulation of toxic metabolites, or high cell density. Continuous perfusion-based culture systems have been developed to circumvent these limitations. In these systems, cells are inoculated into bioreactors with continuously flowing media plus growth factors at a maintained rate. This system is able to expand BM mononuclear cells over 14 days up to 11-fold; LTC-IC are also reproducibly expanded [61]. Van Zant et al. demonstrated that the requirement for feeder layers could be circumvented while using a continuous culture system if a threshold cell inoculum was used ( cells/reactor). They also demonstrated up to 20-fold expansion of LTC-IC over input. In addition, UCB was expanded in perfusion culture fivefold greater when compared to static culture [62]. These results provide support for the use of the bioreactor system when expansion is indicated. Use of in vitro expanded HSCs in humans Initial experience with ex vivo cultured HSCs has demonstrated the efficacy of expanded HSCs in a murine model. One study reported engraftment for up to 9 months without any adverse effect of the culture technique on the ability of these cells to proliferate and repopulate the hematopoietic compartment [63]. Preliminary results in human studies confirm the ex vivo preferential growth characteristics of normal progenitors over CML clones [64] and the ability of hematopoietic progenitors obtained from patients with various malignancies to grow over time in liquid culture [1 3, 47]. These early results suggest the potential for collecting a patient s own stem cells before high dose chemotherapy, manipulating the cells ex vivo for a short 152 Journal of Leukocyte Biology Volume 64, August 1998

7 period of time (possibly up to 1 month), and subsequently reinfusing them. Three recent reports have examined the efficacy of long-term reconstitution in humans after ex vivo expansion. The first study, by Brugger et al., demonstrated the ability of expanded CD34 PB cells grown over 12 days to reconstitute hematopoiesis in a similar pattern compared to historical controls [65]. The second study also utilized CD34 PB cells that had been expanded ex vivo over 8 days and demonstrated no difference in platelet or neutrophil recovery compared to historical controls [66]. Finally, a recent abstract by Stiff et al. reported the efficacy of ex vivo expanded cells in hematopoietic reconstitution after high-dose chemotherapy [67]. In contrast to the other two studies, these authors used a stromal-based perfusion reactor system, demonstrating that this culture method is also effective in obtaining engraftment clinically. It is possible that a significant amount of morbidity and mortality seen with SCT may be eliminated using ex vivo expanded cells, either by decreasing (or eliminating) time to engraftment or enabling cord blood HSCs to be used with their potential for less graft-versus-host disease. Future studies will hopefully be able to answer the question of long-term viability of these ex vivo expanded cells in human subjects. CONCLUSIONS Stem cell transplantation has achieved significant therapeutic success over the last decade, thus raising the prospect of cure for people with previously incurable diseases. The initial potential of SCT has not been fully realized due to many inherent limitations of the procedure. One such limitation is small numbers of primitive hematopoietic cells when umbilical cord blood is used as the donor source. In an attempt to overcome this limitation, work is ongoing to manipulate the primitive cells in an ex vivo artificial environment followed by transplantation. It may now be possible to expand primitive HSCs and HPCs ex vivo and maintain their primitive characteristics. Using stroma or primitive-acting cytokines such as FL or SCF, the absolute number of cells as well as the number of CD34 lin cells may be increased. As yet unknown is whether this increased number is sufficient to engraft a large adult and if so, whether this leads to durable engraftment over time. Careful clinical trials to rigorously yet safely test this hypothesis need to be performed. However, it seems hopeful that with current techniques we are on the verge of successfully expanding permanently reconstituting stem cells for clinical practice. The ability to grow and expand primitive hematopoietic cells and their progeny in liquid culture has many experimental and clinical implications. Although an optimum assay for these cells has not yet been determined, the potential to manipulate these early cell types under defined, controlled conditions would contribute to a greater understanding of the physiology of these elusive cells. With the ever-evolving understanding of the various factors and conditions in the living hematopoietic compartment of both animals and humans, further refinement of ex vivo culture techniques will lead to improved assays for these cells and to improved clinical hematopoietic transplantation in a variety of settings. ACKNOWLEDGMENTS Dr. Stephen Emerson is one of the founders of Aastrom Biosciences, which is currently involved in clinical trials of cord blood ex vivo expansion using proprietary bioreactor technology. REFERENCES 1. Udomsakdi, C., Eaves, C., Swolin, B., Reid, D., Barnett, M., Eaves, A. (1992) Rapid decline of chronic myeloid leukemic cells in long-term culture due to a defect at the leukemic stem cell level. Proc. Natl. Acad. Sci. 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