Ex vivo expansion of haemopoietic progenitor cells

BloodReviews(l996) 0 1996 Pearson

10, 167-176 Professional Ltd

Haematological oncology

Ex vivo expansion of haemopoietic progenitor cells M.J. Alcorn,

T.L. Holyoake

Over the last few years, techniques have become available that allow the extensive proliferation of haemopoietic progenitor cells in ex vivo culture systems. The most commonly used method involves a simple liquid suspension culture system supplemented with a range of cytokines. Alternatively, more complex systems have been devised in which the formation of a stromal layer is required. Large increases in total cell numbers and committed progenitor cells can be readily obtained and, with some techniques, significant expansion of primitive haemopoietic cells has been demonstrated. Although these strategies have several potential applications, few clinical studies have been performed. It has been shown that infusion of ex vivo cultured cells is well tolerated with no associated toxicity. However, it is still unclear whether these culture systems sustain sufficient numbers of long-term repopulating cells to secure durable engraftment following myeloablative therapy. In gene therapy studies, ex vivo expansion of stem cells should improve the efficiency of gene transduction to enable the production of genetically modified cells that are capable of expressing the gene of interest for extended periods of time.

The development of in vitro culture techniques for haemopoietic cells has made a major contribution to our understanding of the control of haemopoiesis2 and allowed identification of an ever-increasing range of regulatory activities,3 for example, the interleukins, which currently number 17 (IL 1-17).4 More recently, virtually unlimited amounts of these growth regulatory factors have become available in recombinant form and this has allowed far more detailed investigation of their activities and potential applications. In parallel with this, several technologies have become available that allow the isolation of relatively pure stem/progenitor cells5 that express the CD34 antigen6 (1 90% CD34 positive). Together, these developments have enabled researchers to investigate the regulatory mechanisms that control haemopoiesis.

INTRODUCTION

For many years, an elusive goal for experimental haematologists has been to manipulate and harness the haemopoietic system. The scale of this system is quite remarkable when one considers that every day some 2 x 10” erythrocytes, 1 x 1O’Ogranulocytes and 4 x 10” platelets enter the circulation, in addition to lymphocytes and monocytes.’ Despite the magnitude of this production system, aberrations are relatively rare. Indeed, external influences can rapidly induce a response to correct any deviation from the norm, e.g. blood loss following major trauma. Just as importantly, the system is sufficiently sensitive to detect when it is appropriate to return blood cell production to normal levels. Clearly, the regulation of haemopoiesis must be exquisite to be able to maintain blood cell counts within a relatively narrow range. It is this control that has long fascinated the experimental haematologist.

MURINE

Not surprisingly, murine studies first demonstrated that primitive haemopoietic cells could proliferate in relatively simple liquid culture systems. Amongst these early reports, Bodine et al cultured bone marrow cells and found that a combination of interleukin-3

Michael J. Alcorn PhD and Tessa L. Holyoake MRCP MRCPath, Leukaemia Research Fund Laboratories, Department of Haematology, Glasgow Royal Infirmary, Glasgow G4 OSF, UK Correspondence

to: Dr M.J.

STUDIES

Alcorn.

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(IL-3) and interleukin-6 (IL-6) was able to increase between five and ten fold the number of primitive precursors as measured by the colony-forming unitspleen (CFU-S) assay.7 This work was subsequently corroborated and extended by several other groups using a variety of growth factor combinations. Expansion of CFU-S was demonstrated using stem cell factor (SCF) + interleukin-la (IL-l CX)~;SCF + IL-69; SCF,+ IL-3 + IL-61°; SCF + IL-lo”; and SCF + IL-1 1.r2 In addition, Muench et al demonstrated the expansion of the multipotential progenitor, high proliferative potential-colony-forming cell (HPP-CFC), ’ l while Ploemacher et al reported the expansion of the more primitive cobblestone area-forming cell (CAFC day 2%35).13 These studies employed relatively simple liquid culture systems supplemented with various haemopoietic growth factors. No attempt was made to incorporate any marrow stromal influence. Whether such stromal-free systems are able to expand cells that can retain the attributes of stem cells is still an area of some controversy. While these studies yielded invaluable information on the regulation of the haemopoietic system and how it could be manipulated in vitro, transplantation studies were required to assess the engraftment potential of such expanded populations. Several studies have demonstrated that transplantation with ex vivo expanded bone marrow (BM) cells can effect accelerated haemopoietic reconstitution in lethally irradiated recipient mice.14-17 Furthermore, ex vivo expansion of bone marrow cells dramatically reduced the number of cells required for radioprotection during the early phase of haemopoietic reconstitution.‘5 In the study of Muench et al, mice were transplanted with BM which had been enriched for early progenitors by the in vivo administration of 5-fluorouracil (%FU BM). All mice survived for at least 30 days when they received 1 x lo6 of these cells. However, similar survival figures were obtained when mice were transplanted with only 5 x lo3 5-FU BM cells that had been expanded in vitro for seven days in suspension culture. In addition, the long-term repopulating ability of cultured cells was not compromised, as demonstrated by sustained donorderived haemopoiesis. In fact, stable secondary transplantations were also achieved. Similarly, Rebel et al reported that enriched stem cell populations (Sea-1’ Lin WGA’) with long-term repopulating ability could be maintained for two weeks in a serum- and stroma-free culture system.18 These data indicated that, in the murine system, ex vivo culture could at least maintain, and perhaps expand, haemopoietic cells with long-term repopulating potential.

POTENTIAL

APPLICATIONS

Clearly, these strategies have several potential clinical applications and these are summarized in the table. Over the last few years, bone marrow transplantation has been largely replaced by the use of mobilized peripheral blood progenitor cells (PBPCS).‘~ If stem/progenitor cells could be expanded in ex vivo culture, then it may be possible to reduce the number and duration of leucapheresis procedures required for autologous transplantation, thus reducing the risk of disease contamination in the apheresis products. Furthermore, recent studies have demonstrated that there is no significant increase in the numbers of residual tumour cells during ex vivo expansion, and this may represent a further purging step.*‘.” One would also anticipate some financial savings, e.g. staff time, if the number of apheresis procedures could be reduced. In some patients, for example those who have received considerable prior chemotherapy, mobilization of PBPCs can be unsatisfactory. The leucapheresis products obtained are frequently deemed to be inadequate for transplantation. At least conceptually, an attractive strategy would be to expand such collections in ex vivo culture systems to produce sufficient numbers of stem/progenitor cells for successful transplantation. This strategy could be extended to include transplantation with umbilical cord blood (UCB) cells. Although the transplant potential of these cells is not in question, reservations exist as to whether there are sufficient numbers of stem cells in UCB to achieve successful transplantation in adults. Ex vivo stem cell expansion may offer a solution to this problem.

Table Potential clinical haemopoietic progenitor Expansion

applications cells

of ex vivo expanded

of stem cells

1. Reduction in the number and duration of leucapheresis procedures, with concomitant purging effect in autologous transplantation; 2. Inadequate PBPC collections may be rendered sufficient for autologous transplantation purposes; 3. Increase the number of stem cells in umbilical cord blood collections for use in adult allogeneic transplantation; 4. Effective vehicles for genetically modified cells. Expansion

of lineage-restricted

cells

1. Re-infusion of populations of myeloid precursor cells to reduce the period of obligate neutropenia following autologous transplantation; 2. Generation of natural killer cells for use in adoptive immunotherapy protocols; 3. Generation of megakaryocyte precursors to alleviate posttransplant-associated thrombocytopenia; 4. Activation of dendritic cells by ex vivo exposure to tumour antigens; possible induction of in vivo tumour immunity.

Ex vivo expansion

High-dose chemotherapy (HDC) with autologous stem cell rescue is indicated for a variety of malignancies. After HDC, there is an obligate period of severe myelosuppression. It may be possible to develop ex vivo culture conditions that preferentially enhance the proliferation of more mature progenitor cells, which, when transplanted, can reduce the duration of pancytopenia and secure more prompt engraftment.22 Finally, over the last few years, there has been intense activity in the development of gene therapy protocols. Clearly, the haemopoietic stem cell would be an ideal candidate for gene transduction since it is able to reconstitute permanently the haemopoietic and immune systems after transplantation. However, to achieve efficient transduction, some degree of stem cell proliferation is necessary.23 Ideally, the aim would be to develop a culture system which could trigger cycling of primitive stem cells and thus facilitate gene transduction, without compromising the self-renewal properties of these cells. The potential applications of ex vivo expanded haemopoietic cells are, therefore, many and varied. The ability to exert significant control over the proliferation of haemopoietic stem cells is clearly an exciting prospect and this is reflected in the large number of studies that have been reported over the last few years. A great deal of effort has gone into defining the optimal conditions for ex vivo culture of haemopoietic cells. The extent of cellular proliferation will be determined by a number of factors, such as the starting cell population, the type of culture system (liquid suspension or stroma-mediated), and the cytokine combination.

SOURCE OF HAEMOPOIETIC

CELLS

A number of haemopoietic tissues have been investigated for their ability to proliferate in culture. Most studies have used PBPCs obtained from leucapheresis collections from patients receiving mobilization regimes (cytokines and/or chemotherapy).24s25 This probably reflects the ease of procuring PBPCs, as well as the fact that these cells are the most widely used in clinical transplantation studies. Alternatively, bone marrow cells have also been used.26J7 More recently, several groups have used cord blood cells28m30 or fetal liver cells.31,32It is unclear whether there are significant differences in the proliferative potential of haemopoietic cells from different sources. For example, Traycoff et al reported that similar numbers of cells and granulocyte-macrophage colony-forming units (CFU-GMs) were generated from cultures initiated with equivalent numbers of either cord blood or bone marrow CD34’ cells.33However, there is a suggestion that cord blood

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may, in fact, be the haemopoietic tissue of choice. This is based on recent reports investigating the length of telomeric DNA in different tissues. In eukaryotic cells, chromosomes end in specialized structures called telomeres. It has been shown that the amount and length of telomeric DNA decreases with ageing.34 Thus, at each cell division, there is a loss of approximately 50 base pairs. This progressive loss of telomeric DNA is thought, eventually, to reach a critical point at which cellular senescence is triggered. It may be that this loss of telomeric DNA is the mechanism by which individual cells are limited to a finite number of cell divisions. Lansdorp’s group reported that cells produced in cytokine-supplemented cultures of purified progenitor cells showed a proliferation-associated loss of telomeric DNA. Furthermore, purified primitive haemopoietic cells (CD34’ CD389 from adult bone marrow had shorter telomeres than cells from either cord blood or fetal liver, reflecting the respective proliferative histories. 35 It would appear; therefore, that these neonatal haemopoietic tissues possess a greater proliferative potential than the ceils from adult tissues. Theoretically then, when compared to adult stem cells, cord blood or fetal liver cells should be capable of more extensive proliferation that can be sustained for longer periods of time. The potential advantages for transplantation studies are obvious. Whatever the source of haemopoietic cells, in most studies, cultures have been initiated with CD34-selected cell populations. These cells can be readily obtained by any one of several technologies. These include fluorescence-activated cell sorting (FACS), immunomagnetic beads (Isolex’, Baxter Biotech, Irvine, CA, USA), or immunoaffinity columns (CEPRATE@, CellPro, Bothell, WA, USA). Indeed, some studies have been performed using more refined subpopulations of CD34’ cells. Several groups have used CD34’ cells that do not express the major histocompatibility complex class II locus, HLA-DR (CD34’ HLA-DR-).j6.37 These CD34’ HLA-DR- cells are enriched for the long-term bone marrow culture-initiating cell (LTBMC-IC) and therefore represent a more primitive subset of CD34’ cells. Alternatively, CD34’ CD38m cells are a similarly primitive subpopulation that have also been used by a number of groups.3S.39

EX WV0 CULTURE

SYSTEMS

The choice of cytokine combination and culture system will largely determine the fate of the cells used to initiate the culture. It is likely that the intended application of expanded cells will influence the culture conditions employed. As discussed earlier, if the aim was to produce extensive proliferation of more mature

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progenitors in an attempt to reduce neutropenia post transplantation, then a particular culture system may be indicated. On the other hand, if the aim was to try to obtain true stem cell expansion for transplantation, then an alternative culture system may be more appropriate. For a more detailed discussion of the technical aspects of the various systems, the reader is referred to a recent review. 4o Generally speaking, there have been two different approaches to the development of culture systems for the ex vivo expansion of haemopoietic cells, each having their own attractions. Cytokine-supplemented

suspension culture

The most widely used method for ex vivo expansion has been a relatively simple liquid suspension culture system that has been supplemented with a combination of various cytokines. It is the very simplicity of these culture systems that is their main attraction. Briefly, CD34’ cells are suspended in culture medium and incubated in an appropriate vessel (tissue culture flasks41 or gas-permeable culture bags42) for between eight and twelve days. The cultured cells can then be harvested with ease and used as required. Furthermore, the medium used can be serum-free (e.g. Progenitor 34 Medium@, Life Technologies). This allows the investigator to use a chemically defined medium supplemented with known amounts of cytokines. Thus, it should be easier to attribute the behaviour of cells in culture to particular cytokine influences without interference from the many undefined activities present in serum. Generally speaking, no stromal influence is incorporated into these systems. This represents a major disadvantage when one considers that, in vivo, blood cell production is thought to be regulated locally by interactions of haemopoietic stem cells with a variety of cell-bound and secreted factors produced by adjacent bone marrow stromal cells. The addition of cytokines to the culture medium is intended to compensate for the absence of stroma-associated activities. Since our understanding of stem cell regulation is incomplete, it is likely that the cytokine combinations currently in use will be inadequate substitutes for stroma. Nevertheless, most cytokine combinations have included SCF as an absolute requirement.43,44 Used on its own, SCF has, at best, only a modest stimulatory activity. However, it has a profound synergistic effect when used in conjunction with other cytokines. Different groups have developed their own preferred combinations, but the cytokines most commonly used include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-lp, IL-3, IL-6, and erythropoietin (Epo). 42,44-49 The degree of ex vivo expansion is

normally assessed by calculating the fold-increase in total cell numbers, committed progenitors (CFU-GM, BFU-E), CD34 cells, and LTBMC-IC with respect to the input cells. Routinely, extensive expansion of cell numbers is obtained. Depending on the duration of culture, this can vary from a 30-fold increase in cell numbers from an eight-day culture,50 up to over lOOO-fold increases with longer time periods of 14 or 21 days. 44.49Similarly, committed progenitor numbers also increase, for example, 41-fold following an eight-day culture,50 up to 190-fold from a 14-day culture. By repeated feeding of cultures, cell numbers can continue to increase for up to 21 days. However, committed progenitor proliferation probably reaches a maximum after between 12 and 14 days of culture.47 This reflects the differentiative influence of the cytokine combinations most commonly used. Thus, primitive cells which are responsive to the cytokines will tend to differentiate to produce committed progenitors. Over a period of weeks in culture, these primitive cells will become exhausted and the production of committed progenitors will decline. Several groups have also reported an increase in the number of cells expressing CD34 following ex vivo culture.47,48 However, this area is somewhat controversial, since other groups have reported a decline in the number of CD34 cells.22,42This discrepancy may be accounted for, at least in part, by technical limitations of CD34 assessment. Stem/progenitor cells are normally identified, by flow cytometry, as cells with low side scatter characteristics that express CD34. During ex vivo culture, the light scatter properties of proliferating cells are altered profoundly, thus making it difficult to measure accurately the number of true CD34’ cells by established criteria.51 The figure shows a typical dot plot from cells analyzed on the FACScan (Becton Dickinson) for CD34 expression and light scatter properties. Input CD34 cells (Panel B) form a discrete cluster exhibiting low side scatter characteristics (upper left quadrant). However, no such cluster is obtained when cultured cells are similarly analyzed (Panel D). Events appear as a diffuse cloud rather than a discrete cluster. There is a high degree of heterogeneity in terms of CD34 expression and light scatter properties, making these data difficult to interpret. Thus, flow cytometric analyses may be an inappropriate technique to measure the CD34 status of cultured cells. Alternatively, immunocytochemical methods may be useful (e.g. alkaline phosphatase anti-alkaline phosphatase (APAAP)). Thus, large numbers of progenitor cells can be readily produced in ex vivo expansion culture systems. These procedures are often referred to generically as ‘stem cell expansion’ thus inferring that the number of stem cells increases during the culture period. Most

Ex vivo expansion of haemopoietic progenitor cells

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Post-selection

CD34+

1

Post-expansion isotype control

1

2.1%

cells

Post-expansion CDS+ ceils

i

Fig. Flow cytometric analyses of CD34 cells selected by the Isolex system (Panels A and B), and cells harvested from suspension culture following eight days’ incubation with growth factors (Panels C and D). In the CD34 selected fraction, the proportion of cells actually expressing CD34 was measured by defining a population of cells with high CD34 expression and low side scatter properties (Panel B, upper left quadrant). The gates were set using an irrelevant isotype control (Panel A). Unlike CD34 cells in the selected fraction which appeared as a discrete cluster (Panel B), cells from expansion culture produced a diffuse cloud (Panel D).

studies indicate that this is not so. In human studies, it is difficult to demonstrate unequivocally the presence of stem cells. In murine studies, true haemopoietic stem cells with long-term repopulating potential can be quantitated readily in a limiting dilution assay.52 Clearly, this sort of strategy is not available for human studies. Instead, stem cells are measured indirectly by the long-term-culture-initiating cell (LTC-IC) assay.

LTC-ICs are able to generate myeloid progenitor cells for at least five weeks in culture and are, therefore, thought to represent a primitive population which contains those cells responsible for sustained in vivo long-term haemopoiesis. In the liquid culture expansion systems discussed so far, there is little evidence of significant LTC-IC proliferation, with, at best, maintenance of LTC-IC numbers over the culture

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period.47,53J4 True stem cell expansion will probably require the development of a culture system that resembles more closely the unique regulatory microenvironment of marrow stroma. One approach would be to investigate other novel cytokines that are thought to play a role in stem cell regulation. Recently, a receptor of the tyrosine kinase type has been described (Flk2lFlt3) that is preferentially expressed on primitive lymphohaemopoietic cells.55x56 The ligand for this receptor, Flt ligand (FL), has subsequently been cloned.j7J8 Although FL has only weak stimulatory activity when used on its own, it can exert a powerful synergistic effect when used in combination with other cytokines such as IL-3, IL-6, G-CSF, GM-CSF and SCF.59,60Interestingly, FL may have a pivotal role to play in recruiting into cell cycle a primitive highly quiescent subpopulation of CD34’ cells. It has been shown that a significant number of LTC-IC are unresponsive to cytokine stimulation and can remain quiescent for up to ten days in cytokinesupplemented culture systems.30 These cells are thought to represent the most primitive stem cells. When used in conjunction with IL-3, IL-6 and SCF, FL has been shown to be capable of inducing proliferation of a primitive and relatively cytokine nonresponsive subpopulation of haemopoietic cells.39 Indeed, using a serum-free suspension culture system supplemented with FL, SCF, IL-3, IL-6, G-CSF, and P-Nerve Growth Factor (P-NGF), Eaves’ group have reported substantial expansion of LTC-IC.38,61 Whether this exciting development represents expansion of true stem cells with long-term in vivo repopulating ability remains to be seen. Other candidate cytokines that are thought to influence stem cell regulation include macrophage inflammatory protein-la (MIP-la), transforming growth factor-p (TGF-P), and leukaemia inhibitory factor (LIF). Although they appear to make only a modest contribution in ex vivo expansion studies, they probably warrant more detailed investigation.62,63 Stromal-based expansion culture As an alternative to these relatively simple suspension culture systems for ex vivo expansion of haemopoietic cells, some groups have developed more complex systems in which stromal elements play a crucial role. This strategy has its origins in the long-term bone marrow culture (LTBMC) technique first described by Dexter et al almost twenty years ago.64 Briefly, in the first few weeks of culture, a complex adherent layer of stromal cells is laid down. This stromal layer comprises fibroblasts, macrophage, adipocytes, endothelial cells and reticular cells. Haemopoiesis can be maintained for months in LTBMC and it is thought that direct

adhesive interactions between haemopoietic cells and various elements of the stroma are crucial to the regulation of primitive haemopoietic cells. Presumably, the complex stromal layer can, to some extent, successfully mimic the unique microenvironment present in the bone marrow. Routinely, LTBMC can sustain haemopoiesis for some months, after which progenitor cell production ceases. However, significant enhancement of haemopoiesis has been obtained by the addition of recombinant cytokines (G-CSF, GM-CSF, IL-3).65,66 Subsequently, Schwartz et al demonstrated that expansion of the progenitor cell pool could be achieved when addition of IL-3, GM-CSF and Epo was combined with daily feeding, whereby half the culture supernatant was removed and replaced with fresh medium.a6 This idea was developed further with the introduction of continuous perfusion cuitures.2i When bone marrow mononuclear cells (BM-MNCs) were inoculated into a large-scale bioreactor system and cultured for 14 days with added cytokines (SCF, Ii~3~ GM-CSF, Epo), a 21-fold expansion in CFU-GM numbers was obtained, More imptXt&rtly, the number of LTC-IC increased 7.5fold. Interestingly, when the input cells were purified (CD34 lin-), and therefore no stroma Was formed, LTC-IC expansion decreased compared to cultures initiated with BM-MNC. These data indicated that LTC-IC expansion was dependent on stroma formation by accessory cells that were present in BM-MNC.67 These findings have subsequently been extended by Zandstra et al, who reported a 7-fold increase in LTC-IC from bone marrow cells cultured in a stirred suspension bioprocess supplemented with FL, SCF, IL-3 and IL-6.68 So, although bone marrow accessory cells were present, there was no formation of an intact, adherent stromal layer. Thus, the major advantage of these stromal-based culture systems is their ability to expand the numbers of primitive haemopoietic cells (i.e. LTC-IC). However, they are far more complex than the relatively simple stroma-free, cytokiae-supplemented suspension culture systems, Ideally, the aim would be to identify and isolate the activities from stroma that are so important in stem cell expansion. It may be possible that primitive cell expansion could be achieved in liquid suspension cultures supplemented with these crucial stromal activities. Such an approach has been taken by Verfaillie’s group. In normal LTBMC, when primitive haemopoietic cells are intimately associated with the stroma (‘stroma contact’), only about 20% of LTC-IC survived a five-week culture period. However, if the haemopoietic progenitors were physically separated

Ex vivo expansion

from the stromal layer by a 0.4~pm microporous filter membrane (‘stroma non-contact’), then primitive progenitors were maintained to a greater extent, with 50% recoverable after five weeks of culture.37 Furthermore, when stroma non-contact cultures were supplemented with IL-3 and MIP-la, then LTC-IC numbers could be maintained (> 100% recovery) for over eight weeks. This extended LTC-IC maintenance was thought to depend on soluble stromal factors diffusing through the porous membrane to interact with the primitive haemopoietic cells. An attempt was made to characterize the factor(s) from stroma. Culture supernatant from the LTC-IC supportive murine marrow stromal fibroblast cell line, M2-10B4, was fractionated by various chromatographic techniques. It was shown that M2- 1OB4-derived heparan sulphate glycosaminoglycans (HSGs) were required for LTC-IC maintenance. This culture system has recently been scaled up, and large numbers (2 x 105) of CD34’ DR- cells have been cultured in gas-permeable Teflon bags in the presence of medium conditioned by allogeneic stromal feeders (SCMs) plus IL-3 or IL-3 and platelet factor-4 (PF-4).70 The number of committed progenitors increased, on average, 12.5-fold, and the number of LTBMC-IC increased almost threefold.

CLINICAL APPLICATIONS EXPANDED CELLS

OF EX VIVO

Over the last few years, a great deal of effort has been invested in the ex vivo expansion of haemopoietic cells. The culture systems discussed have revealed a great deal of information on the regulation of haemopoiesis. This has helped us to devise strategies to try to exploit ex vivo expansion in a clinical situation. Despite the large number of laboratory reports, very few clinical studies have been performed. Initial clinical studies concentrated on safety aspects of infusing cells which had been cultured in the presence of a variety of exogenous cytokines. Silver et al treated five patients (non Hodgkin’s lymphoma (NHL); Hodgkin’s disease (HD)) with a standard bone marrow transplant plus cells that had been expanded in the presence of SCF, IL-3, GM-CSF and Epo in a continuous perfusion culture system.71 The treatment was well tolerated with no associated toxicity. The number of days to engraftment (absolute neutrophil count (ANC) = 500) was comparable to that in a control group who had not received expanded cells. The safety and feasibility of infusing cultured cells has been confirmed by several groups. Brugger et al transplanted four patients (solid tumours) with ex vivo expanded CD3bselected peripheral blood progenitor cells (PBPCs) in tandem

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with uncultured CD34’ cells.“’ The patients engrafted promptly ( ANC 500, day 1l/12) and no toxicity was observed. Similarly, our own group studied a cohort of ten patients (multiple myeloma (MM), NHL, HD, breast carcinoma (Br Ca)).42 In this study, PBPCs were recovered from cryopreservation, CD34’ cells were selected, and ex vivo expansion cultures established. Again, on re-infusion of cultured cells in tandem with unmanipulated PBPC, no toxicity was observed and there were no differences in either neutrophil or platelet recovery compared to historical controls. This lack of toxicity has been corroborated further by two studies of transplantation for breast cancer.22*72 Having demonstrated the safety and feasibility of re-infusing cultured cells in tandem with a standard transplant, it remained to be seen if transplantation with cultured cells alone could secure durable engraftment. This question was first addressed by Brugger et a1.41Following high-dose chemotherapy, six patients, with various solid tumours, were transplanted with ex vivo expanded cells alone. All six showed haemopoietic reconstitution that was identical to historical controls treated with either unseparated mononuclear cells or positively selected CD34’ cells. However, the preparative regimen used in this study was not myeloablative (etoposide 1500 mg/m’; ifosfamide 12 g/m”; carboplatin 750 mg/m*; epirubicin 150 mg/m*) It is likely, therefore, that endogenous precursors made a significant contribution to haemopoietic reconstitution, and so these data do not allow any conclusions to be drawn about the long-term repopulating ability of ex vivo expanded cells. To date, only one study has addressed the engraftment potential of expanded cells following filly myeloablative conditioning.73 In a small study group, two patients (NHL) were conditioned with cyclophosphamide 60 mglkg on two consecutive days/total body irradiation (TBI) 1360 cGy in eight fractions over four days. A further two patients (MM) were conditioned with busulphan 3 mgikg per day for four dayslmelphalan 140 mg/m”. Following re-infusion of cultured cells alone, one patient showed no evidence of neutrophil engraftment and was given unmanipulated ‘back-up’ on day 14 post transplant. The other three patients all showed signs of early neutrophil engraftment, but this was not maintained. Subsequently, they too received back-up PBPCs. In all four cases,following administration of unmanipulated cells, stable engraftment was obtained. These data suggest that, under the culture conditions used in this study, stem cells expanded ex vivo may not have contained sufficient long-term repopulating stem cells to ensure engraftment following myeloablative conditioning. This study strikes a timely note of caution in the clinical use of expanded cells.

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FUTURE

DIRECTIONS

It is worth emphasizing that there are currently no clinical indications for the use of ex vivo expanded haemopoietic cells. Notwithstanding the reservations about the clinical use of expanded cells, this still promises to be an exciting area of research over the next decade. It is to be hoped that refinements to the methodologies may allow reproducible true stem cell expansion to be achieved. As our understanding of the ways in which stem cells can be manipulated increases, further applications are likely to be investigated. Depending on culture conditions and cytokine combinations, it may be possible to produce vast numbers of a particular cell type for a precise application. For example, using an appropriate cytokine combination, large numbers of dendritic cells can be generated from CD34’ cells.74,75These may have a role in vaccination protocols. Similarly, using interleukin-2 (IL-2) and SCF, natural killer cells can be generated from PBPCs or from CD34’ cells.76-7XThese cells may have an application in immunotherapy. A cytokine combination of thrombopoietin, IL-3 and SCF has been shown to stimulate ex vivo expansion of bone marrow megakaryocytes. 7gA potential app Iication could be to treat post-transplant or chemotherapy-associated thrombocytopenia. Finally, the number of gene therapy protocols approved by the National Institutes of Health Recombinant DNA Advisory Committee (NIH RAC) continues to rise (> 100; ASH Meeting, Seattle, December 1995). Although the use of such transduced cells is still in its infancy, there is cautious optimism that genetically modified cells will be able to confer lasting benefit on a wide range of patients.80 One of the major limitations of this technology is the low efficiency of gene transduction. Ex vivo manipulations that could trigger stem cells into cycle or could produce genuine stem cell expansion would greatly improve the efficiency of transduction and make a major contribution to gene therapy protocols.

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