Stem Cells 2003;21:71-78
www.StemCells.com
© 2003 AlphaMed Press
Small-Molecule-Directed Mpl Signaling Can Complement Growth Factors to Selectively Expand Genetically Modified Cord Blood Cells
Robert E. Richard,
C. Anthony Blau
Division of Hematology, Department of Medicine,University of Washington School of Medicine, Seattle, Washington
Key Words. Gene therapy • Hematopoiesis • Cytokines • Thrombopoietin receptor
C. Anthony Blau, M.D., Mailstop 357710, Health Sciences Building, University of Washington, Seattle, Washington 98195, USA. Telephone: 206-685-6873; Fax: 206-543-3560; e-mail:
tblau{at}u.washington.edu
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ABSTRACT
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Efforts toward achieving gene therapy for blood disorders are plagued by low rates of gene transfer into hemopoietic stem cells. Recent studies suggest that this obstacle can be circumvented using selection. One way to achieve selection employs genes that encode receptor-bearing fusion proteins capable of inducing cell growth in response to drugs called chemical inducers of dimerization (CIDs). We have previously shown that genetically modified marrow cells from mice can proliferate for up to a year in culture in response to CID-initiated signals arising from the thrombopoietin receptor (mpl). The sustained growth observed in mouse hemopoietic cells results from an mpl-induced self-renewal of multipotential hemopoietic progenitor cells. In contrast, human hemopoietic cells proliferate only transiently in response to the mpl signal (from differentiation of transduced erythroid and megakaryocytic progenitors), while human myeloid progenitors fail to respond. Here, we show that myeloid progenitors from human cord blood can be induced to proliferate and/or differentiate in response to the mpl signal by providing additional signals via a combination of growth factors. These findings are relevant for the eventual clinical application of CID-regulated cell therapy.
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INTRODUCTION
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The inability to deliver a therapeutic gene to a clinically relevant fraction of human hemopoietic stem cells has proven to be a major obstacle to gene therapy for hemopoietic disorders. Methods to conditionally expand genetically modified blood cells in vivo could substantially increase the safety and efficacy of gene therapy [1, 2]. Toward this end, we have adapted a system that utilizes genes encoding receptor-bearing fusion proteins. Lacking their extracellular domains, these receptors are not activated by endogenous ligands, rather, activation is controlled using drugs that reversibly dimerize the fusion protein. These drugs, termed chemical inducers of dimerization (CIDs), act as artificial growth factors that are specific for genetically modified cells [3]. We have shown that CID-mediated activation of the thrombopoietin receptor (mpl) signaling domain induces transduced murine bone marrow cells to divide extensively in culture [4]. This dramatic expansion is due to the ability of mpl to promote extensive self-renewal of multipotential hemopoietic progenitor cells (MHPCs). The ability to stimulate MHPC self-renewal is not shared by the G-CSF receptor or flt3 ligand (flt3), and thus, appears to be at least somewhat specific for mpl [5].
Initial studies to evaluate CID-induced mpl signaling in human cord blood CD34+ cells showed that growth responses in human cells were less pronounced and less durable than in the mouse [6]. One of the major differences compared with the mouse was that, in cultures of human cells, mpl failed to induce MHPC self-renewal. Instead, mpl signaling in human hemopoietic cells appeared capable only of inducing the differentiation of erythroid and megakaryocytic progenitor cells. Notably, human myeloid progenitors were unresponsive to the mpl signal.
If the effects of mpl in human hemopoietic cells were confined to the differentiation of erythroid and megakaryocytic progenitors, its potential utility would be significantly constrained. We, therefore, tested whether the addition of growth factors to CID would enlarge upon the biological effects of mpl signaling in transduced human hemopoietic cells.
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MATERIALS AND METHODS
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Retroviral Construct and Viral Production
The construction of the F36VmplGFP vector is described in a previous report [7]. The green fluorescent protein-internal ribosomal entry site (GFP-IRES) DNA fragment was isolated from the published construct MSCV-GFP-internal ribosomal entry site-neo (MGIN) [8]. Supernatant from the GP+E-based cell line described in that report was used to transduce the PG13 packaging cell line [7]. Producer clones were screened and sorted based on GFP expression. Titer was determined by GFP expression in transduced HT1080 cells and a clone with a titer of 2 x 105 was isolated.
Transductions of CD34-Selected Cells
CD34-selected cells were isolated from normal human umbilical cord blood scheduled for disposal after delivery, after approval by the Human Subjects Review Board at the University of Washington. Mononuclear cells from total cord blood were separated from red blood cells using density gradient centrifugation (with a density of 1.077) on a cell separation medium (Lymphoprep; Mediatech; Herndon, VA). Immunomagnetic selection of the CD34+ cells was accomplished using the magnetic activated cell sorter system (Miltenyi Biotec; Auburn, CA; http://www.miltenyibiotec.com), and the resulting purity of the CD34-selected cord blood cells was routinely >90%. CD34-selected cadaveric bone marrow cells were a generous gift from S. Heimfeld, Fred Hutchinson Cancer Research Center (Seattle, WA; http://www.fhcrc.org).
We used the method of Henneman et al. for transduction of CD34-selected cord blood cells and peripheral blood cells [9]. Following isolation, cells were placed in prestimulation conditions consisting of Iscove’s modified Dulbecco’s medium (IMDM) supplemented with: 20% Bit9500 (Stem Cell Technologies; Vancouver, Canada; http://www.stemcell.com); 10% low-density lipoproteins (Sigma; St. Louis, MO; http://www.sigmaaldrich.com); 10 ng/ml recombinant human interleukin 6 (hIL-6); 50 ng/ml recombinant human stem cell factor (hSCF); 50 ng/ml recombinant human megakaryocytic growth and differentiation factor (hMGDF), and 20 ng/ml recombinant human flt3 ligand (hFlt3) for 48 hours. The cells were then removed from the prestimulation buffer, brought up in conditioned viral supernatant (IMDM with 10% fetal bovine serum [FBS]) containing the same cytokine mixture, and aliquoted onto nontissue culture plates prepared with retronectin (Panvera; Madison, WI). The retronectin-treated plates were twice preloaded with retroviral supernatant for 30 minutes at room temperature. The cells were then incubated for 24 hours, and the procedure was repeated two more times for a total viral exposure of 72 hours. The cells were then removed and placed in IMDM plus 10% FBS with the same cytokine mixture for 48 hours, subjected to flow cytometry to determine GFP expression, and then used for long-term expansion experiments (day 0).
Long-Term, Cytokine-Supported Cultures
Conditions for ex vivo expansion were similar to those described by Piacibello and colleagues [10-12]. Starting numbers of CD34-selected cord blood cells varied between 3.4 x 104 (Fig. 2
, panel B) and 5 x 105 (Fig. 2, panel E) prior to the transduction. After analysis of baseline GFP expression, cells were placed in the IMDM, 10% FBS (Rehatuin; Intergen; Purchase, NY), 50 ng/ml hSCF, 50 ng/ml hFlt3, 20 ng/ml hMGDF, and 10 ng/ml hIL-6, in the absence or presence of the CID, AP20187. AP20187 was obtained from ARIAD Pharmaceuticals (Cambridge, MA; http://www.ariad.com/regulationkits). Cytokines were obtained from the following sources: hSCF was a generous gift from Dr. Virginia Broudy, hFlt3 was supplied by Immunex (Seattle, WA; http://www.immunex.com), hMGDF was obtained from Amgen (Thousand Oaks, CA; http://www.amgen.com), and hIL-6 was purchased from Peprotech (Rocky Hill, NJ; www.peprotech.com). Cells were demi-depopulated weekly to maintain the cell number in the culture below 1 x 106/ml. In general, the culture was split 1 to 3 or 1 to 4. Cells were periodically subjected to flow cytometry on a BD FacScan, using the following directly phycoerythrin-labeled antibodies: anti-CD34, anti-glycophorin A, anti-CD33, and anti-CD41 (Pharmingen; San Diego, CA; http://www.bdbiosciences.com/pharmingen).
Clonogenic Assays
Clonogenic assays were performed in triplicate using a commercially available methycellulose assay system (Stem Cell Technologies) with the addition of the following cytokines: 5 ng/ml hIL-3 (Peprotech), 50 ng/ml hSCF, and 5 U/ml human erythropoietin. Cells were cultured in a humidified 37°C incubator with 5% CO2. Colonies were counted on day 14. Plasma clot assays (Fig. 1) were performed as previously described [6].
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RESULTS
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We first tested whether human hemopoietic cells transduced with the previously described MSCV-based retroviral vector, F36VmplGFP [7] could be induced to proliferate in response to the FKBP-specific dimerizing drug, AP20187. CD34-selected cells from human cord blood were transduced with F36VmplGFP packaged in the GALV envelope using the cell line PG13 [13]. This bicistronic vector contains an upstream gene encoding a GFP reporter and a downstream gene encoding the F36Vmpl fusion protein, with an interposed encephalomyocarditis IRES sequence [14] (Fig. 1A
). Following transduction, cells were cultured in the absence of added growth factors and in the presence or absence of AP20187 (100 nM). AP20187 produced a transient, 20-fold increase in transduced cord blood cells over 3 weeks of culture (Fig. 1B). The proliferative effect was manifested in transduced erythroid progenitors, in that BFU-E could form in semisolid culture conditions supplemented with AP20187 (Fig. 1C). Glycophorin-A-positive red blood cells were present only in the conditions supplemented with CID (Fig. 1). These results are similar to those we have described previously using AP1903, a different retroviral construct, and an alternative transduction method [6].
We subsequently tested whether selection could occur in the presence of growth factors that support long-term cell expansion and whether the phenotypic response would be modified. CD34+ cells were obtained from cord blood (five experiments) and adult bone marrow (one experiment). Gene transfer rates were assessed by determining the frequency of GFP+ cells immediately post-transduction and by the percentage of GFP+ progenitors as assessed in colony assays. The frequency of GFP+ cells post-transduction ranged from between 64% and 88% (76.6% ± 10.3%, n = 5), while GFP+ progenitor colonies ranged from between 48% and 79% (58.8% ± 14.6%, n = 4). In two experiments, we also assessed the efficiency of gene transfer into nonobese diabetic-severe combined immunodeficient repopulating cells (SRCs) and, in both experiments, gene transfer into 20% of SRCs was attained (data not shown).
Following transduction, cells were cultured in suspension in a combination of growth factors (Flt3, IL-6, SCF, and MGDF), either in the presence or absence of AP20187 (100 nM). Previous studies have shown that this growth factor combination can support the sustained growth of human cord blood CD34+ cells [10-12]. In each of five separate experiments, this cytokine combination supported the growth of cord blood cells for >10 weeks of culture (Figs. 2A-2E, left). In an experiment using CD34-selected cells from cadaveric bone marrow, expansion was limited to 4 weeks (Fig. 2F, left). In five of the six experiments, cells cultured in the presence of growth factors plus AP20187 grew at a slightly more rapid rate during the initial 2 to 4 weeks of culture, whereas AP20187 failed to exert a growth-enhancing effect beyond 4 weeks of culture. In several instances, cell growth in the presence of the CID was slightly lower at later time points of culture (Figs. 2B, 2C, and 2D, left), and in no instance did AP20187 prolong the growth response. The lack of an augmented growth response upon the addition of CID to growth factors suggests the possibility that a specific growth-enhancing effect on transduced cells might be counterbalanced by a nonspecific growth inhibitory effect of AP20187. To test this possibility, mock-transduced CD34+ cord blood cells were cultured using the same growth factor combination, either in the presence or absence of AP20187. As shown in Figure 3, AP20187 had no adverse effect on cell growth.
The percentage of GFP+ cells over time in culture was strongly influenced by the presence of the CID (Fig. 2, right). In all six experiments, the absence of CID was associated with a steady decline in the percentage of GFP+ cells from 64%-88% immediately following transduction, to between 5% and 30% by weeks 4 to 8 of culture (Fig. 2, right, open triangles). In contrast, the addition of CID consistently produced a rise in the percentage of GFP+ cells by the end of the first week of culture (Fig. 2, right, closed triangles). We interpret this early response to be most readily explained by the differentiation of transduced progenitor cells. At later time points, results varied substantially among different experiments. In general, three different patterns of response were observed.
As seen in one experiment in the first pattern (Fig. 2E, right), a rise in the frequency of GFP+ cells over the first 2 weeks of culture was followed by a sharp decline, such that GFP+ cells were undetectable by week 7 of culture. In that experiment, the presence of CID was associated with a somewhat more precipitous decline in GFP+ cells than was observed when cells were cultured in the absence of CID. This finding is consistent with a CID-induced exhaustion of genetically modified cells. We conjecture that, in this experiment, relatively short-lived progenitors were transduced, whereas longer lived (and presumably more primitive) hemopoietic cells were apparently not transduced. The accelerated loss of GFP+ cells in the presence of CID likely arose from an accelerated differentiation of transduced progenitor cells in response to the CID.
In a second pattern, observed in three experiments (Figs. 2C, 2D, and 2F, right), the initial rise in the percentage of GFP+ cells was followed by a decline similar to that observed for cells cultured in the absence of CID. Thereafter, cultures performed in the presence of CID maintained a higher frequency of GFP+ cells than cultures performed in the absence of CID. These findings indicate that, in contrast to the first pattern (Fig. 1E, right), gene transfer into relatively longer lived cells was achieved. The relatively higher frequency of GFP+ cells in the presence of CID could be due either to a CID-induced expansion of transduced progenitor cells, or alternatively, CID exposure might cause transduced progenitors to generate a greater number of progeny compared with their nontransduced counterparts. Transduced progenitors assessed either functionally, in colony assays (Table 1), or phenotypically, as CD34+ cells (Fig. 4A), failed to rise in response to the CID. The higher frequency of GFP+ cells in the bulk culture is, therefore, likely due to an augmented contribution of transduced progenitors to the mature cell pool in response to the CID. This interpretation is supported by our previous observation that CID-mediated mpl signaling synergizes with SCF to dramatically augment progenitor colony size [5].
In a third pattern, seen in two experiments (Figs. 2A and 2B, right), the initial wave of GFP+ cells was followed by a slight decline that was, in turn, followed by a second rise occurring between 5 and 7 weeks of culture. Thereafter, >90% of cells were GFP+ for the remainder of the culture. In these cultures, transduced progenitors rose markedly in response to CID treatment, as reflected by increases in GFP+/CD34+ double-positive cells (Fig. 4B) and GFP+ progenitor colonies (Table 1, experiment A).
Our previously published results demonstrated that CID-induced mpl signaling can support the differentiation of committed erythroid progenitors, but has no effect on myeloid progenitor cells. Erythroid cells were present in cultures containing the growth factor/CID combination (glycophorin A, Fig. 5, bottom). In addition, in the experiment shown in Figure 2A, the combination of growth factors plus CID yielded a dominant population of CD33-expressing cells (Fig. 5 top). These results indicate that myeloid progenitors can be converted to CID responsiveness by supplementing the mpl signal with signals induced by other growth factors.
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Figure 5. Flow cytometry demonstrates selection of both myeloid and erythroid cells in response to CID. Cells expanded for 10 weeks (Fig. 2A) were analyzed by flow cytometry to determine expression of CD33 (top panels), CD34 (middle panels), and glycophorin A (glyA, lower panels). CD19+ B cells and CD41+ megakaryocytic cells were not detected (data not shown). GFP expression of the cell populations was also analyzed. Results from three different culture conditions are shown: mock-transduced cells (Mock), cells transduced with the F36VmplGFP vector cultured in the absence of CID (-CID), cells transduced with the F36VmplGFP vector cultured in the presence of CID (+CID). The percentage of gated events is noted for the upper quadrants.
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DISCUSSION
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A dominant feature of our present studies was the variability in the effect of CID exposure from one experiment to the next. This characteristic distinguishes our current results from those we have reported previously either in the murine system or in cultures using human hemopoietic cells with CID alone. The differences among experiments are, in part, attributable to the variable success in achieving gene transfer into primitive cells with an extensive proliferative capacity. Most notable, in this regard, is the experiment depicted in Figure 2E, where GFP-labeled cells disappeared within 9 weeks of culture, indicating that gene transfer was restricted to relatively mature, committed progenitors that differentiated rapidly in response to CID exposure. In contrast to the other four cord blood experiments, cells that could be maintained for at least 9 weeks of culture were not transduced. Improvements in gene transfer into the human hematopoietic stem cell include vectors based on lentivirus and foamy virus [15, 16]. The use of these vectors to express the CID-dependent fusion protein could result in more reliable selection. This system will also allow the analysis of other CID-dependent growth signals in the presence of other cytokine combinations. In this way, signaling pathways critical for stem cell self-renewal and/or differentiation can be investigated.
Interestingly, variable outcomes were also apparent among the four remaining cord blood experiments in which transduced cells persisted beyond 9 weeks of culture. Within this category, two types of response to CID were observed. In two cultures, CID treatment induced a definite, albeit modest, increase in GFP+ cells in the culture (Figs. 2C and 2D). In that setting, CID treatment failed to induce an expansion of progenitor cells, rather, the apparent effect of CID treatment was to trigger transduced progenitors to make an exaggerated contribution to the bulk culture, presumably by generating more progeny than their nontransduced counterparts. We have previously found that CID-induced mpl signals can synergize with SCF to augment colony size [6]. In the remaining two experiments, GFP+ cells dominated the culture in response to CID exposure, and in those cultures, the CID affected expansion of clonogenic progenitors and CD34+ cells. The differences between these two patterns of CID responsiveness might also be attributable to differences among the types of long-lived progenitors that were transduced: one subset in which the mpl signal leads to differentiation and another subset in which the mpl signal induces both proliferation and differentiation. Alternatively, the observed differences may have arisen from other factors. For example, it is hypothetically possible that the response of a long-lived progenitor to CID treatment might be influenced by the level at which the mpl fusion was expressed. Long-lived progenitors that express the fusion protein at a relatively low level might respond to the mpl signal through differentiation, whereas similar cells expressing higher levels of the fusion protein may exhibit both proliferation and differentiation. We are testing other constructs that should express the fusion protein at a higher level. In these constructs the mpl fusion protein is expressed directly off the long terminal repeat.
In the murine system, CID-mediated mpl signaling induces a sustained self-renewal of multipotential progenitor cells that can last for up to a year of culture [4]. To the contrary, in human cord blood cells, the CID/growth factor combination failed to promote progenitor self-renewal, as evidenced by the finding that, even in the setting of maximal CID responsiveness, CID treatment failed to prolong the period of time over which cells could be expanded in culture.
An unexplained finding associated with the growth factor/CID culture system merits comment. In two experiments (Figs. 2A and 2B), CID exposure promoted a dramatic increase in the frequency of GFP+ cells. Surprisingly, despite this clear influence of CID on the frequency of GFP+ cells, there was no discernible effect of CID on total cell numbers, compared with cultures performed in growth factors alone. This finding initially led us to suspect that the ability of AP20187 to promote growth among genetically modified cells might be counterbalanced by a generalized inhibitory effect, a possibility that was subsequently excluded (Fig. 3). These results indicate that maximal rates of growth had already been accomplished with the cytokine cocktail and could not be further augmented by the CID. The factors that place a ceiling on the maximal rate of cell growth remain to be defined.
Our results demonstrate that human hemopoietic progenitors can expand for prolonged periods in culture in response to CID and emphasize the need to achieve gene delivery into the appropriate cell type. These findings are relevant to the potential clinical application of CID-regulated cell therapy.
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ACKNOWLEDGMENT
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We thank Tim Clackson at ARIAD Pharmaceuticals for AP20187 (www.ariad.com), Hanno Glimm for assistance with transductions, and Shelly Heimfeld for supplying cadaveric CD34-selected bone marrow cells.
This work was supported by grant numbers 5R01DK 52997, 1R01DK57525, 2P01HL53750, 1P01DK 55820, and 2P01DK47754 from the National Institutes of Health, an American Society of Hematology Junior Faculty Scholar Award, and an award from the Fanconi Anemia Research Fund.
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Received June 10, 2002;
accepted for publication August 23, 2002.
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Top
Abstract
Introduction
) or absence (
) of AP20187 (100 nM). Each pair of panels represents a separate experiment. (F) depicts a single experiment performed using CD34-selected cadaveric bone marrow cells. Right panels: percentage of GFP+ cells during the time course of the cultures shown on the left. In the experiment depicted in D, the cells were divided into three separate experiments on day 2 after the transduction and treated as separate experiments. The cell expansion experiment (D, left) is shown with standard error bars (± 0.05). Cultures lacking CID (D, right) are depicted as a single line with standard error bars, while the CID-containing cultures are shown as three separate lines (
, 
, 
). When transduced cord blood cells were plated in plasma clot assays in the presence of CID and in the absence of added cytokines, GFP+ erythroid bursts developed (C). The same colony is pictured under direct microscopy (top) and also viewed under fluorescent microscopy (bottom). D) is a dot plot of transduced cord blood cells cultured with or without CID and subjected to flow cytometry for GFP expression and expression of the erythroid marker, glycophorin A (GlyA). Cells were analyzed after 9 days in culture. In the presence of the CID, a population of glycophorin-A-positive cells emerged.
