Enforced adenoviral vector-mediated expression of HOXB4 in human umbilical cord blood cd34+ cells promotes myeloid differentiation but not proliferation

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doi:10.1016/S1525-0016(03)00237-5

Enforced Adenoviral Vector-Mediated Expression of HOXB4 in Human Umbilical Cord Blood CD34ⴙ Cells Promotes Myeloid Differentiation but Not Proliferation Ann C. M. Brun,1,* Xiaolong Fan,1 Jon Mar Bjo¨rnsson,1 R. Keith Humphries,2 and Stefan Karlsson1 1 2

Department of Molecular Medicine and Gene Therapy, Lund University Hospital, 221 84 Lund, Sweden Terry Fox Laboratory, British Columbia Cancer Research Agency, Vancouver, British Columbia, Canada

*To whom correspondence and reprint requests should be addressed at Molecular Medicine and Gene Therapy, BMC A12, 221 84, Lund, Sweden. Fax: ⫹46 46 222 05 68. E-mail: [email protected].

Retroviral overexpression of the transcription factor HOXB4 results in a rapid increase in proliferation of murine hematopoietic stem cells both in vivo and in vitro. Therefore, we asked whether transient overexpression of HOXB4 would increase proliferation of human primitive hematopoietic progenitors. Transient overexpression of HOXB4 was generated in umbilical cord blood (CB) CD34ⴙ cells by a recombinant adenovirus (AdHOXB4) expressing HOXB4 together with the enhanced green fluorescent protein (GFP). Transduced, GFPⴙ cells were cultured in serum-free medium containing cytokines that primarily support the growth of primitive hematopoietic progenitors. In contrast to previous findings using retroviral overexpression of HOXB4, we did not observe any increase in proliferation of primitive progenitors or increased colony formation of clonogenic progenitors, including progenitor progeny from long-term culture-initiating cells following adenoviral vector overexpression of HOXB4 in CB CD34ⴙ cells. However, enforced expression of HOXB4 by the adenoviral vector significantly increased myeloid differentiation of primitive hematopoietic progenitors. Since retroviral vectors generate low and continuous levels of transgene expression in contrast to the high, transient levels generated by the adenoviral vector, our findings suggest that the high levels of HOXB4 expression generated by AdHOXB4 in human CB CD34ⴙ cells direct the cells toward a myeloid differentiation program rather than increased proliferation. Key Words: human, hematopoietic stem cells, HOXB4, gene therapy, adenovirus

INTRODUCTION The backbone of the hematopoietic machinery is the hematopoietic stem cell (HSC), a rare and mostly dormant cell type that has to be strictly controlled to self-renew and differentiate to produce millions of mature blood cells of all lineages daily throughout life. The control of HSC fate options, i.e., mechanisms that control whether stem cells self-renew, differentiate, undergo apoptosis, or migrate, is poorly understood. However, recent evidence points to the important role of transcription factors that can regulate gene expression and thereby maintain or alter the transcriptional program of hematopoietic cells to control their fate (reviewed in [1]). This complex system of transcription factors can activate or repress genetic pathways depending on the combination and/or concentration of these regulatory molecules. The DNA binding

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homeobox (HOX) transcription factors were identified as key regulators in embryonic development [2,3] and in recent years it has become evident that some Hox genes play an important role in hematopoietic development, pre- and postnatally, controlling fate decisions like selfrenewal, differentiation, and lineage commitment [4 –7]. Expansion of hematopoietic stem and progenitor cells in vivo and in vitro is of importance to achieve successful treatment of many hematological disorders. However, successful expansion of HSC with long-term repopulating ability is still limited using cytokine expansion in standard cell culture systems. Therefore, the development of alternative approaches, including enforced expression of transcription factors, is warranted. One of the genes thought to have a critical role in self-renewal of hematopoietic stem cells is the HOXB4 gene. HOXB4 is selectively expressed in primitive hematopoietic cells but is down-

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regulated in committed progenitors and more mature hematopoietic cells [4,8]. In a murine transplantation model, retroviral overexpression of HOXB4 greatly enhanced the regenerative capacity of the transduced donor hematopoietic stem cells in both primary and secondary recipients. It is noteworthy that the HOXB4-generated expansion continues until steady-state hematopoiesis is observed and the stem cell pool is normalized [6,9]. Furthermore, recent findings demonstrate that an approximately 40-fold expansion of murine repopulating HSCs can be achieved by enforced expression of HOXB4 ex vivo for 10 –14 days [10]. Similarly, our lack-of-function HOXB4 mouse models support the notion that the main role of HOXB4 in vivo is to increase the proliferative capacity of HSCs during hematopoietic stress, whereas the HOXB4 deficiency does not have a significant impact on hematopoietic lineage decisions [11,12]. Together these observations suggest that HOXB4 can provide novel approaches to stimulate the growth of primitive hematopoietic cells. This possibility is further supported by the findings of enhanced growth of human cord blood cells following retrovirally engineered overexpression of HOXB4 [13,14]. Schiedlmeier et al. generated high levels of HOXB4 that were continuously expressed in hematopoietic cells in vivo by a retroviral vector, resulting in increased proliferation of primitive hematopoietic progenitors, while the differentiation of their progeny cells was blocked due to the high levels of HOXB4 [14]. We have recently demonstrated that recombinant adenoviral vectors can efficiently transduce primitive, repopulating, human hematopoietic progenitors [15–17]. Based on the rapid and striking proliferative effects of HOXB4 in primitive hematopoietic cells observed following retroviral gene transfer, we asked whether transient overexpression of HOXB4 mediated by an adenoviral vector in cord blood CD34⫹ cells could also lead to enhanced proliferation of primitive hematopoietic progenitors for beneficial therapeutic applications in cell and gene therapy. In contrast to previous findings showing expansion of primitive progenitors upon retroviral overexpression, the cord blood CD34⫹, CD38⫺ cells expressing high levels of HOXB4 from the adenoviral vector exhibited an increased propensity to differentiate into cells committed to the myeloid lineage.

RESULTS Vector Transduction of Hematopoietic Cells To study the effects of transient HOXB4 expression on the proliferation and differentiation of human CD34⫹ cells, we generated adenoviral vectors containing the HOXB4 and green fluorescent protein (GFP) genes. The adenoviral vector containing the HOXB4 and GFP genes and the control vector containing the GFP gene alone are shown

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in Fig. 1A. A representative transduction and sorting experiment using the adenoviral vectors to transduce cord blood (CB) CD34⫹ cells is depicted in Fig. 1B. The transduction efficiency of the CD34⫹ CB population was 20 – 30% as determined by GFP expression (FACS) for the adenoviral vector containing both HOXB4 and GFP (AdHOXB4) and the control vector (AdGFP). It is noteworthy that the more primitive, lineage-negative, CD34⫹, CD38⫺ population was more efficiently transduced (40 –50%) than the CD34⫹, CD38⫹ fraction, in agreement with our earlier findings [16]. Importantly, high levels of GFP could be detected in the primitive CD34⫹, Lin⫺ fraction 36 h after transduction was initiated, indicating high levels of GFP transgene expression in this primitive cell compartment (Fig. 1B). Very High Levels of HOXB4 Are Generated in AdHOXB4-Transduced CD34ⴙ Cells To quantitate the levels of HOXB4 following transduction, we used quantitative RT-PCR (Q-RT-PCR), which was demonstrated to be in the linear range (Fig. 2A). We demonstrated a close correlation between the levels of HOXB4 mRNA and HOXB4 protein by a quantitative comparison between GFP levels (mean fluorescence intensity), HOXB4 mRNA levels, and HOXB4 protein levels measured in HeLa cells transduced with the adenoviral vectors at different m.o.i. (Fig. 2B). At m.o.i. of 5, the levels of GFP and HOXB4 mRNA and HOXB4 protein were approximately one order of magnitude lower than in cells that were transduced at 50 m.o.i. These findings demonstrate good correlation between the levels of HOXB4 mRNA and protein and hereafter we use HOXB4 mRNA to quantify expression of the transgene. To study the level of HOXB4 transgene expression over time, we transduced cells after 16 h prestimulation and grew them in liquid culture containing stem cell factor (SCF), Flt3 ligand (FL), and thrombopoietin (TPO). We collected cells at different time points and analyzed them for levels of HOXB4 mRNA by Q-RT-PCR (Fig. 3A). An aliquot of each sample was taken on day 3 to determine transduction efficiency. FACS analysis of the cells showed near-equal transduction levels with both viruses used (20 –25% GFPexpressing cells). The level of HOXB4 gene expression was measured relative to the level of actin mRNA and is presented as relative intensity (RI) and followed over time (Figs. 3A and 3B). The level of HOXB4 in the AdHOXB4transduced cells was increased up to the maximum level 24 – 48 h after transduction (604 ⫾ 21 RI, n ⫽ 3, Fig. 3A). Since the above cells represented a mixture of transduced and untransduced cells, we sorted GFP-positive cells on day 4 to determine the HOXB4 level in transduced cells. The findings show a 15-fold higher level of HOXB4 in AdHOXB4-transduced cells compared to AdGFP-transduced cells (Fig. 3B). Next, we asked whether there was a close correlation between expression levels of HOXB4 and

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FIG. 1. Adenoviral vector design and cell sorting. (A) Recombinant adenovirus was generated in an E1/ E3-deleted backbone of adenovirus type 5. The CMV promoter was used to direct expression of the HOXB4 and GFP genes. (B) A representative sorting profile of transduced CB CD34⫹ cells 36 h after initiation of transduction. Cells were transduced by AdHOXB4 or AdGFP. Viable cells, not labeled by 7-AAD (R2), were selected and cells that were CD34 positive, CD38 negative, lineage marker negative (R3), and GFP positive (R4) were sorted.

GFP in transduced cells. We sorted the transduced cells based on their GFP expression level (low or high, Fig. 3C) and analyzed for the expression of HOXB4 by Q-RT-PCR. We found a close correlation between GFP and HOXB4 expression levels in AdHOXB4-transduced cells, and practically no measurable increase in HOXB4 mRNA could be detected in GFP low cells (Fig. 3D). Therefore, the transgene expression of AdHOXB4 is almost immediate and reaches very high levels within 24 h of transduction and the highest levels of HOXB4 are found in the GFP high fraction. Because HOX genes can cross regulate each other [18,19] we asked whether the high HOXB4 transgene expression levels affected the regulation of the neighboring HOX genes and the relevant paralogous HOX genes. We found no difference in the mRNA expression level of HOXA4, HOXA6, HOXA10, HOXB3, HOXB5, HOXB6, or the PBX-1 gene when cells transduced with the AdHOXB4 and control vectors were analyzed (data not shown).

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Adenoviral Vector Overexpression of HOXB4 in CD34ⴙ Cells Promotes Differentiation toward Myeloid Lineages First, we evaluated the effects of high levels of HOXB4 expression on cell proliferation and differentiation in short-term liquid cultures. We cultured transduced sorted (GFP⫹) cells in serum-free medium containing SCF, FL, TPO, and erythropoietin (EPO) for 7–9 days. No difference in total cell expansion between AdHOXB4 and AdGFP transduced cells could be seen (Fig. 4A). To ask whether the high levels of HOXB4 expression would perturb lineage determination, we performed FACS analysis to determine the expression levels of cell surface lineage markers for erythroid (glycophorin A (GpA)) and myeloid (CD14, CD15) cells. AdHOXB4-transduced, GFP⫹ cells showed a significant increase in the number of cells expressing myeloid cell surface markers (22.1 ⫾ 5.2) (P ⫽ 0.004, n ⫽ 6) compared with AdGFP-transduced cells (Fig. 4B). An in-

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FIG. 2. High expression levels of HOXB4 in AdHOXB4-transduced hematopoietic cells. (A) The quantitative RT-PCR analysis is within the linear range. Lanes 1– 6, AdHOXB4 plasmid diluted with genomic DNA to give 10, 1, 0.1, 0.01, 0.001, and 0.0001 copies per cell. Lanes 7 and 9, genomic DNA. Lane 8, genomic DNA mixed with 1:1 ratio of AdGFP. Lane 10, negative control for cDNA synthesis (RT⫺) from AdHOXB4-transduced cells. (B) Western blot of nuclear protein extraction from HeLa cells transduced with 5 or 50 m.o.i. of the AdGFP and AdHOXB4 vectors. High GFP expression correlates well with high relative intensity (RI) of HoxB4 mRNA/cDNA as well as high protein levels.

crease in the number of erythroid cells was also seen, but this was not statistically significant (26.7 ⫾ 13.9 and 17.0 ⫾ 11.4, respectively). Myeloid differentiation was confirmed by May–Gru ¨ nwald–Giemsa staining of cytospin preparations. These findings demonstrate that adenoviral vector-mediated HOXB4 overexpression promotes myeloid differentiation of hematopoietic progenitors. Adenoviral Overexpression of HOXB4 Does Not Increase Proliferation of Primitive Hematopoietic Progenitors Since retroviral vector-mediated overexpression of HOXB4 leads to an increase in proliferation of mouse as well as human primitive hematopoietic progenitors [20,21], we wanted to investigate whether the AdHOXB4transduced cells exhibited a proliferative advantage compared to AdGFP-transduced cells. We sorted AdHOXB4and AdGFP-transduced cells (CD34⫹, CD38⫺, Lin⫺, GFP⫹) and seeded them in Terasaki plates to evaluate survival and proliferation of single cells. Viability (see Materials and Methods) of AdHOXB4- and AdGFP-trans-

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duced clones was similar in a maximum cytokine cocktail, indicating no toxic effect in AdHOXB4-transduced cells (data not shown). To test proliferation recruitment, the sorted cells were plated in Terasaki plates and cultured in SCF alone, SCF and TPO, or a combination of SCF, TPO, and FL from day 0. Wells were scored 10 –11 days after addition of the cytokine mix and the proliferative capacity of the clones was estimated by the total number of clones recruited into proliferation (⬎3 cells/well) and the number of large clones (⬎50 cells/well) [22]. Proliferation recruitment of the AdHOXB4-transduced primitive cells was not increased compared with the AdGFP-transduced cells in any of the cytokine combinations tested (Fig. 5A, data from SCF ⫹ TPO not shown). When cells were sorted into high and low GFP-expressing fractions, there was a strong trend for reduced proliferation recruitment by the AdHOXB4 high-expressing progenitors, in the presence of both TPO alone and SCF, TPO, and FL (Fig. 5B), whereas no difference was observed with progenitors expressing low levels of GFP (and thereby low levels of the HOXB4 transgene as shown in Fig. 3D). A difference was seen between GFP high- and low-expressing cells, indicating that although sorted for the same cell surface markers (Lin⫺, CD34⫹, CD38⫺) high and low GFP-expressing cells could represent slightly different populations. Cumulatively our findings are in sharp contrast to the findings from murine and human primitive hematopoietic cells transduced with HOXB4 retroviral vectors, in which the proliferation of the HOXB4-overexpressing cells was significantly increased [9,13,21]. AdHOXB4 Overexpression Leads to a Reduction in BFU-E, CFU-E, and CFU-GM Colonies Next, we asked whether AdHOXB4 overexpression in CD34⫹ colony-forming progenitors would increase the number of myeloid colonies. We plated sorted cells in methylcellulose and cultured them for 8 days, for CFU-E analysis, and 12 days for BFU-E growth in the presence of SCF and EPO or, for CFU-GM, using IL-3 and GM-CSF [23]. All three assays showed a significant reduction in number of colonies derived from the AdHOXB4-overexpressing cells compared to AdGFP (P ⬍ 0.05, n ⫽ 10, Fig. 6). HOXB4 Overexpression by Adenovirus Leads to a Reduction in Long-Term Culture-Initiating Cell (LTC-IC) Output To investigate the long-term effect of adenoviral overexpression of HOXB4, we plated AdHOXB4-transduced Lin⫺, CD34⫹, CD38⫺, GFP⫹ cells in a 96-well plate on irradiated murine stoma cell lines SL/SL and M210B4, at 50 or 150 cells per well, and cultured them for 6 weeks at 37°C. After 6 weeks, we collected all cells from each well and divided them into two samples containing 20 and 80% of the cells, respectively. We plated each sample in methylcellulose cultures and counted the colonies 12

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FIG. 3. (A) RT-PCR analysis of cultured unsorted CB cells transduced with AdHOXB4 or AdGFP vectors (⬇20% transduction efficiency) and untransduced cells. (B) Transduced cells were sorted for GFP expression on day 4 and analyzed by RT-PCR for HOXB4 expression. (C, D) Transduced cells were sorted for high and low expression levels of GFP and were analyzed for HOXB4 expression using RT-PCR. All the high-expressing HOXB4 cells were found in the GFP high fraction.

days later. The number of colonies derived from 1000 input cells transduced with AdHOXB4 was significantly reduced compared to AdGFP-transduced cells (AdHOXB4 50 ⫾ 13 and AdGFP 189 ⫾ 118; P ⬍ 0.04, n ⫽ 3) (Table 1). Although the high level expression of HOXB4 is present only transiently during the first 7–10 days, its effect, as seen here, has an impact on the fate of the cells over time, compared to control-transduced cells. AdHOXB4 Does Not Increase the Generation of Secondary Clonogenic Progenitors To ask whether generation of secondary hematopoietic progenitors was affected by enforced expression of HOXB4, we plated transduced CD34⫹, CD38⫺, Lin⫺ cells sorted for GFP expression into liquid culture for 6 weeks and plated biweekly aliquots into methylcellulose cul-

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tures. We counted the cells for expansion periodically during the 6-week culture. Initially no significant difference in expansion could be detected between AdHOXB4transduced cells and controls. However, at 6 weeks, we observed a remarkable difference in proliferation potential resulting in a 2163 ⫾ 231-fold expansion for the AdGFP-transduced cells compared to 1126 ⫾ 365 (P ⫽ 0.03, n ⫽ 3) for the AdHOXB4-transduced cells (Fig. 7A). We collected primary colonies from the liquid culture at time 0 through week 6 and washed and replated them to test their ability to form secondary colonies. Sorted AdHOXB4-transduced cells plated into the culture initially (time 0) showed diminished ability to form primary colonies (CFU-GM, 25 ⫾ 7; CFU-E, 1 ⫾ 1 per 500 input cells) compared to AdGFP-transduced cells (CFU-GM, 98 ⫾ 9;

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DISCUSSION

FIG. 4. Adenoviral overexpression of HOXB4 in CD34⫹ cells promotes myeloid differentiation. Expansion and expression of lineage markers on cells transduced with adenoviral vector AdHOXB4- and control vector-transduced cells. (A) Transduced sorted cells (Lin⫺, CD38⫺, 7-AAD⫺, CD34⫹, GFP⫹) were cultured for 12 days and cells were enumerated to evaluate expansion of total cells. No difference in expansion rate could be detected between AdHOXB4transduced and control cells during this time. (B) On day 7–9 cells were analyzed with antibodies against myeloid (CD14, CD15) and erythroid (GpA) lineage markers. Bars represent the average and the standard deviation of the % positive cells for each lineage. The increase in myeloid differentiation following transduction with AdHOXB4 is highly significant (P ⬍ 0.001) while the variation in erythroid differentiation resulted in a nonsignificant difference.

CFU-E, 6 ⫾ 2) (Fig. 7B). The number of secondary colonies was higher for both AdHOXB4- and AdGFP-transduced cells compared to primary colony numbers; however, the majority of the secondary colonies derived from AdHOXB4 cells were of erythroid origin (CFU-GM, 17 ⫾ 3; CFU-E, 93 ⫾ 11 per 500 input cells) while the distribution of colonies derived from the AdGFP-transduced cells showed lower numbers of CFU-E (50 ⫾ 9) and higher numbers of CFU-GM (85 ⫾ 11) colonies. Similarly, after 6 weeks of liquid culture we plated primary and secondary colonies as above. The AdHOXB4-transduced cells could still produce primary (497 ⫾ 56) colonies although at a slightly lower rate than the AdGFP-transduced cells (708 ⫾ 183), but no secondary colonies could be detected from the AdHOXB4-transduced cells, while the AdGFP-transduced cells could produce low levels of secondary colonies (17 ⫾ 6) at this time point (Fig. 7C). These findings suggest that AdHOXB4 does not increase generation of secondary clonogenic progenitors in contrast to HOXB4encoding retroviral vectors [13].

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Given the previously documented stimulatory effects of HOXB4 on the growth of murine and human primitive hematopoietic cells in vitro and in vivo using retroviralmediated gene transfer, we asked whether transient delivery of HOXB4 by adenoviral vectors might have similar effects in human cells in vitro. The rationale of this approach was to avoid the potential complications of permanent HOXB4 overexpression by using adenoviral vectors that can transduce primitive repopulating hematopoietic progenitors efficiently [16]. Efficient transduction and the ability to select cells by GFP expression allowed careful analysis of the fate of the transduced cells. First, we asked whether adenoviral vector overexpression of HOXB4 would expand the pool of primitive human hematopoietic progenitors. In contrast to the findings reported following retroviral delivery, we detected a reduction in colony formation of AdHOXB4-transduced cells and the output from transduced LTC-IC was similarly reduced compared to cells transduced with the AdGFP control vector. Similarly, enforced expression of HOXB4 by adenoviral vectors generated significantly increased numbers of myeloid progeny cells. Previously, Antonchuk et al. have shown that retroviral-mediated transfer of HOXB4 has a growth-promoting effect on colony-forming progenitors and without affecting differentiation decisions following a twofold increase in HOXB4 mRNA [6], which we could confirm using the same vector (data not shown). In the adenoviral vector-transduced hematopoietic cells the levels of the HOXB4 transgene rose sharply and quickly and were an order of magnitude higher than in their control-transduced counterparts. Since we demonstrated that there was a good correlation between the levels of HOXB4 mRNA and protein in AdHOXB4-transduced HeLa cells, we could monitor the levels of HOXB4 mRNA to reflect the levels of HOXB4. Recently, an FMEV retroviral vector containing a tagged HOXB4 was used to transduce human CD34⫹ CB cells, which upon analysis in vivo exhibited a proliferative advantage of primitive hematopoietic progenitors. However, this vector construct generated high expression levels of HOXB4 (approximately 10-fold above controls), rendering substantially impaired myeloerythroid differentiation as well as a reduction in B-cell output in transplanted NOD/SCID mice [14]. Similar findings have been reported for other transcription factors, for which for example cell fate options can be determined by the concentration of GATA-1 [24,25]. Our findings presented here, and the work of Schiedlmeier et al., are consistent with the notion that the absolute levels of HOXB4 may influence the fate of HOXB4-transduced progenitors. Alternatively, transcription factors like HOXB4 may generate effects that are dependent on the differentiation stage of the target cell. For example, enforced expression of HOXB4 may enhance the proliferation of repopulating

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FIG. 5. Adenoviral overexpression of HOXB4 reduces proliferation recruitment in human primitive hematopoietic cells. Sorted CD34⫹, CD38⫺, Lin⫺, GFP⫹ adenoviral vector-transduced cells were plated in Terasaki plates to obtain one cell per well. (A) To investigate cytokine requirements for proliferation recruitment, cells were seeded in TPO or SCF, TPO, and FL, and wells were scored on day 12. The findings are presented as percentage positive wells (average ⫾ SD) as well as distribution of large and small colonies in percentage of positive wells. AdHOXB4- and control-transduced cells were compared and a significant reduction was seen with TPO (P ⬍ 0.01) but not with TPO ⫹ FL ⫹ SCF (P ⫽ 0.072). AdGFP, n ⫽ 10 –11; AdHOXB4, n ⫽ 11–12. (B) Cells sorted for high and low GFP expression levels reveal that high expression of HOXB4 results in reduced proliferation in the cytokine combinations tested (n ⫽ 2).

stem cells but disturb the differentiation program of more differentiated progenitors. Above, we have presented the most likely interpretation of the data, namely that the unexpected effects seen are due to high levels of HOXB4 in primitive hematopoietic progenitors resulting in effects that are totally different from those that ensue when a continuous expression of twofold HOXB4 levels are generated within retrovirally transduced hematopoietic cells. Possible alternative explanations exist. It is possible that hematopoietic progenitors that are efficiently transduced by adenoviral vectors

FIG. 6. Overexpression of HOXB4 by AdHOXB4 in CD34⫹ cells reduces the number of hematopoietic progenitor colonies. The frequency of hematopoietic colonies generated by transduced, sorted Lin⫺, CD38⫺, 7AAD⫺, CD34⫹, GFP⫹ CB cells is shown. Cells were plated in methylcellulose containing SCF 100 ng/ml and EPO 5 U/ml, and scored for CFU-E and BFU-E, or in IL-3 100 U/ml and GM-CSF 200 U/ml and scored for CFU-GM. Data shown are the averages and the standard deviations of the numbers of colonies scored (n ⫽ 10). Colony frequencies generated by the AdHOXB4 vector-transduced cells were significantly reduced (P ⬍ 0.05) compared to controls.

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represent a subpopulation of progenitors with biological properties distinct from those that are less efficiently transduced as may be suggested by the GFP high and low fractions shown in Fig. 5B. This could be due to different adenoviral receptor densities on the progenitors, different levels of post-receptor blockage, or different transgene promoter activities in various progenitor populations. The mechanism of HOXB4 action is poorly understood and it is not clear how enforced expression of HOXB4 may affect transcriptional programming or signal transduction pathways within hematopoietic progenitors. It is of interest, however, that HOXB4 has been shown to be one of the molecules involved in the proto-oncogene c-myc transcription elongation block during differentiation induced by 1,25-dihydroxyvitamin D3 in the promyelocytic leukemia cell line HL-60 [26,27]. This differentiation could be blocked by adding an antisense oligo HOXB4. The mechanism is believed to be caused by binding of the HOXB4 protein to a protein binding site (MIE1) within the first intron of the c-myc gene. When AdHOXB4 enters the cells the level of HOXB4 increases rapidly to very high levels, and it is possible that the excess HOXB4 protein binds to the c-myc MIE1, blocking its expression and thereby inducing differentiation. Enforced expression of HOXB4 may also affect the transcriptional control of other HOX genes. In a study of HOX gene expression during early vertebrate development Hooiveld et al. have shown that an interaction between HOX genes in the same paralog occurs [18]. A specific induction of 5⬘ genes was observed, and at the same time, 3⬘ gene expression

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TABLE 1: Reduced progenitor cell output from AdHOXB4 transduced cells Experiment

Transduction AdGFP

AdHOXB4

1

120

63

2

325

50

3

121

37

Average CFC

189

50

SD

118

13

phoprep (Nycomed Pharma, Oslo, Norway) and enriched for CD34⫹ cells using a CD34 progenitor cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Pooled cells were stored in 20% FCS and 10% DMSO in DMEM (Gibco BRL/Life Technologies, Inc., Paisley, Scotland) in liquid nitrogen. The purity of the CD34⫹ fraction was routinely higher than 80%. Thawed cells were cultured in X-Vivo 15 medium (BioWhittaker, Walkersville, MD) containing 1% detoxified bovine serum albumin (BSA; Stem Cell Technologies, Inc., Vancouver, BC, Canada), 10⫺4 M 2-mercaptoethanol (Sigma Aldrich, Chemie Gmbh, Steinheim, Germany), and 2 mM l-glutamine (Gibco BRL/Life Technologies), hereafter referred to as complete X-Vivo medium. TPO (Kirin Brewery Co. Ltd., Japan), FL (Immunex, Seattle, WA), and SCF (Amgen, Thousand Oaks, CA) were used at 100 ng/ml each and EPO (Eprex, Janssen-Cilag AB, Sollentuna, Sweden) at 3–5 U/ml in different

Six week LTC-IC CFC derived from 1000 input cells. Transduced, sorted cells (Lin⫺, CD38⫺, 7AAD⫺, CD34⫹, GFP⫹) were plated in myelocult containing 10⫺6 M hydrocortisone on irradiated murine stroma cell lines M210B4 and SL/SL and maintained for 6 weeks. Once weekly half the media was replaced by mylecult with freshly prepared hydrocortisone. After six weeks the cells were then plated in methylcellulose medium containing GM-CSF, SCF, G-CSF, IL-3, FL and Epo. Colonies were scored on day 14. The colony frequencies (mean ⫾ SD, n ⫽ 3) generated by AdHOXB4 vector transduced cells were found to be strongly reduced compared to AdGFP transduced cells.

was repressed. Whether this occurs in hematopoietic progenitor cells is unclear, but Care´ et al. have shown a similar pattern for HOXB gene expression in adult T lymphocytes [19]. However, we could not detect any increase or decrease in mRNA levels of neighboring or paralogous HOX genes such as HOXA3, -A4, -B3, or -B5. Furthermore, Krosl et al. have recently shown that suppression of PBX1 by an antisense construct further promotes the effects of retroviral overexpression of HOXB4. In our study we could not detect any changed levels of PBX1 by Q-RTPCR, which could further explain the dramatic effects of the high level of HOXB4 expression from the AdHOXB4 vector [28]. The use of adenoviral delivery of genes to HSCs is an attractive way to target primitive cells in a transient manner. A weaker promoter leading to lower levels of the transgene or a more sophisticated vector with controllable expression levels of the transgene, such as the tetracycline-inducible system, could possibly be used [29]. However, with the inducible vector, control of the level of expression would be preferred, not just the turning on and off of expression. Future studies are needed to determine the mechanism of HOXB4 action in hematopoiesis and following enforced expression in hematopoietic progenitors. Whatever the mechanism, our study suggests that genetic manipulation of HSCs using transcription factor genes like HOXB4 to change HSC fate options requires careful control of the expressed transgene to obtain the desired effect and to avoid potential complications characterized by dysregulated growth and differentiation programs.

MATERIALS

AND

METHODS

Cells. Human umbilical CB cells were harvested following normal birth. Low-density mononuclear cells were recovered by centrifugation on Lym-

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FIG. 7. Reduced CFU-C capacity and expansion of AdHOXB4-transduced hematopoietic cells over time. Transduced sorted cells (Lin⫺, CD38⫺, 7AAD⫺, CD34⫹, GFP⫹) were cultured in liquid culture supplied with cytokines for 6 weeks with weekly half-medium changes. Cells were plated in methylcellulose medium weeks 0, 2, 4, and 6 and colonies were scored after 2 weeks. Secondary colonies were initiated by replating aliquots of primary colonies and scored 2 weeks later. (A) Total cell counts over time are similar for AdHOXB4and AdGFP-transduced cells during the first 4 weeks but are significantly reduced in AdHOXB4-transduced progeny at 6 weeks. (B) Primary and secondary colonies of freshly sorted cells at the initiation of the culture. There was a significant reduction in the number of CFU-GM colonies in the progeny of AdHOXB4-transduced cells (P ⬍ 0.04). (C) Primary and secondary colonies after 6 weeks in liquid culture. No increase in secondary colony formation could be detected in AdHOXB4-transduced progeny cells.

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TABLE 2: Primers used for RT-PCR Primer

Sequence

Primer

Sequence

HOXB3F

GCA GAA AGC CAC CTA CTA CG

HOXA5F

GGC CCG GAC TAC CAG TTG CAT

HOXB3R

CCG TTG AGC TCC TTG CTC TT

HOXA5R

CGC TGG CGC TGG CAG CGT A

HOXB4F

GGA GCC CGG CCA GCG CTG CGA GG

HOXA6F

TTG TGA ATC CCA CTT TCC C

HOXB4R

ACC CGA GCG GAT CTT GGT GTT GGG CAA

HOXA6R

GCG CCA CTG AGG TCC TTA T

HOXB5F

ATT ATG GCA GTG GCA GCT CT

HOXA10F

CCA ACT GGC TCA GGG CAA AGA GTG

HOXB5R

TGT GAA GCT TCC TCA TCC AG

HOXA10R

CGC GTC GCC TGG AGA TTC ATC AG

HOXB6F

CAG GAC AAG GGC TTT GCC ACT

PBX1F

GAC CCC GAT GGA TGT GGA CAA

HOXB6R

CTC GCC GAA CAC GCT CTT GT

PBX1R

GCG CAA AAC CTG GAT TGC TTT TA

HOXB7F

AAC ATG CAC TGC GCG CCC TTT

huAktEx4F

CCA TTG GCA ATG AGC GGTT

HOXB7R

CTT CAT GCG CCG GTT CTG A

huAktEx6R

GCG CTC AGG AGG AGC AA

HOXA4F

AGC GCC CCC GAC CCA GCA

HOXA4R

GCC GCC GCG GTA GCC ATA

combinations. Cells were cultured at a cell density of 105 cells/ml at 37°C in 5% CO2. Adenoviral vector design. Adenoviral vectors (⌬E1E3Ad5) carrying the GFP gene under the control of the human cytomegalovirus (CMV) immediate early promoter alone or in combination with the HOXB4 gene driven by a separate CMV promoter were generated according to standard procedures using low-passage 293 cells (Fig. 1A). Briefly, the HOXB4 cDNA was ligated into the shuttle plasmid pAdTrack-CMV [30]. Five hundred nanograms of linearized (PmeI) pAdTrackCMV-HOXB4 was electroporated together with 0.1 ␮g of pAdEasy into Escherichia coli strain BJ5183 for homologous recombination. Recombinants were confirmed by restriction enzyme digest and correct plasmids were electroporated into E. coli strain DH10B. Final constructs were linearized by PacI digestion and low-passage 293 cells were transfected by calcium chloride precipitation. Adenoviral vector expansion and purification were performed according to standard procedures [31]. The titer was determined by transfection of 293 cells in a 96-well plate at limiting dilution. Virus was stored at ⫺80°C and titers of frozen viral stocks were determined to 5 ⫻ 109 to 1010 infectious units per milliliter. None of the virus stocks contained replication-competent virus as tested on HeLa cells. Transduction and cell sorting. Thawed CD34⫹ CB cells were transduced with the adenoviral vector after 8 –16 h of incubation in complete X-Vivo 15 medium containing 100 ng/ml TPO. Transductions were performed in the same medium at an m.o.i. of 500 for 20 –24 h at 37°C in the presence of 15 ␮g/ml polyamidoamine dendrimer reagent Superfect according to the manufacturer’s instructions (Qiagen, Hilden, Germany) [32]. Mocktransduced cells were treated identically without addition of the vector. Cells were sorted after staining for phycoerythrin-labeled antibodies to CD3, CD14, CD15, CD19, CD20, CD38, CD56, and GpA and allophycocyanin-labeled mAb to CD34 at saturating concentrations for 30 min on ice (all antibodies from Becton–Dickinson, Erembodegem, Belgium). To be able to gate on live cells, 7-amino-actinomycin D (7AAD; Sigma Aldrich) was added after the final wash at 0.1 ␮g/ml, and the cells were incubated 15–20 min prior to sorting on a FACS Vantage cell sorter (Becton–Dickinson, Franklin Lakes, NJ) (Fig. 1B). Western blot. HeLa cells (107) were transduced with 5 or 50 m.o.i. of each adenoviral construct and cells were harvested 24 h later for FACS analysis, Q-RT-PCR, and purification of nuclear proteins. Cells were washed with PBS and pelleted before resuspension in 400 ␮l of ice-cold buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF) and allowed to swell on ice 15 min. Twenty-five microliters of 10% NP-40 was added and the samples were vortexed 10 s followed by 30 s

626

of centrifugation. The pellet was resuspended in 40 ␮l Buffer B (20 mM Hepes, pH 7.9, 0.4 M NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF) and left on ice over night. Thirty-five microliters of the supernatant was loaded into each well in a 12% SDS–PAGE gel, and the gel was electroblotted after electrophoresis onto a PVDF-membrane (Perkin–Elmer Sverige AB, Stockholm, Sweden) for detection of HOXB4 protein using an anti-HOXB4 antibody. The monoclonal rat anti-mouse I12HoxB4 antibody developed by A. Gould and R. Krumlauf was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences (Iowa City, IA 52242). The HoxB4 antibody was detected using a goat anti-rat HRP secondary antibody (Dako Cytomation, Glostrup, Denmark) and enhanced chemiluminescence (ECL Western Blot Luminol Reagent; Santa Cruz Biotechnology, Santa Cruz, CA). RT-PCR. Adenoviral vector-transduced and mock-transduced cells were cultured in complete X-Vivo 15 medium with SCF, FL, and TPO (all at 100 ng/ml) and EPO (3 U/ml). Cell aliquots were harvested at different time points, washed with PBS, and frozen immediately as a dry pellet at ⫺80°C. On day 4 105 cells were sorted for Lin⫺, CD38⫺, 7AAD⫺, CD34⫹, GFP⫹ cells. Cells were lysed and total RNA was extracted (RNeasy kit, Qiagen). cDNA was reversed transcribed (Superscript II; Gibco BRL) and PCR for seven different HOX genes (HOXA4, A6, A10, B3, B4, B5, B6) and PBX-1 was performed, as well as for human actin as reference (Table 2). PCR was performed with just enough cycles for the PCR products to be visible on a 1% agarose gel with ethidium bromide to avoid a saturation in the PCR (varying numbers of cycles for different primer pairs). PCR products were transferred to a nylon membrane (HybondN⫹, Amersham Pharmacia Biotech Ltd., Buckinghamshire, England) and specific [␣-32P]dCTP-labeled cDNA probes were used for hybridization. Band intensity was determined in a Fuji BAS-5000 phosphoimager after exposure to an imaging plate (BAS-SR), and the intensity of the actin bands was used as reference. At later time point we acquired LightCycler equipment (Roche Diagnostics GmbH, Mannheim, Germany) with LightCycler Software version 5.32 and could confirm the HOXB4 data for some of the remaining samples above. The cDNA was analyzed by Q-RT-PCR using SybrGreen I (Sigma Aldrich) for detection of PCR products. Two microliters of cDNA was used in a 15-␮l final volume reaction containing 1 U Platinum Taq DNA polymerase (Invitrogen, Paisley, UK), 1⫻ buffer (provided with the enzyme), 0.8 mM dNTP, 3 mM MgCl2, 0.5 mg/ml BSA, 5% DMSO, 0.5 ␮M forward primer, 0.5 ␮M reverse primer, and 1:20,000 dilution of SybrGreen I. The LightCycler was programmed to measure the buildup of PCR product once each cycle at the end of the extension step. This later method was further used

MOLECULAR THERAPY Vol. 8, No. 4, October 2003 Copyright © The American Society of Gene Therapy

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doi:10.1016/S1525-0016(03)00237-5

for the RT-PCR analysis of cDNA from sorted for high and low GFPexpressing cells. Expression of cell surface lineage markers. Cells were sorted as described above and cultured in complete X-Vivo 15 medium with SCF, FL, and TPO (all at 100 ng/ml) and EPO (3 U/ml). FACS analysis was performed on a FACSCalibur (Becton–Dickinson) on day 7–9 for lineage expression. Myeloid cells were analyzed for expression of CD14 and CD15 and erythroid cells by expression of GpA (all antibodies from Becton–Dickinson). Viability. Cells transduced with adenoviral vectors expressing GFP were sorted (Lin⫺, CD38⫺, 7AAD⫺, CD34⫹, GFP⫹) and seeded in Terasaki plates in complete X-Vivo 15 medium, to obtain single-cell cultures. Control cells were sorted for Lin⫺, CD38⫺, 7AAD⫺, and CD34⫹. To investigate the viability, each sorted cell population was divided into three groups. Groups 1 and 2 were given TPO or SCF at 100 ng/ml for the first 5 days. On day 6, medium containing a cytokine cocktail giving final concentrations of SCF 25 ng/ml, G-CSF (Neupogen; Amgen) 25 ng/ml, IL-3 (Novartis, Basel, Switzerland) 10 ng/ml, TPO 10 ng/ml, IL-6 (Novartis) 25 ng/ml, and EPO 5 U/ml was added to the wells, and the cells were incubated for an additional 10 days before positive wells were scored. The third group was cultured in the complete cytokine cocktail from the start and scored on day 11. Proliferation recruitment. Sorted cells were seeded in Terasaki plates in complete X-Vivo 15 medium at 1 cell per well. Cells were cultured in the presence of (i) TPO (100 ng/ml), (ii) SCF ⫹ TPO (100 ng/ml each), or (iii) SCF ⫹ TPO ⫹ FL (100 ng/ml each) and scored on day 12. CFU-C assay. Sorted cells were plated in methylcellulose medium (H4230; Stem Cell Technologies) supplemented with SCF at 100 ng/ml and Epo at 5 U/ml for growth of erythroid colonies (BFU-E and CFU-E). Myeloid colonies (CFU-GM) were grown in methylcellulose medium containing 100 U/ml IL-3 and 200 U/ml GM-CSF [23]. LTC-IC assay. Sorted cells (Lin⫺, CD38⫺, 7AAD⫺, CD34⫹, GFP⫹) were cultured on a mixture of irradiated (80 Gy) murine stroma cell lines SL/SL and M2-10B4 [33] in Myelocult (H5100; Stem Cell Technologies) with 10⫺6 M hydrocortisone 21-hemisuccinate (Sigma) for 6 weeks with weekly half-medium changes. After 6 weeks, all cells from each well were collected and divided into two plates of methylcellulose cultures (80 and 20%) supplemented with GM-CSF (Novartis), SCF, G-CSF, IL-3, and FL (all at 10 ng/ml) and EPO (5 U/ml) and cultured for an additional 14 days before colonies were scored. Long-term liquid culture. Sorted cells (Lin⫺, CD38⫺, 7AAD⫺, CD34⫹, GFP⫹) were cultured in Iscove’s MDM with a serum substitute (BIT, Stem Cell Technologies) and 10⫺4 M 2-mercaptoethanol, 40 ␮g/ml low-density lipoproteins (Sigma Aldrich), 100 ng/ml FL, 100 ng/ml SCF, 20 ng/ml IL-3, 20 ng/ml IL-6, and 20 ng/ml G-CSF. Cells were counted for expansion at days 7, 14, 21, 28, and 42. Cell morphology was assessed by cytospin preparations stained with May–Gru ¨ nwald–Giemsa. Assays for in vitro colony-forming cells (CFC) were carried out in methylcellulose culture (H4230) supplemented with 50 ng/ml SCF, 3 U/ml EPO, and 20 ng/ml each IL-3, IL-6, GM-CSF, and G-CSF [34]. Primary CFCs were plated on days 0, 14, 28, and 42. Secondary progenitor assays were performed by harvesting all cells from the primary plates at day 14 and replating aliquots of cells in fresh methylcellulose medium as above. Secondary progenitor assays were scored 14 days later. Statistical methods. Statistical calculations were done using the Student t test or Mann–Whitney rank sum test (SPSS 10.0, SPSS, Inc., Chicago, IL). P ⬍ 0.05 was considered statistically significant. Data are presented as averages ⫾ standard deviations.

ACKNOWLEDGMENTS The authors thank Saemundur Gudmundsson and the staff at the Department of Obstetrics and Gynecology, Malmo¨ University Hospital, for collecting umbilical cord blood samples; Sten Eirik Jacobsen for discussions and advice; Ingbritt ˚ strand Grundstro¨m for assessment of cytospins; Zhi Ma and Anna Fossum for A

MOLECULAR THERAPY Vol. 8, No. 4, October 2003 Copyright © The American Society of Gene Therapy

skillful cell sorting; Karin Leandersson for Western blot expertise; and Jonas Larsson and Johan Richter for helpful suggestions on the manuscript. This project has been funded by Cancerfonden, Sweden; The Swedish Gene Therapy Program; a Clinical Research Award (ALF) from Lund University Hospital; and the National Cancer Institute of Canada with funds from the Terry Fox Foundation. RECEIVED FOR PUBLICATION MAY 5, 2003; ACCEPTED JULY 5, 2003.

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MOLECULAR THERAPY Vol. 8, No. 4, October 2003 Copyright © The American Society of Gene Therapy