Enhanced self-renewal of hematopoietic stem/progenitor cells mediated by the stem cell gene Sall4

Given the selective expression pattern of SALL4 proteins in hematopoietic stem/progenitor cell compartments [16], we asked whether increased expression of SALL4 isoforms may affect HSC/HPC phenotypes. To study this, mouse HSC/HPC s (Lin-, Sca-1 +, c-Kit +; LSK cells) were isolated and transduced with lentiviruses carrying either GFP alone (control), or together with Sall4A or Sall4B isoform. All GFP positive cells were determined by either fluorescence microscope inspection or flow cytometric analysis (Additional file 1: Figure. S1a, and data not shown). We next performed western analysis to assess the expression of Sall4 isoforms in infected NIH3T3 fibroblast cells. It was found that these cells expressed the appropriate Sall4A or Sall4B protein at 160 and 90 kDa. As negative control, the empty GFP vector did not generate Sall4A or Sall4B bands (Additional file 1: Figure. S1b).

In culture, all LSK cells with and without Sall4 transduction were strictly cytokine dependent, which were not able to survive more than 5 days in the absence of cytokines (100 ng/ml mSCF, 6 ng/ml mIL-3 and 10 ng/ml hIL-6) or in each alone (data not shown). Thus, Sall4 overexpression did not convert cells to cytokine independence. In the combination of cytokines, the Sall4A or Sall4B transduced cells exhibited a ~2 fold increase in the rate of proliferation relative to the GFP control group at day 15 and thereafter (Figure 1a). During the second week, a small part of adherent cells were seen on the bottom of GFP control culture dishes, many floating cells resembled granulocytes and exhibited various shapes and different sizes, while these were barely observed in Sall4A or Sall4B infected cultures, in which cells were non-adherent, uniquely round, spherical and at similar size (Figure 1b). We performed flow cytometry assay at day 14 to test their surface antigens. It was found that most Sall4 treated HSC/HPCs retained immature surface phenotypes (Sall4A group: Sca-1+c-Kit+, 71.2% and Lin+, 10.8%; Sall4B group: Sca-1+c-Kit+, 64.5% and Lin+, 14.3%). By contrast, many GFP treated HSC/HPC cells exhibited differentiating markers (Sca-1+c-Kit +, 42% and Lin+, 37.5%) (Figure 2). After three weeks, the GFP treated LSK cells gradually stopped proliferating and were no longer viable after day 30. However, both Sall4A and Sall4B transduced cells continued to proliferate at a stable doubling time of about 48 hours and proliferated indefinitely in culture (> 4 months). These in vitro studies were repeated in two additional experiments and similar results were obtained.

Next, we conducted reconstitution assays to evaluate Sall4-expanded HSC/HPC function in vivo. After 10-day cultures, Sall4 or GFP treated cells were collected, and 2 × 10⁶ cells (Ly5.2+) were intravenously transfused into sublethally irradiated (500 rad) Ly5.1 mice (GFP group, N = 4; Sall4A group, N = 4; and Sall4B group, N = 3). Two weeks after transfusion, the percentages of transplanted Ly5.2+ cells in peripheral blood mononuclear cells (PBMCs) were similar among all groups, as detected by flow cytometry assays with multiple lineage-specific antibodies (GFP group, 9.28%; Sall4A group, 10.0%; and Sall4B group, 12.3%, Figure 3a). However, the Ly5.2+ blood cells in recipient mice deriving from Sall4 transduced cells rose over months and surpassed the contribution from the GFP control-transduced HSC/HPCs by week 20 and week 50 (Figure 3b). Further more, these Ly5.2+ cells were able to reconstitute granulocyte, T cells, and B cells, as well as immature blood cells, as indicated by expression of GR-1, CD4/8, B220 and Sca-1 in BM cells respectively (Figure 4).

We further performed competitive repopulation assays to determine the activity of Sall4 transduced HSC/HPCs with day14 ex vivo cultured cells. Varying doses of cells baring Ly5.2 antigens were mixed with a constant number of non-transduced competitive BM cells (2 × 10⁵, Ly5.1+), and injected into lethally irradiated (900 rad) congenic mice (Ly5.1). After 3 months, the contributions of control or Sall4 transduced HSC/HPCs to the cellularities of mature lineages were measured by Ly5.2 antigen expression of peripheral blood cells. In each group, proportion of recipients exhibiting at least 5% donor-derived (Ly5.1+) leukocytes was determined and scored as positive only in which donor-derived (test) cells were detectable among B220+, CD4/CD8+ and Gr-1+ compartments. As shown in Figure 5a, when 2 × 10⁴ donor cells were mixed with 2 × 10⁵ competitive cells, Sall4A transduced LSK cells contributed to the repopulation of 3 of 4 recipients. Similarly, Sall4B transduced LSK cells contributed substantially to the repopulation of 4 of 4 recipients (Ly5.2+ cell percentage > 5%), whereas repopulation mediated by GFP control cells was not detected. When the number of infused cells was raised to 2 × 10⁵ cells, both Sall4A and Sall4B transduced cells repopulated 4 of 4 recipients, whereas GFP transduced cells did not. Taken together, these data indicated that overexpression of Sall4 increased the repopulation activity of HSC/HPCs.

Of note, however, though HSC/HPCs were expanded by sustained ectopic expression of Sall4 isoforms and exhibited enhanced repopulating activity, when transplanted into lethally or sublethally irradiated syngeneic recipients, the mice grew normally and were apparently healthy. Analysis of percent chimerism of donor cells in each hematopoietic lineage confirmed that Sall4-transduced HSC/HPCs retained full differentiation capacity and no dramatic changes of lineage percentages were found (Figure 5b, 4 and data not shown).

To evaluate long-term safety issues such as myeloproliferative neoplasm (MPN)-like features, we transduced SALL4 isoforms to mouse LSK cells and then performed syngeneic transplantations to allow for long-term follow-up. As shown in Table 1, no MPN-like phenotypes were exhibited for more than 11 months (more than 50% lifespan in mice) or even more than 17 months post syngeneic transplantation (n = 18). Our studies are consistent with the finding that ex vivo expansion of HSC/HPCs mediated by SALL4 is still cytokine dependent, which is also consistent with that seen in HoxB4 mediated HSC/HPC expansion.

We previously showed that Sall4 was exclusively expressed in CD34 positive hematopoietic stem/progenitor cells [16]. To further test the effect of Sall4 in LSK cells, we performed a Sall4 knockdown study in isolated mouse BM LSK cells by an RNA interference strategy as previously described [20]. After 10 days culture under puromycine selection (1.2 μg/ml), a ~62% reduction of Sall4 mRNA levels by #7412 expressing retrovirus was confirmed by qRT-PCR (Figure 6a). We found that Sall4-reduction LSK cells exhibited a decreased proliferation rate relative to the pRS control or non-treated cells (Figure 6b). In addition, reduction of endogenous Sall4 in LSK cells caused increased lineage differentiation markers (Sca-1+: 48 ± 7% versus pRS control: 63.3 ± 9%; cKit+:40 ± 6% versus pRS control: 54 ± 6%, and Lin+:42 ± 3% versus pRS control: 25 ± 3%, N = 3) during the two weeks of culture in dishes (Figure 7). These results suggest that proper expression of endogenous Sall4 is required for the self-renewal activity of the HSC/HPCs.

We have mapped SALL4 global gene targets using chromatin-immunoprecipitation followed by microarray hybridization (ChIP-chip) in myeloid leukemic NB4 cells as well as normal human CD34+ BM cells ([20], and unpublished data). Bmi1, TPO, Runx1, c-Myc, CD34, Meis1 and Nf-ya were identified as potential Sall4 downstream targets in both cell types. To further test if Sall4 is able to regulate these gene expressions, we performed qRT-PCR assays. We detected mRNA levels of Sall4 isoforms as well as some important HSC/HPC regulatory genes at day 7 post lentiviral transduction. Cells were collected and total RNAs were prepared and subjected to qRT-PCR assays. Results showed that in Sall4A or Sall4B transduced LSK cells, there were ~40 and ~20 fold increases of relevant Sall4A or Sall4B mRNA levels as compared with GFP control transduced cells, (this further confirms a successful Sall4 lentiviral transduction). Of note, transcripts of Bmi1, HoxB4, Notch1, Runx1, CD34, Meis1 and Nf-ya were all significantly activated, exhibiting a 2.5~15 fold up-regulation when cells were treated with Sall4 isoforms (Figure 8). ALL of these genes have been reported to play active roles in maintaining short term or long term HSC proliferation. In addition, Sall4 isoforms dramatically stimulate several molecules that are implicated in the Wnt/beta-catenin signaling pathway which is essential for HSC growth, including c-Myc, cyclin D1 and cyclin D2.

We have performed in vitro colony forming assay to evaluate effects of Sall4 isoforms on hematopoietic cell growth and progenitor differentiation. Mouse BM cells were isolated and infected with Sall4- expressing lentivirus. After four days, 10⁴ cells were mixed with MethoCult^® medium and seeded in dishes for colony formation. As shown in Figure 9a and 9b, both Sall4A and Sall4B transduced cells could generate various colonies including CFU-GM, CFU-GEMM, and BFU-E since day 9. However, the total numbers of each type of colony are lower than that from the GFP control infected cells. Two additional experiments with various initial seeding cell numbers showed similar results (data not shown). These experiments indicate that over-expression of Sall4 isoforms down-modulate myeloid differentiation versus proliferation from early primitive cells.

We next extended our study to a myeloid progenitor cell line (32D), to determine how Sall4 may affect hematopoietic progenitor cell differentiation. 32D cells were cultured and infected with empty GFP control or Sall4 expressing lentiviruses. The transduction efficiency was more than 80% as judged by fluorescent observation as well as flow cytometry assays (Additional file 2: Figure S2). During culture, we found that the 32D cells with or without transduction of Sall4 isoforms are all strictly IL-3 dependent, which are unable to grow when removed from IL-3. Thus, over-expression of Sall4 isoforms did not convert the 32D cells to cytokine independence. In agreement with previous reports [21, 22], 32D cells proliferate as undifferentiated blasts when maintained in IL-3, but differentiate into mature granulocytes when stimulated with G-CSF (Figure 10a. i, ii). However, when cultured with G-CSF but in the absence of IL-3, control 32D cells died after 5-6 days. By contrast, the Sall4 A or Sall4 B transduced 32D cells proliferated at a 3 to 6-fold higher rate than the control counterparts in the same media and then grew indefinitely (Figure 10b). In addition, these cells retained undifferentiated blast morphology in the presence of G-CSF but lack of IL-3 (Figure 10a, iii). This study indicates that constitutive expression of Sall4 inhibits G-CSF induced granulocytic differentiation and permits expansion of undifferentiated cells in 32D myeloid progenitors.

C57Bl/6J (Ly5.2) and congenic C57Bl/6.SJL-Ly5.1-Pep3b (Ly5.1) mice at 8-12 weeks of age were obtained from The Jackson Laboratory (Bar Harbor, ME). All animal experiments were preapproved by the Office of Laboratory Animal Welfare, Institutional Animal Care and Use Committee.

The full-length cDNA of mouse Sall4A and Sall4B were cloned into the SalI/NotI site of entry vector pENTR3C (Invitrogen, Carlsbad, CA). A Gateway LR reaction was carried out to subclone the cDNA into the lentiviral mammalian expression vector pDEST-CMVFG12 [35], which contains reading frame of the enhanced green fluorescent protein (EGFP). The obtained lentiviral constructs were confirmed by PCR reaction, enzyme digestion and gene sequencing. To generate lentivirus, the expression vectors were transfected into 293FT packaging cells (Invitrogen) along with pSPAX2 and pMD2.G plasmids (Addgene Inc., Cambridge, MA) for 48 hrs, then pooled filtered supernatants were used to infect cells in the presence of polybrene (8 μg/ml, Millipore, Billerica, MA).

For lentivirus transduction, the isolated LSK or Lin-/Sca-1+ cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) in the presence of mSCF (100 ng/ml), mIL-3 (6 ng/ml) and hIL-6 (10 ng/ml). The cells (~1 × 10⁶ per ml) were then incubated with high titer lentivirus (~1 × 10⁶ per ml) for 24 hours before being replaced with fresh media. For cultured 32D cells, lentivirus was added in conditioned medium (RMI1640 supplemented with 10% FBS) in the presence of mIL-3 (1 ng/ml) for 24 hours, then fresh media was used thereafter. Cytokines were purchased from ProSpec-Tany TechnoGene Ltd (Rehovot, Israel).

Total cell lysates prepared from 1 × 10⁵ wide type or lentiviral infected cells were electrophoresed through 7% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and then immunoblotted using the anti-Sall4 monoclonal antibody (Abcam, Cambridge, United Kingdom). Immunostained proteins were detected using enhanced chemilluminescence blot reagents (Thermo Fisher Scientific, Rockford, IL). Blots were detected by a Kodak Image Station 2000 MM (Kodak, Rochester, NY).

Cells were isolated from whole BM by immunostaining with either magnetic bead isolation or fluorescence-activated cell sorting. To obtain BM, 8- to 12- week-old mice were euthanized by carbon dioxide inhalation and the femurs and tibias removed. A 25-gauge needle was used to expel the marrow by a buffer solution contained phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA. For depletion of mature hematopoietic cells, the Lineage Cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used. The lineage (CD5, CD45R, CD11b, Ter119, and GR-1) negative cells were collected through mini MACS separation columns (Miltenyi Biotech) while in a magnetic field. For positive purification, the collected lineage negative cell fraction were dual stained with fluorescein isothiocyanate (FITC) conjugated anti Sca-1 and anti-FITC MicroBeads (Miltenyi Biotech) and separated again to yield HSC/HPCs (Lin-/Sca-1+). At this step, we confirmed that more than 95% of the separated cells were Lin-/Sca-1+ cells by flow cytometric analysis.

In some cases, fluorescence-activated cell sorting of Lin-/Sca-1+/c-Kit+ (LSK) was performed on the Reflection Cell sorter (iCyt, Champaign, IL). Antibodies were purchased from BD Pharmingen (San Diego, CA). Cells sorted were propidium iodide (PI)-negative, Lin (CD5, B220, Ter119, Mac1, and Gr-1) negative, c-Kit, Sca-1 and GFP positive.

Peripheral blood was obtained from each mouse by tail vein bleeding. One hundred microliters of blood was incubated for 10 minutes at room temperature with 3 mL ice-cold erythrocyte lysing solution (150 mM NH4Cl, 10 mM NaHCO3, 1 mM EDTA, pH 7.4), washed with PBS and resuspended in PBS and 1% paraformaldehyde (Sigma, St Louis, MO) and kept at 4°C until analysis.

Freshly isolated cells or cultured cells were stained by described fluorescence-labeled antibodies. The flow cytometry data were collected by using a FACScan or FACSCalibur machine (Becton Dickinson, Franklin Lakes, NJ) and analyzed by using FLOWJO or CELLQUEST software.

RNAi mediated Sall4 knockdown and qRT-PCR analyses were performed as reported previously [20]. The primer sets are described in Additional file 3: Table S1.

The 32D cells were cultured as previously described [36]. For granulocytic differentiation, IL-3 was removed by washing cells 3 times, then G-CSF (R&D Systems, Minneapolis, MN) was added to cells at a final concentration of 200 ng/ml.

Tubes of MethoCult^® GF M3434 (Stem Cell Technologies, Vancouver, BC, Canada) medium were thawed overnight in a 4°C refrigerator. Sall4 or GFP-transduced BM cells were prepared at 10 × the final concentration required. Cell suspensions of 1 × 10⁵ cells per mL were prepared and 0.3 mL of cells were added to 3 mL of MethoCult^® medium for duplicate cultures. 1.1 mL of cells was dispensed per 35 mm dish. The cells were incubated for 8-12 days at 37°C with 5% CO₂ and ≥95% humidity. The BFU-E, CFU-GM and CFU-GEMM colonies were observed with bright field and fluorescent microscopy. CFUs were counted under the microscope 9 days after the cells were plated in MethoCult^® medium. A colony with more than 100 cells was counted as a positive colony.