Enhanced self-renewal of hematopoietic stem/progenitor cells mediated by the stem cell gene Sall4
© Yang et al; licensee BioMed Central Ltd. 2011
Received: 27 August 2011
Accepted: 23 September 2011
Published: 23 September 2011
Sall4 is a key factor for the maintenance of pluripotency and self-renewal of embryonic stem cells (ESCs). Our previous studies have shown that Sall4 is a robust stimulator for human hematopoietic stem and progenitor cell (HSC/HPC) expansion. The purpose of the current study is to further evaluate how Sall4 may affect HSC/HPC activities in a murine system.
Lentiviral vectors expressing Sall4A or Sall4B isoform were used to transduce mouse bone marrow Lin-/Sca1+/c-Kit+ (LSK) cells and HSC/HPC self-renewal and differentiation were evaluated.
Forced expression of Sall4 isoforms led to sustained ex vivo proliferation of LSK cells. In addition, Sall4 expanded HSC/HPCs exhibited increased in vivo repopulating abilities after bone marrow transplantation. These activities were associated with dramatic upregulation of multiple HSC/HPC regulatory genes including HoxB4, Notch1, Bmi1, Runx1, Meis1 and Nf-ya. Consistently, downregulation of endogenous Sall4 expression led to reduced LSK cell proliferation and accelerated cell differentiation. Moreover, in myeloid progenitor cells (32D), overexpression of Sall4 isoforms inhibited granulocytic differentiation and permitted expansion of undifferentiated cells with defined cytokines, consistent with the known functions of Sall4 in the ES cell system.
Sall4 is a potent regulator for HSC/HPC self-renewal, likely by increasing self-renewal activity and inhibiting differentiation. Our work provides further support that Sall4 manipulation may be a new model for expanding clinically transplantable stem cells.
Hematopoietic stem cells (HSCs) are rare cells defined by their unique ability to self-renew and their ability to replenish all blood cell types in the body. Under normal conditions however, only a small number of HSCs enter cell division to generate daughter cells and supply mature lineages. Thus a key question is how these HSCs are regulated for their self-renewal and multipotency properties. Scientists have tried to expand clinically transplantable HSCs ex vivo, mainly by optimizing the use of bioactive proteins and various hematopoietic cytokines. The few genes that have been reported to effectively expand HSCs ex vivo include the transcription factor homeobox B4 (HoxB4), Notch family receptors, as well as Wnt signaling proteins [1–3]. However, the long term outcome for clinical therapy using HSCs treated with these factors still needs to be further elucidated.
Sall4 is a zinc-finger transcription factor and is essential for developmental events [4, 5]. We and others have previously reported that Sall4 plays important roles in maintaining the properties of embryonic stem cells (ESCs) by interacting with transcription factors Oct4 and Nanog [6–9]. In stem cells, Sall4 functions as both an activator and a repressor of gene transcription depending on the cell context. It suppresses important differentiation genes and activates key pluripotency genes [9, 10]. Sall4 also plays positive roles in the reprogramming of differentiated cells to ESC-like cells, and generation of induced pluripotent stem cells (iPS)[11–13]. We and others have determined that Sall4 exists as two isoforms (Sall4A and Sall4B), and they have unique and overlapping functions [14–16]. Interestingly, Sall4 is one of the few genes that are also involved in adult tissue stem cells [17, 18], and its protein expression is always correlated with the presence of stem and progenitor cell populations in various organ systems including bone marrow (BM) . Importantly, SALL4 has been identified as a robust expanding factor for human HSCs . We have recently studied the roles of Sall4 isoforms in controlling murine HSC/HPC properties. Our data indicate that a certain level of expression of Sall4 isoforms is necessary for normal HSC/HPC activity. Moreover, both Sall4A and Sall4B isoforms act as potent regulators of HSC/HPC self-renewal.
Sall4 isoforms enhance and support murine LSK cell proliferation
Given the selective expression pattern of SALL4 proteins in hematopoietic stem/progenitor cell compartments , 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).
To date, only several factors have been reported to expand HSC/HPCs in vitro while their stem cell characteristics were not impaired. To test whether Sall4- expanded HSC/HPCs may retain immature properties after long term culture, we repeated flow cytometry assays after 6 weeks of culture. We found that there were more than 70% of expanded cells retaining immature surface features (Sall4A group: Sca-1+, 79%, c-Kit +, 64% and Lin+, 21%; Sall4B group: Sca-1+, 75%, ckit+, 48% and Lin+, 22%). In addition, more than sixty percent of these cells were still Sca-1 positive even after 3 months culture (data not shown). These results indicate that overexpression of Sall4 isoforms is capable of enhancing and supporting primitive hematopoietic cells in the presence of combined cytokine stimulations.
Enhanced HSC/HPC activity by forced expression of Sall4 isoforms
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).
Status of mice after being transplanted with Sall4 expanded cells
Number of injected cells
Sall4A expanded cells, 2 × 106
Sall4A expanded cells, 4 × 106
Sall4A expanded cells, 6 × 106
Sall4B expanded cells, 4 × 106
Sall4B expanded cells, 6 × 106
GFP-infected cells, 4 × 106
GFP-infected cells, 6 × 106
Down-regulation of Sall4 accelerated differentiation of HSC/HPCs
Sall4 up-regulates multiple important HSC/HPC regulators
Sall4 inhibits myeloid progenitor differentiation
We recently reported that SALL4 is a robust expanding factor for human HSC/HPCs . In the current study, we demonstrate that both Sall4A and Sall4B isoforms also stimulate sustained cell proliferation of murine HSC/HPCs (> 4 months in culture) while their capacity of multi-lineage differentiation was not impaired. Similarly, when Sall4 expanded cells were transfused into irradiated mouse recipients, no abnormal hematopoietic or leukemic features were observed even after 17 months of BM transplantation (n = 18). This study is consistent with the finding that HSC/HPC expansion mediated by Sall4 is still under cytokine control. In fact, in our previous transgenic mouse studies of Sall4B overexpression controlled by a universal CMV promoter, there was an increased incidence of leukemic formation in older mice . Similar studies with Sall4A overexpression under the same promoter, however, were free of leukemic formation (N = 170, five transgenic mouse lines, data not shown) after 2 years of observation. This could be due to an abnormal niche resulting from dysregulated SALL4B expression in various mouse tissues throughout the mouse development with this universal CMV promoter and/or SALL4B may bear oncogenic potential.
In isolated mouse LSK cells, forced expression of Sall4 isoforms dramatically stimulate multiple known HSC regulators, such as Notch1, HOXB4, cMyc, CyclinD1, CyclinD2, Bmi1, Nfya, Runx1and Meis1. This is of great interest since all of these factors play important roles in regulating HSC activity [23–26]. Specifically, Notch1 and HOXB4 are thought to be highly interesting candidates for therapeutic stem cell expansion . cMyc may act as a downstream mediator of both factors in murine HSCs, while the trimetric transcription factor Nfya is able to activate multiple HSC regulatory genes including HOXB4 [28, 29]. The transcription factors Meis1, Runx1 and the poly comb gene Bmi1 are all expressed at high levels in HSCs and required for cell activity [30–32]. Though whether/how Sall4 directly regulates other factors is unresolved, in our previous ChIP-chip study on mouse ESCs, Sall4 bound approximately twice as many annotated genes within promoter regions as Nanog and approximately four times as many as Oct4 . Moreover, Sall4 seems to act as a "central tower of pluripotency" in ES cells in association with Oct3 ⁄ 4, Sox2 and Nanog [33, 34]. Further detailed studies are required to elucidate whether it may also function as a master controller in modulating genetic networks within the HSC system.
Another discovery is the finding of how overexpression of Sall4 isoforms affects G-CSF induced granulocytic differentiation. The results obtained here were similar as compared with the Sall4 activities in the human system . We have hypothesized that the Sall4 gene is also involved in mediating cell fate decisions during hematopoiesis, helping to regulate the exquisite balance among self-renewal, differentiation, and proliferation required for normal blood formation. Our findings demonstrate that sustained activation of both Sall4A and Sall4B isoforms inhibits granulocytic differentiation of 32D myeloid progenitors, supporting the view that Sall4 is capable of influencing cell fate determination in hematopoietic cells. This may also explain, at least in part, why Sall4 isoforms are expressed preferentially in HSCs, down-regulated rapidly in HPCs, and absent in the differentiated lineage populations.
In summary, our work demonstrates that the potent stem cell factor Sall4, which is preferentially expressed within the HSC/HPC pool during hematopoiesis, activates and integrates multiple genetic pathways responsible for the precisely controlled proliferation and differentiation of HSC/HPCs. Sall4 is thus an excellent candidate for the genetic manipulation of HSC/HPC self-renewal in vivo and in vitro.
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.
Gene cloning and lentiviral transduction
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 , 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).
SDS-PAGE and western blotting
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).
Isolation of mouse BM hematopoietic stem/progenitor cells
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.
Preparation of peripheral blood
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.
Immunostaining and flow cytometry
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.
RNA interference (RNAi) and quantitative reverse transcription (qRT-PCR) Analyses
32D cell culture and induction of differentiation
The 32D cells were cultured as previously described . 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.
CFU assay of BM cells
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.
Support and financial disclosure declaration
This work is supported in part by Leukemia & Lymphoma Society Special Fellow Award (3366-09) (J.Y.), Department of Defense Grant W81XWH-10-0046 (LMF), and National Institutes of Health Grant NIH R01HL087948 (Y.M.).
Financial Disclosure Declaration: Y.M. is a scientific consultant to MarrowSource Therapeutics International LLC.
We thank the Genomics Core Facility of the University of Nevada, Las Vegas for their service in performing flow cytometry.
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