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Blood, 1 March 2005, Vol. 105, No. 5, pp. 1905-1915. Prepublished online as a Blood First Edition Paper on October 21, 2004; DOI 10.1182/blood-2004-03-0881.
HEMATOPOIESIS Smad7 alters cell fate decisions of human hematopoietic repopulating cellsFrom the Krembil Centre for Stem Cell Biology, Robarts Research Institute; and the Department of Microbiology and Immunology, University of Western Ontario; London, ON, Canada.
Intracellular Smad proteins mediate signal transduction of the transforming growth factor-  (TGF-) superfamily that play pleiotropic roles in hematopoietic development, suggesting that intracellular Smad proteins may play key roles in hematopoietic regulation. Although inhibitory Smad7, which negatively regulates TGF- signaling, has been implicated in the development of mature hematopoietic cells, a role for Smad7 in regulating more primitive hematopoietic cells has yet to be examined. Here, Smad7 was overexpressed in primary human severe combined immunodeficient (SCID) repopulating cells (SRCs), representing a common myeloid/lymphoid precursor cell with the functional capacity to repopulate the bone marrow of nonobese diabetic (NOD)/SCID recipient mice. Retroviral transduction of Smad7 into human umbilical cord blood (CB)-SRCs caused a shift from lymphoid dominant engraftment toward increased myeloid contribution, and increased the myeloid-committed clonogenic progenitor frequency in reconstituted mice. Neither myeloid nor B-lymphoid lineage developmental stages were compromised by Smad7 overexpression, suggesting Smad7 regulates cell fate commitment decisions of myeloid/lymphoid precursors by augmenting myeloid differentiation at the expense of lymphoid commitment. In addition, global gene expression analysis using microarray was used to identify potential target genes regulated by Smad7 in primitive hematopoietic cells that may control this process. Our study demonstrates a novel and unexpected role for Smad7 in modulating the cell fate decisions of primary multipotent human repopulating cells and establishes a role for Smad7 in the development of primitive human hematopoietic cells.
The transforming growth factor- (TGF-) superfamily of extracellular ligands, including the TGF-s, activins, and bone morphogenetic proteins (BMPs), are responsible for mediating a wide array of cellular responses ranging from proliferation and differentiation to embryonic patterning.1-3 Multiple TGF- family ligands are involved in embryonic and adult hematopoietic development. TGF- is best characterized as negative regulator of primitive hematopoietic cells,4,5 while BMP-4 is involved in the induction of hematopoietic tissue from embryonic mesoderm,6,7 and has also been shown to enhance the survival and proliferation of human hematopoietic stem cells following ex vivo culture.8,9
Ligands of the TGF-
Given the importance of multiple TGF-
Smad7 transcription is also induced in response to signaling by various ligands upstream of nuclear factor- To understand the biological role of Smad7 in primitive human hematopoiesis, we have used a retroviral gene transfer approach to examine the effect of Smad7 on candidate human HSCs capable of repopulating the bone marrow (BM) of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice.30,31 In comparison with control vector–transduced SCID repopulating cells (SRCs), Smad7 SRCs exhibited a shift from a B-lymphoid dominant graft toward a graft with a higher frequency of myeloid progeny within the murine bone marrow (BM). This phenotypic increase in myeloid progeny was coupled with a higher frequency of functional clonogenic myeloid progenitors isolated from the mouse BM. Our data suggest that Smad7 is capable of altering the balance of cell fate commitment decisions within human SRCs, which represent a common lymphoid/myeloid precursor cell with in vivo repopulating function. This study identifies a novel role for Smad7 in cell fate commitment decisions in primary human multipotent cells.
Reverse transcription–polymerase chain reaction analysis
mRNA was extracted from purified cells using an mRNA extraction kit, and was reverse-transcribed into cDNA using a first-strand cDNA synthesis kit, according to the manufacturer's instructions (Amersham Biosciences, Baie d'Urfé, QC, Canada). Reverse transcription–polymerase chain reaction (RT-PCR) reactions were used to detect Smad7 and Retroviral vectors A BamHI linker was subcloned into the HpaI-SnaBI sites of the MIEV vector32 (a kind gift from Robert Hawley, George Washington University Medical Center, Washington, DC). A 2.9-kb EcoRI-BamHI fragment coding for the full-length human Smad7 gene14 (from pCMV5-Smad7, a kind gift from Jeffrey Wrana, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada), was subcloned into the EcoRI-BamHI sites of the modified MIEV vector, upstream of an internal ribosomal entry site (IRES) and an enhanced green fluorescent protein (GFP) reporter gene, as previously described (K.C., K. Jay, B. Murdoch, M.B., manuscript submitted). Retroviral packaging lines The bicistronic Smad7 vector and control MIEV vector were transfected into the amphotrophic PA317 packaging line33 using Fugene6 (Roche, Laval, QC, Canada). Transient retroviral supernatant was harvested, supplemented with 8 µg/mL polybrene (Sigma, St Louis, MO), and used to infect the gibbon ape leukemia virus (GaLV)–pseudotyped PG13 packaging line34 to create stable producer lines releasing Smad7 or control vector retroviral particles. Fluorescence-activated cell-sorting (FACS) was used to isolate stable producer lines expressing the GFP transgene through 2 sequential sorts for GFP+ cells. Bulk cultures of selected PG13-Smad7 and PG13-vector cells were confirmed to produce retroviruses capable of transducing human cells by titration on HeLa cells with titers of 8 x 105 to 9 x 105 infectious particles/mL (K.C., K. Jay, B. Murdoch, M.B., manuscript submitted). Retroviral transduction of human hematopoietic cell lines Human megakaryocytic leukemia lines MBA.135 and M-O7e36 were grown in Iscove modified Dulbecco medium (IMDM) with 10% fetal bovine serum (FBS) and 100 U/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (M-O7e only). For transduction with Smad7 or vector retroviruses, cells were resuspended once in 0.45 µm filtered virus-containing supernatants, collected in IMDM with 10% FBS and 300 U/mL GM-CSF, and supplemented with 8 ng/mL polybrene. Two days after retroviral transduction, cultured cells were washed with phosphate-buffered saline (PBS), and GFP+ cells were selected by fluorescence-activated cell sorting for subsequent expansion. Assessment of Smad7 overexpression in retrovirally transduced cells PG13 packaging lines and MBA.1 cells transduced with vector or Smad7 retrovirus, sorted for GFP+ expression, were analyzed by Western blotting for detection of Smad7 protein. Cell lysates were prepared using lysis buffer containing 10% Triton, 1M Tris (tris(hydroxymethyl)aminomethane), 0.5M EDTA (ethylenediaminetetraacetic acid) and protease inhibitors leupeptin and aprotinin at 10 mg/mL, and proteins were separated using 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. Equal amounts of protein were loaded per lane in all experiments as determined by Coomassie staining of parallel gels used for subsequent Western blotting. Smad7 protein was detected using the Smad7 antibody H-79 (Santa Cruz Biotechnologies, Santa Cruz, CA), a goat anti-rabbit–horseradish peroxidase (HRP) 2° antibody (Santa Cruz Biotechnologies) and enhanced chemiluminescence system (ECL; Amersham Pharmacia), and Kodak X-omat film (Kodak, Eastman, NY).
TGF-
M-O7e cells were cultured in IMDM with 10% FBS and 300 U/mL GM-CSF (Amgen, Thousand Oaks, CA) in 96-well suspension plates (BD Biosciences, Mississauga, ON, Canada). Smad7–M-O7e or vector–M-O7e GFP+ cells (20 000 each) were seeded in triplicate for each experiment. Cells were treated with 1 ng/mL TGF- Human cell purification Samples of full-term human umbilical cord blood (CB) were obtained in conjunction with local ethical and biohazard authorities of the University of Western Ontario and London Heath Sciences Centre. CB samples were diluted 1:3 in PBS, and mononuclear cells (MNCs) were collected by separation on Ficoll-paque (Amersham Biosciences). Contaminating red blood cells were lysed with a 0.8% ammonium chloride solution. Lineage depleted (Lin–) cells were purified by negative selection using a StemSep device (StemCell Technologies, Vancouver, BC, Canada) as previously described.37 Cells expressing lineage commitment markers (Lin+) were separately eluted from the column for use as accessory cells during transplantation.38 CB Lin– and Lin+ populations were cryopreserved in fetal bovine serum (FBS) and 10% DMSO (dimethyl sulfoxide), and stored in liquid nitrogen. Production of retrovirus-conditioned supernatants
PG13 packaging lines stably transduced with either the control or Smad7 retroviral vector were grown in Retroviral transduction of CB Lin– cells
CB Lin– cells were thawed and placed into serum-free (SF) liquid culture (SF BIT media) consisting of IMDM supplemented with 20% bovine serum albumin, insulin, and transferrin (BIT) 9500 (StemCell Technologies), 10-4 M For transduction of Lin– cells, 35-mm dishes were precoated with fibronectin (Sigma) at 5 µg/cm2 for 4 to 12 hours. Fibronectin-coated dishes were preloaded twice with 1 mL serum-rich VCS for 30 minutes at room temperature. Prestimulated Lin– cells were harvested, resuspended in serum-depleted VCS supplemented with hematopoietic cytokines as listed for SF BIT media, and placed onto fibronectin-coated dishes preloaded with viral supernatant. Cells were transduced in this manner once every 24 hours for 3 days. Less than 12 hours after the final transduction, cells were harvested for transplantation into NOD/SCID mice, and analysis of gene transfer efficiency. Analysis of gene transfer efficiency into CB Lin– cells An aliquot of 2 to 5 x 104 cells was removed from each well prior to transplantation, and were cultured for an additional 1.5 days to allow for GFP expression, for analysis of gene transfer efficiency into the bulk CB Lin– population. Cells were analyzed by flow cytometry for GFP expression in combination with CD34, on a FACSCalibur flow cytometer (BD Biosciences). The gene transfer efficiency was determined as the percentage of cells expressing GFP 2 days after the final VCS exposure. Transplantation of human cells into NOD/SCID mice
After 4.5 days of retroviral transduction, the entire contents of wells initially seeded with 4 x 105 CB Lin– cells were transplanted into sublethally irradiated (3.5 Gy [350 rad] cesium 137 [137Cs]) NOD/LtSz-Prkdcscid/Prkdcscid (NOD/SCID) or Flow cytometric analysis of human cell engraftment and gene marking Cells harvested from the mouse BM were prepared for flow cytometry by lysing contaminating red blood cells with a 0.8% ammonium chloride solution, and subsequent washing with PBS with 3% FBS. Cells were resuspended at 106 cells/mL in PBS + 3% FBS for staining with fluorochrome conjugated antibodies at 5 µg/mL (all purchased from BD Biosciences or Beckman Coulter, Mississauga, ON, Canada). Cells were stained for 30 minutes at 4°C, and washed 3 times with PBS + 3% FBS before being analyzed on a FACSCalibur or FACSVantage SE using Cell Quest software (BD Biosciences). At least 20 000 CD45+ events were acquired for each analysis. For analysis of human cell engraftment, cells were stained with a human-specific pan-leukocyte marker CD45, or the corresponding mouse immunoglobulin (Ig) G1 isotype control. Mice engrafted with human (CD45+) cells were further analyzed for expression of the retroviral transgene by detection of the GFP reporter gene in FL1. In pairs of mice engrafted with CB Lin–-derived SCID repopulating cells (SRCs) transduced with control and Smad7 vectors, and expressing GFP+ cells at sufficient levels, the human graft was further analyzed for myeloid/lymphoid hematopoietic reconstitution. Transduced human cells gated on CD45 expression were analyzed for the presence of primitive hematopoietic cells (CD34), committed myeloid cells (CD33, CD13, CD14, CD15), and committed B-lymphoid cells (CD19, CD10), in combination with GFP expression. Analysis of clonogenic progenitors from NOD/SCID mice In mice with sufficient levels of engraftment and retroviral marking, clonogenic progenitors arising from transduced SRCs were assessed in semisolid methylcellulose culture. Mouse BM cells were sorted on a FACSVantage SE, and primitive human cells expressing the transgene were isolated based on the phenotype CD45+CD34+GFP+. As a control, CD45+CD34+GFP– cells were also isolated from the same mice. In some instances, cells were also selected based on exclusion of the 7-aminoactinomycin D (7-AAD) viability dye. Five thousand CD45+CD34+GFP+ or GFP– cells were placed into Methocult H4320 (StemCell Technologies) supplemented with the following recombinant human cytokines: 50 ng/mL SCF, 10 ng/mL GM-CSF, 10 ng/mL IL-3, and 3 U/mL erythropoietin (epo), and maintained at 37°C with 5% CO2 in a humidified atmosphere. Differential colony counts were performed at 10 to 14 days, according to standard protocols, on an Axiovert 25 microscope (Zeiss, North York, ON, Canada) using a 20 x/0.40 Ph2 objective. Expression of the GFP transgene was assessed after 7 days with a mercury burner and GFP filter on the Axiovert 25. Light and fluorescent micrographs were acquired with a Sony DXC-950 3CCD color video camera (Sony of Canada, Toronto, ON, Canada) and Northern Eclipse Image Editing Software v2.1 (Empix Imaging, Mississauga, ON, Canada). Quantitative polymerase chain reaction
Expression of Smad7 (forward primer, 5'AGAAGGTGCGGAGCAAAAT3' and reverse primer, 5'GTGTGGCGGACTTGATGA3') was quantified by quantitative polymerase chain reaction (Q-PCR; MX4000; Stratagene, La Jolla, CA) using SYBRGREEN (Stratagene) DNA binding dye. The Q-PCR condition was 2 mM MgCl, 0.4 mM dNTP (deoxyribonucleoside triphosphate), 8% glycerol, 3% DMSO, 150 nM of each primer, 0.375 µL of 1:500 dilution of reference dye, and 2.5 µL of 1:2000 dilution of SYBRGREEN. The Q-PCR reaction conditions were primary denaturation at 95°C for 1 minute and 40 cycles of PCR consisting of 95°C for 10 seconds, 60°C for 1 minute, and 72°C for 30 seconds, followed by analyzing the amplified products using dissociation curve. The signal intensities were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH; forward primer, 5'TGCACCA CCAACTGCTTAGC3', and reverse primer, 5'GGCATGGACTGTGGTCA TGAC 3') and the 2– Amplification of RNA for microarray analysis The MessageAmp aRNA kit (Ambion, Austin, TX) was used for RNA amplification as described elsewhere.43 Briefly, total RNA, extracted from transgenic Smad7 and vector cells, was used as the starting material for generating aRNA. First-strand cDNAs were synthesized using poly deoxythymidine (dT) primers followed by second-strand cDNA synthesis. The double-stranded cDNA was used as a template for in vitro transcription (IVT) reaction to generate antisense RNA (aRNA). Biotin-16–uridine-5'-triphosphate (UTP) (Roche) and Biotin-11–cytidine triphosphate (CTP) (Perkin Elmer) nucleotides were incorporated to the newly synthesized RNA during IVT reaction. The quality and quantity of aRNA were evaluated with a spectrophotometer (Fisher Scientific, Hampton, NH) and with an Agilent bioanalyzer (Agilent Technologies, Mississauga, ON, Canada) at the London Regional Genomic Center (LRGC; Robarts Research Institute, London, ON, Canada). Microarray analysis
Human HG-U133A chips (Affymetrix, Santa Clara, CA) were used for hybridization reactions. Probe preparations, hybridization, washing, and data acquisition were performed at the LRGC according to standard protocol provided by Affymetrix. The quality of array experiments was primarily evaluated by looking at the number of present and absent calls and the ratio of signal intensity of 3' to 5' end of housekeeping genes such as GAPDH and Statistical analysis Values shown represent the mean ± SEM for "n" number of experiments. Statistical analyses were performed using a 2-tailed, paired Student t test. Results were reported as significant if P was less than or equal to .05.
De novo expression of Smad7 in human hematopoietic tissues, construction and evaluation of Smad7 retroviral vector
To determine whether Smad7 might regulate TGF-
To examine the biological role of Smad7 in human hematopoiesis, retroviral gene transfer was used to overexpress Smad7 in putative human hematopoietic stem cells, defined functionally by their ability to repopulate the bone marrow of NOD/SCID mice with human hematopoietic cells of multiple committed lineages.44,45 The human Smad7 gene was subcloned upstream of an enhanced green fluorescent protein (GFP) reporter to create a bicistronic retroviral vector for Smad7 overexpression (Figure 1D). Stable GaLV-pseudotyped retroviral producer lines expressing the Smad7 and control vector retroviruses were constructed as described (K.C., K. Jay, B. Murdoch, M.B., manuscript submitted). The GFP-selected producer lines were confirmed to produce infectious retrovirus by transduction of the human megakaryocytic leukemia cell lines MBA.135 and M-O7e.36 PG13-Smad7, MBA.1-Smad7, and M-O7e–Smad7 GFP+ cell lines were morphologically indistinguishable from their vector control counterparts (Figure 1E, and data not shown), suggesting that Smad7 did not alter cellular morphology in stably transduced cells. Western blot analysis of GFP+ PG13-Smad7 and MBA.1-Smad7 cell lysates demonstrated an approximate 2-fold increase in Smad7 protein compared with endogenous levels of Smad7 protein detected in PG13-vector and MBA.1-vector cells, respectively (Figure 1F). In addition, Smad7 was expressed at approximately 2- to 3-fold greater levels in HeLa-Smad7 and M-O7e–Smad7 compared with HeLa-vector and M-O7e vector cells, respectively, as detected using amplified RNA (Figure 1G, and data not shown). These results indicate that transduction of Smad7 causes an increase in both Smad7 mRNA and protein in transduced cells.
The M-O7e megakaryocytic cell line has previously been shown to be sensitive to TGF- Ex vivo transduction of Smad7 into primitive human hematopoietic cells The CB Lin– cells used in this study are enriched for primitive hematopoietic cells with in vitro progenitor and in vivo repopulating function, present mostly within the CD34+CD38– subfraction,30 and have been shown to express Smad7 mRNA (Figure 1B). Efficiency of myeloid and B-cell lineage depletion is shown in a representative example of CB MNCs before and after lineage depletion using CD33 and CD15 for myeloid cells and CD19 and CD20 for B-lymphoid cells (Figure 2A). CB Lin– cells were transduced with either the Smad7 or vector retrovirus as outlined in Figure 2B. To determine the effect of Smad7 in human hematopoietic SRCs and their differentiated progeny, the total cells cultured from 4 x 105 Lin– cells were transplanted into each sublethally irradiated NOD/SCID mouse.
To assess the gene transfer efficiency into the bulk Lin– population, a fraction of Lin– cells were cultured for an additional 36 hours after transduction to allow for retroviral integration and GFP expression. Two days after the final transduction, the gene transfer efficiency into the bulk Smad7 and vector Lin– populations was not significantly different, as assessed by flow cytometry. Approximately 17% of vector-transduced Lin– cells and 15% of Smad7 transduced Lin– cells were GFP+ as determined by comparison to GFP– Lin– cells exposed to supernatant not containing retroviral particles (Figure 2C). To determine if Smad7 altered the primitive state of Lin– cells, transduced (GFP+) cells were examined for expression of the primitive hematopoietic marker CD34. Approximately 20% of the GFP+ cells 36 hours after gene transfer remained primitive based on retention of CD34 expression, regardless of the vector treatment (Figure 2D), suggesting that Smad7 did not enhance or inhibit the differentiation of the transduced Lin– cells in vitro. In addition, cells transduced with Smad7 versus vector alone demonstrated a more than 3-fold increase in expression of Smad7 (Figure 2E), thereby supporting effective Smad7 overexpression in transgenic human hematopoietic cells to be used in subsequent functional assays. Effect of Smad7 on CB hematopoietic repopulating cell capacity Previous studies using retroviral gene marking have determined that the cells within the Lin– population that engraft the BM of NOD/SCID mice with differentiated myeloid and lymphoid progeny, termed SRCs, are distinct from the less-primitive hematopoietic progenitors detected in the in vitro colony-forming unit (CFU) assay.31,47 To evaluate whether Smad7 plays a role in regulating the human multilineage repopulating cells detected in this assay, sublethally irradiated NOD/SCID mice were given transplants of equivalent numbers of transduced Smad7 and vector Lin– cells. Mice were killed 5 to 8 weeks after transplantation for analysis of human hematopoietic reconstitution by flow cytometric analysis of the bone marrow (BM) using the human-specific CD45 panleukocyte marker (Figure 3Ai), and those human cells expressing the transgene were detected by GFP fluorescence as shown (Figure 3Aii). From 5 independent CB samples transplanted into 13 mice, the level of human engraftment did not differ significantly between SRCs transduced with Smad7 compared with vector alone (Figure 3B). In addition, the fraction of gene-marked (GFP+) human (CD45+) cells engrafting the NOD/SCID mouse was similar under vector and Smad7 treatments (Figure 3C), suggesting that expression of Smad7 did not alter the level of hematopoietic chimerism. Since mice were given transplants of similar numbers of vector- or Smad7-transduced cells, this data suggests that overexpression of Smad7 did not result in increased expansion or proliferation of SRCs.
Smad7 alters the developmental capacity of human hematopoietic repopulating cells
When Lin– populations containing SRCs engraft the BM of NOD/SCID mice, these primitive cells divide and differentiate to give rise to committed cells of multiple hematopoietic lineages in a distinctive pattern of hematopoietic engraftment consisting of a predominantly B-lymphoid graft, with lesser contributions from committed myeloid cells and primitive cells.41 To determine if Smad7 affects the hematopoietic developmental capacity of transduced SRCs, mice successfully engrafted with gene-marked SRCs, which then gave rise to GFP+CD45+ cells in the mouse BM, were further analyzed for the presence of multilineage cell surface markers. B-lymphoid (CD19+), myeloid (CD33+), and primitive (CD34+) lineages were examined in combination with CD45 and GFP expression to assess the contribution of GFP+ cells to each of these hematopoietic lineages. In a representative experiment (Figure 3A), vector-transduced SRCs gave rise to committed cells with a typical pattern of engraftment,41 where the majority of hematopoietic cells within the graft expressed the B-lymphoid marker CD19 (Figure 3Aiii) and a smaller population of cells expressed the pan-myeloid marker CD33 (Figure 3Aiv). However, reconstitution of Smad7 SRCs demonstrated equal lymphoid to myeloid graft composition. Overall, in 13 mice from 5 independent CB samples, a shift from the lymphoid (CD19+ cells) to myeloid (CD33+ cells) arm of the hematopoietic system was observed, representing 20% of the bulk GFP+ population derived from Smad7 SRCs (vector 73.2: 26.3% vs Smad7, 53.0: 44.9%, lymphoid: myeloid progeny, P To address any potential variability between independent primary human CB samples, the percentages of lymphoid and myeloid cells were analyzed from paired mice given transplants of SRCs from 5 separate CB samples (Figure 3F). In all 5 CB samples, the engraftment pattern demonstrates a shift toward increased myeloid composition in Smad7 GFP+ cells. Despite the changes in lymphoid and myeloid contributions to the human hematopoietic graft, the proportion of primitive CD34+ cells was not altered in Smad7 mice, regardless of GFP expression (Figure 3D), suggesting that Smad7 alters the lineage commitment decision during hematopoiesis, but does not alter the total levels of differentiation or cellular expansion. Smad7 only affects in vivo reconstitution arising from the most primitive long-term hematopoietic repopulating cells engrafting the NOD/SCID mouse
Smad7-transduced Lin– cells were transplanted into Smad7 does not alter hematopoietic maturation subsequent to lineage commitment To determine if the increase in myeloid cells and decrease in lymphoid cells is due to impaired development of myeloid cells, and hence accumulation of primitive blastlike myeloid cells, progression down the myeloid lineage was examined in greater detail by flow cytometric analysis of NOD/SCID mice engrafted from a representative CB sample (Figure 4A). Bipotent granulocyte/monocyte precursors (CD33+CD15–), as well as mature monocytic and granulocytic progeny determined by the expression of CD13, CD14, and CD15 in addition to CD33 expression, were observed within both vector and Smad7 GFP+ human grafts, suggesting that Smad7 does not impair normal myeloid differentiation and maturation patterns. Furthermore, the fold increases in the various myeloid lineages in Smad7-versus vector-transduced cells were similar (1.5-2.2 fold; Table 1), suggesting that Smad7 overexpression does not favor, or inhibit, the development of any particular subset of committed myeloid cells. Since all subsets of committed myeloid cells were altered similarly by Smad7 expression, this suggests that Smad7 overexpression in SRCs prior to myeloid cell fate commitment does not impair the ability of committed myeloid cells to differentiate normally.
Alternatively, to determine if the increase in myeloid progeny could be caused by a relative decrease in lymphoid cells due to an early block in lymphoid commitment, thus preventing cellular maturation to the pro–B cell stage associated with expression of CD19, we also examined GFP+ human cells for the expression of CD10, a marker expressed on uncommitted lymphoid precursors. The fold decrease in lymphoid cells in Smad7 versus vector SRCs was comparable based on expression of CD10 and CD19 (1.96-fold compared with 2.27-fold, respectively; Figure 4B, Table 1), and was reproducible in all independent CB samples examined. These results suggest that Smad7 does not block progression of uncommitted lymphoid cells toward the B-cell lineage prior to CD19 acquisition, but instead reduces the frequency of lymphoid cell fate commitment decisions prior to CD10 acquisition during SRC differentiation into lineage-restricted progeny. Mice engrafted with Smad7 SRCs demonstrate an increased frequency of functional myeloid progenitors Our data suggest that mice engrafted with Smad7 SRCs give rise to a greater proportion of committed myeloid cells compared with vector SRCs based on expression of myeloid cell surface markers. To determine if Smad7 also affects the frequency of functional myeloid progenitors, primitive CD34+ cells fractionated on GFP expression were isolated from the BM of engrafted mice by FACS (Figure 5A). Aliquots of 5000 selected CD34+GFP+ or CD34+GFP– cells were placed into clonogenic progenitor assays to detect myeloid and erythroid CFUs arising from SRCs that had engrafted the NOD/SCID mouse, here termed CFU-SRCs. Representative granulocytic vector and Smad7 GFP+ CFU-SRCs are shown in Figure 5B, as visualized by light and fluorescent microscopy. Both colonies demonstrate a similar morphology, suggesting that Smad7 did not alter the process of myeloid maturation. Figure 5C summarizes the CFU-SRC capacity of vector compared with Smad7 GFP+ cells. In 4 out of 5 experiments, mice given transplants of Smad7 SRCs gave rise to more CFU-SRCs per 5000 CD34+GFP+ cells than did vector SRCs from the same CB sample (average, 4.6-fold increase). On average, Smad7 SRCs gave rise to 41 GFP+ CFU-SRCs, compared with only 18 CFU-SRCs from the corresponding vector control. Similar to phenotypic data, the increased frequency of CFU-SRCs was apparent only in GFP+ Smad7 cells, as demonstrated by similar frequencies of GFP– Smad7 and vector CFU-SRCs, suggesting the effect is specific to overexpression of the Smad7 transgene (Figure 5C, inset). Both vector- and Smad7-transduced cell populations gave rise to the full distribution of erythroid and myeloid CFU-SRC types at approximately equal proportions, both in the GFP+ population (Figure 5D) as well as the GFP– population (Figure 5D, inset), suggesting that while Smad7 may influence the frequency of myeloid progenitors, Smad7 does not alter subsequent maturation of committed myeloid colony types.
Potential gene targets of Smad7 in primitive human hematopoietic cells
Downstream gene regulation by Smad7 has not been characterized in primitive human hematopoietic cells. Given the effects of Smad7 on repopulating cells, we isolated primitive Smad7-transduced hematopoietic cells (CD34+SMAD7–GFP+) and compared their gene expression profile with similar cells transduced with vector alone (CD34+vector–GFP+). Genes associated with the TGF-
It is well documented that numerous ligands of the TGF- superfamily are involved in various stages of hematopoietic regulation, both in humans and small animal models. However, the effects of downstream regulators of this signaling pathway by Smad effectors and inhibitors have yet to be evaluated. Here, we examined the role of a TGF- superfamily inhibitor, Smad7, in primitive human hematopoiesis through retroviral modification of multipotent human hematopoietic cells with in vivo lymphoid and myeloid repopulating function (SRCs). Surprisingly, overexpression of Smad7 in CB Lin– cells caused a lineage shift of SRC reconstitution toward myelopoiesis (Figure 3D,F).41 This observation suggests that Smad7 either alters the cell fate decisions of multipotent cells with myeloid and lymphoid differentiation capacity, or influences a subset of committed myeloid or lymphoid progeny to produce altered frequencies of mature cells. To determine at which stage of hematopoiesis Smad7 was acting, Smad7-transduced SRCs were transplanted into NOD/SCID/2m–/– mice39,40 that allow preferential engraftment of more mature STR hematopoietic progenitors, while the parental NOD/SCID strain only permits the engraftment of the more primitive LTR hematopoietic cells.48 NOD/SCID/2m–/– mice given transplants of Smad7-transduced Lin– cells gave rise to myeloid/lymphoid progeny in a repopulation pattern indistinguishable from vector control cells 6 to 8 weeks after transplantation (Figure 3E), suggesting that Smad7 has no effect on this more mature STR hematopoietic progenitor population. This suggests that Smad7 influences cell fate commitment decisions only in the most primitive human hematopoietic repopulating cells within the Lin– subfraction of CB MNCs. Our study indicates that Smad7 did not block myeloid progression of SRCs to cause an accumulation of early CD33+ myeloid blasts. Consistent with the phenotypic data supporting an increased production of normal myeloid cells from Smad7 SRCs, we also observed an increase in the frequency of functional myeloid progenitors arising from the CD34+ cells within the BM of SRC-repopulated mice overexpressing Smad7 (Figure 5C). Since the early stages of lymphoid commitment and B-lymphoid maturation were not compromised in Smad7 SRCs, and the decrease in committed lymphoid cells was due to a decrease in the frequency of lymphoid cell fate commitment decisions, our data suggest that Smad7 does not alter the behavior of cells once they have committed to a particular hematopoietic lineage. We suggest that Smad7 acts only to alter hematopoietic cell fate commitment decisions in the most primitive cells with in vivo lymphoid and myeloid differentiation potential. Since the NOD/SCID assays for human repopulating cells does not readily detect differentiation into the T-cell lineage, use of alternative recipients capable of supporting human T lymphopoiesis will be required to determine whether Smad7 effects lymphoid T-cell development. This role for Smad7 in human SRCs is similar in principle to observations of the role of Smad7 in Xenopus embryogenesis, where the ectopic expression of Smad7 in Xenopus embryos and animal pole explants was found to inhibit BMP-4– and activin-driven mesoderm formation, instead promoting/inducing neural cell fate decisions.27,50 There is no previous indication from the literature that Smad7 can alter cell fate commitment decisions in such a fashion in primary mammalian tissue, or outside of early embryonic development, thus identifying a novel role for Smad7 in multipotent human hematopoietic stem cell populations. Our hypothesis that Smad7 plays an important role in stem cell regulation is supported by the recent report that Smad7 expression is enhanced in murine multipotent HSC and pluripotent ESC populations, identified through oligonucleotide microarray analysis of genes preferentially expressed in various stem cell populations.28,29 Only 2 other genes, both encoding leukemogenic transcription factors,51-53 have been shown to alter the hematopoietic balance from committed lymphoid to myeloid progeny in mice repopulated with retrovirally transduced human SRCs. Retroviral-mediated overexpression of HoxA10 in SRCs gave rise to an engraftment pattern with increased myeloid and decreased lymphoid graft contribution, coupled with an absence of in vitro erythroid CFUs and an increase in the frequency of SRC-derived blast-CFUs incapable of proper myeloid differentiation.54 In our study, the myeloid differentiation capacity of cells arising from Smad7-transduced SRCs was unimpaired, as demonstrated by the expression of mature myeloid markers at equal proportions compared with vector-transduced cells, and by the presence of multiple lineages of mature myeloid CFU-SRCs (including erythroid CFUs) isolated from the BM of engrafted mice. Thus, Smad7 does not likely operate upstream of a committed myeloid factor such as HoxA10. Overexpression of the dominant negative Ikaros 6 (Ik6) isoform in SRCs resulted in a decrease in lymphoid cells and concomitant increase in myeloid cells55 in a pattern superficially similar to that seen by Smad7 expression in SRCs. Since Ikaros is believed to be an essential transcription factor for regulating the proliferation and differentiation of cells within the T and B lymphoid lineages,53 Ik6 overexpression was proposed to cause an inhibition of B-cell development, thus leading to the development of a relative increase in the proportion of committed myeloid cells.55 As the clonogenic capacity of myeloid CFU-SRCs was increased with Smad7 overexpression, it seems unlikely that Smad7 operates upstream of genes responsible for regulating committed lymphoid lineages, but rather acts in a novel manner to modulate the expression of genes involved in the earliest hematopoietic lineage commitment decisions. The mechanism by which Smad7 achieves this effect on cell fate decisions remains to be elucidated and these efforts will be facilitated by preliminary microarray data that identify regulated genes in response to Smad7 overexpression.
We thank Dr Jeffrey Wrana for providing us with the Smad7 construct, Dr Robert Hawley for providing us with the MIEV retroviral vector, and Drs Kysta Levac and Francis Karanu for their helpful discussions and insights toward completion of this work.
Submitted March 8, 2004; accepted September 30, 2004.
Prepublished online as Blood First Edition Paper, October 21, 2004; DOI 10.1182/blood-2004-03-0881.
Supported by a research grant from the Canadian Institutes of Health Research (CIHR) and Krembil Foundation, Canada Research Chair in Stem Cell Biology and Regenerative Medicine (M.B.), and a postgraduate scholarship award from CIHR to K.C.
K.C. and F.S. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Mickie Bhatia, Krembil Centre for Stem Cell Biology, Robarts Research Institute, 100 Perth Dr, London, ON N6A 5K8, Canada; e-mail: mbhatia{at}robarts.ca.
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| Copyright © 2005 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
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Abstract
Introduction
B (NF-
–minimal essential medium (MEM) with 7% FBS. For collection of virus-conditioned supernatant (VCS), cells at near confluence were grown in IMDM with 10% FBS (serum-rich VCS) or 2.5% FBS (serum-deficient VCS) for 24 hours. Fresh VCS was harvested and filtered with a 0.45-µm syringe filter before transduction. 
] compared with Lin–CD34+CD38+ cells [
], right) and (C) in mature human CB subsets containing the myeloid (
.01. Error bars indicate SEM.
