A Road Map Toward Defining the Role of Smad Signaling in Hematopoietic Stem Cell - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:08 |只看该作者 |倒序浏览 |打印

作者：Taiju Utsugisawaa, Jennifer L. Moodya, Marie Asplinga, Eva Nilssona, Leif Carlssonb, Stefan Karlssona作者单位：a Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and The Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University Hospital, Lund, Sweden;b Ume Center for Molecular Medicine, Ume University, Ume, Sweden

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      【摘要】; ?1 b) x& Q+ E4 X1 u
   The transforming growth factor-&szlig; (TGF-&szlig;) superfamily encompasses the ligands and receptors for TGF-&szlig;, bone morphogenic proteins (BMPs), and Activins. Cellular response to ligand is context-dependent and may be controlled by specificity and/or redundancy of expression of these superfamily members. Several pathways within this family have been implicated in the proliferation, differentiation, and renewal of hematopoietic stem cells (HSCs); however, their roles and redundancies at the molecular level are poorly understood in the rare HSC. Here we have characterized the expression of TGF-&szlig; superfamily ligands, receptors, and Smads in murine HSCs and in the Lhx2-hematopoietic progenitor cell (Lhx2-HPC) line. We demonstrate a remarkable likeness between these two cell types with regard to expression of the majority of receptors and Smads necessary for the transduction of signals from TGF-&szlig;, BMP, and Activin. We have also evaluated the response of these two cell types to various ligands in proliferation assays. In this regard, primary cells and the Lhx2-HPC line behave similarly, revealing a suppressive effect of Activin-A that is similar to that of TGF-&szlig; in bulk cultures and no effect of BMP-4 on proliferation. Signaling studies that verify the phosphorylation of Smad2 (Activin and TGF-&szlig;) and Smad1/5 (BMP) confirm cytosolic responses to these ligands. In addition to providing a thorough characterization of TGF-&szlig; superfamily expression in HSCs, our results define the Lhx2-HPC line as an appropriate model for molecular characterization of Smad signaling. , p' E+ [" q2 |3 d. K7 Q8 Q7 _; p% L
      【关键词】 Hematopoietic stem cells Smad signaling Transforming growth factor- Bone morphogenic proteins Activin: {0 p  e* [9 H; ]! |: g
   INTRODUCTION# g& l# v6 ~$ m2 ]1 h' w
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The transforming growth factor-ß (TGF-ß) superfamily consists of more than 35 structurally related members, including TGF-ßs, bone morphogenic proteins (BMPs), and Activins .
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The above-described linearity with which these ligands transduce their signals may be valid for particular cell types and contexts but is likely to be an oversimplification if applied to all cells. Overlapping receptor and Smad usage by different TGF-ß superfamily ligands is one level complexity of the signaling system . Consequently, comprehensive expression analyses of the molecules related to TGF-ß signaling pathways are a first step in understanding the orchestration of Smad signaling in a particular cell type.

A large number of studies on both human and murine cells have shown the potent growth inhibitory effect of TGF-ß1 on early hematopoietic progenitors , very little is understood about its direct effects on murine adult hematopoiesis.
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Overall, the molecular mechanisms of receptor/Smad signal transduction in HSCs are poorly characterized, mainly due to their rarity in the bone marrow and the requirement of large numbers of cells for the functional analysis of signal transduction. The Lhx2 HSC-like hematopoietic progenitor cell (Lhx2-HPC) line was previously generated by immortalizing mouse bone marrow (BM) HSCs using a retroviral vector to drive the expression of the LIM-homeo box gene Lhx2 . We therefore sought to characterize the similarity of these cells to primary murine HSCs, specifically with respect to expression of TGF-ß superfamily members and response to ligand. Our data maps the players of these important pathways in HSCs and reveals fidelity of the Lhx2-HPC line to their primary counterparts, thereby demonstrating their value as a tool for the assessment of signal transduction events.1 x9 V; I$ Y7 \5 X- p5 G0 i8 Y

MATERIALS AND METHODS
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Single-Cell Cultures
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All animal procedures were performed with consent from the local ethics committee at Lund University. Mouse BM cells were obtained from 10¨C12-week-old C57Bl/6xSJL mice and treated with ammonium chloride (NH4Cl; Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) and then incubated in a lineage antibody cocktail (CD4, CD8, CD5, Gr1, Mac1, B220, and TER119; all antibodies were from BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen). After washing, sheep anti-rat immunoglobulin G (Fc)-conjugated immunomagnetic beads (Dynal, Oslo, Norway) were added, and lineage-positive cells were removed with a magnetic particle concentrator (MPC-6; Dynal). Lin¨C/lo cells were stained with fluorescein isothiocyanate-conjugated anti-CD34, allophycocyanin (APC)-conjugated anti-c-kit and phycoerythrin-conjugated Sca1 antibodies with 7AAD (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) added to exclude dead cells. The LSKCD34¨C cells were sorted on a FACS-Vantage Cell Sorter (Becton, Dickinson and Company, San Jose, CA, http://www.bd.com) and seeded into Terasaki plates (Nunc, Naperville, IL, http://www.nuncbrand.com) at a concentration of one cell per well in 20 µl of serum-free medium (X-vivo 15; BioWhittaker Molecular Applications, Verviers, Belgium, http://www.bmaproducts.com) supplemented with 1% bovine serum albumin (Stem Cell Technologies), 2 mM L-glutamine (Gibco-Invitrogen, Carlsbad, CA), 1.5 x 10¨C4 M monothioglycerol (Sigma), 50 ng/ml recombinant murine SCF (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), 50 ng/ml recombinant murine interleukin (IL)-6, and 10 ng/ml recombinant murine IL-3. TGF-µ1, BMP-4, and Activin-A (R&D Systems) were added to obtain final concentrations as indicated. The number of proliferating clones was assessed after 12 days in culture.& y0 V  `4 W$ g

Bulk Liquid Cell Cultures
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The LSKCD34¨C cells (500 cells in 0.1 ml) and Lhx2-HPC line (5 x 105 in 1 ml) were cultured in serum free medium, supplemented as described above. Fresh media supplemented with either TGF-µ1, BMP-4, or Activin-A at the indicated concentrations were added every 72 hours to re-establish the original plating density.0 M1 b3 _; C7 R6 k- y" V6 b

Colony Formation Assay
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The LSKCD34¨C cells (1,000 cells in 0.2 ml) were cultured in bulk liquid culture condition as described above with or without BMP-4 at the indicated concentrations for 3 or 6 days. The cells were then plated in 3 ml of 0.8% methylcellulose (M3231; Stem Cell Technologies) (200 cells/ml after 3 days of culture and 300 cells/ml after 6 days of culture) supplemented with 50 ng/ml SCF, 10 ng/ml IL-6, and 10 ng/ml IL-3. Cells were plated in 35-mm Petri dishes (Nunc, Inc.) and placed in a humidified box at 37¡ãC with 5% CO2. Colonies were counted after 6 days using an inverted microscope. A colony was defined as a cluster of more than 40 cells.% k% f0 u  y: _1 F

Annexin-V Staining+ H4 A# G5 ~7 B

Quantitation of apoptotic cells was assessed by Annexin-V-APC staining and fluorescence-activated cell sorter (FACS) analysis, according to the manufacturer¡¯s instructions (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen).) G9 [# n( j. k: ?
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Western Blot Analysis
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Mouse BM cells, BM Lin¨C cells (depleted as described above), and the HPC line (5 x 105 cells each) were treated with TGF-ß1 (20 ng/ml), BMP-4 (25 ng/ml), and Activin-A (25 ng/ml) for 1 hour at 37¡ãC. Total cellular proteins were extracted by boiling in sample buffer containing 60 mM Tris HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 5% vol/vol glycerol, 2% ß-mercaptoethanol, and 20 mM dithiothreitol. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and were transferred to a polyvinyliden fluoride membrane (Hybond-P; Amersham Bioscience, Uppsala, Sweden). Antibodies specific for Smad1, Smad5, phosphorylated Smad2, phosphorylated Smad1/5, and TGF-ßRI (ALK5) were from Cell Signaling Technology (Beverly, MA). Antibodies for Smad2/3 and Smad4 were from BD Pharmingen. Antibodies for Smad7 and TGF-ßRII were from Santa Cruz Biotechnology (Santa Cruz, CA, http://www.scbt.com), and the endoglin-specific antibody (clone MJ7/18) was obtained from Southern-Biotech (Birmingham, AL, http://www.southernbiotech.com) and Immunkemi (Stockholm, Sweden, http://www.immunkemi.se). Proteins were visualized using chemiluminescence reagents (Western Lightning; PerkinElmer Life Sciences, Boston, http://www.perkinelmer.com) according to the manufacturer¡¯s protocol.

Quantitative Real-Time Polymerase Chain Reaction (Q-RT-PCR)

Cell populations were sorted directly into RLT buffer (RNeasy micro RNA isolation kit) containing 2-mercaptoethanol (10,000 cells/75 µl of buffer) and were flash frozen on dry ice and stored at ¨C80¡ãC. Total RNA was isolated using the RNeasy micro RNA isolation kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) according to the manufacturer¡¯s protocol. cDNA was reverse transcribed using random hexamers and SuperscriptIII (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Q-RT-PCR was performed using the Taq-Man system (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com) according to the manufacturer¡¯s protocol and was analyzed on an ABI Prism 7700 sequence detection system. Primers for all molecules were obtained from Applied Biosystems Assays on Demand/By Design. Expression values were normalized to murine hypoxanthine guanine phosphoribosyltransferase (HPRT) that was run simultaneously in each reaction, using the formula 2¨C(CT specific-CT HPRT). cDNA obtained from mouse embryonic endothelial cells  verified primer functionality in cases where expression could not be detected in the primary cells or the Lhx2 cell line. In the case of Alk-6, which was not detectable in mouse embryonic endothelial cells (MEECs), primer functionality was verified using cDNA obtained from human cord blood CD34  cells.% p& `- \" N+ e+ g  j% k# G$ a

Statistical Analysis
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Statistical analysis was performed using Student¡¯s t test. A p value of less than .05 was considered significant.
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RESULTS

Several TGF-ß Superfamily Ligands and the Majority of Receptors and Smad mRNAs Are Expressed by Purified HSCs and the Lhx2-HPC Line: n& \' X# m, D/ X/ O; S

To characterize which components of the TGF-ß superfamily are expressed in both primary HSCs and the Lhx2-HPC line, the mRNA expression levels of ligands, receptors, and Smads were quantified by Q-RT-PCR. To obtain purified HSCs, LSKCD34¨C cells, a population shown to contain primitive and long-term repopulating cells,  were sorted. Also sorted were equal numbers of the Lhx2-HPC line, as well as a wild-type MEEC line and CD34  human cord blood cells, with the latter two populations acting to verify primer functionality. The absence of expression in HSCs or the Lhx2-HPC line was only concluded for molecules where primer functionality could be confirmed in one of our control cell populations.& z. \+ L2 X7 H9 D; w7 o6 f) j
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Interestingly, the expression patterns of all ligands, receptors, and Smads were similar in both LSKCD34¨C cells and the Lhx2-HPC line. However, due to a higher level of HPRT expression in the Lhx2-HPC cells, the relative expression levels of all components were lower in those than in primary cells. With respect to ligand expression, both LSKCD34¨C cells and the Lhx2-HPC line showed high expression of TGF-ß1, detectable expression of BMP-4, and very low to undetectable levels of Activin-A (Fig. 1A, 1B). TGF-ß2, TGF-ß3, BMP-2, and BMP-7 were not detected (data not shown). Of the type I receptors, considerable levels of ALK5 (TGF-ßRI), ALK2 (ActRI), and ALK4 (ActRIB) were expressed in both cell types, whereas ALK1, ALK3 (BMPRIA), and ALK6 (BMPRIB) levels were extremely low to absent (Fig. 1C, 1D). mRNA for the type II receptors TGF-ßRII, BMPRII, ActRIIA, and ActRIIB were expressed, although levels of ActRIIA were very low in the cell line (Fig. 1C, 1D). Endoglin, the accessory receptor for TGF-ß recently reported as present on long-term reconstituting stem cells , was indeed detectable in both cell types (Fig. 1C, 1D).

Figure 1. Expression of mRNA for TGF-ß superfamily ligands, receptors, and Smads. Total RNA was isolated from LSKCD34¨C cells (A, C, E) and the Lhx2-HPC line (B, D, F). The expression levels of the ligands (A, B), receptors (C, D), and Smads (E, F) are shown relative to the HPRT expression values that were run simultaneously with the samples. Data are pooled averages from two independent sorting experiments, each one assessed in triplicate ¡À SEM. Abbreviations: BMP, bone morphogenic protein; TGF-ß, transforming growth factor-ß; HPC, hematopoietic progenitor cell; HPRT, hypoxanthine guanine phosphoribosyltransferase.* ?0 I+ {# n6 w( U8 t' ]$ T4 \4 N

All R-Smad mRNA species, with the exception of Smad8, were expressed in both cell types, and levels of the co-Smad, Smad4, were particularly abundant (Fig. 1E, 1F). Examination of I-Smads levels revealed that Smad7 was expressed in both cell sources, albeit with some variability in levels in the LSKCD34¨C cells, whereas Smad6 expression was very low to undetectable in both cell types (Fig. 1E, 1F). Therefore, with the exception of ALK1, ALK3, and ALK6, primary HSCs and the Lhx2-HPC cell line express all the molecules required to transmit signals from the various TGF-ß family ligands and share a common expression pattern with respect to TGF-ß superfamily members.
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We utilized the Lhx2-HPC cell line to verify our results at the protein level and demonstrated that several receptors and Smads were readily detectable by Western blot (Fig. 2).

Figure 2. Production of TGF-ß superfamily receptors and Smads in Lhx2-HPC cells. Western analysis confirms the expression profiles for selected TGF-ß superfamily receptors and Smads. Total lysates obtained from Lhx2-HPC line were analyzed by Western blot using specific antibodies as indicated. Abbreviations: HPC, hematopoietic progenitor cell; TGF-ß, transforming growth factor-ß.  f4 A' x9 ]) Z. C. D/ Y( k
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TGF-ß and Activin-A Both Suppress HSC Proliferation by Induction of Apoptosis, Whereas BMP-4 Neither Enhances nor Inhibits Proliferation- ~* v6 q* ^1 @: ]- I! i% E

Expression of the bulk of the machinery required for transduction through TGF-ß, Activin, and BMPs suggest that HSCs could be responsive to all three types of ligands. Indeed, studies characterizing TGF-ß inhibition on the initial stages of proliferation of murine and human long-term repopulating HSCs  are well established. However, the effects of BMP-4 and Activin-A on murine purified HSCs have not been as well characterized. To investigate the effect of TGF-ß1, BMP-4, and Activin-A on the recruitment of primitive murine HSCs into proliferation, LSKCD34¨C cells were purified by FACS analysis and grown as single cells in serum-free medium with IL-6, IL-3, and SCF, along with various ligand concentrations. The numbers of proliferating clones were scored after 12 days. The addition of TGF-ß1 to these cultures, as expected, markedly reduced the numbers of both total clones (>10; more than 10 cells) and large clones (>100; more than 100 cells) in a dose-dependent manner (Fig. 3A). Activin-A also appeared to transmit inhibitory signals to these cells, albeit less potently than TGF-ß1, showing a modest suppressive effect at concentrations of 2.5 ng/ml or higher (Fig. 3B). Interestingly, BMP-4 had no significant proliferative or inhibitory effect on LSKCD34¨C cells at any concentration (Fig. 3C).' d7 k" S8 }" x2 f9 ^

Figure 3. Single-cell and bulk culture of primitive HSCs reveal similar roles for TGF-ß and Activin-A in the suppression of proliferation. Single LSKCD34¨C cells were seeded in Terasaki plates in serum-free medium supplemented with SCF, IL-6, and IL-3. TGF-ß1 (A), Activin-A (B), and BMP-4 (C) were added to obtain final concentration as indicated. The numbers of total proliferating colonies (>10, more than 10 cells) and high proliferative colonies (>100, more than 100 cells) per 60 wells were scored at day 12. Data represent the mean percentage of proliferating wells and are the combined data from cells sorted from six individual mice assessed independently in two experiments ¡À SEM. (D): LSKCD34¨C cells were cultured in serum-free medium supplemented with SCF, IL-6, and IL-3. TGF-ß1, BMP-4, and Activin-A were added to obtain final concentration as indicated. Each time point represents the average of two independent experiments in which cells from three individual mice were assessed independently ¡À SEM. (E): LSKCD34¨C cells were cultured under bulk liquid culture conditions for 3 or 6 days with BMP-4 added to obtain the final concentrations as indicated. The cells were seeded in methylcellulose supplemented with SCF, IL-6, and IL-3. Total colony-forming units-C were counted after 6 days. Data represent the mean colony number per 100 cells plated and are the combined data from three experiments ¡À SEM. Abbreviations: BMP, bone morphogenic protein; HPC, hematopoietic progenitor cell; IL, interleukin; SCF, stem cell factor; TGF-ß, transforming growth factor-ß.
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To study the effects of these ligands under bulk cell conditions, the LSKCD34¨C cells were cultured in serum-free medium with the same growth promoting cytokines as above and with the highest concentration of the ligands used for the single-cell assay. Again as expected, the addition of TGF-ß1 completely inhibited the proliferation of these cells (Fig. 3D). Activin-A similarly inhibited the proliferative response at early points in the assay, but it did, however, allow for mild proliferation at a later time point (day 7, Fig. 3D), perhaps suggesting a less potent effect on cells undergoing differentiation. Once more, BMP-4 appeared to have no effect on proliferation of LSKCD34¨C cells compared to the control serum-free conditions.
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The lack of a proliferative effect of BMP-4 on murine HSCs contrasts with studies that indicated that certain doses of BMP-4 promote proliferation and clonogenic expansion of human cord blood cells . These studies also suggested that high doses of BMP-4 preserved primitive cell surface phenotypes, whereas low doses promoted differentiation. As such, we also examined the effects of BMP-4 on these parameters in our cells. No significant difference in clonogenic capacity was read out in methylcellulose assays that supported CFU-GM growth after 3 or 6 days of culture in control cytokines compared with addition of 2.5 or 25 ng/ml of BMP-4 (Fig. 3E). Furthermore, both the low and high doses of BMP-4 did not affect the differentiation of LSKCD34¨C cells assessed by FACS analysis of c-kit, sca-1, CD34, Mac1, and Gr-1 after 12 days culture (data not shown). This data suggests that the effects of BMP-4 on human cord blood cells are not paralleled on adult mouse HSCs.& u0 B8 }! g: W- ?

As the growth response of the Lhx2-HPC line is density-dependent in the absence of a multitude of cytokines , the response of the cell line to these ligands was only assessed in bulk cultures. Whereas Activin-A and TGF-ß1 both had suppressive effects on the proliferative response of the cells, TGF-ß1 again demonstrated the more potent inhibition (Fig. 4A). Likewise, BMP-4 did not alter the cell growth of the Lhx2-HPC line (Fig. 4A). After 24 hours of culture with these ligands, cells were assessed by Annexin-V staining and FACS analysis. The induction of apoptosis by TGF-ß1 could be clearly demonstrated, with up to 43% of the population being positive for Annexin-V staining after 24 hours of stimulation (Fig. 4B). Activin-A also induced apoptosis in these cells, albeit less potently than TGF-ß1, and this was in line with the results of our proliferation assays (Fig. 4B). Also in keeping with our other results, the percentages of Annexin-V-positive cells in BMP-4-treated cultures were very similar to the those in the control cultures (Fig. 4B). Thus, the proliferative response of Lhx2-HPC cell line is suppressed by TGF-ß1 and Activin-A and is seemingly unaffected by BMP-4, similar to the effects on primary HSCs.
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Figure 4. TGF-ß1 and Activin-A suppress proliferation of the Lhx2-HPC line by inducing apoptosis. (A): The Lhx2-HPC line was cultured in serum-free medium supplemented with SCF, IL-6, and IL-3 with the addition of TGF-ß1, BMP-4, or Activin-A to obtain a final concentration as indicated. The data are presented as the average of three independent experiments ¡À SEM. (B): The Lhx2-HPC line was cultured in serum-free medium, supplemented as described above. Quantitation of apoptosis was assessed by Annexin-V staining and FACS analysis after 24 hours. FACS plots are representative of three independent experiments. Abbreviations: BMP, bone morphogenic protein; FACS, fluorescence-activated cell sorter; HPC, hematopoietic progenitor cell; SCF, stem cell factor; TGF-ß, transforming growth factor-ß.
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Activation of the Smad Pathways by TGF-ß1, BMP-4, and Activin-A' m/ G8 Q0 L& _' Z9 H9 w* k/ n
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The phosphorylation of molecules as an indicator for pathway activation has become a standard molecular method that is precluded in populations of rare primary cells. A cell line that could mimic the expression of molecules in primary HSCs as well as behave in a functionally similar manner would be a valuable tool for these types of studies. Given the previous functional characterization of the Lhx2-HPC line  and the above-described similarity with respect to the TGF-ß superfamily pathways, we hypothesized that these cells may serve this purpose well. As such, Western blotting was performed to examine whether the R-Smads are phosphorylated efficiently by their respective ligands. For comparison, we performed the same stimulation and analysis on primary lineage marker-negative (Lin¨C) cells and whole bone marrow cells. In general, stronger signals were obtained from Lhx2-HPC lysates (Fig. 5). Expression of Smad1/5 and Smad2 protein was detected in Lin¨C cells, non-sorted BM cells, and the Lhx2-HPC line. The phosphorylation of Smad1/5 was detected in primary cells and the Lhx2-HPC line with BMP-4 treatment, but it was absent in response to TGF-ß and Activin-A. Conversely, TGF-ß and Activin-A elicited the phosphorylation of Smad2 in all cell sources, and this response was not invoked by BMP-4 stimulation (Fig. 5). Thus, TGF-ß and Activin-A are able to propagate their growth suppressive signals through the phosphorylation of Smad2. Furthermore, despite the fact that BMP-4 did not alter cell proliferation of either mouse HSCs or the Lhx2-HPC line, and despite the absence of ALK6 and ALK3, a BMP-specific signal was detectably transduced through Smad1/5.

Figure 5. Ligand stimulation of HSCs induces Smad phosphorylation. The Lhx2-HPC line, Lineage¨C cells (Lin¨C) and total mouse bone marrow cells (BM) were untreated (C) and treated with 20 ng/ml of TGF-µ1, 25 ng/ml BMP-4, and 25ng/ml Activin A for 1 hour. Total lysates were analyzed by Western blot using anti-Smad1, anti-phospho-Smad1 (p-Smad1), anti-Smad2, and anti-phospho-Smad2 (p-Smad2) antibodies. Abbreviations: BM, bone marrow; BMP, bone morphogenic protein; C, untreated control; HPC, hematopoietic progenitor cell; TGF-ß, transforming growth factor-ß.

DISCUSSION

The potent inhibitory effect of exogenous TGF-ß1 on human and mouse hematopoietic progenitors and long-term repopulating cells has been well documented . These studies have raised interesting questions regarding the role for TGF-ß in in vivo hematopoiesis, yet the possibility that other signaling pathways are able to compensate for TGF-ß in these settings cannot be excluded. The Activin and BMP pathways could be likely candidates for compensatory roles given the convergence of their signals on the Smad pathway. We have thus characterized the components of these pathways to determine the potential relevancy of these molecules to HSCs.

It has been shown that, in vitro, TGF-ß1 is produced by immature human hematopoietic cells, and that the inhibition of autocrine TGF-ß1 can stimulate the growth of human and mouse hematopoietic progenitor cells . It is therefore intriguing that ALK1 is not expressed in HSCs, suggesting that endoglin may play a novel role in these cells.
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Activin-A was not considerably expressed by either the LSKCD34¨C cells or the Lhx2-HPC line, similar to studies on human primitive HSCs  effects on the proliferation of human HSCs, the direct effects on purified murine HSCs have not been shown. Our results demonstrate an inhibitory effect of Activin-A on proliferation of murine HSCs. In single-cell primary cultures, the suppressive effect was modest compared to that of TGF-ß1, especially with regards to the inhibition of large colony formation, suggesting a limited effect on the most primitive progenitors. The suppressive effects of Activin-A were more impressive in the bulk cultures, allowing for proliferation only late in the assay. The discrepancy between these experiments may be attributable to instability of the ligand in the single-cell cultures, as it was added only at initial seeding versus every 72 hours in the bulk cell culture system.

The amount of mRNA for BMP-4 produced by both types of cells was not copious and was also rather variable, possibly reflecting differences in cellular response to autocrine or paracrine factors at the time of analysis. It has been shown that BMP can signal through three distinct type II receptors: BMPRII, ActRII, and ActRIIB . Our results suggest that BMPRII/ALK2 may be a receptor complex for BMP-4 signaling in murine HSCs.' u( w% L* w0 H2 a* b/ l! G

A previous study using human primitive HSCs purified from cord blood (CB), has shown that BMP-4 can enhance the proliferation of HSCs in bulk culture and in clonogenic assays . Indeed, our data revealed that murine BM HSCs lack expression of these two type I receptors. Therefore, human CB HSCs may represent a unique population of HSCs that respond to BMP-4 in a proliferative manner that is distinct from cells in the bone marrow. Although BMP-4 did not alter the cell growth of murine HSCs, the phosphorylation of downstream target Smad1/5 was induced. It is therefore possible that BMP-4 signaling contributes to another yet uncharacterized aspect of HSC function.2 W6 X! N5 W" l- N

The fact that both TGF-ß1 and Activin-A induced similar suppressive effects on the primary cells and the cell line by causing apoptosis and that both ligands also induced the phosphorylation of Smad2 raises the question of whether there is cellular discernment between these ligand signals or whether they act in a redundant manner. Although R-Smads act as transcriptional activators or repressors in the nucleus ; therefore, Smad-independent effects are likely to be factors in cellular responses. An important future goal will be to evaluate the downstream consequences specific to particular ligands, and in this regard the Lhx2-HPC line is likely to be of great value.

One of the major problems for molecular analysis of HSCs is their rarity; for instance, there are fewer than 3 LSKCD34¨C cells per 106 mouse BM cells . Here we have shown that these cells resemble primary LSKCD34¨C cells with respect to expression of TGF-ß superfamily ligands, receptors, and Smads; that they respond similarly to TGF-ß, Activin-A, and BMP-4; and most importantly that they can clearly be used to measure Smad activation at the phosphorylation level in response to stimulation. They can thus act as a surrogate for primary HSCs to help define the complexity of signaling through Smads and perhaps other pathways. Furthermore, as it is possible to establish these lines by transducing mouse primary bone marrow, transduction of conditional or inducible cells from various transgenic mouse strains could create useful in vitro tools with which to study the role of various genes in HSC signal transduction.

ACKNOWLEDGMENTS

We thank Anna Fossum and Zhi Ma for expert assistance with cell sorting and Ulrika Blank, Göran Karlsson, and Sofie Singbrant for helpful discussions. This work was supported by a grant from Åke Wibergs Stiftelse to J.L.M. and by grants to S.K. from the Swedish Cancer Society, the European Commission (INHERINET and CONSERT), the Swedish Gene Therapy Program, the Swedish Medical Research Council, and the Swedish Children Cancer Foundation, Lund University Hospital (a clinical research award), the Joint Program on Stem Cell Research (supported by The Juvenile Diabetes Research Foundation), and the Swedish Medical Research Council. The Lund Stem Cell Center is supported by a Center of Excellence grant in life sciences from the Swedish Foundation for Strategic Research. T.U. and J.L.M. contributed equally to this work." b+ `5 Y% @) Z0 b) h0 P
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DISCLOSURES

The authors indicate no potential conflicts of interest.
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