Low expression of stem cell antigen-1 on mouse haematopoietic precursors is associated with erythroid differentiation

Cellular Immunology 279 (2012) 187–195

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Low expression of stem cell antigen-1 on mouse haematopoietic precursors is associated with erythroid differentiation Mirna Azalea-Romero 1, Marianela González-Mendoza 1, Alejandro Arturo Cáceres-Pérez, Eleazar Lara-Padilla, Julio Roberto Cáceres-Cortés ⇑ Laboratory of Cancer and Hematopoiesis, Superior School of Medicine, National Polytechnic Institute, C.P. 11340 México, Mexico

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Article history: Received 18 April 2012 Accepted 4 October 2012 Available online 29 October 2012 Keywords: Sca1+ cells Erythroid differentiation Transcription factors

a b s t r a c t Sca1 is a surface marker of haematopoietic stem cell but its role in erythropoiesis is still largely unknown. In this work we evaluated the ability of Sca1+ cells to differentiate into cells of the erythrocytic lineage. We performed FACS analysis of complete and purified Sca1+ bone marrow cells from C3H/HeNHsd mice and measured the expression of CD71 and Terr119 to evaluate the stages in erythroid development. Definitive erythropoiesis was evident within the complete bone marrow, while only proerythroblasts were found in Sca1+ cells, suggesting that Sca1 is a negative regulator of erythropoiesis. We also used FDCP-mix cells and their PU.1 and SCL transfectants. The PU.1 transfectant showed significantly increased expression of Sca1 and was not induced to differentiate into red blood cells, while the SCL transfectant showed significantly lower expression of Sca1 and produced red blood cells. The results of this study suggest that increased Sca1 expression on erythropoietic precursors inhibits erythroid differentiation. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Mouse haematopoietic stem cells have previously been identified and isolated from the bone marrow and are characterised by their lack of expression of various antigens found on mature cell types and their positive expression of two cell-surface proteins, Sca1 and c-kit [1,2]. Despite its wide spread use as a marker, the function of Sca1 in haematopoiesis remains poorly defined [3]. Sca1 is a member of the cysteine-rich Ly-6 family encoding glycosyl-phosphatidylinositol (GPI)-anchored surface membrane proteins [4–6], and is associated with protein tyrosine kinases [7]. Sca1 is expressed mainly by activated T and B lymphocytes, natural killer (NK) cells and haematopoietic stem cells in bone marrow, and is also detected in the kidney, heart and brain [8,9]. Furthermore Dr. Grange’s study indicates that Sca1+ cells can generate mammary tumors in mice [10]. Some reports have suggested a role for the Sca1 protein in cell signalling [11,12] and cell adhesion [13–15]. Horejsí et al. have demonstrated instances of GPI-anchored proteins like Sca1 assembled within membrane lipid raft structures that play important roles in the initiation of signalling via immunoreceptors [16]. Sca1 plays a role in the fate of haematopoietic progenitors/stem ⇑ Corresponding author. Address: Laboratory of Cancer and Hematopoiesis, Superior School of Medicine, National Polytechnic Institute, Plan de San Luis y Díaz Mirón s/n, Col. Casco de Santo Tomas, Delegación Miguel Hidalgo, C.P. 11340 México, DF, Mexico. Fax: +52 55 5729 6000x62806. E-mail address: [email protected] (J.R. Cáceres-Cortés). 1 These authors contributed equally to this work. 0008-8749/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2012.10.006

cells and there is evidence that its expression can modulate the expression of c-Kit, whose role in maintaining quiescent stem cells in steady state adult haematopoiesis has previously been recognized [17,18]. As reported by Bradfute et al. conditional knockout approach revealed that Sca1 / mice are viable however, they exhibit lymphocytosis and thrombocytopaenia [2,17], and Sca1 / T-cells isolated from these mice undergo prolonged hyperproliferation with stimulation in vitro [19]. The number of haematopoietic progenitor cells is reduced in Sca1-null murine bone marrow and Sca1-null cells show a reduced haematopoietic repopulating ability in lethally irradiated mice [2,17]. Erythropoietic commitment is modulated by hormones and transcription factors. Erythropoietin has a role in the regulation of the rate of erythropoiesis [20], and hydrocortisone (10 6 mol/l) stimulates erythroid colony growth and could play a role in the physiological regulation of erythropoiesis [21]. Molecular and cellular observations support the contention that the SCL transcription factor is a positive regulator of erythroid differentiation [22]. However, the role of Sca1, another molecular regulator of erythropoiesis, has not been studied in detail. The present study was undertaken to investigate how variations in the level of mouse haematopoietic Sca1 affect the fate of haematopoietic progenitor/stem cells. 2. Materials and methods 2.1. Animals All mice were maintained in a conventional mouse facility at Escuela Superior de Medicina-IPN (Mexico City, Mexico). Eight to

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12-week-old-mice were used for all primary assays. The C3H/HeNHsd mice were obtained from Harlan (Madison, WI, USA) and bred in-house. Animals of both sexes were housed in the same specific pathogen-free room in separate filter-topped cages. 2.2. Flow cytometry Single cell suspensions were prepared from bone marrow (femoral). Cells were washed once in Iscove’s modified Dulbecco’s medium (IMDM), counted, and resuspended in phosphate-buffered saline (PBS) plus 2% foetal bovine serum (FBS) (staining medium [SM]) at 5  106 cells/mL. Then, 1  106 cells were incubated with mAbs for 30 min, washed twice in SM, and resuspended in 0.2 mL SM for analysis by means of a Becton Dickinson FACSCalibur (Mountain View, CA) flow cytometer. The following primary antibodies were used: anti-CD71-PE, anti-Terr119-FITC, anti-Mac1PE, anti-Gr1-FITC and anti-Sca1-FITC (1:100 dilution; Cedarlane Laboratories, ON), and primary rabbit polyclonal SF antibody (1:20 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Aliquots of 1  106 cells/sample of ‘‘Factor-dependent cell Paterson mixed potential’’ FDCP-mix cells and transfectants were stained for surface expression of Sca1. The proportion of positive cells was determined by comparison with cells stained with fluorescein isothiocyanate (FITC)-conjugated secondary antibody alone. Erythrocytic precursors were classified by flow cytometry into different maturation stages. Live cells were gated according to their forward light scatter (FSC) and side light scatter profiles after staining with PI. Data were analysed using CellQuest software. To examine whether erythroid expansion proceeds in distinct Epo-mediated phases in Sca1+ cells, we sorted them using a BD FACSAria flow cytometer. 2.3. Progenitor cells and cell lines FDCP-mix is a multipotential IL-3-dependent cell line maintained in Fischer’s medium (Gibco, Grand Island, NY) supplemented with 15% (vol/vol) WEHI conditioned medium as a source of IL-3 and 20% (vol/vol) horse serum (Gibco), with penicillin (100 U/mL) and streptomycin (50 lg/mL) at 37 °C in a humidified atmosphere with 5% CO2. WEH1 cell-conditioned medium 10% (vol/vol) was added as a source of IL-3, which is essential for the survival and proliferation of these cells. The cells were routinely subcultured twice weekly to give 2  105 cells/mL. The WEHI cell line is a myelomonocytic leukemic cell line maintained in McCoy’s medium (Gibco) supplemented with 5% (vol/vol) foetal calf serum (FCS; Gibco). FDCPmix, FDCP-PU.1 and FDCP-SCL cells were obtained from Dr. T. Hoang, of the Institute for Research in Immunology and Cancer (IRIC), Quebec, Montreal, Canada. 2.4. Evaluation of progenitor cells BFU-E (Burst forming unit-erythroid), CFU-GM (Colony forming unit-granulocyte, macrophage) and CFU-GEMM (Colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte) colonies were assessed microscopically to determine whether Sca1 levels affect progenitor fate. We evaluated colonies according to standard guidelines as follows: (a) BFU-E: the size of the colony can be described as small (3–8 clusters), intermediate (9–16 clusters), or large (more than 16 clusters) according to the number of clusters present. These are primitive erythroid progenitors that have a high proliferative capacity (multiple centres, typical red colour, dense core); (b) CFU-GM: progenitors that give rise to colonies containing a heterogeneous population of macrophages and granulocytes. Their morphology is similar to CFU-M and CFU-G. The typical CFU-GM colony displays a starburst pattern of both granulocytes (smaller densely packed) and macrophages (large, round

and refractile). The core is dense, but the individual cells at the edge of the core are still identifiable. Large, round and refractile macrophages are evident at the periphery of the colony; (c) CFUGEMM: multi-lineage progenitors that give rise to erythroid, granulocyte, macrophage and megakaryocyte lineages. The round, bright, and refractile macrophages are clearly associated with the dense brownish-red erythroid core of the colony. 2.5. In vitro progenitor assays Methylcellulose and reagents for clonogenic progenitor assays were obtained from StemCell Technologies (Vancouver, Canada). Cells (4  104) were added to 1 mL methylcellulose (1%) medium (MethoCult M3434) containing 15% FBS, 1% BSA, 10 lg/mL Insulin, and 200 lg/mL Transferrin, supplemented by the manufacturer with 50 ng/mL rm-SCF, 10 ng/mL rm-IL-3, 10 ng/mL rh-IL-6 and 3 U/mL rh-EpO. Cell mixtures were plated in 35-mm suspension culture dishes (Nunc, Roskilde, Denmark), and incubated at 37 °C. After 10 days, colonies (>100 cells) were enumerated in each of three triplicate plates. Different culture conditions were used for FDCP cells as follows. Aliquots of 4  104 FDCP cells/mL were seeded in 1 mL methylcellulose (2%), Iscove’s medium 2 (GIBCO) with 0.01 ng/mL IL-3, in erythroid differentiation conditions (3 U/mL EpO, 45 lM haemin), or 10 ng/mL GM-CSF, and 10% (vol:vol) foetal calf serum. We had previously confirmed that when FDCP-mix cells are cultured in 10 ng/mL IL-3 the cells undergo preferential self-renewal, irrespective of the presence of other growth factors. 2.6. Benzidine staining and determination The percentage of cells staining for haemoglobin was estimated by staining with benzidine/H2O2 as previously described [23]. Briefly, benzidine stain was freshly prepared before use by adding 10 lL of 30% hydrogen peroxide to 2.5 mL of a stock solution containing 0.2% benzidine (benzidine dihydrochloride, B0386, Sigma– Aldrich, St. Louis, MO)/0.5 M acetic acid; 50 lL of this solution was used per 50 lL of cell suspension. The blue-stained haemoglobinpositive cells were counted using a haemocytometer and visualised by light microscopy. At least 500 cells were counted for each sample. We also performed spectrometric determinations of haemoglobin from red cell colonies in methylcellulose cultures. Petri dishes containing methylcellulose cultures at different time points were air-dried for 2–3 min after which 1 mL of the stain solution was added. Cells that contained haemoglobin turned blue within 5–10 min. The culture was then collected, the methylcellulose was washed off, the cell suspension was centrifuged at 100g for 3 min and the cell pellet was washed once in normal saline. The cells were lysed by suspension in 3 volumes of lysing buffer (50 mM Tris, pH 7.0, 25 mM KCl, 5 mM MgCl2, 1 mM 2mercaptoethanol and 0.3% Triton X-100) at 4 °C and vortexed briefly. The lysed cell suspension was centrifuged in an Eppendorf centrifuge at maximum speed (8000g) for 10–15 min using microcentrifuge tubes. The supernatant liquid was collected, and absorbance was measured in a spectrophotometer at 420 nm using the lysing buffer as a blank. Haemoglobin levels were determined as previously described [24]. 2.7. Long-term culture-initiating cell (LTC-IC) assay of murine cells using primary marrow feeders Aliquots of 2  106 bone marrow cells/mL were resuspended in murine myeloid long-term culture medium LTCM (MyeloCult™ M5300 with 10 6 M hydrocortisone, STEMCELL Technologies Vancouver, BC) and placed in 2.0-cm2 well (24-well plate) culture plates. Cultures were incubated at 33 °C in a humidified incubator

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(P95%) with 5% CO2. Half of the medium was removed weekly and replaced with fresh LTCM until an approximately 80% confluent adherent layer was established. We inactivated the haematopoietic progenitor cells within the feeder/stromal layer by irradiation. The feeder layer was irradiated at 15 Gy using a 137Cs c-irradiation source. We proceeded to add the test cells to the irradiated feeder layers. The test cell suspension of unseparated or purified murine cells was resuspended in an appropriate volume of LTCM (MyeloCult™ M5300 with 10 6 M hydrocortisone) and added to the test wells in a 0.5 mL volume. Cultures were incubated at 33 °C in a humidified incubator (P95%) with 5% CO2 for 4 weeks with weekly changes of half of the medium. We proceeded to harvest LTC-ICs. Both adherent and non-adherent cells were resuspended in 1 mL methylcellulose (MethoCult M3434), as indicated for in vitro progenitor assays. Colonies were counted on day 10. 2.8. Thymidine suicide assay The number of clonogenic cells cycling in response to growth factor was determined using the thymidine suicide assay [25]. Briefly, cells were exposed to IL-3 for 16 h, washed twice, and exposed to [3H]thymidine (Amersham Corp., Arlington Heights, IL) (25 Ci/mmol, sp act; 200 lCi/mL) for 20 min. The reaction was stopped by the addition of cold thymidine and the cells were washed twice in medium containing FCS before plating. Control cells were exposed to the whole procedure without radioactive thymidine. 2.9. Microscopy studies Studies were performed using a Leica IV/05 inverted microscope equipped with a Hitachi HV-F22 colour camera and Empix Imaging Northern Eclipse (Mississauga, ON) software for analysis and image processing. 2.10. Statistics Data were analysed for statistical significance using the Student’s t test. Differences were considered significant at p < .05. 3. Results 3.1. Purified Sca1+ bone marrow cells lack the capacity to differentiate into mature erythroid cells We purified Sca1+ cells from 4  107 mouse bone marrow cells. These cells represented 1.2% of the total mononuclear bone marrow population and strongly fluoresced with anti-Sca1-FITC (Fig. 1, central photo). We performed flow cytometric analysis of total and purified Sca1+ bone marrow cells after a 12-day induction with differentiation-inducing factors SCF (50 ng/mL), IL-3 (10 ng/mL), IL-6 (10 ng/mL) and EPO (3 U/mL) to evaluate erythroid and macrophage–granulocyte differentiation. The erythrocytic lineage was identified by the differential expression of fluorescein isothiocyanate-conjugated anti-Terr119 and phycoerythrin-conjugated anti-CD71 monoclonal antibodies, while that of the macrophage– granulocyte lineage was identified by the expression of Mac-1 and Gr-1 detected using specific monoclonal antibodies. We obtained high CD71-low Terr119, high CD71-high Terr119 and low CD71-high Terr119 fluorescent cells from the total bone marrow cells (Fig. 1a), hinting at the presence of proerythroblasts, basophilic and orthochromatophilic erythroblasts respectively representative of normal erythroid differentiation [26], as well as cells that stained strongly with anti Mac-1 and Gr-1 (Fig. 1b), pointing to the presence of normal macrophage and granulocyte differentia-

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tion. On the other hand, purified Sca1+ bone marrow cells exhibited neither high CD71-high Terr119 nor high Terr119-low CD71 positive cells (Fig. 1c) suggesting a lack of cells in the different stages of erythroid differentiation. They did, however, contain high Mac-1- and high Gr1-expressing cells (Fig. 1d), pointing to the presence of cells in the mature stages of the granulocyte and macrophage lineage. In order to further evaluate the possible inability of Sca1+ cells to differentiate into erythrocytes, we performed LTC-IC assays. Aliquots of 5  104 cells/mL of total and 1  104 cells/mL of Sca1+ cells were cultured in liquid cultures in the presence of a stromal feeder layer positive for membrane steel factor (Fig. 2A and B) to which 10 6 M hydrocortisone was added for 10 days. Thus, our primary mouse bone marrow stromal cells produce and important hematopoietic cytokine and are in conditions to support hematopoiesis in long-term cultures. In the first part of the assay, the complete bone marrow cells produced a significant number of BFU-E (Fig. 2C) whereas the Sca1+ cells produced none (Fig. 2D). Our results provide evidence that differentiation into the red cell lineage could be absent or blocked in Sca1+ cells during the first part of the LTC-IC assay. The Sca1-FITC-positive regions observed in different areas of the cells were quantitated using EMPIX imaging software 4 weeks before plating onto methylcellulose (second part of LTCIC). We observed decreased expression of Sca1: 6.04% (Fig. 2E) in comparison to the positive control: 29.92% (photo of sorted Sca1+ cells, Fig. 1). The culture conditions used in the present study were similar to those previously used and yielded similar numbers of LTC-IC: 1 cell/40,000 seeded cells in adult mouse bone marrow, and 1 cell/60 seeded cells in sorted Sca1+ cells [27]. Our results indicate that Sca1+ cells are enriched and remain undifferentiated in LTC-IC assays but tend to lose antigen expression thereby opening up the option for differentiation into red cells. In order to measure the content of red blood cells in methylcellulose cultures as a function of time we lysed the cells in the colonies formed by either total bone marrow cells, Sca1+ cells, FDCP-mix cells or their transfectants (FDCP-PU.1 and FDCP-SCL) which represent the macrophage–granulocyte lineage and red blood cells, respectively. Using benzidine as a marker for haemoglobin content, our results showed that the production of red cells in the total bone marrow increased after 5 days until the 10th day, whereas there was a complete absence of red cell production in Sca1+ bone marrow cells (Fig. 2F) and in the PU.1 transfectant (Fig. 2G). Absorbance at 420 nm, at the peak of each curve, was multiplied by 0.0945 to give the haemoglobin concentration in milligrams per millilitre, as follows: 0.1496 mg/mL haemoglobin in bone marrow cells, 0.003 mg/mL in Sca1+ cells, 0.1632 mg/mL in FDCP-SCL, 0.1457 mg/mL in FDCP-mix and 0.003 mg/mL in FDCP-PU.1 cells. Our results thus provide further evidence that the level of Sca1 positivity on myeloid precursors could determine their commitment to the erythroid lineage in such a way that cells with low Sca1 expression could be forced into erythroid differentiation while those with high levels are not. 3.2. FDCP-mix cells transfected with PU.1 (FDCP-PU.1) showed significantly higher expression of Sca1, whereas those transfected with SCL (FDCP-SCL) showed significantly lower expression To further evaluate the function of Sca1 in erythropoiesis, we used a known Sca1+ multipotent cell line (FDCP-mix) that is capable of differentiating into all cells of the myeloid lineage in order to evaluate whether or not the level of Sca1 could be modified. For this purpose we used the SCL and PU.1 transcription factors, which are deterministic for red cells and macrophages–granulocytes, respectively. One million FDCP-mix cells and FDCP-mix transfectants (FDCPSCL and FDCP-PU.1) were stained for surface expression of Sca1.

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Fig. 1. (A) Flow cytometry analysis of mouse bone marrow cells. Panel a shows a quantitative flow cytometry analysis that assessed bone marrow erythroblast differentiation. Cells were double-labelled for erythroid-specific TER119 and non-erythroid-specific transferrin receptor (CD71) and analysed by flow cytometry. Dead cells (propidium iodide-positive) and debris (low forward scatter) were excluded from the analysis. Regions R1 to R3 were defined by characteristic staining of the cells, including CD71highTER119low, CD71highTER119high, and CD71lowTER119high, respectively. Panel b shows macrophage–granulocyte abundance in the bone marrow. (B) The expression of CD71 and TER119 tracks erythroid differentiation of Sca1+ cells in vitro. A typical dual staining is shown in the biparametric histograms. We exposed the cells to haematopoietic growth factors for 12 days and then performed FACS analysis. Sca1+ cells demonstrate to undergo blocked terminal erythroid differentiation, as verified by the absence of the CD71highTER119high and CD71lowTER119high groups (c). Nevertheless, macrophage–granulocyte differentiation continues normally (d).

Our results showed low expression of Sca1 by FDCP-SCL (45%) and high expression by FDCP-PU.1 (87%), compared with the parental cell line (Fig. 3A). These results highlight the fact that Sca1 positivity is not only related to the inhibition of red cell differentiation in multipotent precursors but that its level could determine its commitment to either erythropoiesis or myelopoiesis. In order to evaluate the cell differentiation tendency in clonogenic assays we analysed flow cytometry data over time and create diagrams of three components (Fig. 3B): Sca1, Terr 119 and Gr1+Mac1. From the results observed in Fig. 3B, there was the suggestion that PU.1, which increases Sca1, appears to be more critically required for differentiation into mature granulocytes and macrophages. In contrast SCL, which decreases Sca1, influences progenitors to develop red cells. 3.3. FDCP-SCL cells maintained the ability to form erythroid colonies in vitro while FDCP-PU.1 did not In order to evaluate whether the FDCP-SCL transfectant showing low expression of Sca1 and the FDCP-PU.1 transfectant showing high expression of Sca1 could influence erythropoiesis, we seeded 2  104 cells of each group into 1 mL methylcellulose (2.0%) for 10 days and evaluated colony formation and their staining. Our results showed that whereas the SCL transfectant produced red blood cells (Fig. 4d), the PU.1 transfectant did not

(Fig. 4e). We then proceeded to evaluate the colony content of each culture. Our results showed that the bone marrow-derived cells as well as the FDCP-mix cells and the FDCP-SCL transfectant had a similar proportion of cell colonies, whereas the Sca1+ bone marrow cells and the FDCP-PU.1 transfectant had no BFU-E or CFU-GEMM colonies (Fig. 4B). Our results thus point to a possible blockage of erythropoiesis in cells with high levels of Sca1. In order to visualise the erythroid colonies, the methylcellulose cultures were sprinkled with benzidine, an indicator of the presence of haemoglobin in the cells. Cells in culture from the FDCP-mix cell line and the FDCP-SCL transfectant stained positive for benzidine, whereas FDCP-PU.1 cultures and purified Sca1 + cell cultures contained no benzidine-positive cells (Fig. 4a–f, C). 3.4. Thymidine killing of FDCP-mix cells was lower than that of the FDCP-PU.1 transfectant but higher than that of FDCP-SCL and significantly lower than purified Sca1+ bone marrow cells Since Sca1 is expressed in haematopoietic stem cells and these cells are known to have a low rate of proliferation, we examined whether or not the level of Sca1 in our cells and cell lines could be associated with cell quiescence. For this purpose 1  104 cells/ mL of either Sca1+, FDCP-mix, FDCP-PU.1 or FDCP-SCL were

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Fig. 2. Erythropoiesis in long-term suspension cultures. Panel A shows the mouse stromal cells, membrane Steel Factor (mSF+), used in long-term cultures. Panel B shows the flow cytometric profile (mSF+) of mouse stromal cells. Expression of the steel factor ligand was determined by flow cytometry using an anti-SF antibody. Picture C shows aggregates (BFU-E) found floating in situ at 7 days of long-term cell culture of bone marrow following the addition of hydroxycortisone. The erythroid colony-forming inability of Sca1+ cells exposed to erythropoietin or hydroxycortisone in long-term cultures is apparent (D). LTC-IC (4 weeks) were labelled immunohistochemically for the Sca1 protein marker (E) for comparison see picture in Fig. 1 of the freshly isolated cells taken as a positive control). The kinetic of the haemoglobin production was evaluated by the absorbance of benzidine (at 420 nm) (F, G). Results in A–G are representative of more than three independent experiments.

exposed to 10 ng/mL IL-3 for 16 h, washed twice, and exposed to 200 lCi/mL [3H]thymidine (25.5 Ci/mmol, Amersham, Arlington Heights, IL) for 20 min to ensure that all dividing cells incorporated the radioactive material and died. At the end of the thymidine suicide assay a complete methylcellulose culture was performed. The Sca1+ bone marrow cells produced the highest number of colonies and thus showed the lowest death rate followed by the FDCP-PU.1 transfectant, whereas the FDCP-SCL transfectant produced the lowest number of colonies and thus the highest death rate (Fig. 5). Our results could indicate that the higher the expression of Sca1 on a cell precursor the higher its quiescence. Given that in this work we have shown that precursor cells with low Sca1 expression are able to differentiate towards the erythroid lineage we can speculate that these cells have higher proliferation rates than those with high levels of Sca1.

4. Discussion It is well accepted that erythroid cell growth and differentiation may be regulated by many autocrine and paracrine factors secreted by progenitor cells and other cells present in the bone marrow environment. In this study we determined that a low level of Sca1 expression is determinant for mouse erythroid differentiation. In vivo, erythropoietin presumably initiates enzymatic actions that convert multipotent cells into cells capable of erythrocyte proliferation. Once this has occurred, the cells are considered to have been released from the stem cell compartment.

We investigated the cellular differentiation of Sca1+ cells from bone marrow. Our results were consistent with a previous report that 1.62% of total bone marrow cells are Sca1+ in C3H mice [28]. We performed a quantitative flow cytometry analysis of mouse C3H bone marrow cells based on the expression of CD71 (transferrin receptor) and Terr119 (glycophorin A) to evaluate stages of erythroid development and compared this to the results obtained from Sca1+ bone marrow cells. While C3H bone marrow cells differentiate normally into different stages of the erythroid lineage, Sca1+ cells lack the most mature stages of differentiation. To elucidate whether the presence of Sca1 might interfere with erythroblast differentiation we used the FDCP-mix cell line and FDCP-PU.1 and FDCP-SCL transfectants, which represent the macrophage–granulocyte lineage and red blood cells, respectively [22]. Consistent with the blockage of erythroid blast differentiation found in isolated Sca1+ cells, the FDCP-mix cells that were upregulated for Sca1 expression (FDCP-PU.1) did not produce erythroid cells in semisolid cultures whereas cells that had lower values than the control (FDCP-SCL) did produce such cells over time. The LTCIC cultures seemed to confirm that Sca1 and membrane Kit ligand are involved in maintaining the viability of Sca1+ cells without a significant increase in cell number. Nevertheless, Sca1+ cells started to lose their marker very late in culture. Our data thus indicate that variations in the level of mouse haematopoietic Sca1 affect the fate of erythropoietic progenitor stem cells. In our opinion, it is the low level of expression of Sca1 in LTC-IC (4 weeks) (Fig. 2E) that allows these cells to differentiate into red blood cells and others (CFC) when they are plated in methylcellulose complete

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Fig. 3. (A) FACS profiles of FDCP-mix cells transfected with SCL and PU.1 transcription factors. The figure shows the FACS analysis of clone FDCP-SCL with an average downmodulation of the mean fluorescence intensity of 45.03% for Sca1 compared to wild-type and irrelevant-transfected FDCP-mix cells. Clone FDCP-PU.1 shows an average upmodulation of Sca1 with a mean fluorescence intensity of 86.59%. Profiles are from one of three independent experiments with superimposable results. First control graph: cells labelled with secondary antibody alone; thin line: cells labelled with anti Sca1-FITC. (B) Diagram of three hematopoietic cell surface markers expression. Panel b shows a quantitative diagram that assessed FDCP mix, FDCP PU.1 and FDCP SCL cell lines differentiation over time in the presence of hematopoietic growth factors in Methylcellulose for clonogenic progenitor assays described in Section 2. Cells were harvested at different times (day 0 (d0) to day seven (d7)) and analysed by flow cytometry for the detection of Sca1, Terr 119 and Gr1+Mac1 antigens. Data shown are typical of two independent experiments for each sample. The dots positions are based on the MCF (mean channel fluorescence) values.

medium (second part of LTC-IC assay). Cells maintained with Kit ligand (herein membrane Steel Factor) retain SCL expression, which is required for their survival response to Kit ligand [29]. Several lines of evidence point to SCL as an important regulator of erythropoiesis and one of the few genes required for primitive erythropoiesis [30]. Analysis of haematopoietic precursors has shown that SCL is highly expressed in committed erythroid progenitors whereas it becomes downregulated in terminally differentiated red cells [22,31]. The work of Aplan et al. [32] provided valuable data on the physiological role played by SCL in erythroid differentiation. SCL mRNA is expressed in all erythroid tissues and cell lines examined to date, and SCL mRNA increases upon induced differentiation of murine erythroleukaemia (MEL) cells by DMSO. It has been demonstrated that forced SCL expression in haematopoietic cell lines and primary bone marrow cells favours erythroid differentiation [33], but little is known about its effects on other surface molecules. In our hands Sca1 was downregulated as a consequence of SCL transfection as well as at the end of the LTC-IC assay. If Sca1 acts as an inhibitor of erythropoiesis it would be logical to think that it must be low in such cells. We provide

evidence indicating that the SCL gene has a dominant role in erythropoiesis by eliminating high levels of the negative regulator. It is possible that the marker is expressed in response to a particular transcription factor. In our hands, Sca1 was maintained by the action of PU.1 and downregulated by the SCL transcription factor. Specific interactions between proteins in mammalian cells are the basis of many essential biological processes. The transcriptional circuitry, which SCL and PU.1 operate, is an important regulator of hematopoiesis. There is increasing evidence that hematopoiesis is controlled by networks of interacting transcription factors, and that subtle variations in protein partners may have profound consequences for gene expression programs [34]. The PU.1 gene blocks GATA1 [35], an essential lineage-specific transcription factor for erythroid development, and is implicated in the inhibition of erythropoiesis by stimulating lysozyme enzyme expression in macrophages. In contrast, SCL inhibits lysozyme production (Herblot S. Personal communication). Importantly, Sca1 is a direct target of PU.1 as determined by CHIP-Seq [36]. Le Clech et al. [37] showed that PU.1 binds to the human SCL silencer to mediate its activity. Lahlil provides detailed experimental results

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C Benzidine-positive cells (%) PU.1 transfectant

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FDCP neo FDCP mix

Fig. 4. (A) Erythroid differentiation studies. Pictures depict colony appearance in semisolid cultures. Benzidine stains for haemoglobin (blue), a hallmark of terminal erythroid differentiation. (a) In vitro response to growth factors of C3H bone marrow (CFU-GEMM); (b, c) macrophage-colony formation of Sca1+ cells; (d) FDCP SCL red cell colony; (e) macrophage-colony formation of PU-1 transfectant; (f) SCF, IL-3, IL-6 and EpO promoted the formation of CFU-GEMM in FDCP-mix. SCF, IL-3, IL-6 and EpO promoted macrophage–granulocyte differentiation of Sca1+ cells and FDCP PU.1 but were unable to stimulate red cell production. The internal control (FDCP-neo clone) rendered similar results to those of the parental FDCP-mix cell line in all experiments. (B) Potential clonogenic ability of normal bone marrow, Sca1+ cells, FDCP-SCL, FDCP-PU.1 and FDCP-mix cells. Colonies were counted after 7 days using an inverted microscope. A colony was defined as a cluster of more than 100 cells. (C) Benzidine staining. Benzidine staining of Sca1+ cells, SCL transfectant, PU.1 transfectant, neo clone, and the parental FDCP-mix cells stimulated with SCF 50 ng/mL, IL-3 10 ng/mL, IL-6 10 ng/mL and EpO 3 U/mL. Cells were harvested for benzidine staining on day 5. Data shown are the means of duplicate counts of at least 500 cells each.

Thymidine killing assay of Sca1+ bone marrow cells, FDCP mix and transfectants SCL or PU.1

45 40 35 30 25 20 15 10 5 0

FDCP mix

Sca1+

FDCP SCL

FDCP PU.1

Fig. 5. Growth of clonogenic cells after exposure to IL-3 in suspension culture followed by thymidine suicide. Cells (FDCP mix, SCL transfectant, PU.1 transfectant, and Sca1+ cells) were exposed to IL-3 (10 ng/mL) in suspension cultures for 16 h, at a concentration of 2  104 cells/mL. The cells were then washed and exposed to [3H]TdR before plating. Methylcellulose cultures were set up at 104 cells/well with SCF 50 ng/mL, IL-3 10 ng/mL, IL-6 10 ng/mL and EpO 3 U/mL. Data shown are typical of two distinct experiments.

for erythroid cell-specific glycophorin A gene (GPA) as a target of SCL in primary hematopoietic cells [38]. Pulford et al. have demonstrated instances of SCL expressed in hematopoietic stem cells, as well as multipotent, erythroid, and megakaryocytic progenitors [39]. Loss- and gain-of-function studies with different vertebrate models have shown that SCL is essential for the establishment of

the hematopoietic system and that it can specify the hematopoietic cell fate when ectopically expressed [40]. In a recent report authors concluded that SCL regulates the quiescence and long-term ability for reconstitution of haematopoietic stem cells [41]. Since Sca1 expression is typical in haematopoietic stem cells and these cells are usually dormant, we thought it might be linked to the state of quiescence because the transfectants and parental cell line did not have similar doubling times in culture, a situation that was earlier interpreted as being due to inhibition mechanisms and cellular destruction. Copious evidence exists to support the inhibitory role of Sca1 in the signal transduction of T lymphocyte responses. It is noteworthy that T cells from Sca1deficient animals exhibit a prolonged proliferative response to antigen stimulation [19]. Sca1 is known for its modulation of c-Kit expression [17], another determinant of common progenitor whose action prevents programmed cell death and does not induce cell differentiation or proliferation [18,29]. In this way the presence of Sca1 on the surface of the stem cells ensures their primitiveness. Although it represents less than 0.1% of total murine bone marrow cells, virtually all haematopoietic stem cell activity has been shown to be contained within the Sca1+c-Kit+ compartment [42]. To evaluate the fraction of the stem cell pool that is actively dividing in each group of cells, we performed a thymidine suicide assay. We found a correlation between the level of Sca1 and the percentage of nondividing cells. We believe that Sca1 has a functional role in haematopoietic stem cell proliferation, differentiation and activation, and that it could be considered a ‘‘longevity’’ protein expressed by a small subpopulation of haematopoietic stem cells. Our observations suggest two possibilities, the first being that high levels of Sca1 inhibit erythrocyte differentiation and the

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second being that Sca1-negative cells are important in the function of the Sca1-positive population leading to erythrocyte differentiation. Once Sca1-negative cells are eliminated by selection the Sca1+ cells would not work properly. In total bone marrow the Sca1 is an exclusive marker of haematopoietic stem cells and is absent in mesenchymal stem cells [43]. If mesenchymal stem cells are important for erythroid differentiation then the haematopoietic progenitor cell line FDCP-mix would not have been able to produce red cells because FDCP-mix cells, by definition, are not composed of mesenchymal stem cells. Thus we can rule out the possibility that mesenchymal stem cells are important for red cell production. An attempt was made to correlate colony formation observations with a kinetic model of erythropoiesis. We believe the curves can be explained by the differences in Sca1 expression on the precursors. Competitive progenitor cell stimulation from related cellular systems of the haematopoietic system, different from mesenchymal stem cells, is lacking on Sca1+-purified cells. On the other hand, it has been suggested that there exists a condition termed ineffective erythropoiesis [44], whereby cells go through one or two divisions and die. It appears that this process is regulatory and possibly under some specific control, which might conceivably include Sca1 even in vitro.

5. Conclusions The regulation of cell differentiation and cell death is crucial to the generation of haematopoietic cells both in vitro and in vivo. The biologic role of stem cell antigen-1 (Sca1) in haematopoietic cell development is not well known. We monitored the differentiation of mouse haematopoietic cells: bone marrow, purified Sca1+ cells, FDCP-mix cells and their PU.1 and SCL transfectants in the presence of a cocktail of haematopoietic growth factors. Examination of colony formation, haemoglobin content, cell surface phenotype, and thymidine killing indicated that Sca1 is mainly an inhibitor for red cell differentiation. Our results show that Sca1 maintains cells in an ‘‘undifferentiated’’ state. Committed erythroid progenitors get terminal differentiation under SCL direction that provokes Sca1 decrease. Under PU.1 conditions, that increase Sca1, maturing granulocytic-monocytic cells can be recovered. Together, our data indicate that Sca1 induces an inactive character in haematopoietic progenitors. Furthermore, Sca1 favours the development of macrophage-granulocytic progenitors over that of erythroid progenitors. In the absence of later acting factors, cells that progress beyond the CFU-s stages lose Sca1 expression. Finally, our data suggest that preserving signals by Sca1 are crucial during the differentiative process of erythrocytes, giving strength to the deterministic model. Our results implied the importance of considering the Sca1 presence while evaluating the role of haematopoietic growth factors.

Conflict of interest statement This research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgments The authors’ own work was supported by a grant from the National Science and Technology Council of Mexico (CONACYT) 66928 and The National Polytechnic Institute (IPN)-SIP 20120715 and 20121237. We thank English Journal Experts for manuscript preparation and acknowledge the generous support and encouragement of Louis Robichaud.

References [1] E.C. Forsberg, E. Passegué, S.S. Prohaska, A.J. Wagers, M. Koeva, J.M. Stuart, I.L. Weissman, Molecular signatures of quiescent, mobilized and leukemiainitiating hematopoietic stem cells, PLoS ONE 5 (2010) e8785. [2] M. van de Rijn, S. Heimfeld, G.J. Spangrude, I.L. Weissman, Mouse hematopoietic stem-cell antigen Sca-1 is a member of the Ly-6 antigen family, Proc. Natl. Acad. Sci. USA 86 (1989) 4634–4638. [3] C. Holmes, W.L. Stanford, Concise review: stem cell antigen-1: expression, function, and enigma, Stem Cells 25 (2007) 1339–1347. [4] C.Y. Ito, C.Y. Li, A. Bernstein, J.E. Dick, W.L. Stanford, Hematopoietic stem cell and progenitor defects in Sca-1/Ly-6A-null mice, Blood 101 (2003) 517–523. [5] X. Ma, C. Robin, K. Ottersbach, E. Dzierzak, The Ly-6A (Sca-1) GFP transgene is expressed in all adult mouse hematopoietic stem cells, Stem Cells 20 (2002) 514–521. [6] P. Hanson, V. Mathews, S.H. Marrus, T.A. Graubert, Enhanced green fluorescent protein targeted to the Sca-1 (Ly-6A) locus in transgenic mice results in efficient marking of hematopoietic stem cells in vivo, Exp. Hematol. 31 (2003) 159–167. [7] I. Stefanova, V. Horejsi, I.J. Ansotegui, W. Knapp, H. Stockinger, GPI-anchored cell-surface molecules complexed to protein tyrosine kinases, Science 254 (1991) 1016–1019. [8] E.M. Shevach, P.E. Korty, Ly-6: a multigene family in search of a function, Immunol. Today 10 (1989) 195–200. [9] A. Bamezai, Mouse Ly-6 proteins and their extended family: markers of cell differentiation and regulators of cell signaling, Arch. Immunol. Ther. Exp. (Warsz) 52 (2004) 255–266. [10] C. Grange, S. Lanzardo, F. Cavallo, G. Camussi, B. Bussolati, Sca-1 identifies the tumor-initiating cells in mammary tumors of BALB-neuT transgenic mice, Neoplasia 10 (2008) 1433–1443. [11] K.L. Rock, E.T. Yeh, C.F. Gramm, S.I. Haber, H. Reiser, B. Benacerraf, TAP, a novel T cell-activating protein involved in the stimulation of MHC restricted T lymphocytes, J. Exp. Med. 163 (1986) 315–333. [12] T.R. Malek, G. Ortega, R.A. Chan, R.A. Kroczek, E.M. Shevach, Role of Ly-6 in lymphocyte activation. II. Induction of T cell activation by monoclonal anti-Ly6 antibodies, J. Exp. Med. 164 (1986) 709–722. [13] A. Bamezai, K.L. Rock, Overexpressed Ly-6A.2 mediates cell–cell adhesion by binding a ligand expressed on lymphoid cells, Proc. Natl. Acad. Sci. USA 92 (1995) 4294–4298. [14] R.H. Brakenhoff, M. Gerretsen, E.M. Knippels, M. van Dijk, H. van Essen, D.O. Weghuis, R.J. Sinke, G.B. Snow, G.A. van Dongen, The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell–cell adhesion, J. Cell Biol. 129 (1995) 1677–1689. [15] B.J. Classon, R.L. Boyd, Thymic-shared antigen-1 (TSA-1): a lymphostromal cell membrane Ly-6 superfamily molecule with a putative role in cellular adhesion, Dev. Immunol. 6 (1998) 149–156. [16] V. Horejsí, K. Drbal, M. Cebecauer, J. Cerny´, T. Brdicka, P. Angelisová, H. Stockinger, GPI-microdomains: a role in signalling via immunoreceptors, Immunol. Today 20 (1999) 356–361. [17] S.B. Bradfute, T.A. Graubert, M.A. Goodell, Roles of Sca-1 in hematopoietic stem/progenitor cell function, Exp. Hematol. 33 (2005) 836–843. [18] L.A. Thorén, K. Liuba, D. Bryder, J.M. Nygren, C.T. Jensen, H. Qian, J. Antonchuk, S.E. Jacobsen, Kit regulates maintenance of quiescent hematopoietic stem cells, J. Immunol. 180 (2008) 2045–2053. [19] W.L. Stanford, S. Haque, R. Alexander, X. Liu, A.M. Latour, H.R. Snodgrass, B.H. Koller, P.M. Flood, Altered proliferative response by T lymphocytes of Ly-6A (Sca-1) null mice, J. Exp. Med. 186 (1997) 705–717. [20] W. Fried, Erythropoietin and erythropoiesis, Exp. Hematol. 37 (2009) 1007– 1015. [21] D.J. King, M. Koekebakker, R.D. Barr, Modulation of human erythropoiesis by hydrocortisone in vitro, Eur. J. Haematol. 38 (1987) 137–140. [22] T. Hoang, E. Paradis, G. Brady, F. Billia, K. Nakahara, N.N. Iscove, I.R. Kirsch, Opposing effects of the basic helix-loop-helix transcription factor SCL on erythroid and monocytic differentiation, Blood 87 (1996) 102–111. [23] Z.C. Yi, Z. Wang, H.X. Li, M.J. Liu, R.C. Wu, X.H. Wang, Effects of chebulinic acid on differentiation of human leukemia K562 cells, Acta Pharmacol. Sin. 25 (2004) 231–238. [24] T.V. Gopalakrishnan, W.F. Anderson, Mouse erythroleukemia cells, Methods Enzymol. 58 (1979) 506–511. [25] T. Hoang, B. Levy, N. Onetto, A. Haman, J.C. Rodriguez-Cimadevilla, Tumor necrosis factor alpha stimulates the growth of the clonogenic cells of acute myeloblastic leukemia in synergy with granulocyte/macrophage colonystimulating factor, J. Exp. Med. 170 (1989) 15–26. [26] J. Zhang, M. Socolovsky, A.W. Gross, H.F. Lodish, Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry-based novel culture system, Blood 102 (2003) 3938– 3946. [27] L. Gammaitoni, S. Bruno, F. Sanavio, M. Gunetti, O. Kollet, G. Cavalloni, M. Falda, F. Fagioli, T. Lapidot, M. Aglietta, W. Piacibello, Ex vivo expansion of human adult stem cells capable of primary and secondary hemopoietic reconstitution, Exp. Hematol. 31 (2003) 261–270. [28] G.J. Spangrude, D.M. Brooks, Mouse strain variability in the expression of the hematopoietic stem cell antigen Ly-6A/E by bone marrow cells, Blood 82 (1993) 3327–3332.

M. Azalea-Romero et al. / Cellular Immunology 279 (2012) 187–195 [29] J.R. Caceres-Cortes, G. Krosl, N. Tessier, P. Hugo, T. Hoang, Steel factor sustains SCL expression and the survival of purified CD34+ bone marrow cells in the absence of detectable cell differentiation, Stem Cells 19 (2001) 59–70. [30] L.C. Redmond, C.I. Dumur, K.J. Archer, J.L. Haar, J.A. Lloyd, Identification of erythroid-enriched gene expression in the mouse embryonic yolk sac using microdissected cells, Dev. Dyn. 237 (2008) 436–446. [31] Y. Zhang, K.J. Payne, Y. Zhu, M.A. Price, Y.K. Parrish, E. Zielinska, L.W. Barsky, G.M. Crooks, SCL expression at critical points in human hematopoietic lineage commitment, Stem Cells 23 (2005) 852–860. [32] P.D. Aplan, K. Nakahara, S.H. Orkin, I.R. Kirsch, The SCL gene product: a positive regulator of erythroid differentiation, EMBO J. 11 (1992) 4073–4081. [33] N.J. Elwood, H. Zogos, D.S. Pereira, J.E. Dick, C.G. Begley, Enhanced megakaryocyte and erythroid development from normal human CD34(+) cells: consequence of enforced expression of SCL, Blood 91 (1998) 3756–3765. [34] M.H. Sieweke, T. Graf, A transcription factor party during blood cell differentiation, Curr. Opin. Genet. Dev. 8 (1998) 545–551. [35] P. Zhang, X. Zhang, A. Iwama, C. Yu, K.A. Smith, B.U. Mueller, S. Narravula, B.E. Torbett, S.H. Orkin, D.G. Tenen, PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding, Blood 96 (2000) 2641– 2648. [36] A.M. Smith, F.J. Calero-Nieto, J. Schütte, S. Kinston, R.T. Timms, N.K. Wilson, R.L. Hannah, J.R. Landry, B. Göttgens, Integration of Elf-4 into stem/progenitor and erythroid regulatory networks through locus-wide chromatin studies coupled with in vivo functional validation, Mol. Cell. Biol. 32 (2012) 763–773.

195

[37] M. Le Clech, E. Chalhoub, C. Dohet, V. Roure, S. Fichelson, F. Moreau-Gachelin, D. Mathieu, PU.1/Spi-1 binds to the human TAL-1 silencer to mediate its activity, J. Mol. Biol. 355 (2006) 9–19. [38] R. Lahlil, E. Lécuyer, S. Herblot, T. Hoang, SCL assembles a multifactorial complex that determines glycophorin A expression, Mol. Cell. Biol. 24 (2004) 1439–1452. [39] K. Pulford, N. Lecointe, K. Leroy-Viard, M. Jones, D. Mathieu-Mahul, D.Y. Mason, Expression of TAL-1 proteins in human tissues, Blood 85 (1995) 675–684. [40] R.A. Shivdasani, E.L. Mayer, S.H. Orkin, Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL, Nature 373 (1995) 432– 434. [41] J. Lacombe, S. Herblot, S. Rojas-Sutterlin, A. Haman, S. Barakat, N.N. Iscove, G. Sauvageau, T. Hoang, Scl regulates the quiescence and the long-term competence of hematopoietic stem cells, Blood 115 (2010) 792–803. [42] L. Yang, D. Bryder, J. Adolfsson, J. Nygren, R. Månsson, M. Sigvardsson, S.E. Jacobsen, Identification of Lin( )Sca1(+)kit(+)CD34(+)Flt3 short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients, Blood 105 (2005) 2717–2723. [43] S. Hombach-Klonisch, S. Panigrahi, I. Rashedi, A. Seifert, E. Alberti, P. Pocar, M. Kurpisz, K. Schulze-Osthoff, A. Mackiewicz, M. Los, Adult stem cells and their trans-differentiation potential – perspectives and therapeutic applications, J. Mol. Med. 86 (2008) 1301–1314. [44] S. Rivella, Ineffective erythropoiesis and thalassemias, Curr. Opin. Hematol. 16 (2009) 187–194.