Stromal cell-mediated inhibition of erythropoiesis can be attenuated by Sotatercept (ACE-011), an activin receptor type II ligand trap
2012; Elsevier BV; Volume: 41; Issue: 2 Linguagem: Inglês
10.1016/j.exphem.2012.12.002
ISSN1873-2399
AutoresCamelia Iancu‐Rubin, Goar Mosoyan, Jiapeng Wang, Thomas Kraus, Victoria Sung, Ronald Hoffman,
Tópico(s)Pancreatic and Hepatic Oncology Research
ResumoRed cell production is primarily determined by the action of erythropoietin. Additional erythropoiesis-regulatory factors include molecules and cellular interactions occurring within the bone marrow (BM) microenvironment. Sotatercept (ACE-011) is an activin receptor ligand trap that binds several members of the TGF-β superfamily. Treatment with ACE-011 reverses bone loss and reduces the degree of osteoporosis, but it is accompanied by elevated hemoglobin and hematocrit levels. The mechanisms underlying the beneficial effects of ACE-011 on red cell production remain unknown. This study explores the means by which ACE-011 promotes erythropoiesis. We showed that ACE-011 does not directly affect erythroid differentiation of human CD34+ cells in vitro. We next tested whether ACE-011 acts indirectly by affecting BM accessory cells. Conditioned media produced by BM stromal cells (SCs) inhibited erythroid differentiation of CD34+ cells while maintained their ability to proliferate. However, conditioned media from SCs treated with ACE-011 partially restored erythropoiesis, coinciding with changes in the molecular and secretory profile of SCs, including the expression and secretion of erythropoiesis-modulatory factors. We conclude that inhibitory factors produced by BM SCs in vitro might control erythropoiesis in vivo and that agents that reverse these microenvironmental signals could provide an approach to attenuate anemia in clinical conditions. Red cell production is primarily determined by the action of erythropoietin. Additional erythropoiesis-regulatory factors include molecules and cellular interactions occurring within the bone marrow (BM) microenvironment. Sotatercept (ACE-011) is an activin receptor ligand trap that binds several members of the TGF-β superfamily. Treatment with ACE-011 reverses bone loss and reduces the degree of osteoporosis, but it is accompanied by elevated hemoglobin and hematocrit levels. The mechanisms underlying the beneficial effects of ACE-011 on red cell production remain unknown. This study explores the means by which ACE-011 promotes erythropoiesis. We showed that ACE-011 does not directly affect erythroid differentiation of human CD34+ cells in vitro. We next tested whether ACE-011 acts indirectly by affecting BM accessory cells. Conditioned media produced by BM stromal cells (SCs) inhibited erythroid differentiation of CD34+ cells while maintained their ability to proliferate. However, conditioned media from SCs treated with ACE-011 partially restored erythropoiesis, coinciding with changes in the molecular and secretory profile of SCs, including the expression and secretion of erythropoiesis-modulatory factors. We conclude that inhibitory factors produced by BM SCs in vitro might control erythropoiesis in vivo and that agents that reverse these microenvironmental signals could provide an approach to attenuate anemia in clinical conditions. Activin and several other members of the TGF-β family, including the bone morphogenetic proteins (BMPs), have a role in the regulation of erythropoiesis, acting either directly on erythroid progenitor or precursor cells or by modifying the functions of bone marrow (BM) accessory cells [1Yu J. Maderazo L. Shao L.E. et al.Specific roles of activin/inhibin in human erythropoiesis in vitro.Ann N Y Acad Sci. 1991; 628: 199-211Crossref PubMed Scopus (11) Google Scholar, 2Zermati Y. Fichelson S. Valensi F. et al.Transforming growth factor inhibits erythropoiesis by blocking proliferation and accelerating differentiation of erythroid progenitors.Exp Hematol. 2000; 28: 885-894Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 3Maguer-Satta V. Bartholin L. Jeanpierre S. et al.Regulation of human erythropoiesis by activin A, BMP2, and BMP4, members of the TGFbeta family.Exp Cell Res. 2003; 282: 110-120Crossref PubMed Scopus (88) Google Scholar]. Although the exact mechanisms by which activin and the TGF-β family members affect erythropoiesis remain unclear, these actions appear to be mediated by the SMAD protein signaling pathway initiated by type I and type II serine–threonine kinase receptors (ActRI and II) [4Massague J. How cells read TGF-beta signals.Nat Rev Mol Cell Biol. 2000; 1: 169-178Crossref PubMed Scopus (1635) Google Scholar]. Ligand-activated receptors induce the phosphorylation of SMAD proteins signaling cascade, which eventually affects erythroid-specific gene expression [5Blank U. Karlsson S. The role of Smad signaling in hematopoiesis and translational hematology.Leukemia. 2011; 25: 1379-1388Crossref PubMed Scopus (84) Google Scholar]. Aberrant signaling through the TGF-β–related pathway has been associated with impaired erythroid development [6Miller K.L. Carlino J.A. Ogawa Y. Avis P.D. Carroll K.G. Alterations in erythropoiesis in TGF-beta 1-treated mice.Exp Hematol. 1992; 20: 951-956PubMed Google Scholar, 7Bruno E. Horrigan S.K. Van Den Berg D. et al.The Smad5 gene is involved in the intracellular signaling pathways that mediate the inhibitory effects of transforming growth factor-beta on human hematopoiesis.Blood. 1998; 91: 1917-1923Crossref PubMed Google Scholar, 8Kang Y.J. Shin J.W. Yoon J.H. et al.Inhibition of erythropoiesis by Smad6 in human cord blood hematopoietic stem cells.Biochem Biophys Res Commun. 2011; 423: 750-756Crossref Scopus (19) Google Scholar] and has been a target for drug development to promote erythropoiesis. Recently, SMAD-mediated inhibition of BMPs has been shown to result in attenuation of the degree of anemia associated with inflammation [9Steinbicker A.U. Sachidanandan C. Vonner A.J. et al.Inhibition of bone morphogenetic protein signaling attenuates anemia associated with inflammation.Blood. 2011; 117: 4915-4923Crossref PubMed Scopus (140) Google Scholar], whereas inhibition of TGF-β signaling in an experimental murine system of BM failure has been shown to accelerate differentiation of human progenitor cells from patients with myelodysplasia [10Zhou L. Nguyen A.N. Sohal D. et al.Inhibition of the TGF-beta receptor I kinase promotes hematopoiesis in MDS.Blood. 2008; 112: 3434-3443Crossref PubMed Scopus (135) Google Scholar].ACE-011 is a chimeric protein in which the extracellular domain of the ActRIIA receptor is fused to the Fc portion of the human IgG1 antibody. ACE-011 antagonizes activin and several other members of the TGF-β superfamily that signal through the ActRIIA [11Raje N. Vallet S. Sotatercept, a soluble activin receptor type 2A IgG-Fc fusion protein for the treatment of anemia and bone loss.Curr Opin Mol Ther. 2010; 12: 586-597PubMed Google Scholar]. The administration of ACE-011, and its murine counterpart RAP-011, to cynomolgus monkeys or mice has resulted in the reversal of bone loss and osteoporosis [12Lotinun S. Pearsall R.S. Davies M.V. et al.A soluble activin receptor Type IIA fusion protein (ACE-011) increases bone mass via a dual anabolic-antiresorptive effect in Cynomolgus monkeys.Bone. 2010; 46: 1082-1088Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 13Vallet S. Mukherjee S. Vaghela N. et al.Activin A promotes multiple myeloma-induced osteolysis and is a promising target for myeloma bone disease.Proc Natl Acad Sci U S A. 2010; 107: 5124-5129Crossref PubMed Scopus (64) Google Scholar, 14Pearsall R.S. Canalis E. Cornwall-Brady M. et al.A soluble activin type IIA receptor induces bone formation and improves skeletal integrity.Proc Natl Acad Sci U S A. 2008; 105: 7082-7087Crossref PubMed Scopus (158) Google Scholar]. In a phase I trial in healthy postmenopausal women, the administration of this drug was accompanied by enhanced bone formation and decreased bone resorption [15Ruckle J. Jacobs M. Kramer W. et al.Single-dose, randomized, double-blind, placebo-controlled study of ACE-011 (ActRIIA-IgG1) in postmenopausal women.J Bone Miner Res. 2009; 24: 744-752Crossref PubMed Scopus (48) Google Scholar]. Surprisingly, the beneficial effects of ACE-011 in this study were associated with an unanticipated increase in the baseline hemoglobin and hematocrit levels. These findings were also confirmed by observations in murine models in which treatment with RAP-011 prevented chemotherapy-induced anemia [16Mulivor A. Barbosa D. Kumar R. Sherman M.L. Seehra J. Pearsall A.E. RAP-011, a soluble activin receptor type IIa murine IgG-Fc fusion protein, prevents chemotherapy induced anemia.Blood. 2009; (Abstract 161): 114Google Scholar]. In a phase II clinical trial in multiple patients with myeloma treated with ACE-011, increased hemoglobin and hematocrit levels were also observed and paralleled the improvement in bone lesions [17Abdulkadyrov K. Salogub G. Khuazheva N. et al.ACE-011, a soluble activin receptor type IIa IgG-FC fusion protein, increases hemoglobin (Hb) and improves bone lesions in multiple myeloma patients receiving myelosuppressive chemotherapy: preliminary analysis.Blood. 2009; (Abstract 749): 114Google Scholar]. These studies led to ACE-011 being currently evaluated in anemic patients with end-stage renal disease and patients with Diamond-Blackfan anemia (ClinicalTrials.gov identifiers NCT01146574 and NCT01464164).Although the stimulatory effects of ACE-011 on bone formation are believed to be mediated through its interaction with activin [18Lotinun S. Pearsall R.S. Horne W.C. Baron R. Activin receptor signaling: a potential therapeutic target for osteoporosis.Curr Mol Pharmacol. 2012; 5: 195-204Crossref PubMed Google Scholar], the mechanism underlying its effect on erythropoiesis remains unknown. The reported ability of activin to favor in vitro erythroid differentiation [19Yu J. Shao L. Vaughan J. Vale W. Yu A.L. Characterization of the potentiation effect of activin on human erythroid colony formation in vitro.Blood. 1989; 73: 952-960Crossref PubMed Google Scholar, 20Shao L. Frigon Jr., N.L. Young A.L. et al.Effect of activin A on globin gene expression in purified human erythroid progenitors.Blood. 1992; 79: 773-781Crossref PubMed Google Scholar] is difficult to reconcile with the clinical observations that an activin antagonist such as ACE-011 stimulates erythropoiesis. These seemingly contradictory findings indicate a more complex mechanism of action by which ACE-011 favorably affects red cell production in vivo. To clarify these effects, we examined the direct and indirect effects of ACE-011 in vitro on human erythropoiesis.MethodsCell culturesErythroid cells were generated from adult human BM, peripheral blood (PB), and cord blood (CB) CD34+ cells (All Cells, Emeryville, CA, USA) using a two-step liquid culture system as described previously [21Chen J. Peterson K.R. Iancu-Rubin C. Bieker J.J. Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo.Proc Natl Acad Sci U S A. 2010; 107: 16846-16851Crossref PubMed Scopus (16) Google Scholar]. K562 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Human BM stromal cells (SCs; catalog no. 2M-302C) were purchased from Lonza (Allendale, NJ, USA). These cells represent a heterogeneous population of adherent BM cells obtained after 3–4 weeks of culture then cryopreserved. After thawing, BM-SCs were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal bovine serum for 24 hours and then replated in IMDM supplemented with 1% penicillin/streptomycin, 1% L-Glutamine (Invitrogen, Grand Island, NY, USA), 20 mmol/L B-mercaptoethanol, 1% bovine serum albumin (Fraction V; Sigma, St. Louis, MO, USA) and 30% serum substitute BIT 9500 (Stem Cell Technologies, Vancouver, Canada) without further passage. Conditioned media (CM) was collected after 7 days, centrifuged at 2,100 × g, aliquoted, and stored at −80°C. Serial dilutions of CM generated in the absence or presence of ACE-011 were initially tested, and a 1:1 dilution (CM:IMDM) was used for the entire study.ReagentsActivin was purchased from Sigma. ACE-011 was provided by the Celgene Corporation (Warren, NJ, USA). ACE-011 was tested at various concentrations, ranging from 5–1,000 μg/mL.Hematopoietic colony assaysBurst-forming unit erythrocytes (BFU-Es) were assayed using the MethoCult system (Stem Cell Technologies), and colonies were enumerated under an inverted microscope after 14–16 days of culture as described previously [7Bruno E. Horrigan S.K. Van Den Berg D. et al.The Smad5 gene is involved in the intracellular signaling pathways that mediate the inhibitory effects of transforming growth factor-beta on human hematopoiesis.Blood. 1998; 91: 1917-1923Crossref PubMed Google Scholar].Flow cytometric analysisCells were labeled with CD34, CD235a (glycophorin A [GPA]), CD36, CD33, CD45, CD14, or CD61 fluorochrome-conjugated antibodies (Becton Dickinson, Mountain Blue, CA, USA) followed by incubation with 7-amino-actinomycin D (7-AAD; Sigma, St. Louis, MO, USA). Data acquisition and analysis was performed using a BD FACSCanto II (Becton Dickinson) flow cytometer and FACS Diva software. Nonviable 7-AAD+ cells were excluded from flow cytometric analysis.Q-PCR and microarray assaysTotal RNA was purified using an RNeasy purification kit and reverse-transcribed with an Omniscript kit (Qiagen Sciences, Maryland, MD, USA). Quantitative polymerase chain reaction was performed using IQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) and primers specific for γ- and β-globin and for glyceraldehyde-3-phosphate dehydrogenase (SABiosciences, Frederick, MD, USA) in a RealPlex MasterCycler (Eppendorf, Hauppage, NY, USA). RNA microarray assays and analyses were performed at The Keck Microarray Facility at Yale University Center for Genome Analysis (New Haven, CT, USA) using an Illumina Human HT-12 version 4.0 Expression BeadChip and GenomeStudio Software according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). The quantile normalization method was used for the data analysis. Fold change calculation and significance were determined based on the normalized average values of biological replicates, each performed in duplicate. Gene function annotation and pathway analyses were performed utilizing MetaCoreT version 6.8 Database and Software (GeneGo, Carlsbad, CA, USA). Heat maps and hierarchical clustering were generated with Partek Genomics Suite (Partek Incorporated, St. Louis, MO, USA). Complete datasets were deposited into the GEO database and are available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41708.Cytokine profilingActivin, BMP2, BMP6, and oncostatin levels present in SC CM were measured using Quantikine ELISA Kits according to manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Vascular endothelial growth factor (VEGF), interleukin (IL) 1, IL-6, and IL-10, IGFBP2 and 5, CCL8 and 13, and MMP1, 9, 12 and 13 were measured using Milliplex MAP multiplex biomarker panels and a Luminex xMAP platform (EMD Millipore, Billerica, MA, USA).Statistical analysisUnless otherwise specified, all experiments were performed independently at least three times. Data were expressed as mean ± SD and analyzed using a Student t test; p < 0.05 was considered statistically significant.ResultsEffects of ACE-011 on erythroid differentiation of CD34+ cellsThe direct effects of ACE-011 on CD34+ cells were initially examined in standard methylcellulose cultures containing erythropoietin (EPO), stem cell factor (SCF), and Il-3 under serum-free conditions. Figure 1A illustrates that the number of BFU-E colonies assayed from CB-, BM-, or PB-derived CD34+ cells in the presence of 50 and 100 μmol/L/mL of ACE-011 did not differ from that formed in the absence of ACE-011. Microscopic evaluation of colonies revealed that neither the size nor the morphologic appearance of the BFU-E colonies were affected by ACE-011 treatment. The direct effects of ACE-011 on the ability of CD34+ cells to generate erythroid cells were also evaluated in a two-step liquid culture system [21Chen J. Peterson K.R. Iancu-Rubin C. Bieker J.J. Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo.Proc Natl Acad Sci U S A. 2010; 107: 16846-16851Crossref PubMed Scopus (16) Google Scholar]. 1.25 × 105 CD34+ cells were cultured for 7 days in serum-free media with EPO and SCF. Next, the generated cells were enumerated and analyzed flow cytometrically for the expression of thrombospondin receptor (CD36) and GPA. Both control and ACE-011–treated cultures contained similar numbers of viable cells (e.g., 6.9 ± 1.1 × 105 in control vs. 6 ± 1.9 × 105 cells in ACE-011–treated cultures) suggesting that ACE-011 treatment did not interfere with the ability of CD34+ cells to proliferate. The fraction of CD36+/GPA+ cells generated in the presence of 100 μg/mL ACE-011 was comparable to that observed in control cultures (e.g., 37.2 ± 10.2% in ACE-011 treated cultures vs. 46.5 ± 0.7% in untreated cultures; p = 0.4194; Fig. 1B). These observations were confirmed by microscopic evaluation of cytospin preparations showing that after 7 days incubation, both control and ACE-011 cultures contained both erythroblasts at various stages of differentiation and undifferentiated elements (Fig. 1B). These results led us to examine whether ACE-011 treatment affected erythroid maturation during the terminal stages. Cultures generated after the initial 7 days were incubated for an additional 7 days in serum-free media supplemented with EPO only in the presence or absence of 100 μg/mL ACE-011. As illustrated in representative histograms (Fig. 1C), ACE-011 treatment did not affect terminal erythroid differentiation because similar numbers of GPA+ cells were generated in the absence and presence of the drug. The erythroid cells generated under either condition consisted of both erythroblasts and terminally differentiated red cells (Fig. 1C). Because ACE-011 is able to bind activin, a putative erythroid regulator [20Shao L. Frigon Jr., N.L. Young A.L. et al.Effect of activin A on globin gene expression in purified human erythroid progenitors.Blood. 1992; 79: 773-781Crossref PubMed Google Scholar, 22Frigon Jr., N.L. Shao L. Young A.L. Maderazo L. Yu J. Regulation of globin gene expression in human K562 cells by recombinant activin A.Blood. 1992; 79: 765-772Crossref PubMed Google Scholar], identical experiments were performed using a combination of ACE-011 and activin. The results showed that neither activin alone nor activin plus ACE-011 significantly affects primary erythropoiesis (Supplementary Material and Supplementary Figure 1, online only, available at www.exphem.org). These observations demonstrate that ACE-011 does not affect in vitro erythropoiesis directly or through its interaction with activin.Effects of CM from ACE-011–treated BM cells on erythropoiesisThe observation that ACE-011 did not affect in vitro erythroid differentiation directly, led us to hypothesize that in vivo activity might be mediated by BM accessory cells or factors within the hematopoietic microenvironment. We used a surrogate in vitro system to recapitulate the BM microenvironment by culturing CD34+ cells with CM produced by BM-derived SCs. BM-SCs are known to secrete a number of cytokines and regulatory molecules, including activin and members of the TGF-β family [23Shao L. Frigon Jr., N.L. Sehy D.W. et al.Regulation of production of activin A in human marrow stromal cells and monocytes.Exp Hematol. 1992; 20: 1235-1242PubMed Google Scholar, 24Uchimaru K. Motokura T. Takahashi S. Sakurai T. Asano S. Yamashita T. Bone marrow stromal cells produce and respond to activin A: interactions with basic fibroblast growth factor and platelet-derived growth factor.Exp Hematol. 1995; 23: 613-618PubMed Google Scholar]. SCs derived from BM of healthy donors were cultured in the presence or absence of 100 μg/mL ACE-011 in serum-free conditions. The CM generated was collected and tested for its effects on erythroid differentiation. Viable cells generated from 1.25 × 105 CD34+ cells incubated for 7 days in erythroid cultures in the presence or absence of SC-CM, were enumerated and characterized phenotypically. As illustrated in Figure 2A, the ability of CD34+ cells to proliferate was not affected by the addition of CM generated from untreated SCs, whereas it was modestly increased in the presence of SC-CM treated with ACE-011. When CD34+ cells were maintained in cultures to which EPO was not added, the ability of cultured cells to proliferate was preserved, suggesting that ACE-011–treated SC-CM supports cellular proliferation independent of EPO (Fig. 2A). We then evaluated the phenotype of the cells expanded in each condition and found that the addition of CM resulted in up to 80% reduction in the number of CD36+/GPA+ cells (Fig. 2B). This inhibitory effect was accompanied by a doubling in the percentage of CD34+ cells (Fig. 2B), suggesting that the CM inhibited erythroid differentiation while maintaining CD34+ cells in an undifferentiated state. Gene expression profiling of cells differentiated in control cultures and in cultures containing SC-CM was also performed, and the results confirmed the phenotypical observations (Supplementary Figure 2 and Supplementary Table 1, online only, available at www.exphem.org). However, when CD34+ cells were cultured in the presence of SC-CM treated with ACE-011, the percentage of CD36+/GPA+ cells was partially restored compared with the cultures containing untreated SC-CM (Fig. 2C). Data resulting from five independent experiments showed that CM+ACE-011 led to a significant 1.7-fold increase in the fraction of CD36+/GPA+ cells (Fig. 2D). However, this partial rescue of erythroid differentiation was dependent on the presence of EPO because SC-CM+ACE-011 cultures lacking EPO failed to support the generation of CD36+/GPA+ cells (Fig. 2D). Importantly, the restoration of erythroid phenotype was paralleled by a diminished ability of CD34+ cells to retain their undifferentiated state. There were more CD34+/CD36+ and CD34−/CD36+ cells in SC-CM+ACE-011 cultures than in SC-CM cultures (Fig. 2C). These changes were confirmed by microscopic evaluation of Wright-Giemsa–stained cytospin preparations. Representative microphotographs illustrate that the vast majority of cells differentiated under standard conditions were erythroblast and reticulocytes at different stages of maturation (Fig. 2E, a and d) whereas most of the cells cultured in the presence of SC-CM resembled blastlike cells and cells with myeloid-like features (Fig. 2E, b and e). In contrast, the population of cells differentiated in the presence of SC-CM–ACE-011 included blasts, myeloid elements, erythroblasts, and enucleated red cells, further supporting that ACE-011–treated SC-CM rescued erythropoiesis (Fig. 2E, c and f). We also assessed the expression of nonerythroid markers in these cultures and found that CD33, but not the expression of CD61 or CD14, was enhanced by the SC-CM compared with control cultures (i.e., up to fivefold more CD33+ cells were generated in the presence of CM [Supplementary Figure 3, online only, available at www.exphem.org]), suggesting that primary SCs released factors that favored myeloid differentiation. The effect of SC-CM on CD33 expression, however, was not influenced by SC-CM treated with ACE-011. These observations are consistent with the presence of cells with myeloid morphologic characteristics in the cultures differentiated in the presence of SC-SM (Fig. 2E). Finally, the effects of SC-CM untreated or SC-CM treated with ACE-011 on the ability of CD34+ cells to form BFU-E colonies was also evaluated. CD34+ cells exposed to SC-CM treated with ACE-011 formed 1.4-fold greater numbers of BFU-E than that observed in the presence of untreated SC-CM (208.5 ± 158.9 BFU-Es formed in cultures containing CM vs. 293.25 ± 138.2 BFU-Es formed in cultures containing CM+ACE-011; p = 0.0564; data not shown). These findings are consistent with the stimulatory effects of ACE-011 on cellular proliferation observed in liquid culture. Collectively, these results indicate that CM generated by BM-derived SC inhibits erythropoiesis and that this effect is partially eliminated by ACE-011 treatment of SC.Figure 2Evaluation of erythropoiesis in the presence of CM produced by BM-SCs. (A) Quantification of viable cells generated from 1.25 × 105 CD34+ cells cultured for 7 days in standard liquid culture system (control group, IMDM-based media with EPO and SCF), in the presence of CM from untreated SCs (SC-CM) or in the presence of CM from SCs treated with ACE-011 (SC-CM+ACE-011). Cells generated in the cultures that contained SC-CM+ACE-011 but lacked EPO (SC-CM+ACE-011 [no EPO]) were quantified in an identical fashion. (B) Quantification of CD36+/GPA+ cells (left graph) and CD34+ cells (right graph) in cultures generated from CD34+ cells cultured for 7 days under standard conditions (control group) or in the presence of media conditioned by untreated SCs (SC-CM). The results represent the fold change in the number of CD36+/GPA+ or CD34+ cells as determined after flow cytometric analyses of cells from five independent experiments. (C) Representative dot plot histograms illustrating GPA/CD36 expression (upper panels) and CD36/CD34 expression (lower panels) by cells generated under standard cultures (control group), in cultures containing CM from untreated SCs (SC-CM) or in cultures containing CM derived from SCs treated with ACE-011 (SC-CM+ACE-011). (D) Quantification of CD36+/GPA+ cells in cultures generated in the presence of CM from untreated SCs (SC-CM), from SCs treated with ACE-011 (SC-CM+ACE-011), or in the cultures containing SC-CM+ACE-011 but lacking EPO (SC-CM+ACE-011 [no EPO]). The data represents the mean ± SD of five experiments performed independently using CD34+ cells from different donors. (E) Morphologic analysis of cells generated after 7 days (upper panels) and 14 days (lower panels) of differentiation under standard conditions (control group) in cultures containing CM from untreated SCs (SC-CM) or containing CM derived from SCs treated with ACE-011 (SC-CM+ACE-011). Images were obtained using an Olympus BX40 microscope and an Olympus DP25 camera. Original magnification ×40/0.75. Ery = erythroblasts; My = myeloid-like cells; Ret = reticulocytes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Evaluation of erythropoiesis in the presence of CM produced by BM-SCs. (A) Quantification of viable cells generated from 1.25 × 105 CD34+ cells cultured for 7 days in standard liquid culture system (control group, IMDM-based media with EPO and SCF), in the presence of CM from untreated SCs (SC-CM) or in the presence of CM from SCs treated with ACE-011 (SC-CM+ACE-011). Cells generated in the cultures that contained SC-CM+ACE-011 but lacked EPO (SC-CM+ACE-011 [no EPO]) were quantified in an identical fashion. (B) Quantification of CD36+/GPA+ cells (left graph) and CD34+ cells (right graph) in cultures generated from CD34+ cells cultured for 7 days under standard conditions (control group) or in the presence of media conditioned by untreated SCs (SC-CM). The results represent the fold change in the number of CD36+/GPA+ or CD34+ cells as determined after flow cytometric analyses of cells from five independent experiments. (C) Representative dot plot histograms illustrating GPA/CD36 expression (upper panels) and CD36/CD34 expression (lower panels) by cells generated under standard cultures (control group), in cultures containing CM from untreated SCs (SC-CM) or in cultures containing CM derived from SCs treated with ACE-011 (SC-CM+ACE-011). (D) Quantification of CD36+/GPA+ cells in cultures generated in the presence of CM from untreated SCs (SC-CM), from SCs treated with ACE-011 (SC-CM+ACE-011), or in the cultures containing SC-CM+ACE-011 but lacking EPO (SC-CM+ACE-011 [no EPO]). The data represents the mean ± SD of five experiments performed independently using CD34+ cells from different donors. (E) Morphologic analysis of cells generated after 7 days (upper panels) and 14 days (lower panels) of differentiation under standard conditions (control group) in cultures containing CM from untreated SCs (SC-CM) or containing CM derived from SCs treated with ACE-011 (SC-CM+ACE-011). Images were obtained using an Olympus BX40 microscope and an Olympus DP25 camera. Original magnification ×40/0.75. Ery = erythroblasts; My = myeloid-like cells; Ret = reticulocytes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Gene expression profiling of BM-derived SC. Messenger RNA (mRNA) extracted from two different SCs (i.e., derived from independent donors, SC77 and SC97) that were either untreated (SC77-UN and SC97-UN) or treated with 100 μg/mL ACE-011 (SC77-T and SC97-T) was evaluated for differentially expressed gene transcripts using a Human Illumina HT-12 version 4.0 microarray platform. The heat map illustrates the expression levels of the top 50 genes that were differentially expressed in response to ACE-011 treatment. The results represent the normalized mean expression values obtained from analyses of mRNA isolated from two different sources of SC, each performed in biological duplicates. A description of the genes depicted and their corresponding expression values are provided in Supplementary Table 2 (online only, available at www.exphem.org), and the complete data sets are available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41708.View Large Im
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