Bcl2 enhances induced hematopoietic differentiation of murine embryonic stem cells
2007; Elsevier BV; Volume: 36; Issue: 2 Linguagem: Inglês
10.1016/j.exphem.2007.09.004
ISSN1873-2399
AutoresYan-Yi Wang, Xingming Deng, Lijun Xu, Fengqin Gao, Tammy Flagg, W. Stratford May,
Tópico(s)Cancer-related Molecular Pathways
ResumoBcl2 is a potent antiapoptotic gene that can increase resistance of adult bone marrow hematopoietic progenitor cells to lethal irradiation, and thereby preserve their ability to differentiate. However, the effect of Bcl2 on murine embryonic stem (ES) cells induced to undergo hematopoietic differentiation in the absence of a toxic stress is not known. To test this, murine CCE-ES cells that can be induced to undergo hematopoietic differentiation in a two-step process that results in upregulation of Bcl2 were used. Upregulation of Bcl2 precedes formation of hematopoietic embryoid bodies (EB) and their further differentiation into hematopoietic colony-forming units, when plated as single cells in methylcellulose. ES cells stably expressing a Bcl2 siRNA plasmid to “knock-down” endogenous expression or cells expressing wild-type (WT) Bcl2 or phosphomimetic Bcl2 mutants were examined. ES cells expressing the Bcl2 siRNA or those expressing a dominant-negative, nonphosphorylatable Bcl2 display a strikingly reduced capacity to form hematopoietic EBs and colony-forming units compared to cells expressing WT or phosphomimetic Bcl2 that demonstrate an increased capacity. Bcl2's effect on induced-hematopoietic differentiation of ES cells does not result from either decreased apoptosis or a reduced number of cells. Rather, Bcl2-enhances hematopoietic differentiation of ES cells by upregulating p27, which results in retardation of the cell cycle at G1/G0. Thus siRNA silencing of p27 reverts Bcl2's enhancement phenotype in a manner similar to that of Bcl2 “silencing” or expression of a nonphosphorylable Bcl2. In addition to Bcl2's well-described antiapoptotic and cell-cycle retardant effect on somatic cells, Bcl2 may also function to enhance induced hematopoietic cell differentiation of murine ES cells. These findings may have potential relevance for expanding hematopoietic stem/progenitor cell numbers from an ES cell source for stem cell transplantation applications. Bcl2 is a potent antiapoptotic gene that can increase resistance of adult bone marrow hematopoietic progenitor cells to lethal irradiation, and thereby preserve their ability to differentiate. However, the effect of Bcl2 on murine embryonic stem (ES) cells induced to undergo hematopoietic differentiation in the absence of a toxic stress is not known. To test this, murine CCE-ES cells that can be induced to undergo hematopoietic differentiation in a two-step process that results in upregulation of Bcl2 were used. Upregulation of Bcl2 precedes formation of hematopoietic embryoid bodies (EB) and their further differentiation into hematopoietic colony-forming units, when plated as single cells in methylcellulose. ES cells stably expressing a Bcl2 siRNA plasmid to “knock-down” endogenous expression or cells expressing wild-type (WT) Bcl2 or phosphomimetic Bcl2 mutants were examined. ES cells expressing the Bcl2 siRNA or those expressing a dominant-negative, nonphosphorylatable Bcl2 display a strikingly reduced capacity to form hematopoietic EBs and colony-forming units compared to cells expressing WT or phosphomimetic Bcl2 that demonstrate an increased capacity. Bcl2's effect on induced-hematopoietic differentiation of ES cells does not result from either decreased apoptosis or a reduced number of cells. Rather, Bcl2-enhances hematopoietic differentiation of ES cells by upregulating p27, which results in retardation of the cell cycle at G1/G0. Thus siRNA silencing of p27 reverts Bcl2's enhancement phenotype in a manner similar to that of Bcl2 “silencing” or expression of a nonphosphorylable Bcl2. In addition to Bcl2's well-described antiapoptotic and cell-cycle retardant effect on somatic cells, Bcl2 may also function to enhance induced hematopoietic cell differentiation of murine ES cells. These findings may have potential relevance for expanding hematopoietic stem/progenitor cell numbers from an ES cell source for stem cell transplantation applications. Bcl2 family members have been reported to regulate somatic cell differentiation, but little is known about whether or how these apoptotic genes may be involved in embryonic stem cell (ES) differentiation. Some antiapoptotic members, including Bcl2, BclxL, and MCL1 [1Townsend K.J. Zhou P. Qian L. et al.Regulation of MCL1 through a serum response factor/Elk-1-mediated mechanism links expression of a viability-promoting member of the BCL2 family to the induction of hematopoietic cell differentiation.J Biol Chem. 1999; 274: 1801-1813Crossref PubMed Scopus (117) Google Scholar, 2Kozopas K.M. Yang T. Buchan H.L. et al.MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2.Proc Natl Acad Sci U S A. 1993; 90: 3516-3520Crossref PubMed Scopus (876) Google Scholar] have been reported to support neuronal and sensory neuron cell differentiation and maturation [3Zhang K.Z. Westberg J.A. Holtta E. et al.BCL2 regulates neural differentiation.Proc Natl Acad Sci U S A. 1996; 93: 4504-4508Crossref PubMed Scopus (132) Google Scholar, 4Rolletschek A. Chang H. Guan K. et al.Differentiation of embryonic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors.Mech Dev. 2001; 105: 93-104Crossref PubMed Scopus (109) Google Scholar, 5Middleton G. Pinon L.G. Wyatt S. et al.Bcl-2 accelerates the maturation of early sensory neurons.J Neurosci. 1998; 18: 3344-3350Crossref PubMed Google Scholar], and have a role in restricting lineage choice during differentiation of multipotent hematopoietic progenitor cells [6Haughn L. Hawley R.G. Morrison D.K. et al.BCL-2 and BCL-XL restrict lineage choice during hematopoietic differentiation.J Biol Chem. 2003; 278: 25158-25165Crossref PubMed Scopus (44) Google Scholar, 7Marone M. Bonanno G. Rutella S. et al.Survival and cell cycle control in early hematopoiesis: role of bcl-2, and the cyclin dependent kinase inhibitors P27 and P21.Leuk Lymphoma. 2002; 43: 51-57Crossref PubMed Scopus (19) Google Scholar]. Interestingly, inhibition of Bcl2 expression by antisense oligodeoxynbonucleotide technology has been reported to block neuronal differentiation, but the mechanism is not clear [3Zhang K.Z. Westberg J.A. Holtta E. et al.BCL2 regulates neural differentiation.Proc Natl Acad Sci U S A. 1996; 93: 4504-4508Crossref PubMed Scopus (132) Google Scholar]. Bcl2 has also been reported to influence the morphological transition of “undifferentiated” ES cells to “committed” precursor cells in both adult and embryonic nonhematopoietic tissues [8Lu Q.L. Poulsom R. Wong L. et al.Bcl-2 expression in adult and embryonic non-haematopoietic tissues.J Pathol. 1993; 169: 431-437Crossref PubMed Scopus (301) Google Scholar]. Collectively, these reports suggest that Bcl2 may be involved in regulating/influencing cell differentiation as well as tissue fate through its antiapoptotic function. Indeed, expression of exogenous Bcl2 in adult hematopoietic stem/progenitor cells of transgenic mice mediates enhanced resistance to irradiation [9Domen J. Gandy K.L. Weissman I.L. Systemic overexpression of BCL-2 in the hematopoietic system protects transgenic mice from the consequences of lethal irradiation.Blood. 1998; 91: 2272-2282Crossref PubMed Google Scholar]. Although Bcl2 is not expressed in early adult hematopoietic stem cells [10Park J.R. Bernstein I.D. Hockenbery D.M. Primitive human hematopoietic precursors express Bcl-x but not Bcl-2.Blood. 1995; 86: 868-876Crossref PubMed Google Scholar], when exogenous Bcl2 is expressed, it can function to protect these cells against stresses that induce apoptosis. However, Bcl2's effect on hematopoietic differentiation of ES cells, if any, is not known. Recently, it was discovered that phosphorylation is required for Bcl2's functional antiapoptotic and cell-cycle retardant activity and its role in delaying DNA damage repair [11Deng X. Gao F. Flagg T. et al.Mono- and multisite phosphorylation enhances Bcl2's antiapoptotic function and inhibition of cell cycle entry functions.Proc Natl Acad Sci U S A. 2004; 101: 153-158Crossref PubMed Scopus (132) Google Scholar, 12Korhonen L. Belluardo N. Mudo G. et al.Increase in Bcl-2 phosphorylation and reduced levels of BH3-only Bcl-2 family proteins in kainic acid-mediated neuronal death in the rat brain.Eur J Neurosci. 2003; 18: 1121-1134Crossref PubMed Scopus (46) Google Scholar, 13Deng X. Gao F. May Jr., W.S. Bcl2 retards G1/S cell cycle transition by regulating intracellular ROS.Blood. 2003; 102: 3179-3185Crossref PubMed Scopus (111) Google Scholar, 14Youn C.K. Cho H.J. Kim S.H. et al.Bcl-2 expression suppresses mismatch repair activity through inhibition of E2F transcriptional activity.Nat Cell Biol. 2005; 7: 137-147Crossref PubMed Scopus (67) Google Scholar, 15Jin Z. May W.S. Gao F. et al.Bcl2 suppresses DNA repair by enhancing c-Myc transcriptional activity.J Biol Chem. 2006; 281: 14446-14456Crossref PubMed Scopus (55) Google Scholar]. Dynamic phosphorylation of Bcl2 occurs on the S70 site located in the flexible loop domain. Phosphorylation can also occur on two other potential sites, T69 and S87, which are also located in the flexible loop domain. Either mono- or multiple-site phosphorylation of Bcl2 enhances its function [11Deng X. Gao F. Flagg T. et al.Mono- and multisite phosphorylation enhances Bcl2's antiapoptotic function and inhibition of cell cycle entry functions.Proc Natl Acad Sci U S A. 2004; 101: 153-158Crossref PubMed Scopus (132) Google Scholar, 12Korhonen L. Belluardo N. Mudo G. et al.Increase in Bcl-2 phosphorylation and reduced levels of BH3-only Bcl-2 family proteins in kainic acid-mediated neuronal death in the rat brain.Eur J Neurosci. 2003; 18: 1121-1134Crossref PubMed Scopus (46) Google Scholar, 13Deng X. Gao F. May Jr., W.S. Bcl2 retards G1/S cell cycle transition by regulating intracellular ROS.Blood. 2003; 102: 3179-3185Crossref PubMed Scopus (111) Google Scholar, 14Youn C.K. Cho H.J. Kim S.H. et al.Bcl-2 expression suppresses mismatch repair activity through inhibition of E2F transcriptional activity.Nat Cell Biol. 2005; 7: 137-147Crossref PubMed Scopus (67) Google Scholar, 15Jin Z. May W.S. Gao F. et al.Bcl2 suppresses DNA repair by enhancing c-Myc transcriptional activity.J Biol Chem. 2006; 281: 14446-14456Crossref PubMed Scopus (55) Google Scholar, 16Ruvolo P.P. Deng X. May W.S. Phosphorylation of Bcl2 and regulation of apoptosis.Leukemia. 2001; 15: 515-522Crossref PubMed Scopus (375) Google Scholar]. Importantly, Bcl2 phosphorylation at S70 is physiologically induced by growth factors that mediate cell growth and survival, including interleukin (IL)-3, erythropoietin, and nerve growth factor [17May W.S. Tyler P.G. Ito T. et al.Interleukin-3 and bryostatin-1 mediate hyperphosphorylation of BCL2 alpha in association with suppression of apoptosis.J Biol Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar, 18Ito T. Deng X. Carr B. et al.Bcl-2 phosphorylation required for anti-apoptosis function.J Biol Chem. 1997; 272: 11671-11673Crossref PubMed Scopus (494) Google Scholar, 19Deng X. Ruvolo P. Carr B. et al.Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases.Proc Natl Acad Sci U S A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar, 20Horiuchi M. Hayashida W. Kambe T. et al.Angiotensin type 2 receptor dephosphorylates Bcl-2 by activating mitogen-activated protein kinase phosphatase-1 and induces apoptosis.J Biol Chem. 1997; 272: 19022-19026Crossref PubMed Scopus (264) Google Scholar]. Regardless of the inducing agent, Bcl2 phosphorylation enhances its antiapoptotic function because the nonphosphorylatable S70A or the AAA Bcl2 mutant displays a loss of function phenotype and fails to block apoptosis or retard cell-cycle progression [11Deng X. Gao F. Flagg T. et al.Mono- and multisite phosphorylation enhances Bcl2's antiapoptotic function and inhibition of cell cycle entry functions.Proc Natl Acad Sci U S A. 2004; 101: 153-158Crossref PubMed Scopus (132) Google Scholar]. While not yet clinically achievable, ES cells are envisioned to represent a novel and potentially limitless source of functional hematopoietic stem and progenitor cells for transplantation of patients with bone marrow failure diseases, including leukemia and aplastic anemia, who may not have a living, related, or unrelated donor match. Therefore, we tested the effect of silencing endogenous Bcl2 expression or expressing wild-type (WT), phosphomimetic or nonphosphorylatable Bcl2 mutants on induced hematopoietic differentiation of murine CCE-ES cells. Results reveal that Bcl2 has a stimulatory effect on stem cell fate during induced hematopoietic differentiation that is not the result of its potent antiapoptotic function. Murine Bcl2 cDNA was cloned in pUC19 plasmid. Nucleotides corresponding to each serine (S) or threonine (T) residue were substituted to create a conservative alteration to alanine (A) or glutamic acid (E) with a site-directed mutagenesis kit (Clontech, Palo Alto, CA, USA) as described [18Ito T. Deng X. Carr B. et al.Bcl-2 phosphorylation required for anti-apoptosis function.J Biol Chem. 1997; 272: 11671-11673Crossref PubMed Scopus (494) Google Scholar]. Each single mutant was confirmed by sequencing the cDNA and subsequently cloning into the pMigR1 retroviral expression vector [21Pear W.S. Miller J.P. Xu L. et al.Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow.Blood. 1998; 92: 3780-3792Crossref PubMed Google Scholar]. The MIGR1 retrovirus contains the murine stem cell virus promoter and an internal ribosomal entry site element followed by the green fluorescent protein (GFP) gene. The MIGR1 vector expresses both the Bcl2 insert and the GFP marker protein from a single internal ribosomal entry site–containing message driven by the murine stem cell virus retroviral promoter/enhancer (Fig. 2A).Figure 2Stable Bcl2 transgene expression in murine CCE-embryonic stem (ES) cells during hematopoietic differentiation. (A) Map of retroviral Expression Vector MigR1 containing Bcl2 transgene. (B) Stable expression of Bcl2 transgenes in ES cells determined by Western blotting. (C) Western blot analysis of Bcl2 transgene expression in day 0 ES through day-11 embryoid bodies (EBs) cells. (D) Stable expression of green fluorescent protein (GFP) in cells comprising day-11 EBs (1) and in the day-10 hematopoietic colony-forming units (CFUs) (2) derived in Step 2. Expression of GFP in cells of EBs and CFUs indicates stable expression of the Bcl2 transgenes. Colonies formed by cells expressing AAA Bcl2 in differentiation Step 2 are EB (1), but few can be induced to hematopoietic CFUs in Step 2 (2).View Large Image Figure ViewerDownload (PPT) Retroviral supernatants were generated by transient transfection of BOSC23 cells and titered on NIH 3T3 cells before use [22Pear W.S. Nolan G.P. Scott M.L. et al.Production of high-titer helper-free retroviruses by transient transfection.Proc Natl Acad Sci U S A. 1993; 90: 8392-8396Crossref PubMed Scopus (2291) Google Scholar]. These were immediately used for transduction of murine ES cells (CCE) on gelatinized six-well plates [23Helgason C.D. Sauvageau G. Lawrence H.J. et al.Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro.Blood. 1996; 87: 2740-2749PubMed Google Scholar]. Infected ES cells were traced by their expression of GFP and sorted using fluorescein-activated cell sorting (FACS). Stably expressing batch cultures of ES cells were expanded and used in the following experiments. Bcl2 or p27 gene target sequence GCTGCACCTGACGCCCTTC or GTGGAATTTCGACTTTCAG was identified as a template for producing the siRNA as determined using the Ambion siRNA Target Finder and the mouse Bcl2 or p27 cDNA sequence. The Bcl2 or p27-specific hairpin siRNA insert (sense-loop-antisense) was determined using a computerized insert design tool based on a target sequence following instructions on the Ambion Web site. The oligonucleotide encoding the Bcl2 or p27-specific hairpin siRNA insert was synthesized and ligated into the pSilencer 2.1-U6 hygro vector (Ambion, Austin, TX, USA). The pSilencer 2.1-U6 hygro plasmid bearing the siRNA insert was used to transfect CCE-ES cells using Lipofectamine 2000 according to manufacturer's instructions. A scrambled control siRNA that is not homologous to any known gene was used as a negative control. Stable clones expressing Bcl2 or p27 or control siRNA are selected in hygromycin and the level of Bcl2 or p27 silence was determined by Western blot. Murine CCE-ES cells were grown on gelatinized dishes in Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY, USA, cat. no. 11965-084) supplemented with leukemia inhibitory factor, 15% fetal bovine serum (GIBCO, cat. no. 16141-061), l-glutamine (Cellgro, cat. no. 25-005-CI), nonessential amino acid, and β-mercaptoethanol (Sigma, St Louis, MO, USA). Unless otherwise stated, all reagents for cell culture and in vitro differentiation of ES cells were purchased from StemCell Technologies Inc (Vancouver, BC, Canada). All cell culture was performed in humidified incubators at 37°C with a mixture of 5% CO2 in air. Murine CCE-ES cells can undergo induced hematopoietic differentiation in vitro in a two-step process that represents a modification of the method described by Keller et al. [24Keller G. Kennedy M. Papayannopoulou T. et al.Hematopoietic commitment during embryonic stem cell differentiation in culture.Mol Cell Biol. 1993; 13: 473-486Crossref PubMed Scopus (767) Google Scholar] and as detailed in the Technical Manual of StemCell Technologies, Inc. (www.stemcell.com; Fig. 1A). Briefly, for Step 1, induced hematopoietic differentiation, murine CCE ES cells (500 cells/mL) are cultured in Iscove's modified Dulbecco's medium (StemCell, cat. no. 36150) supplemented with 1% methylcellulose (StemCell, cat. no. 03120), 15% fetal bovine serum (GIBCO, cat. no. 16141-061), 2 mM l-glutamine (Cellgro, cat. no. 25-005-CI), 150 μM monothioglycerol (Sigma), and 40 ng/mL murine stem cell factor plated in low-adherence 35-mm dishes (StemCell Technologies Inc., cat. no. 27100). Cultures are placed into a covered Petri dish, along with an open 35-mm dish containing sterile water for humidification and incubated at 5% CO2 at 37°C. Cultures are re-fed on day 6 with “feed medium” containing 10 μg/mL stem cell factor, IL-3, human IL-6, and 3 U/mL erythropoietin as described in the manual. For Step 2, the resulting day-11 embryoid bodies (EBs) that develop are collected and made into a single cell suspension that is plated at 2 × 104 cells/mL in methylcellulose-based, hematopoietic “differentiation medium” containing stem cell factor, IL-3, IL-6, erythropoietin, and insulin and transferrin in 35-mm dishes. Cultures are incubated in 5% CO2 in 37°C at 95% humidity for 10 days in order to enumerate hematopoietic colonies (colony-forming units [CFUs]) that form, including burst-forming unit erythroid, CFU granulocyte-macrophage, and CFU-MIX. The EBs formed during Step 1 are scored as “hematopoietic” or “nonhematopoietic” EBs. “Hematopoietic” EBs are morphologically identified using a dissecting microscope by the visual presence of macrophages, erythroid cells, and, occasionally, granulocytic cells at the edges of the EB. Hemoglobinization of the erythroid cells is often visible. The “efficiency of EB formation” = total EBs scored per dish/number of ES cells plated per dish. The “% hematopoietic EBs” = number of hematopoietic EBs scored per dish/total number of EBs identified per dish. Hematopoietic differentiation was further documented and quantified by the percentage of CD41+ cells that formed in the EBs during Step 1 of induced hematopoietic differentiation. The CFUs formed during Step 2 are identified and scored visually using a microscope as defined in the manual. Day 11 EBs from Step 1 are disaggregated into single cell suspensions by washing with phosphate-buffered saline (PBS) and resuspending in 0.1% trypsin in PBS and pipetting the mixture up and down. Cells are strained through a filter mesh (Miltenyi Biotec, Auburn, CA, USA, cat. no. 130-041-407) to remove clumps and collected by low-speed centrifugation at 300g. For the Cell Viability Assay, Annexin-V–CY5 (BD Pharmingen, San Diego, CA, USA) and 7-amino-actinomycin D (Sigma) or propidium iodide (Sigma) staining was done at room temperature for 15 minutes according to manufacturer's specifications (BD Pharmingen, San Diego, CA, USA). For dual staining of Annexin-V–CY5 and CD41-phycoerythrin (PE) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), after Annexin-V–CY5 staining, cells were transferred to 4°C, and CD41-PE antibody was added. After 20 minutes, samples were diluted with Annexin-V binding buffer containing 7-amino-actinomycin D and analyzed by FACS [25Kyba M. Perlingeiro R.C. Hoover R.R. et al.Enhanced hematopoietic differentiation of embryonic stem cells conditionally expressing Stat5.Proc Natl Acad Sci U S A. 2003; 100: 11904-11910Crossref PubMed Scopus (56) Google Scholar]. For hematopoietic differentiation and cell-cycle analysis, single viable cells were separated by Ficoll centrifugation (Amersham Biosciences, Uppsala, Sweden) and stained with a primary SSEA-1 antibody (Chemicon, cat. no. MAB 4301) for 30 minutes at 4°C after blockade of the Fc receptor using mCD16/CD32 (BD Biosciences Pharmingen, San Diego, CA, USA), a secondary mouse IgM-allophycocyanin antibody (BD Biosciences Pharmingen) was added and incubated for 30 minutes at 4°C. Simultaneously, cells were stained with a CD41-PE antibody used to assess hematopoietic differentiation, as reported [25Kyba M. Perlingeiro R.C. Hoover R.R. et al.Enhanced hematopoietic differentiation of embryonic stem cells conditionally expressing Stat5.Proc Natl Acad Sci U S A. 2003; 100: 11904-11910Crossref PubMed Scopus (56) Google Scholar]. Cells were fixed in 90% methanol for 60 minutes at 4°C and stained with 50 μg/mL propidium iodide (Sigma) to determine cell-cycle distribution. For detection of phosphorylated Bcl2, Ficoll-separated cells were fixed and permeabilized using BD Cytofix/Cytoperm Kit according to manufacturer's instructions (BD Biosciences Pharmingen; cat. no. 554715). Permeabilized cells were incubated with 0.5% bovine serum albumin in PBS for 10 minutes and stained with a primary anti-Phospho-Bcl-2 (Ser70) specific antibody (Cell Signaling; cat. no. 2871) followed by incubation with a secondary PE-conjugated donkey anti-rabbit antibody for 30 minutes at 4°C. Cells were washed and analyzed by FACS. FDC-P1/ER cells expressing Bcl2 were incubated with 10 nM Bryostatin 1 (Sigma) to induce Bcl2 phosphorylation [17May W.S. Tyler P.G. Ito T. et al.Interleukin-3 and bryostatin-1 mediate hyperphosphorylation of BCL2 alpha in association with suppression of apoptosis.J Biol Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar] and used as a positive control for the phosphospecific antibody. Cells were washed with ice-cold PBS and homogenized in ice-cold lysis buffer (50 mM Tris-HCl [pH 8.0], 0.5% NP-40; 1 mM ethylene diamine tetraacetic acid; 150 mM NaCl; 10% glycerol; 1 mM sodium vanadate; 50 mM sodium fluoride; 10 mM sodium pyrophosphate; 1 mM β-mercaptoethanol) containing a protease inhibitor cocktail set I (Calbiochem, La Jolla, CA, USA; cat. no. 539131). Cell extracts were clarified by centrifugation at 14,000 for 10 minutes and an aliquot of the supernatant removed for total protein determination using Bio-Rad Protein assays. An aliquot corresponding to 100 μg total protein is then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and transferred electrophoretically to a nitrocellulose filter. Nonspecific binding of antibody is initially blocked by incubation of the filter in 5% nonfat dry milk containing 0.1% Tween 20 in PBS for 2 hours at room temperature. Immunoblotting was carried out using several antibodies against Bcl2, p27, and p21 (Santa Cruz Biotechnology) followed by addition of peroxidase-conjugated secondary anti-immunoglobulin antibodies. Blots were developed using the enhanced chemiluminescence method (Amersham Biosciences, Buckinghamshire, England). Murine ES cells can be induced to undergo hematopoietic differentiation in a two-step process (Fig. 1A). By definition, induced ES cells first form “hematopoietic EBs” during Step 1 after 11 days of culture and then, when dispersed as single cells in methylcellulose, secondary form colonies of the burst-forming unit erythroid, CFU-GM, and CFU-MIX type in Step 2 as described in Materials and Methods. To test the effect of Bcl2 on hematopoietic differentiation, the expression of endogenous Bcl2 was efficiently silenced using siRNA technology. Because the capacity of the control or siRNA Bcl2-expressing ES cells to form EBs and the total number of EBs formed in Step 1 was not significantly changed, these data indicate that silencing of endogenous Bcl2 expression does not affect ES cell proliferation or EB formation under these conditions (Fig. 1B, C, and D). However, the percent of “hemopoietic” EBs (Step 1) and the number of CFUs formed per 500 ES cells plated is drastically reduced, by >50% (Fig. 1E). This reduced capacity is apparently not due entirely to any increased apoptosis, because EB cell viability is only reduced by ∼12% (i.e., 78.9% of control vs 66.5% from Bcl2 siRNA, Fig. 1F). Hematopoietic differentiation was determined using FACS analysis to compare undifferentiated ES cells in the EBs formed during hematopoietic differentiation. SSEA-1 was used as a stage-specific embryonic antigen to detect undifferentiated ES cells and CD41 expression was used to detect “hematopoietic” differentiation [25Kyba M. Perlingeiro R.C. Hoover R.R. et al.Enhanced hematopoietic differentiation of embryonic stem cells conditionally expressing Stat5.Proc Natl Acad Sci U S A. 2003; 100: 11904-11910Crossref PubMed Scopus (56) Google Scholar, 26Mitjavila-Garcia M.T. Cailleret M. Godin I. et al.Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells.Development. 2002; 129: 2003-2013PubMed Google Scholar, 27Mikkola H.K. Fujiwara Y. Schlaeger T.M. et al.Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo.Blood. 2003; 101: 508-516Crossref PubMed Scopus (301) Google Scholar]. While only 15.7% of control cells expressed SSEA-1 and 26.3% expressed CD41, 42.7% of Bcl2 silenced cells continue to express SSEA-1 and only 7.7% continue to express CD41 (Fig. 1G). These data indicate that silencing of endogenous Bcl2 significantly inhibits induced hematopoietic cell differentiation of ES cells. Because silencing of Bcl2 inhibits induced hematopoietic differentiation of ES cells, we tested whether Bcl2 phosphorylation affected this process. We compared expression of WT, mono-or multisite phosphomimetic [S70E, EEE (T69E/S70E/S87E)] or nonphosphorylatable [S70A or AAA (T69A/S70A/S87A)] Bcl2 mutants. WT or each of the Bcl2 mutants was expressed in ES cells using the MIGR1 retroviral expression system as described in Materials and Methods (Fig. 2A). Retrovirally infected, batch cultures of ES cells that stably express similar levels of the Bcl2 transgene (Fig. 2B and C) were then used to compare to their capacity to undergo induced hematopoietic differentiation. Importantly, during both Step 1 that leads to hematopoietic EB formation and Step 2 where CFUs are formed, the transgenes were continuously expressed as detected by both Western blot and the continued monitoring green fluorescence of cells comprising either EBs or CFUs (Fig. 2D; panel 1 represents EBs and panel 2 represents a CFU). Thus, any differential effect observed for Bcl2 is not considered to result from a silencing or loss of expression of the transgene during differentiation. Results reveal that cells expressing the mono- or multisite phosphomimetic Bcl2 (S70E or EEE) mutant displayed a marked increase (up to two- or threefold) in formation of “hematopoietic” EBs and CFUs (including BFU-E, CFU-GM, and CFU-MIX; Fig. 3C and E). Again the efficiency of hematopoietic EB formation or total cell number per 500 ES cells initially plated is not significantly different (Fig. 3A and B). Furthermore, the loss of viability of CD41-expressing, differentiated cells remains 1.4% and is not altered (Fig. 3D), indicating that the effect of Bcl2 on induced hematopoietic differentiation of ES cells is not likely due to its antiapoptotic function. By contrast, ES cells expressing either of the nonphosphorylatable Bcl2 mutants S70A or AAA, display markedly reduced total numbers of hematopoietic EBs in Step 1 and CFUs in Step 2. However, any effect of the Bcl2 mutants in enhancing or reducing hematopoietic EB or CFU formation does not appear to be due to a change in cell viability, because the percent of Annexin-V–expressing cells obtained from EBs is not significantly changed for any Bcl2 mutant expressing cells (Fig. 3D). In addition, the failure to express CD41 appears to be balanced by the continuous expression of SSEA-1 by the AAA Bcl2 expressing cells from Step 1 (Fig. 3F). Again, more of the Bcl2-expressing cells display CD41 and less express SSEA-1 compared to vector-only cells, indicating an enhanced capacity of such cells to undergo induced hematopoietic differentiation (Fig. 3F). Furthermore, the EEE Bcl2-expressing cells demonstrate an even greater capacity to undergo differentiation with increased numbers of progeny expressing CD41 and a reduced number expressing SSEA-1 (Fig. 3F). By contrast, the AAA Bcl2–expressing cells reciprocally show reduced expression of CD41 and increased expression of SSEA-1. This enhanced stimulatory effect of EEE Bcl2 over WT Bcl2 likely occurs because the
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