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Colloquium Enhanced hematopoietic differentiation of embryonic stem cells conditionally expressing Stats Michael Kybat, Rita C. R. Perlingeirot$, Russell R. Hoovert, Chi-Wei Lut, Jonathan Piercet, and George Q. Dales iWhitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142; and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, and Division of Pediatric Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute, Boston, MA 02115 . . . The signal transducer Stats plays a key role in the regulation of hematopoietic differentiation and hematopoietic stem cell func- tion. To evaluate the effects of Stats signaling in the earliest hematopoietic progenitors, we have generated an embryonic stem cell line in which Stats signaling can be induced with doxycycline. Ectopic Stats activation at the point of origin of the hematopoietic lineage (from day 4 to day 6 of embryoid body differentiation) significantly enhances the number of hematopoietic progenitors with colony-forming potential. It does so without significantly altering total numbers or apoptosis of hematopoietic cells, sug- gesting a cell-intrinsic effect of Stats on either the developmental potential or clonogenicity of this population. From day-6 embryoid bodies, under the influence of Stats signaling, a population of semiadherent cells can be expanded on OP9 stromal cells that is comprised of primitive hematopoietic blast cells with ongoing, mainly myeloid, differentiation. When these cells are injected into lethally irradiated mice, they engraft transiently in a doxycycline- dependent manner. These results demonstrate that the hemato- poietic commitment of embryonic stem cells may be augmented by a Stats-mediated signal, and highlight the utility of manipulating individual components of signaling pathways for engineering tissue-specific differentiation of stem cells. hematopoiesis When differentiated as suspension aggregates (embryoid bodies, EBs), embryonic stem (ES) cells will readily give rise to differentiated hematopoietic cells (1) as well as colony- forming cells (CFCs) that can be assayed in secondary semisolid hematopoietic cultures (24. However, ES cell differentiation does not efficiently generate hematopoietic stem cells (HSCs) capable of repopulating the hematopoietic system of lethally irradiated adult recipients. In this regard, ES cells recapitulate the development of the earliest embryonic hematopoietic tissue, the yolk sac. Analysis of knockout mice has implicated several genes in the embryonic development of the definitive HSC (3-5~; however, a detailed understanding of the extracellular signals that guide development from the pluripotent state to the lineage- restricted HSC state is lacking. In an effort to evaluate signals that promote hematopoietic differentiation of pluripotent cells, as well as the generation of cells with hematopoietic repopulating potential, we sought to test the effects of Stats activation during in vitro differentiation of ES cells. Stats are cytoplasmic signal transducers that are recruited by ligand-activated receptors via Src homology 2- mediated interactions with receptor-bound Janus kineses. This interaction results in Stat protein phosphorylation, disengage- ment, dimerization, and translocation to the nucleus where these proteins then function as a transcription factors, binding to and activating the transcription of target genes (6~. Stats is encoded by two genes, StatSa and StatSb, with 95% sequence identity (7) and is activated by engagement of numerous hematopoietic and 11904-11910 1 PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1 nonhematopoietic receptors (8-11~. Stats signaling has been implicated in cellular proliferation (12, 13), resistance to apo- ptosis (14-16), and differentiation (17, 18~. Mice genetically null for both Stat5a and Stat5b display obvious defects in response to growth hormone and prolactin (15) and subtler defects in embryonic hematopoietic develop- ment (144. Although definitive HSCs develop in Stats knockout mice, they display a profound defect in competitive repopulation (19-21), suggesting that Stats may be interacting cooperatively and redundantly with other signal transducers in HSC regula- tion. In the classic HSC pathology, chronic myeloid leukemia, regulation of proliferation is disrupted by the oncogene Bcr/Abl, with concomitant activation of Stats (22-254. Moreover, dom- inant negative Stats mutants can block transformation by Bcr/ Abl (26), indicating that inappropriate activation of StatS can have dramatic consequences for HSC regulation. Because of the pivotal role of Stats signaling in hematopoiesis and HSC homeostasis, we selected this pathway for study during the earliest stages of hematopoietic specification in an in vitro system of ES cell differentiation. For this purpose, we have generated an ES cell line with a tetracycline-inducible, domi- nant-active allele of Stats. We report that induction of Stats signaling during EB development dramatically enhances hema- topoiesis. Furthermore, on OP9 stromal cell coculture, Stats promotes the expansion of a blast cell population from day-6 EBs. Cultures expanded in this way are rich in primitive, undifferentiated cells, with surface marker similarities to HSCs, and have the capacity to engraft lethally irradiated adult mice in a transient, Stats-dependent manner. --rib - r~r~ _ ~ Materials and Methods Generation of Stat5CA Inducible ES Cells. The cDNA for the constitutively active mutant of Stats (H299R/S711F, a gift from T. Kitamura, University of Tokyo, Tokyo) was subcloned on an EcoRI-NotI fragment from murine stem cell virus (MSCV)- Stat5CA-iresGFP (16) into pLox. This was then co-electropo- rated along with pSalk-CRE (a gift from S. O'Gorman, The Salk Institute, San Diego) into the targeting cell line Ainvl5. The targeting ES cell line and targeting plasmid, pLox, have been described (274. The resulting cell line was selected and expanded This paper results from the Arthur M. Sackier Colloquium of the National Acaclemy of Sciences, "Regenerative Meclicine," held October 18-22, 2002, at the Arnold and Mabel Beckman Center of the National Acaclemies of Science and Engineering in Irvine, CA. Abbreviations: ES, embryonic stem; ED, embryoicl bocly; HSC, hematopoietic stem cell; CFC, colony-forming cell; TRE, tetracycline response element; TPO, thrombopoietin; SCF, stem cell factor; VEGF, vascular enclothelial growth factor; IFS, inactivated fetal serum; MSCV, murine stem cell virus; IMDM, Iscove's modified Dulbecco's meclium. "Present aciciress: ViaCell, Inc., 26 LanciscJowne Street 580, Cambricige, MA 02139-4216. Present aciciress: Vertex Pharmaceuticals, 130 Waverly Street, Cambricige, MA 02139-4242. Vito whom correspondence should be aciciressecl. E-mail: claley@wi.mit.eclu. 2003 by The National Acaclemy of Sciences of the USA www.pnas.org/cgi/cdoi/10. 1 073/pnas. 1734140100

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a b / pLoxStatSCA ~1 Chromosome 6 +1 ~ X ~ X-chromosome plasma seq. ~ X o O O4 As ~ ~ ~ ~ - ~ V . ~~ I, 1 , X o VO _1 ~.4 Fig. 1. Generation of the Stats-inducible ES cell line. (a) Integration of pLoxStat5CA into the LoxP site on the X chromosome of Ainv15 ES cells places the cDNA for Stat5CA underthe control of the tetracycline response element. Recombination between the chromosomal and plasmid LoxP sites, denoted by X, is mediated by transient expression of Cre recombinase. Reconstitution of neo gene function by the promoter-ATG sequence 5' to the loxP site allows for selection of successfu I i nteg ration events. rtTA, reverse tetracycl i ne tra nsactivator. (b) Exposu re of iStat5CA ES cel Is to doxycycl i ne, but not the pa renta I cel I I i ne Al nv 1 5, resu Its in the bandshift of a probe containing Stats binding sites. Arrowhead denotes the Stats-specific bandshift. in 400 ,ug/ml G418 (Sigma). ES cells were maintained on irradiated mouse embryonic fibroblasts in DME/15% inacti- vated fetal serum (IFS)/0. 1 mM nonessential amino acids (GIBCO/BRL)/2 mM glutamine/50 units/ml penicillin/50 1lg/ml streptomycin (GIBCO/BRL)/0.1 mM 2-mercaptoethanol (Sigma)/1,000 units/ml leukemia inhibitory factor (PeproTech, Boston). To induce StatSCA expression in ES cells, 2 ,ug/ml doxycycline (Sigma) was added to the culture medium. Bandshift Assay. To generate whole cell extracts, cells were washed in ice-cold PBS and lysed in EMSA lysis buffer (150 mM NaCl/20 mM Tris HCl, pH 7.4/1 mM EDTA/10 mM Na3VO4/1 mM MgCl2/1% Nonidet P-40/1 mM phenylmethyl-sulfonyl fluoride/10% glycerol) on ice for 10 min. Cells and lysate were scraped with a sterile cell scraper, collected, and spun for 10 min at max at 4C on a benchtop centrifuge. Complimentary oligo- nucleotides that contained a Stats consensus binding site from the ,B-casein promoter (5'-AGATTTCTAGGAATTCAATCC- 3') were annealed and radiolabeled with ty-32PJATP by using T4 polynucleotide kinase (New England Biolabs). Approximately 20,000 cpm (0.2 ng) of probe was incubated with 20 ,ug of whole cell extract in 20 ,ul of 10 mM Hepes, pH 7.9/50 mM KCl/0.2 mM DTT/10% glycerol/0.05% Nonidet P-40/1 ,ug poly~dIdC) (Sigma) for 20 min at room temperature. Resulting DNA/ protein complexes were resolved on a 5% nondenaturing poly- acrylamide gel. EB Differentiation. ES cells were trypsinized, collected in EBD tIscove's modified Dulbecco's medium (IMDM)/15% IFS/200 ~g/ml iron-saturated transferrin (Sigma)/4.5 mM monothioglyc- erol (Sigma)/50 ,u~g/ml ascorbic acid (Sigma)/2 mM glutamine] and plated onto fresh T25 flasks (Corning) for 45 min to allow mouse embryonic fibroblasts to adhere. Nonadherent cells were collected and plated in hanging drops at 100 cells per 10-~l drop in an inverted bacterial Petri dish, and cultured for 2 days. They were then collected from the hanging drops and further cultured in 10 ml of EBD in slowly rotating 10-cm bacterial Petri dishes. At day 4, EBs were fed by exchanging half of their spent medium for fresh EBD. In some cultures, doxycycline was added at day 4 at 2 ,ug/ml to induce expression of Stat5CA. CFC Assay. Day-6 EBs were dissociated by trypsinization, col- lected, and resuspended in IMDM/10% IFS at a concentration of 5 x 105 cells per ml. A total of 100 ~l of this cell suspension Kyba et a/. was added to 1.5 ml of complete methylcellulose for murine colonies (StemCell Technologies, Vancouver, catalog no. 3434~. Methylcellulose suspension cultures were not supplemented with doxycycline. EryP colonies were counted on day 6 of methyl- cellulose culture, all other colonies were counted at day 10. OP9 Coculture. Day-6 EBs were trypsinized to a single cell suspension and plated on a semiconfluent monolayer of OP9 cells (a gift of T. Nakano, University of Osaka, Osaka) at a density of 200,000 cells per well of a six-well dish in IMDM, 10% IFS supplemented with 2 mM glutamine, 50 units/ml penicillin, 50 ,ug/ml streptomycin (GIBCO/BRL), 0.1 mM 2-mercapto- ethanol (Sigma), cytokines t40 ng/ml vascular endothelial growth factor (VEGF), 40 ng/ml thrombopoietin (TPO), 100 ng/ml stem cell factor (SCF), and 100 ng/ml Flt-3 ligandi, and doxycycline at 1 ~g/ml. When cells became confluent, they were passaged by trypsinization onto fresh OP9. Fluorescent Staining and FACS Analysis. Staining of day-6 EB cells. EBs were disaggregated by washing once with PBS followed by resuspension in 0.1% trypsin/PBS and pipetting for 30 s. Trypsin was blocked with IMDM/10% IFS, the cells were strained to remove clumps and collected by centrifugation. Annexin V- phycoerythrin staining was done at room temperature for 15 min according to the manufacturer's specifications (CLONTECH). After staining, cells were transferred to 4C, and 1 ~l of FITC-conjugated CD41 antibody was added. After 20 min, samples were diluted with annexin V binding buffer containing propidium iodide and analyzed by FACS. Staining of OP9 cultures. Cells were collected by trypsinization and resuspended in PBS containing 5% IFS. Samples of one million cells in 100 ,ul were blocked with 1 ,ul of Fc block (PharMingen) and stained with 1 ,ul of phycoerythrin- or FITC-conjugated antibody for 20 min at 4C. Samples were washed twice with PBS/5% IFS, and resuspended in PBS/5% IFS containing propidium iodide. All antibodies were purchased from Phar- Mingen. FACS analyses were performed on a Becton Dickinson FACSCalibur. Dead cells were excluded from phycoerythrin- stained cells by gating on FL2 vs. FL3. RT-PCR. The following primers were used: actin~f) 5'-GT- GGGGCGCCCCAGGCACCA-3'; actintr) 5'-CTCCTTAATGT- CACGCACGATTTC-3'; ,l3-Hl~f) 5'-AGTCCCCATGGAGT- CAAAGA-3'; b-Hltr) 5'-CTCAAGGAGACCTTTGCTCA-3'; PNAS | September 30, 2003 | vo~. 100 | supp~. ~ | 11905

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13-maj(f) 5'-CTGACAGATGCTCTCTTGGG-3'; `(3-maj(r) 5'- a 4o CACAACCCCAGAAACAGACA-3'. Cycle conditions were as follows: 2 min at 96C; 30 cycles of 45 s at 95C, 1 min at 60C, and ~ 35 45 s at 72C; and then 5 min at 72C. c' 30. o 25 To O 20 a) 15 Q At) 1 0 Retroviral Labeling with GFP. GFP retroviral supernatants were produced by FUGENE transfection of 293 cells with pMSC~i- resGFP (28) and pCL-Eco, a packaging-defective helper plasmid (294. 293 cells were grown in DME/10% IFS, and medium was replaced on the day after transfection Forty-eight hours after transfection, supernatants were collected, filtered, plated onto iStatSCA blast cells growing on OP9 at 3 ml per well of a six-well dish, supplemented with 4 ,ug/ml polybrene and cytokines (100 ng/ml SCF, 40 ng/ml VEGF, 40 ng/ml TPO, 100 ng/ml Flt-3 ligand), and spin-infected at 2,500 rpm for 90 min in a Beckman GS-6R centrifuge. After several days of growth, GFP-positive cells were separated by FACS and cultured on fresh OP9. Filtered supernatants from GFP-positive cells expanded on OP9 were tested for lateral transfer to 10T1/2 cells and found to be negative. Mouse Transplantation. Two- to four-month-old 12901a/Hsd (Harlan Breeders, Indianapolis) mice were exposed to 2 x 500 cGy of y-irradiation, separated by 4 h, and injected with 1.75 x 106 cells in 500 ,ul of IMDM/10% IFS via lateral tail vein. Mice that received doxycycline were provided drinking water supple- mented with 500 ,ug/ml doxycycline hydrochloride (Sigma) and 5% sucrose. Results Stats Signaling During EB Development. To generate an ES cell line with inducible StatS signaling, we made use of the Tet-On targeting cell line, Ainvl5 (27~. These cells constitutively express the reverse tetracycline transactivator from the ROSA26 locus, and have a tetracycline response element (TRE) integrated into the transcriptionally open chromatin 5' to the HPRT gene on the X chromosome. Downstream of the TRE is a single LoxP site, into which we integrated, by Cre-Lox recombination, the cDNA c for StatSCA, a constitutively active mutant of Stat5a (a double mutant of H299R and S711F, also known as 1*6) (12) (Fig. la). The resulting ES cell line, named iStatSCA, as well as its differentiated progeny, express this mutant cDNA when exposed to doxycycline. Expression results in binding of Stat5CA to DNA as measured by bandshifting activity against an oligonucleotide probe encoding a StatS DNA-binding consensus sequence, in the lysate of doxycycline-treated iStat5CA ES cells (Fig. lb). We used this cell line to generate EBs and applied doxycycline to the cultures for 48 h, from day 4 to day 6 of differentiation. This time window was chosen based on the kinetics of colony formation in EBs: the bipotent precursor to the hematopoietic and endothelial lineages, the hemangioblast, peaks at day 3.75 and is no longer present by day 5 (30), whereas hematopoietic CFC, particularly m~xed erythroid-myeloid colonies, first be- come detectable between days 5 and 6. Thus, StatS signaling was induced at the time of specification of the hematopoietic lineage. At day 6, the EBs were disaggregated into single cells and assayed for hematopoietic CFC content by plating in methylcel- lulose suspension medium with hematopoietic cytokines. As shown in Fig. 2a, exposure of EBs to doxycycline increased the numbers of all types of hematopoietic colonies assayed between 2- and 4-fold. To investigate the mechanism of StatS-mediated hematopoi- etic enhancement, we analyzed cells from doxycycline-treated or untreated day-6 EBs for apoptosis. Preliminary results (not shown) demonstrated that StatS activation modestly decreased the number of apoptotic (annexin V-positive) cells from day-6 EBs; however, a general reduction in apoptosis would increase CFC frequency only if the hematopoietic lineage were subject to 11906 1 www.pnas.org/cgi/doi/10.1073/pnas.1734140100 |ONO dox 1 1~ Dox d4-6 CD41 C No dox CD41 1 ' ._ y C~ . . . . . . . . CD41 CD41 Dox d4-6 Dox d4-6 1 11 0.5 Fig. 2. Effects of Stats signaling during EB differentiation. EBs were grown for 6 days, either exposed or not exposed to doxycycline from day 4 to day 6. (a) CFC assay: day-6 EB cells were disaggregated and plated into methylcellu- lose suspension culture with hematopoietic cytokines. Filled bars denote colony number from doxycycline-treated EBs; open bars denote colony num- ber from untreated EBs. Standard errors for three independent experiments are shown. (n = 3 for each bar; P < 0.05 for combined CFCs.) (b) Apoptosis assay: day-6 EB cells were disaggregated and stained with annexin V (yaxis) to label apoptotic cells and anti-CD41 (x axis) to label hematopoietic cells. The percentage of cells falling into single- and double-positive quadrants is shown. (c) Hematopoietic compartment quantitation: day-6 EB cells were disaggregated and stained with antibodies to c-Kit (y axis) and CD41 (x axis). The percentage of cells falling within the double-positive rectangular gate is shown. higher levels of apoptosis than the other nonhematopoietic lineages that arise in a day-6 EB. To assay the levels of apoptosis in hematopoietic vs. nonhematopoietic cells, we stained EB cells with both annexin V and a pan-hematopoietic antibody. Studies of adult hematopoiesis commonly use the CD45 antigen as a pan-hematopoietic marker; however, this marker is not univer- sally expressed by the earliest embryonic hematopoietic progen- itors. The recent discovery that the adult megakaryocytic anti- gen CD41 is actually pan-hematopoietic in very early embryos as well as in day-6 EBs (31, 32) prompted us to use this marker rather than CD45 for this purpose. To our surprise, this assay revealed that apoptosis in day-6 EBs is almost completely restricted to the nonhematopoietic population (Fig. 2b). Kyba et a/.

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a Q 10 He = ~ O a) ~ at_ ~ 6 in Vitro Growth Air 9 8 . h ~ ~ O O A ~ _ _I BOX - + IL-3 a" C Day I: ii I O. + DOX ., ... Fig. 3. Stats signaling promotes blast cell outgrowth. (a) Cumulative cell number from OP9 stromal cell cocultures initiated by 2 x 105 cells. (b) Stats DNA-binding activity was seen in OP9 cocultures of iStat5CA day-6 EB cells grown in the presence of doxycycline, but not in the residual growth that appeared in the absence of doxycycline. For comparison, the Stats bandshift from BaF/3 cells growing exponentially in the presence of IL-3, or BaF/3 cells infected with a MSCV retrovirus expressing Stat5CA are shown. Arrowhead denotes Stats-specific bandshift product. (c) Cytospin of iStat5CA day-6 EB cells expanded on OP9. Cells were spun onto glass slides and stained with Wright-Giemsa. An alternative to a reduction in apoptosis would be overpro- liferation of hematopoietic cells in response to StatS. Because hematopoietic CFCs from the day-6 EB are CD41 and c-Kit double positive (31, 32), we assayed the frequency of this population in the presence vs. absence of Stats induction (Fig. 2c). We observed only a very modest increase with doxycycline treatment, which was not sufficient to account for the increase in CFCs. We therefore conclude that StatS activation is either influencing development within the hematopoietic compart- ment, such that it contains a higher ratio of CFCs to more differentiated cells, or enhancing the clonogenicity of the CFCs that are present. Stats Signaling During OP9 Stromal Cell Coculture of Day-6 EB Cells in Vitro. Cells from day-6 EBs that had been exposed to doxycycline for 48 h were also plated on OP9 stromal cells with a cytokine mixture tailored for HSC expansion, consisting of TPO, SCF, Flt-3 ligand, and VEGF. In the absence of doxycycline, there was minimal growth, whereas in the presence of doxycycline, there was a dramatic expansion of a semiadherent cell type growing attached to the OP9 feeder layer. These semiadherent cells could be passaged by trypsinization and expanded exponentially (Fig. 3a). Whole cell protein extracts from cells growing on OP9 in the presence of doxycycline, but not in its absence, contained StatS-specific DNA-binding activity (Fig. 3b). The intensity of the StatS bandshift was similar to that observed in the pro-B cell line BaF/3 growing in the presence of IL-3, but much less than the bandshift observed in BaF/3 cells infected with a retrovirus expressing StatSCA (Fig. 3b). This demonstrates that the level of StatS activation achieved by exposure to doxycycline approxi- mates the physiological level that hematopoietic cells experience when growing in the presence of cytokines. The dominant cell type in the OP9 expansion cultures was a primitive hematopoietic blast; however, other cell types could also be detected, particularly differentiated myeloid cells (Fig. 3c). We analyzed these cells for surface antigen expression by flow cytometry (Fig. 4 a and b). Consistent with the blast cell morphology, the majority of cells were negative for markers of hematopoietic differentiation. Of those cells that were positive Kyba et a/. for lineage markers, the majority expressed the myeloid markers, Gr-1 and Mac-1, but a small number expressed markers of lymphoid (B220) and erythroid (Ter-119) differentiation. We observed strong positivity for the hematopoietic stem cell mark- ers c-Kit and Sca-1. The cells were negative for CD45 but positive for CD41, consistent with an early embryonic hematopoietic character, and the majority of both the c-Kit- and Sca-1-positive cells were double positive for CD41. The cells were also strongly positive for CD31, a marker displayed by hematopoietic stem cells, some differentiated hematopoietic cells, as well as endo- thelial cells. This expression is likely hematopoietic in origin as opposed to endothelial given the compression of CD41. This profile is suggestive of the expansion of an undifferentiated embryonic hematopoietic progenitor with many characteristics of the HSC, which undergoes concomitant differentiation mainly along the myeloid lineage in vitro. We assayed globin gene expression in these cells and com- pared it to that seen in a similar cell population obtained by expression of HoxB4, which we have previously described (27~. Whereas embryonic (,B-H1) globin is almost undetectable in the HoxB4-expanded cells, it is clearly present in those expanded by Stats. The embryonic globin signal is weak compared with adult (,B-major) globin; however, its presence suggests that StatS signaling does not efficiently drive primitive to definitive hema- topoietic switching in the same way that HoxB4 appears to. Stats-Dependent Engraftment in Vivo. To determine the capacity of these cells to undergo differentiation in salvo, they were marked with GFP by retroviral infection with the virus MSCViresGFP (28~. Cells were then injected into 10 irradiated isogenic recip- ient adult mice, in two independent experiments, and the peripheral blood was sampled periodically for GFP positivity. We observed no engraftment in mice not treated with doxycy- cline, even at time points as early as 2 weeks. However, when mice were fed drinking water supplemented with doxycycline, we observed transient donor cell contribution to the peripheral blood, liver, spleen, and marrow, which was exhausted by 8 weeks after transplantation. Although we observed contribution to the spleen, we did not observe donor-derived CFU-S. Engraftment was best when OP9 cocultures were injected as soon as sufficient PNAS I September30, 2003 1 vol. 100 1 supple. ~ I 11907

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CD4 0.5% .`,,.,,, I, ...... Ter-119 2.1% 66.71 ._ By ~ ' ~ .' ~ . : In m ~ PA O ~ 4 - M actor CD41 CD41 m U m ~ ~ ~ TIC 0 ~ O p-H1 p-maj CD31 50 7%1 Fig. 4. Characterization of Stats-induced blast cells growing on OP9. (a) Surface antigen expression. Day-6 iStat5CA EB cells expanded on OP9 were labeled with the antibodies indicated and analyzed by flow cytometry. Cell number is plotted on the y axis; fluorescence intensity is plotted on the x axis. The first plot, labeled "iso," is staining by an isotype control, nonspecific antibody. Percentages of cells positive for each marker are indicated. (b) Double staining. The same cells were costained with antibodies against CD41 (x axis) vs. c-Kit, Sca-1, or CD31 (y axis). The percentage of cells falling into each single- and double-positive quadrant is shown. (c) Globin gene expression. RNA was derived from cells expanded on OP9 with Stat5CA induction or with HoxB4-induction. RT-PCR for actin, f-H1 globin, and p-major globin was performed. M represents the molecular mass marker lane. cells were available, and was gradually lost with extensive passage in vitro. FACS analysis of the bone marrow of a typical recipient 1 month after transplantation is shown in Fig. 5. Although we observed low levels of GFP positive cells overall, they counter- stained with markers representing differentiation into all three hematopoietic lineages: lymphoid (B220 and CD4), myeloid (Gr-1 and Mac-1), and erythroid (Ter-119), demonstrating that these cells have a broad differentiation potential in vivo. In one case, we observed an animal succumb to a GFP-positive myeloid leukemia 2 months after transplant, suggesting that at some frequency, continual activation of Stats signaling can result in the eventual outgrowth of a malignant population, as has been seen after retroviral transduction of Stat5CA in bone marrow transplant models (334. However, because donor cells were eventually lost by the majority of recipients, our results demon- strate that this cell population has limited self-renewal potential in viva, even with maintenance of induction of Stats signaling. DIscusslon ES cells are competent to differentiate into cells of all embryonic and adult lineages, as evidenced by the derivation of chimeric animals from blastocysts injected with ES cells (344. This potency 11908 1 www.pnas.org/cgi/doi/10. 1 073/pnas. 1734140100 makes ES cells promising source material for regenerative medicine. However, putting this potential into practice in adults, as opposed to embryos, will require the derivation of adult- repopulating stem cells from ES cells in vitro. In the case of the hematopoietic system, this has proven to be more difficult than expected, given that ES cells will readily generate blood in vitro when differentiated as EBs (1~. ES cells seem predisposed to an embryonic mode of blood differentiation akin to that of the early extraembryonic yolk sac, producing mainly primitive erythro- cytes and myeloid progenitors (2, 35), but lacking adult- repopulating cells that are thought to arise in the embryo proper (36, 37~. The HSC for this primitive (embryonic) mode of hematopoi- esis seems to have the latent potential to generate definitive (adult) lineages. When adult engraftment is enabled by trans- formation with Bcr/Abl, an oncogene with specific growth- promoting effects on the HSC, contribution to these lineages can be observed (38~. Adult engraftment and multilineage hemato- poiesis has also been observed when EB-derived cells are made to overexpress the transcription factor HoxB4 (27~. This tran- scription factor has growth-promoting effects on the HSC similar to but less transforming than Bcr/Abl. HoxB4 also induces a switch in the expression pattern of several markers that distin- Kyba et a/.

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uninjected, iso n on to C) ~ ~ . , . , . . 10 10 1o2 103 1 4 Cal CON V' , \ so o ~ ISO 1 _ _ 1 ~ No 100 1o1 1o2 103 104 o o/o > GFP B220 At' I . . T I . ~ 1 . .... '.~1 . . ~ ~ 8 , 10 10 10 10- 10 Gr-1 o.5 onto .. .. .. ~ ~ ~ At ~ ~ - 1~ 1o1 1o2 103 104 o o/o it ~ 2 3 4 10 10 10 10 10 Mac- 1 .. i.... 0.6 onto Ter-119 ~ .~ ~ 0.6 GO 100 1 . , .l . . 4 100 1 1 1 o2 l o3 1 ( 14 Fig. 5. FACS analysis of bone marrow of a doxycycline-treated recipient mouse. Bone marrow cells were harvested 1 month after transplantation, stained with the indicated antibodies, and subjected to flow cytometry. GFP fluorescence is plotted on the x axis; antibody staining is plotted on the y axis. "iso" represents an isotype control nonspecific antibody. The upper left plot is from a control, uninfected mouse; all others are from the experimental mouse. In each plot, the percentage of double-positive cells is shown in the upper right quadrant. Double positives represent donor cells expressing a given antigen. guish primitive from definitive hematopoiesis, suggesting the possibility that it may promote a primitive-definitive switch at the level of the embryonic HSC. Under conditions that favor expansion of undifferentiated hematopoietic progenitors, namely coculture with OP9 stromal cells, and the cytokines SCF, TPO, Flt-3 ligand, and VEGF, maintenance of Stats signaling in day-6 EB cells enables the outgrowth of a very primitive cell population expressing many of the markers of the HSC. The levels of Stats activation in these cells are typical of those seen in hematopoietic cells activating Stats for physiological reasons, such as growing in the presence of IL-3. This finding implies that the effects we have observed are not caused by superphysiological levels of StatS activating spu- rious target genes. Cells expanded by Stats activation have similarities to both the Bcr/Abl-expanded cells and the HoxB4- expanded cells that we have previously described. Morphologi- cally, all three populations consist mainly of undifferentiated blast cells. By analysis of surface marker expression, the StatS- expanded cells described here are closer in type to those expanded by HoxB4, in particular in their expression of CD31, CD41, and c-Kit, and in the presence of differentiating cells expressing myeloid lineage markers. The Bcr/Abl-expanded cells were negative for all lineage markers tested, although we did observe lineage marker expression on some cells in the earliest phase of expansion. It may be the case that Bcr/Abl drives expansion of the most primitive cells more efficiently, and is better able to suppress their differentiation in vitro. In contrast to the HoxB4-expanded cells, the in viva engraftment potential of the Stats-expanded cells is quite distinct. Their engraftment is strictly doxycycline-dependent, and temporally limited, whereas HoxB4-expanded cells engraft without the need for maintenance of HoxB4 expression in vivo, and readily contribute to long-term hematopoiesis. It may be the case that Stats is driving self-renewal of lineage-committed progenitors or possi- bly short-term-repopulating HSCs in vitro, as opposed to long- Kyba et a/. term-repopulating HSCs. Given their maintenance of embryonic globin expression, it is also likely that the Stats-expanded cells are not undergoing a primitive-definitive hematopoietic switch, and that their inability to reconstitute adult hematopoiesis is reflective of their similarity to the earliest hematopoietic pro- genitors of the yolk sac, which suffer a similar defect in adult repopulation. Although genetic modification can enable engraftment, it should in principle be possible to guide the differentiation of unmodified ES cells into definitive HSCs. To achieve this, it will be necessary to recapitulate, in a temporally appropriate man- ner, all of the extracellular signals that an ES cell and its lineage-restricted progeny experience from the point of blasto- cyst injection to the point of differentiation to fetal liver-stage HSC. This is a daunting task, not only because of the large number of known extracellular signaling molecules and combi- natorial possibilities, but also because the relevant molecules may not be known at present. Given that many extracellular signaling molecules share common intracellular mediators, the endeavor may be simplified by focusing on these signal trans- ducers. Focusing on individual mediators has the additional advantage of bypassing or deconvoluting the complexity of cell surface receptor-mediated signaling, in which multiple pathways are commonly activated by binding of a single ligand. For example, signaling by the leukemia inhibitory factor receptor promotes ES cell self-renewal through activation of Stat3 (39~; however, it also activates the mitogen-activated protein kineses, ERK1 and ERK2, resulting a counteractive signal that attenu- ates self-renewal (40~. By interfering with or activating individual pathways, unique outcomes can be selected from the manifold of effects initiated by receptor activation. We chose to study the effects of StatS activation on the process of hematopoietic differentiation of ES cells, in part because StatS is a major downstream mediator of Bcr/Abl signaling, which we have previously shown induces the expansion of an PNAS | September 30, 2003 | vol. 100 | supple. ~ | 11909

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~ -I' embryonic hematopoietic stem cell population from EBs (38), and in part because of the critical role that Stats plays in normal hematopoiesis by acting to transduce signals from a variety of cytokine receptors. Our results demonstrate that at the point of origin of the hematopoietic lineage, day 4 to day 6 of EB differentiation, the precursors of this lineage are competent to respond to Stats activation. We have determined that the enhanced hematopoiesis driven by Stats is not the result of the rescue of hematopoietic precursors that were destined for apo- ptosis, nor is it the result of over proliferation of the hemato- poietic compartment. An attractive possibility is that Stats modulates the rate of progression from stem cell to committed progenitor (CFC) to differentiated progeny within the early embryonic hematopoietic compartment such that stem cells and progenitors accumulate to greater numbers in the presence of . . slgna 1ng. The fact that Stats null embryos generate hematopoietic tissue means that Stats is not essential for hematopoietic development (15~. However, the fetal anemia seen in these embryos, which has been attributed to a defect in erythropoietin signaling (14) might also be due in part to reduction of the stem cell pool in the absence of Stats. It is noteworthy that StatS functions in a partially redundant manner in the regulation of the definitive HSC: Stats null HSCs are capable of repopulating irradiated recipients, but they are at a tremendous disadvantage when placed into competition with wild-type HSCs. If Stats has a 1. Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W. & Kemler, R. (1985) J. Embryol. Exp. Morphol. 87, 27-45. 2. Keller, G., Kennedy, M., Papayannopoulou, T. & Wiles, M. V. (1993) Mol. Cell. Biol. 13, 473-486. 3. Porcher, C., Swat, W., Rockwell, K., Fujiwara, Y., Alt, F. W. & Orkin, S. H. (1996) Cell 86, 47-57. 4. North, T., Gu, T. 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