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Research Article 1445 Introduction Human pluripotent stem cells (hPSCs), encompassing human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the unique capacity to give rise to all cell types of the body, including hematopoietic cells (Murry and Keller, 2008). Thus, hPSCs have been suggested as an alternative source of cells for hematopoietic cell-based therapies, instead of adult sources such as umbilical cord blood (UCB), bone marrow and mobilized peripheral blood, for in vivo reconstitution of the hematopoietic system (Bhatia, 2007). The recently discovered hiPSCs, possessing pluripotent capacity, have potential advantages over hESCs, owing to their immunological competence for use in an autologous transplant setting (Takahashi et al., 2007; Yu et al., 2007). However, specifying hematopoietic differentiation from either hESCs or hiPSCs remains the major barrier in the therapeutical use of hematopoietic cells. Although several factors that influence both the emergence of hemogenic cells from hPSCs and the subsequent hematopoietic development have been identified (Chadwick et al., 2003; Vijayaragavan et al., 2009; Wang et al., 2004), specific pathways and genes responsible for inhibiting, in contrast with inducing, hematopoietic development, have yet to be identified and investigated. Hematopoietic development is a complex and highly coordinated process that is controlled by spatial and temporal expression of a plethora of genes involved in specification and differentiation of blood cells (Orkin and Zon, 2008). Inhibitor of differentiation (ID) genes are helix-loop-helix (HLH) transcription factors and are ubiquitously expressed in many tissues (Benezra et al., 1990; Yokota, 2001). Mechanistically, ID proteins act as dominant- negative regulators of transcription factors and form complexes with ubiquitous E proteins (E2A, HEB and E2-2), preventing E protein homodimerization or heterodimerization with tissue- restricted basic HLH proteins, leading to cell cycle promotion and disparate regulation of terminal differentiation in various cell types (Jen et al., 1992; Norton et al., 1998). ID genes are constitutively expressed in murine and human hematopoietic cells (Buitenhuis et al., 2005; Cooper et al., 1997; Jankovic et al., 2007; Kreider et al., 1992; Leeanansaksiri and Dechsukhum, 2006; Lister et al., 1995; Martin et al., 2008). Previous gain-of-function studies have revealed that ectopic expression of ID1 enhanced myeloid and neutrophil differentiation in mouse hematopoietic stem cells (HSCs) and CD34+ cells from human UCB, respectively (Buitenhuis et al., 2005; Kreider et al., 1992). Moreover, downregulation of ID1 in mouse erythroleukemia cells inhibited terminal erythroid differentiation (Lister et al., 1995). These studies demonstrated that early hematopoietic commitment and differentiation towards myeloid and erythroid lineages could be regulated by ID proteins. ID genes are known to be direct targets of bone morphogenetic protein 4 (BMP4). BMP4 has been shown to regulate mesoderm patterning and, hence, blood development in mammals (Hollnagel et al., 1999; Miyazono and Miyazawa, 2002; Sadlon et al., 2004), and is known to promote hematopoietic differentiation of hESCs. Most interestingly, BMP4, independently of hematopoietic cytokines, supports the maintenance of primitive progenitors with Summary Mechanisms that govern hematopoietic lineage specification, as opposed to the expansion of committed hematopoietic progenitors, from human pluripotent stem cells (hPSCs) have yet to be fully defined. Here, we show that within the family of genes called inhibitors of differentiation (ID), ID1 and ID3 negatively regulate the transition from lineage-specified hemogenic cells to committed hematopoietic progenitors during hematopoiesis of both human embryonic stem cells (hESCs) and human induced pluripotent stem cell (hiPSCs). Upon hematopoietic induction of hPSCs, levels of ID1 and ID3 transcripts rapidly increase, peaking at the stage of hemogenic precursor emergence, and then exclusively decrease during subsequent hematopoietic commitment. Suppression of ID1 and ID3 expression in hemogenic precursors using specific small interfering RNAs augments differentiation into committed hematopoietic progenitors, with dual suppression of ID1 and ID3 further increasing hematopoietic induction compared with upon knockdown of each gene alone. This inhibitory role of ID1 and ID3 directly affects hemogenic precursors and is not dependent on non-hemogenic cells of other lineages within developing human embryoid bodies from hESCs or hiPSCs. Our study uniquely identifies ID1 and ID3 as negative regulators of the hPSC–hematopoietic transition from a hemogenic to a committed hematopoietic fate, and demonstrates that this is conserved between hESCs and hiPSCs. Key words: Human embryonic stem cell, Human induced pluripotent stem cell, ID, Hematopoiesis Accepted 17 December 2010 Journal of Cell Science 124, 1445-1452 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.077511 ID1 and ID3 represent conserved negative regulators of human embryonic and induced pluripotent stem cell hematopoiesis Seok-Ho Hong*, Jong-Hee Lee*, Jung Bok Lee, Junfeng Ji and Mickie Bhatia McMaster Stem Cell and Cancer Research Institute, Faculty of Health Sciences, McMaster University, 1200 Main Street West, MDCL 5029, Hamilton, ON L8N 3Z5, Canada *These authors contributed equally to this work Author for correspondence ([email protected]) Journal of Cell Science

ID1 and ID3 represent conserved negative regulators of ...Suppression of ID1 and ID3 expression in hemogenic precursors using specific small interfering RNAs augments differentiation

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Research Article 1445

IntroductionHuman pluripotent stem cells (hPSCs), encompassing humanembryonic stem cells (hESCs) and human induced pluripotentstem cells (hiPSCs), have the unique capacity to give rise to all celltypes of the body, including hematopoietic cells (Murry and Keller,2008). Thus, hPSCs have been suggested as an alternative sourceof cells for hematopoietic cell-based therapies, instead of adultsources such as umbilical cord blood (UCB), bone marrow andmobilized peripheral blood, for in vivo reconstitution of thehematopoietic system (Bhatia, 2007). The recently discoveredhiPSCs, possessing pluripotent capacity, have potential advantagesover hESCs, owing to their immunological competence for use inan autologous transplant setting (Takahashi et al., 2007; Yu et al.,2007). However, specifying hematopoietic differentiation fromeither hESCs or hiPSCs remains the major barrier in thetherapeutical use of hematopoietic cells. Although several factorsthat influence both the emergence of hemogenic cells from hPSCsand the subsequent hematopoietic development have been identified(Chadwick et al., 2003; Vijayaragavan et al., 2009; Wang et al.,2004), specific pathways and genes responsible for inhibiting, incontrast with inducing, hematopoietic development, have yet to beidentified and investigated.

Hematopoietic development is a complex and highly coordinatedprocess that is controlled by spatial and temporal expression of aplethora of genes involved in specification and differentiation ofblood cells (Orkin and Zon, 2008). Inhibitor of differentiation (ID)genes are helix-loop-helix (HLH) transcription factors and are

ubiquitously expressed in many tissues (Benezra et al., 1990;Yokota, 2001). Mechanistically, ID proteins act as dominant-negative regulators of transcription factors and form complexeswith ubiquitous E proteins (E2A, HEB and E2-2), preventing Eprotein homodimerization or heterodimerization with tissue-restricted basic HLH proteins, leading to cell cycle promotion anddisparate regulation of terminal differentiation in various cell types(Jen et al., 1992; Norton et al., 1998). ID genes are constitutivelyexpressed in murine and human hematopoietic cells (Buitenhuis etal., 2005; Cooper et al., 1997; Jankovic et al., 2007; Kreider et al.,1992; Leeanansaksiri and Dechsukhum, 2006; Lister et al., 1995;Martin et al., 2008). Previous gain-of-function studies have revealedthat ectopic expression of ID1 enhanced myeloid and neutrophildifferentiation in mouse hematopoietic stem cells (HSCs) andCD34+ cells from human UCB, respectively (Buitenhuis et al.,2005; Kreider et al., 1992). Moreover, downregulation of ID1 inmouse erythroleukemia cells inhibited terminal erythroiddifferentiation (Lister et al., 1995). These studies demonstratedthat early hematopoietic commitment and differentiation towardsmyeloid and erythroid lineages could be regulated by ID proteins.ID genes are known to be direct targets of bone morphogeneticprotein 4 (BMP4). BMP4 has been shown to regulate mesodermpatterning and, hence, blood development in mammals (Hollnagelet al., 1999; Miyazono and Miyazawa, 2002; Sadlon et al., 2004),and is known to promote hematopoietic differentiation of hESCs.Most interestingly, BMP4, independently of hematopoieticcytokines, supports the maintenance of primitive progenitors with

SummaryMechanisms that govern hematopoietic lineage specification, as opposed to the expansion of committed hematopoietic progenitors,from human pluripotent stem cells (hPSCs) have yet to be fully defined. Here, we show that within the family of genes called inhibitorsof differentiation (ID), ID1 and ID3 negatively regulate the transition from lineage-specified hemogenic cells to committed hematopoieticprogenitors during hematopoiesis of both human embryonic stem cells (hESCs) and human induced pluripotent stem cell (hiPSCs).Upon hematopoietic induction of hPSCs, levels of ID1 and ID3 transcripts rapidly increase, peaking at the stage of hemogenicprecursor emergence, and then exclusively decrease during subsequent hematopoietic commitment. Suppression of ID1 and ID3expression in hemogenic precursors using specific small interfering RNAs augments differentiation into committed hematopoieticprogenitors, with dual suppression of ID1 and ID3 further increasing hematopoietic induction compared with upon knockdown of eachgene alone. This inhibitory role of ID1 and ID3 directly affects hemogenic precursors and is not dependent on non-hemogenic cellsof other lineages within developing human embryoid bodies from hESCs or hiPSCs. Our study uniquely identifies ID1 and ID3 asnegative regulators of the hPSC–hematopoietic transition from a hemogenic to a committed hematopoietic fate, and demonstrates thatthis is conserved between hESCs and hiPSCs.

Key words: Human embryonic stem cell, Human induced pluripotent stem cell, ID, Hematopoiesis

Accepted 17 December 2010Journal of Cell Science 124, 1445-1452 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.077511

ID1 and ID3 represent conserved negative regulatorsof human embryonic and induced pluripotent stemcell hematopoiesisSeok-Ho Hong*, Jong-Hee Lee*, Jung Bok Lee, Junfeng Ji and Mickie Bhatia‡

McMaster Stem Cell and Cancer Research Institute, Faculty of Health Sciences, McMaster University, 1200 Main Street West, MDCL 5029,Hamilton, ON L8N 3Z5, Canada*These authors contributed equally to this work‡Author for correspondence ([email protected])

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enhanced self-renewal capacity in differentiating hESCs (Chadwicket al., 2003). Collectively these studies suggest ID genes might benecessary for hematopoietic development, underscoring theirpotential importance in hPSC hematopoiesis that has not beenexamined to date.

Here, we reveal a distinct temporal expression of ID1 and ID3during hematopoietic specification compared with hematopoieticcommitment, and demonstrate that ID1 and ID3 act as directnegative regulators of the hemogenic precursor transition to thehematopoietic lineage in both hESCs and human fibroblast-derivediPSCs.

ResultsBMP4 augments hematopoietic differentiation of hPSCsPrevious studies have demonstrated that BMP4 promoteshematopoietic differentiation from hESCs (Chadwick et al., 2003;Pick et al., 2007). To characterize further the cellular response ofBMP4 on hESC-derived hematopoiesis, we first examined thespatiotemporal expression of the putative BMP4 receptor ALK3(officially known as ACVR1B), in human embryoid bodies (hEBs)grown under hematopoietic-inducing culture conditions. Aspreviously established (Vijayaragavan et al., 2009), hierarchicalstages of hematopoiesis from hPSCs can be divided into twophases of hEB development: phase I (day 0–7) is characterized bythe emergence and specification of bipotential hemogenicprecursors (CD45negPFV); phase II (day 7–15) is the commitmentphase, characterized as the period where committed hematopoieticprogenitors (CD34+ CD45+) and mature hematopoietic (CD34–

CD45+) cells are detected (Fig. 1A). Expression of ALK3 wasmaintained during both hemogenic specification and subsequenthematopoietic commitment (Fig. 1B). Both CD31 (a hemogenicprecursor marker) and CD45 (a committed hematopoieticprogenitor marker) were colocalized with ALK3-positive cellswithin hEBs (Fig. 1Bi–iii). This suggests that the majority of cellswithin the differentiating hEBs, which express ALK3, could bereceptive to BMP4 treatment and subsequent mesodermaldifferentiation.

To demonstrate the specific role of BMP4 during hematopoieticdifferentiation from hESCs, we repressed the effect of BMP4through its natural antagonist noggin. BMP4-treated hEBs exhibiteda 4-fold increase in hemogenic precursors (Fig. 1C) and a 3.5-foldincrease in hematopoietic progenitors (Fig. 1D), indicating thatBMP4 treatment augments hematopoietic output. Treatment ofhEBs continuously with noggin from day 0 to day 15 had no effecton the output of hemogenic precursors or committed hematopoieticcells in the absence of BMP4 (Fig. 1C–E). However, the enhancedhematopoietic differentiation induced by BMP4 addition wasdramatically inhibited by noggin treatment (Fig. 1C–E).Collectively, the results indicate that exogenous BMP4 augmentshematopoietic differentiation, but that hematopoietic output mightnot be dependent on endogenous BMP4 in hEBs given that addingnoggin in the differentiation system without exogenous BMP4 hadno effect on blood development.

Recent studies have shown that, similar to hESC-derivedhematopoietic cells, hiPSC-derived cells can also gave rise to alltypes of hematopoietic colonies (Choi et al., 2009). Given the role

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Fig. 1. Effect of BMP4 in hPSC-derived hematopoiesis.(A)Schematic diagram of hematopoietic development fromhPSCs, divided into two phases: phase I (day 0–7),characterized by specification of hPSCs as hemogenicprecursors (CD45negPFV), and phase II (day 7–15), thecommitment phase, defined as the period where committedhematopoietic progenitors (CD34+ CD45+ or CD34– CD45+)are detected. (B)Spatiotemporal expression pattern of ALK3(red) in hESC-derived hEBs developed under hematopoieticculture conditions. hEBs were dual-immunostained to detectcoexpression of ALK3 with CD31 (green, i and ii) or ALK3with CD45 (green, iii and iv). hEBs were harvested at theindicated days (d) and cryosectioned. Scale bars: 500m.(C–E) Effects of BMP4 on the production efficiency ofhemogenic precursors (C), hematopoietic progenitors (D) andmature hematopoietic cells (CD34– CD45+) (E) from hESC-derived hEBs at day 15. hEBs were cultured in hEBdifferentiation medium alone or supplemented with differentcombinations of hGFs, BMP4 and noggin. All error barsindicate mean+s.d. from hESCs from both H1 and H9 linesused for these experiments. (F)Effects of BMP4 on thepercentage of hemogenic precursors, hematopoieticprogenitors and mature hematopoietic cells from dissociatedhiPSC-derived hEBs at day 15 of hematopoieticdifferentiation. (G)Total cell number of differentiated hiPSCs.(H)Effects of BMP4 on total number of hemogenicprecursors, hematopoietic progenitors and maturehematopoietic cells at day 15 of hematopoietic differentiation.hEBs were cultured in hEB differentiation medium alone orsupplemented with different combinations of hGFs andBMP4. a* highlights the difference between BMP4supplement or not in the presence of hGFs (P<0.05). Allresults are means+s.d. *P<0.05; **P<0.01 (compared withthat in hEB differentiation medium alone).

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of BMP4 in inducing hematopoietic differentiation from hESCs,we examined whether its functional role was conserved in human-fibroblast-reprogrammed iPSCs. Using a hiPSC line (MchiPSC1.1)established and characterized in our laboratory by transduction ofhuman dermal fibroblasts with OCT4, SOX2, NANOG and LIN28(supplementary material Fig. S1), we examined the role of BMP4in hiPSC-derived hematopoiesis. We found that BMP4 had noeffect on the number of hemogenic precursors at day 15 ofdifferentiation, independent of the inclusion of hematopoieticgrowth factors (hGFs) (Fig. 1F). However, treatment with BMP4or BMP4 plus hGFs substantially increased the proportion ofcommitted hematopoietic cells compared with that of the control(Fig. 1F). Although the total number of cells within hEBs was notinfluenced by the addition of hGFs and BMP4 (Fig. 1G), the totalyield of committed hematopoietic cell types was substantiallyhigher in hEBs treated with BMP4 and BMP4 plus hGFs comparedwith that of the control (Fig. 1H). Addition of BMP4 plus hGFsfurther augments hematopoietic differentiation (Fig. 1F,H),indicating a specific role for BMP4 in hematopoietic commitment.Overall, these data suggested a conserved role for BMP4 in hESCs-and hiPSC-derived hematopoiesis.

Next, we treated hEBs with BMP4 at specific phases in order toexamine whether BMP4 is effective at a specific phase ofhematopoietic specification and commitment. Treatment withBMP4 at phase I or phase II of hEB development revealed thatBMP4 increased both hemogenic precursor and committedhematopoietic cells only when present during the early commitmentphase I (supplementary material Fig. S2). However, the enhancedeffect on hematopoiesis observed upon BMP4 addition wascompletely blocked by addition of its natural antagonist noggin,suggesting a direct function of BMP4 on hEB development,especially during phase I. These results indicate that the BMP4acts on hematopoietic differentiation by targeting cells at hemogenicspecification phase I. Collectively, these data underscore theimportance of BMP4 signals, and its downstream targets, duringhematopoietic differentiation from hPSCs, thus prompting us toexamine the expression profile of BMP4-targeted ID genes duringhPSC-derived hematopoietic development.

Differential regulation of ID1 and ID3 transcripts betweentwo hematopoietic phases of hPSCsAs ID genes are known to be direct targets of BMP4, a ventralmesoderm inducer that promotes hematopoietic differentiation ofhESCs (Chadwick et al., 2003; Hollnagel et al., 1999; Miyazonoand Miyazawa, 2002), we investigated the temporal expression ofthe ID family of genes (ID1–ID4) during this process. We initiallyexamined the expression profile of ID genes in undifferentiatedhESCs and hiPSCs to determine the baseline expression. BothhESCs and hiPSCs exhibited identical expression profiles for theID genes; in the undifferentiated state higher levels of ID3 transcriptwere observed with no detectable expression of ID4 (Fig. 2A,B).Upon differentiation of hESCs and hiPSCs under hematopoieticculture conditions, OCT4 expression rapidly decreased, whereasID1 and ID3 transcript levels increased and peaked at day 7,coincidental to the hemogenic specification phase (phase I; days0–7) (Fig. 2C,D; supplementary material Fig. S3A). ID2 expressionwas also increased in the initial 7 days of differentiation; however,during the subsequent 7 days, its levels were constant, indicatingthat ID2 might represent a general marker of differentiation thatis expressed independently of the hematopoietic lineage. Bycontrast, ID1 and ID3 transcripts were downregulated during the

hematopoietic commitment phase (phase II; days 7–15) (Fig. 2C,D).To examine whether the ID genes are regulated by BMP4, thetranscripts levels of ID genes were determined upon supplement ofBMP4 without hGFs. The expression profiles of the ID genes weresimilar to that with BMP4 plus hGFs (Fig. 2C,D; supplementarymaterial Fig. S3B), suggesting that the association of BMP with IDtranscription during hematopoietic differentiation. Interestingly,ID4 expression was not detectable in either undifferentiated hPSCsor during hematopoietic development (Fig. 2A–D; supplementarymaterial Fig. S3), suggesting that ID4 is not involved with hESCself-renewal or differentiation. The differential expression profile

1447ID genes regulate hPSC-derived hematopoiesis

Fig. 2. Temporal expression patterns of ID family genes during hPSC-derived hematopoietic development. (A,B)Quantitative RT-PCR todetermine the expression profile of ID family genes in undifferentiated hESCs(A, black bars) and hiPSCs (B, green bars). The expression of ID genes wasnormalized to GAPDH levels. (C,D)Temporal expression patterns of ID andOCT4 transcripts during the hematopoietic differentiation timeline. hEBs wereharvested on the indicated day (D4, 7, 10 or 15) and analyzed by quantitativeRT-PCR. Expression of ID genes was calculated relative to their levels inundifferentiated (UN) hESCs (C, black lines) and hiPSCs (D, green lines).Results are mean±s.d. for three independent experiments. ND, not detected.*P<0.05 for the increase in ID genes expression compared with the levels inundifferentiated hESCs. (E–G) Hematopoietic lineage subsets were isolatedfrom hEBs at day (d) 10, 15 and 20 of hematopoietic differentiation. Relativelevels of ID1 and ID3 transcripts in hematopoietic precursors (E),hematopoietic progenitors (F) and mature hematopoietic cells (G) comparedwith that in the remaining non-hemogenic hEB cells (broken horizontal line).Results are means+s.d. for three independent experiments.

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of ID1 and ID3 implies that these genes are uniquely regulatedduring the specification compared with the commitment phase inboth hESC- and hiPSC-derived hematopoiesis.

To determine whether changes in ID transcript levels areassociated with the hematopoietic commitment of hPSCs, wecompared the levels of ID1 and ID3 transcripts betweenhematopoietic lineage populations and the remaining unspecified(non-hematopoietic) cells. Hemogenic precursors, hematopoieticprogenitors and mature hematopoietic cells, along with theremaining hEB cells, were isolated from day 10, 15 and 20 hEBs.Analysis of highly purified subsets of different cell populationswithin hEBs revealed that the levels of ID1 and ID3 transcriptswere diminished in hemogenic precursors and committedhematopoietic cells (Fig. 2E–G). In day 15 hEBs, although an~50% decrease of ID1 and ID3 transcripts was seen in hemogenicprecursors in comparison with that in the remaining hEB cells, a80% decrease was seen in committed hematopoietic cells. Thesedata indicate downregulation of ID1 and ID3 transcripts inhematopoietic populations and suggest that ID1 and ID3 mighthave unique regulatory roles during hematopoietic commitment ofhPSCs.

ID1 and ID3 act as negative regulators during thehematopoietic commitment phase of hESCs and hiPSCsOn the basis of their distinct temporal expression patterns andeffect on hematopoietic differentiation, we aimed to functionallyevaluate the role of ID1, ID2 and ID3 in hESC-derivedhematopoiesis by using small interfering RNA (siRNA) to silenceexpression at two periods of hEB hematopoietic development (day0–2 and day 8–10) (Fig. 3A; supplementary material Fig. S4).Specific siRNA-mediated downregulation of ID1, ID2 and ID3resulted in a 30–40% decrease in the level of the transcripts withoutchanging the total cell number and viability of hEBs (Fig. 3B,C;supplementary material Fig. S4A). Knockdown using siRNAsagainst ID1, ID2 and ID3 at the early hEB stage (day 0–2) had noeffects on the percentage of either hemogenic precursors or

committed hematopoietic cells at day 15 of differentiation(supplementary material Fig. S4B), indicating that either hemogenicspecification or subsequent hematopoietic commitment wasrestrained by the expression of ID genes during the hemogenicspecification phase (phase I). However, treatment with siRNAsagainst ID1 and ID3 at the late hEB stage (day 8–10; phase II)resulted in a substantial increase in the generation of maturehematopoietic cells, whereas ID2 knockdown had no effects on thepercentage of either hemogenic precursors or committedhematopoietic cells (Fig. 3D). These results provide functionalevidence for the active role of ID1 and ID3 as negative regulatorsduring hESC hematopoiesis and indicate that they function duringthe commitment phase of hEB hematopoietic maturation. Dualsuppression of ID1 and ID3 at phase II further increasedhematopoietic development from hPSCs (supplementary materialFig. S5A). Western blotting analysis further verified that siRNAeffectively downregulated ID transcription and resultant proteinexpression as compared with when using a scrambled control(supplementary material Fig. S5B). These data suggest that ID1and ID3 might regulate commitment through unique targets andpathways of hematopoietic maturation.

On the basis of the observed effects of single and dual siRNAknockdown of ID1 and ID3 during hematopoietic commitment inhESCs, we similarly treated hiPSC-derived hEBs at phase II ofdifferentiation (day 8–10; Fig. 3E–G) with siRNA against ID1,ID2 and ID3. As observed using hESCs, siRNA treatment decreasedthe level of the transcripts by 30–40% in the hiPSC-derived hEBswithout changing the total cell number and viability of the hEBs(Fig. 3E,F). Downregulation of ID1 and ID3 resulted in an increasein the generation of committed hematopoietic (CD34+ CD45+ andCD34–CD45+) cells, whereas ID2 knockdown had no effect (Fig.3G). These results reveal that the negative regulatory role of ID1and ID3 during hematopoietic commitment phase is conserved inboth hESCs and hiPSCs.

Using the colony forming unit (CFU) assay for primitivehematopoietic cells as a measure of functional progenitors, we

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Fig. 3. siRNA treatment targeting ID1 and ID3transcripts results in an increase in committedhematopoietic cells. (A)Schematic diagram showing theknockdown of ID transcripts during the transitionbetween bipotent hemogenic precursor emergence andhematopoietic commitment, and hematopoietic analysis.(B–G) siRNA targeting ID1, ID2 and ID3 transcripts inhESC- (black bars) and hiPSC- (green bars) derived EBs.Knockdown efficiencies of ID transcripts are shown in Band E. Changes in total cell number and viability uponsiRNA treatment are shown in C and F. Knockdown ofID1 and ID3 transcripts between day 8 and 10significantly increased the output of committedhematopoietic cells from hESCs (D) and hiPSCs (G).Results are means+s.d. for three independentexperiments. The results are expressed as the levelrelative to that in control siRNA-treated hEBs (dashedhorizontal line).

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evaluated the multi-lineage potential of the hematopoieticprogenitors from differentiating hESC or hiPSC-derived hEBs.siRNA knockdown of ID1 or ID3 in hESC- or hiPSC-derivedhEBs, and subsequent isolation of the hematopoietic progenitors(CD34+ CD45+ cells), resulted in a substantially higher totalnumber of CFUs produced by CD34+ CD45+ cells compared withthat by control cells. ID2 siRNA did not affect the CFU output(Fig. 4A,B). Furthermore, ID gene knockdown did not have aneffect on the distribution of CFU types generated from eitherhESCs- or hiPSCs-derived hematopoietic progenitors (Fig. 4C–E),suggesting the effects of ID genes are not at the level ofhematopoietic lineage development, but at the point of cell fatecommitment to the hematopoietic lineage.

The negative regulatory role of ID1 and ID3 is exclusivelyrestricted to hemogenic populationsOur observations indicating enhanced hematopoietic differentiationupon suppression of ID1 and ID3 expression was limited to analysisof whole hEBs, and therefore it remains unclear whether thenegative regulatory function of ID1 and ID3 is exclusively restrictedto direct effects on hemogenic precursors or whether they actindirectly on cells comprising hEBs. Accordingly, we prospectivelyisolated hemogenic precursors and the remaining non-hemogeniccells from hESC- and hiPSC-derived hEBs (Fig. 5A,B). As thebipotent precursors emerge between days 8–10 of hEBhematopoietic differentiation (Wang et al., 2004), we isolated cellsfrom day 12 hEBs. These subsets were then treated with ID1 and

ID3 siRNAs, and effects on hematopoietic output were measured.Treatment with siRNAs decreased ID1 and ID3 expression by 40–50% in the sorted subsets of cells (Fig. 5C). Notably, knockdownof ID1 and ID3 in hemogenic precursors resulted in substantialincreases in the generation of committed hematopoietic cells,whereas downregulation of ID1 and ID3 in the remaining controlnon-hemogenic cells had no effect, as expected (Fig. 5D). Theseresults demonstrate that the negative regulatory role of ID1 andID3 is direct and restricted to hemogenic precursors committedtowards the hematopoietic lineage, and that it is not dependent onthe presence of non-hemogenic cells in whole hEBs.

DiscussionIdentification of the cellular and molecular processes regulatinghematopoietic development in vitro from hPSCs is an importantbasis for understanding complicated in utero lineage specificationand applications of cell-based therapies. Our current study revealsthat ID1 and ID3 regulate hematopoietic commitment, and that thisregulation is under stringent temporal regulation by extrinsicstimuli, such as BMP4 and growth factors. hPSC-derivedhematopoiesis can be divided into two developmental phases,hemogenic specification and hematopoietic commitment, whichare temporally regulated by distinct signaling factors (Vijayaragavanet al., 2009). Using hierarchical stages of blood development fromhPSCs, our study reveals that BMP4 affected hematopoieticdifferentiation by targeting cells during hemogenic specificationand demonstrates a unique negative regulatory role for ID1 andID3 during hematopoietic commitment from hPSCs.

ID proteins are know to act as a negative regulators by blockingDNA binding by transcription factors and the downstream activityof basic HLH proteins, and ID proteins are required for maturationand terminal differentiation in various cell types (Yokota, 2001).This inhibitory function of ID genes has been identified duringboth lymphoid and myeloid lineage commitment in adult humanHSCs (Buitenhuis et al., 2005; Cooper et al., 1997; Jankovic et al.,2007; Kreider et al., 1992; Leeanansaksiri and Dechsukhum, 2006;Lister et al., 1995). More specifically, during hPSC cell fateregulation, ID genes have been shown to negatively regulatedevelopment of T cells from hESC-derived hematopoieticprogenitors on the basis of constitutive expression of ID genesduring differentiation (Martin et al., 2008). On the basis of ourresults, we propose a model to illustrate the role of ID1 and ID3in the context of hPSC-hematopoietic development (Fig. 5E).

In both hESCs and hiPSCs, the regulatory role of ID1 and ID3was restricted to the commitment phase of blood development(from hemogenic to hematopoietic progenitors) (Fig. 5E). Thenegative role for ID1 and ID3 during blood development wasconserved between hESCs and hiPSCs, suggesting thattranscriptional regulation of these genes is maintained duringmesodermal differentiation of pluripotent cells in the human.Furthermore, studies have shown that ID genes also function duringmouse ESC differentiation, where ID1 and ID3 transcripts aredownregulated during the development of primitive blast-colonyforming cells (considered to be the in vitro equivalent of thehemangioblast) into mature myeloid cells (Nogueira et al., 2000).ID1 and ID3 have been shown to regulate crucial steps duringangiogenesis and vascularization (Lyden et al., 1999). Recently,James et al. (James et al., 2010) have shown that ID1 is requiredfor expansion and maintenance of hESC-derived endothelial cellsand preservation of endothelial cell commitment (James et al.,2010). As the hemogenic precursors isolated in our current study

1449ID genes regulate hPSC-derived hematopoiesis

Fig. 4. Suppression of ID1 and ID3 transcripts results in an increase inclonogenic progenitors. (A,B)Assessment of the hematopoietic multi-lineagepotential of hEBs treated with siRNA against ID1, ID2 and ID3. The totalnumber of CFUs produced from hESC- (A) or hiPSC- (B) derived hEBsfollowing ID1, ID2 and ID3 siRNA knockdown between day 8 and 10compared with that in control. *P<0.05 compared with that in control siRNA-treated hEBs. (C,D)Distribution of CFU types (CFU-E, erythroid; CFU-M;macrophage; and CFU-G, granulocyte) following siRNA knockdown of ID1,ID2 and ID3 in hESC- (C) and hiPSC- (D) derived hEBs compared with thatin control siRNA treated hEBs. All results are means (+s.d. for A,B) for threeindependent experiments. (E)Representative CFU colony images derivedfrom hESCs (left) and hiPSCs (right). Scale bars: 100m.

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originate as hemogenic endothelial precursors and continue tomature along the endothelial lineage, the increased hematopoieticpotential induced by downregulation of ID1 might be due to aninhibition of endothelial development that indirectly increaseshematopoietic capacity. This suggests that ID3, as well as ID1,could influence endothelial commitment. Thus, our results suggestthat BMP4 activates expression of ID genes at the hemogenicspecification phase, and that other extrinsic stimuli, such as growthfactors, determine the fate of hemogenic precursors as endothelialcells, whereas subsequent maturation to a hematopoietic faterequires downregulation of ID1 and ID3 for complete blood celldevelopment. On this point, a recent study demonstrated that E2-2, one of the basic HLH family of transcription factors, negativelyregulates endothelial cell specification by blocking activation ofID1 and its downstream effectors, VEGFR2, integrin-b4 and MMP2(Tanaka et al., 2010). In addition, it has been reported that activatedNotch and its downstream effector Herp2 antagonize BMP andID1 expression (Itoh et al., 2004). Accordingly, it is possible thatthese molecules cooperate with ID genes to tightly regulatetransitions from hemogenic endothelial, as well as hematopoieticdifferentiation. Additional studies will be required to clarify themechanisms of action involving ID1 and ID3 during regulation ofhemogenic endothelial specification to the hematopoietic lineage.

Unlike ID1 and ID3, ID2 levels were not altered during eitherhESC- or hiPSC-derived hematopoiesis. Knockdown of ID2 hadno effect on the emergence of hemogenic cells or the production

of committed hematopoietic cells. Moreover, levels of ID2transcript between hemogenic precursors and committedhematopoietic cells were maintained (data not shown). In mouse-ESC-derived hematopoiesis, ID2 expression is maintained duringblast cell colony differentiation, contrary to expression of ID1 andID3 (Nogueira et al., 2000). Consistently, ID2 mRNA levels arenot downregulated upon induction of differentiation of humanmyeloid blasts toward either granulocytes or macrophage; however,inhibition of ID2 expression blocked granulocyte differentiationfrom human UCB-derived CD34+ cells (Buitenhuis et al., 2005;Ishiguro et al., 1996). The differential expression profile andfunction of ID2, as distinguished from those of ID1 and ID3,suggest that ID2 might have a unique function during myeloiddifferentiation and have no effect during early blood developmentevents. However, the exact role of ID2 during the hierarchicalstages of hematopoiesis remains elusive.

Our current study reveals a negative regulatory role for ID1 andID3 during hematopoietic commitment that is highly conserved inboth hESC- and hiPSC-derived blood development, therebydemonstrating that ID genes are able to control early hematopoieticevents in the human. Collectively, these findings suggest thatintrinsic transcription factors are under stringent temporal regulationas a direct consequence of extrinsic stimuli, such as growth factors(BMP4) and co-culture conditions, during the derivation andexpansion of hematopoietic cells. The ultimate goal is to apply thisknowledge towards cell-based therapies by generating sufficient

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Fig. 5. Negative regulatory role of ID1 and ID3 in hemogenic cells.(A)Isolation of hemogenic precursors (CD45negPFV) and the remainingnon-hemogenic (CD31– CD34– CD45–) cells from hESC- and hiPSC-derived hEBs at day 12 (d12) for siRNA treatment and hematopoieticanalysis. Sorted subsets were plated (8,000 cells per well), transfected andcultured for 7 days. (B)Representative FACS plots of CD45negPFV andthe remaining hEB cells. Gates (R1 and R2) and post-purity are shown.FSC, forward scatter; SSC, side scatter; 7AAD, 7-aminoactinomycin D.(C)Knockdown efficiency of ID1 and ID3 transcripts. (D)Knockdown ofID1 and ID3 transcripts exclusively increased the output of maturehematopoietic cells from hemogenic precursors. Similar results wereobtained for hESCs and hiPSCs. Graphs represent combined data fromhESCs and hiPSCs. *P<0.05 compared with results in control siRNA-treated cells (broken horizontal line). Results are means+s.d. for threeindependent experiments. (E)Our hypothetical model suggests that ID1and ID3 act as negative regulators during the hematopoietic commitmentphase and their role is conserved in hESC- and hiPSC-derivedhematopoiesis.

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numbers and appropriately programmed hematopoietic cells fromeither hESCs or immunocompetent hiPSCs that have the potentialfor in vivo reconstitution of the hematopoietic system similar tothat observed for human stem cells.

Materials and MethodshESC cultureUndifferentiated hESC lines (H1 and H9) were maintained in feeder-free culture aspreviously described (Chadwick et al., 2003). Briefly, hESCs were cultured onMatrigel (BD Biosciences)-coated six-well plates with mouse embryonic fibroblastconditioned medium (MEF-CM) supplemented with 8 ng/ml human basic fibroblastgrowth factor (hbFGF) (Invitrogen). In order to maintain the undifferentiated state,the medium was changed daily and hESCs were passaged at a 1:2 split ratio every6–7 days by enzymatic dissociation with 200 units/ml collagenase IV (Invitrogen).hESC culture was carried out at 37°C in a humidified atmosphere containing 5%CO2. H1 and H9 hESC lines were used for all experiments.

Establishment and maintenance of hiPSCsAdult dermal fibroblasts were reprogrammed as described previously (Yu et al.,2007). In brief, lentiviruses expressing transgenes (OCT4, SOX2, NANOG andLIN28) were produced using the human embryonic kidney (HEK)-293FT cell line.Lentiviral transduction was performed with human adult dermal fibroblasts(ScienCell) for 48 hours, and then the cells were re-plated on 1:15 Matrigel-coatedsix-well plates in MEF-CM supplemented with 16 ng/ml hbFGF and 30 ng/ml IGF-II (Millipore). At between day 20 and 25 post-transduction of lentiviruses, iPSCcolonies (Mc-hiPSC) emerged, and were expanded and maintained in the sameconditions as hESCs and used for all experiments. To examine the presence of thetransgenes and the pluripotency genes in the generated hiPSCs, specific primer setswere used to measure the coding regions of both the endogeneous gene and thetransgene (Total), the 3� untranslated region (Endogenous) or the region of the viraltransgenes (Exogenous). A list of primers can be found in supplementary materialTable S1. To examine in vivo differentiation potency, hiPSC cultures (1.0�106 cellsper mouse) were injected into the testicle of NOD-SCID mice (approved by theAnimal Research and Ethics Board of McMaster University), as described previously(Werbowetski-Ogilvie et al., 2009). After 8 weeks, mouse testicles were harvested,sectioned and stained with hematoxylin and eosin. Images were acquired using aScanScope CS digital slide scanner (Aperio, CA, USA).

hEB formation and hematopoietic differentiationEmbryoid bodies (hEBs) were generated by suspension culture, as previouslydescribed (Cerdan et al., 2007; Hong et al., 2010). The medium was exchanged withhEB differentiation medium [80% Dulbecco’s modified essential amino acids(DMEM) base medium plus 20% FBS supplemented with 1% non-essential aminoacids, 1 mM L-glutamine and 0.1 mM b-mercaptoethanol] supplemented withhematopoietic growth factors (hGFs) [50 ng/ml granulocyte colony stimulatingfactor (G-CSF; Stem Cell Technologies), 300 ng/ml stem cell factor (SCF; Stem CellTechnologies), 10 ng/ml interleukin-3 (IL-3; R&D Systems), 10 ng/ml interleukin-6 (IL-6; R&D Systems), 25 ng/ml BMP4 (R&D Systems) and 300 ng/ml Flt-3 ligand(Flt-3L: R&D Systems)]. The hEBs were cultured for 15–20 days, with thehematopoietic differentiation medium, including the hGFs, changed at 3-day intervals.To investigate the effect of BMP4 on hESC-derived hematopoiesis, we blockedBMP4 signals using noggin (500 ng/ml; R&D systems) in hEB differentiationmedium.

Colony forming unit assayThe colony forming unit (CFU) assay was performed by plating single-cellsuspensions from dissociated hEBs into methylcellulose H4230 (Stem CellTechnologies), as previously described (Chadwick et al., 2003). Briefly, hEBs weredissociated with collagenase B and cell dissociation buffer, followed by filtrationthrough a 40-m cell strainer to obtain a single-cell suspension. Cells derived fromdissociated hEBs were counted, and 10,000 cells were plated into methylcelluloseH4230 supplemented with recombinant human growth factors [50 ng/ml SCF, 3units/ml erythropoietin, 10 ng/ml granulocyte monocyte-colony stimulating growthfactor and 10 ng/ml IL-3]. After 14 days, differential colony counts were performedon the basis of morphology.

Flow cytometry analysisSingle-cell suspensions from dissociated hEBs were resuspended at ~2�105–4�105

cells/ml in 3% fetal bovine serum (FBS) in PBS and were stained for 1 hour at 4°Cwith fluorochrome-conjugated monoclonal antibodies against: CD31 (phycoerythrin-conjugated; BD Pharmingen), CD34 (FITC-conjugated; Miltenyi Biotech, BergischGladbach, Germany) and CD45 (allophycocyanin-conjugated; Miltenyi Biotech) ortheir corresponding isotype controls. Stained cells were washed twice in 3% FBS inPBS and stained with the viability dye 7-aminoactinomycin D (7-AAD)(Immunotech). Live cells were analyzed for surface marker expression using aFACSCalibur cell analyzer (Becton Dickinson Immunocytometry Systems, San Jose,CA) and Cell Quest software (BDIS).

Sorting of hEBsTo isolate CD45negPFV, CD34+ CD45+, CD34– CD45+, and the remaining hEBcells, hEBs were dissociated with collagenase B and stained with the fluorochrome-conjugated antibodies as above and 7AAD. Each cellular subset was sorted on aFACSAria (BD Pharmingen) as previously described (Wang et al., 2004).

Immunohistochemistry of hEB sectionsFor staining ALK3, CD31, and CD45, hEBs were washed twice with 3% FBS inPBS, fixed with 4% paraformaldehyde in PBS for 2 hours, embedded, then snap-frozen in liquid nitrogen and stored at –80°C. Cryostat sections were stained forALK3, CD31 and CD45 proteins for detection within the same hEB region. Thefollowing primary antibody and dilutions were used: rabbit anti-ALK3 antibody(1:100; Abcam), mouse anti-CD31 antibody (1:100; BD Pharmingen) and mouseanti-human CD45 antibody (1:100; BD Pharmingen). Sections were incubated withprimary antibodies for 2 hours at room temperature. Subsequently, the sections werestained for 30 minutes with Alexa-Fluor-594-conjugated goat anti-(rabbit IgG)(Molecular Probes) for ALK3 and Alexa-Fluor-488-conjugated goat anti-(mouseIgG) (Molecular Probes) for CD45 and CD31. Slides were mounted andcounterstained using VECTASHIELD HardSet Mounting Medium with DAPI (VectorLabs). Images were captured with a Photometrix Cool Snap HQ2 camera andanalyzed using Image-Pro 3DA version 6.0.

Western blottingCells were treated as indicated, washed with cold PBS, and then lysed for 45 minuteson ice, using Triton lysis buffer (20 mM Tris-HCl pH 7.4, 137 mM NaCl, 25 mMb-glycerophosphate, 2 mM sodium pyrophosphate, 2 mM EDTA, 1 mM Na3VO4,1% Triton X-100, 10% glycerol, 0.5 mM dithiothreitol and protease inhibitors). Thelysates were clarified by centrifugation at 18,000 g for 15 minutes, and thesupernatants were stored at –80°C until use. The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes, For western blotting, bandswere visualized using ECL solutions.

Quantitative RT-PCRTotal RNA was extracted from hEBs and sorted cell populations using the AbsolutelyRNA microprep kit (Stratagene). cDNA was made with 1 g of total RNA using thefirst-strand cDNA synthesis kit (Invitrogen) and subsequent quantitative real-timePCR (Q-PCR) were carried out in triplicate, using SYBR Green RT-PCR reagents(Applied Biosystems) on an Mx3000P Q-PCR System according to manufacturerinstructions (Stratagene). Amplifications were performed using the followingconditions: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and60°C for 1 minute. All data were normalized to GAPDH. The primer sequences usedare shown in supplementary material Table S2.

Knockdown of ID1, ID2 and ID3 transcripts using siRNA transfectionWhole hEBs and sorted subsets (hemogenic precursors and remaining hEB cells)were transfected with ON-TARGETplusSMARTpool siRNA targeting ID1 (100 nM,L-005051-00, Dharmacon), ID2 (100 nM, L-009864-00) or ID3 (100 nM, L-009905-00) for 24 hours using the Silencer siRNA Transfection II kit (Ambion) in KO-DMEM medium, according to the manufacturer’s instructions. Non-targeting siRNA(Ambion) was used as a negative control. After siRNA transfection, culture mediumwas changed to fresh hEB differentiation medium. The hEBs were allowed to growfor 24 hours and lysed for quantitative RT-PCR.

Statistical analysisAll results are expressed as means±s.d. and were generated from at least threeindependent experiments. Statistical significance was determined using Student’s t-tests and differences were considered significant when P<0.05.

We would like to thank Eva Szabo and Chantal Cerdan for helpfuldiscussions on the manuscript. This work was supported by grantsfrom the Canadian Institutes of Health Research and Canada ResearchChair Program (to M.B.).

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/9/1445/DC1

ReferencesBenezra, R., Davis, R. L., Lockshon, D., Turner, D. L. and Weintraub, H. (1990). The

protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell 61, 49-59.

Bhatia, M. (2007). Hematopoietic development from human embryonic stem cells.Hematol. Am. Soc. Hematol. Educ. Program 2007, 11-16.

Buitenhuis, M., van Deutekom, H. W., Verhagen, L. P., Castor, A., Jacobsen, S. E.,Lammers, J. W., Koenderman, L. and Coffer, P. J. (2005). Differential regulation ofgranulopoiesis by the basic helix-loop-helix transcriptional inhibitors Id1 and Id2.Blood 105, 4272-4281.

Cerdan, C., Hong, S. H. and Bhatia, M. (2007). Formation and hematopoieticdifferentiation of human embryoid bodies by suspension and hanging drop cultures.Curr. Protoc. Stem Cell Biol. Chapter 1, Unit 1D 2.

1451ID genes regulate hPSC-derived hematopoiesis

Jour

nal o

f Cel

l Sci

ence

Chadwick, K., Wang, L., Li, L., Menendez, P., Murdoch, B., Rouleau, A. and Bhatia,M. (2003). Cytokines and BMP-4 promote hematopoietic differentiation of humanembryonic stem cells. Blood 102, 906-915.

Choi, K. D., Yu, J., Smuga-Otto, K., Salvagiotto, G., Rehrauer, W., Vodyanik, M.,Thomson, J. and Slukvin, I. (2009). Hematopoietic and endothelial differentiation ofhuman induced pluripotent stem cells. Stem Cells 27, 559-567.

Cooper, C. L., Brady, G., Bilia, F., Iscove, N. N. and Quesenberry, P. J. (1997).Expression of the Id family helix-loop-helix regulators during growth and developmentin the hematopoietic system. Blood 89, 3155-3165.

Hollnagel, A., Oehlmann, V., Heymer, J., Ruther, U. and Nordheim, A. (1999). Idgenes are direct targets of bone morphogenetic protein induction in embryonic stemcells. J. Biol. Chem. 274, 19838-19845.

Hong, S. H., Werbowetski-Ogilvie, T., Ramos-Mejia, V., Lee, J. B. and Bhatia, M.(2010). Multiparameter comparisons of embryoid body differentiation toward humanstem cell applications. Stem Cell Res. 5, 120-130.

Ishiguro, A., Spirin, K. S., Shiohara, M., Tobler, A., Gombart, A. F., Israel, M. A.,Norton, J. D. and Koeffler, H. P. (1996). Id2 expression increases with differentiationof human myeloid cells. Blood 87, 5225-5231.

Itoh, F., Itoh, S., Goumans, M. J., Valdimarsdottir, G., Iso, T., Dotto, G. P., Hamamori,Y., Kedes, L., Kato, M. and ten Dijke Pt, P. (2004). Synergy and antagonism betweenNotch and BMP receptor signaling pathways in endothelial cells. EMBO J. 23, 541-551.

James, D., Nam, H. S., Seandel, M., Nolan, D., Janovitz, T., Tomishima, M., Studer,L., Lee, G., Lyden, D., Benezra, R. et al. (2010). Expansion and maintenance ofhuman embryonic stem cell-derived endothelial cells by TGFbeta inhibition is Id1dependent. Nat. Biotechnol. 28, 161-166.

Jankovic, V., Ciarrocchi, A., Boccuni, P., DeBlasio, T., Benezra, R. and Nimer, S. D.(2007). Id1 restrains myeloid commitment, maintaining the self-renewal capacity ofhematopoietic stem cells. Proc. Natl. Acad. Sci. USA 104, 1260-1265.

Jen, Y., Weintraub, H. and Benezra, R. (1992). Overexpression of Id protein inhibits themuscle differentiation program: in vivo association of Id with E2A proteins. Genes Dev.6, 1466-1479.

Kreider, B. L., Benezra, R., Rovera, G. and Kadesch, T. (1992). Inhibition of myeloiddifferentiation by the helix-loop-helix protein Id. Science 255, 1700-1702.

Leeanansaksiri, W. and Dechsukhum, C. (2006). Regulation of stem cell fate inhematopoietic development. J. Med. Assoc. Thai. 89, 1788-1797.

Lister, J., Forrester, W. C. and Baron, M. H. (1995). Inhibition of an erythroiddifferentiation switch by the helix-loop-helix protein Id1. J. Biol. Chem. 270, 17939-17946.

Lyden, D., Young, A. Z., Zagzag, D., Yan, W., Gerald, W., O’Reilly, R., Bader, B. L.,Hynes, R. O., Zhuang, Y., Manova, K. et al. (1999). Id1 and Id3 are required forneurogenesis, angiogenesis and vascularization of tumour xenografts. Nature 401, 670-677.

Martin, C. H., Woll, P. S., Ni, Z., Zuniga-Pflucker, J. C. and Kaufman, D. S. (2008).Differences in lymphocyte developmental potential between human embryonic stem

cell and umbilical cord blood-derived hematopoietic progenitor cells. Blood 112, 2730-2737.

Miyazono, K. and Miyazawa, K. (2002). Id: a target of BMP signaling. Sci. STKE 2002,PE40.

Murry, C. E. and Keller, G. (2008). Differentiation of embryonic stem cells to clinicallyrelevant populations: lessons from embryonic development. Cell 132, 661-680.

Nogueira, M. M., Mitjavila-Garcia, M. T., Le Pesteur, F., Filippi, M. D., Vainchenker,W., Dubart Kupperschmitt, A. and Sainteny, F. (2000). Regulation of Id geneexpression during embryonic stem cell-derived hematopoietic differentiation. Biochem.Biophys. Res. Commun. 276, 803-812.

Norton, J. D., Deed, R. W., Craggs, G. and Sablitzky, F. (1998). Id helix-loop-helixproteins in cell growth and differentiation. Trends Cell Biol. 8, 58-65.

Orkin, S. H. and Zon, L. I. (2008). Hematopoiesis: an evolving paradigm for stem cellbiology. Cell 132, 631-644.

Pick, M., Azzola, L., Mossman, A., Stanley, E. G. and Elefanty, A. G. (2007).Differentiation of human embryonic stem cells in serum-free medium reveals distinctroles for bone morphogenetic protein 4, vascular endothelial growth factor, stemcell factor, and fibroblast growth factor 2 in hematopoiesis. Stem Cells 25, 2206-2214.

Sadlon, T. J., Lewis, I. D. and D’Andrea, R. J. (2004). BMP4: its role in developmentof the hematopoietic system and potential as a hematopoietic growth factor. Stem Cells22, 457-474.

Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K. andYamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblastsby defined factors. Cell 131, 861-872.

Tanaka, A., Itoh, F., Nishiyama, K., Takezawa, T., Kurihara, H., Itoh, S. and Kato,M. (2010). Inhibition of endothelial cell activation by bHLH protein E2-2 and itsimpairment of angiogenesis. Blood 115, 4138-4147.

Vijayaragavan, K., Szabo, E., Bosse, M., Ramos-Mejia, V., Moon, R. T. and Bhatia,M. (2009). Noncanonical Wnt signaling orchestrates early developmental eventstoward hematopoietic cell fate from human embryonic stem cells. Cell Stem Cell 4,248-262.

Wang, L., Li, L., Shojaei, F., Levac, K., Cerdan, C., Menendez, P., Martin, T.,Rouleau, A. and Bhatia, M. (2004). Endothelial and hematopoietic cell fate of humanembryonic stem cells originates from primitive endothelium with hemangioblasticproperties. Immunity 21, 31-41.

Werbowetski-Ogilvie, T. E., Bosse, M., Stewart, M., Schnerch, A., Ramos-Mejia, V.,Rouleau, A., Wynder, T., Smith, M. J., Dingwall, S., Carter, T. et al. (2009).Characterization of human embryonic stem cells with features of neoplastic progression.Nat. Biotechnol. 27, 91-97.

Yokota, Y. (2001). Id and development. Oncogene 20, 8290-8298.Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian,

S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R. et al. (2007). Induced pluripotentstem cell lines derived from human somatic cells. Science 318, 1917-1920.

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