Identification of Endoglin As a Functional Marker That Defines Long-Term Repopulating Hematopoietic Stem Cells

Identification of endoglin as a functional marker that defines long-term repopulating hematopoietic stem cells

Chang-Zheng Chen*, Min Li*, David de Graaf†, Stefano Monti†, Berthold Go¨ ttgens‡, Maria-Jose Sanchez‡, Eric S. Lander*†§, Todd R. Golub†¶ʈ**, Anthony R. Green‡, and Harvey F. Lodish*§††

*Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142; †Whitehead Institute͞Massachusetts Institute of Technology Center for Genome Research, Cambridge, MA 02142; ‡Cambridge Institute for Medical Research, Cambridge University, Hills Road, Cambridge CB2 2XY, United Kingdom; §Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142; ¶Department of Pediatric Oncology, Dana–Farber Cancer Institute, Boston, MA 02115; ʈHarvard Medical School, Boston, MA 02115; and **Division of Pediatric Hematology͞Oncology, Children’s Hospital, Boston, MA 02115

Contributed by Harvey F. Lodish, October 10, 2002 We describe a strategy to obtain highly enriched long-term repop- used to purify long-term repopulating (LTR) HSCs have re- ulating (LTR) hematopoietic stem cells (HSCs) from bone marrow sulted from these screens. The application of cDNA͞ side-population (SP) cells by using a transgenic reporter gene oligonucleotide microarray technology to identification of HSC- driven by a stem cell enhancer. To analyze the gene-expression specific genes has been prohibited by the amount of RNA profile of the rare HSC population, we developed an amplification required: 50–100 ␮g of total RNA and 2–5 ␮g of poly(A) RNA protocol termed ‘‘constant-ratio PCR,’’ in which sample and control (11). A number of amplification protocols, particularly antisense cDNAs are amplified in the same PCR. This protocol allowed us to RNA (aRNA) amplification, were used for amplifying limiting identify genes differentially expressed in the enriched LTR-HSC amount of RNA for microarray analysis (12, 13). However, population by oligonucleotide microarray analysis using as little as application of these protocols to amplify extremely low amounts 1 ng of total RNA. Endoglin, an ancillary transforming growth of RNA (Ͻ10 ng) for oligonucleotide microarray analysis was factor ␤ receptor, was differentially expressed by the enriched not assessed (14). Recently two groups independently carried out HSCs. Importantly, endoglin-positive cells, which account for 20% comprehensive expression profile analyses on enriched HSC of total SP cells, contain all the LTR-HSC activity within bone populations and other stem cells (15, 16). Although they gen- marrow SP. Our results demonstrate that endoglin, which plays erated a number of genes that appear to be differentially important roles in angiogenesis and hematopoiesis, is a functional expressed in HSCs or other stem cell types, their differential marker that defines LTR HSCs. Our overall strategy may be appli- expression on actual stem cells was not validated (15, 16). cable for the identification of markers for other tissue-specific stem Here we describe an approach to enrich HSCs from the SP cells. cells by using a transgenic stem cell marker and a method for identifying stem cell-specific genes by oligonucleotide microar- n mammals, hematopoietic stem cells (HSCs) are responsible ray analysis. The strategy was validated by the identification Ifor the daily production of billions of mature cells of all blood and demonstration of endoglin as a functional surface marker lineages throughout adult life. HSCs are defined by their ability that defines LTR HSCs. These results suggest that the overall to self-renew and differentiate into all blood cell types (1–3). strategy can be applied for the identification of markers for other Their extraordinary ability to self-renew and differentiate was tissue-specific stem cells. demonstrated by repopulation of the entire blood system by a Materials and Methods single stem cell (3, 4). To better understand the mechanisms that ͞ control HSC self-renewal and differentiation, it is essential to Mouse Strains. Nonobese diabetic severe combined immunode- ͞ ͞ identify and purify these cells unambiguously and identify genes ficient (NOD-SCID) mice, FVB, and C57BL 6J (ly5.2),B6 SJL that are involved specifically in regulating HSC activity. This is (Ly5.1) congenic mice, 6–8 weeks of age, were purchased from ␤ a difficult task because of the extremely low frequency of HSCs The Jackson Laboratory. The transgenic construct 6E5- geo- Ј in normal bone marrow, Ϸ1in105 (5). A variety of methods have 3 En was generated by replacing the lacZ gene in the pGL2– ͞ ͞ Ј ␤ been developed to enrich this rare pluripotent HSC population. 6E5 lacZ 3 En vector (17) with the geo (lacZneo) reporter For example, mouse bone marrow HSCs can be enriched sig- gene. Transgenic mice were analyzed by whole-mount 5-bromo- ␤ nificantly as c-Kit, Sca-1, Thy-1-positive, lineage marker low or 4-chloro-3-indolyl -D-galactoside staining and fluorescence- ␤ negative (c-Kitpos, Thy-1pos, Sca-1pos, Linneg/low) cells (6). HSCs activated cell sorter (FACS) using the -galactosidase substrate ␤ can be enriched also as side-population (SP) cells based on the fluorescein di- -galactopyranoside (FDG) as described (17). differential efflux of the fluorescent dye Hoechst 33342 (7). Irradiated animals were provided with antibiotic and acidified Despite significant enrichment for HSC activity, the c-Kitpos, water for the first 2 weeks posttransplantation. All animals were Thy-1pos, Sca-1pos, Linneg/low and SP cells remain heterogeneous maintained at the Whitehead Institute animal facility according (7, 8), and additional surface markers are required to further to institutional guidelines. enrich HSCs to homogeneity. Moreover, markers that are functionally important for HSC activity may shed light on the FACS Analysis. Murine bone marrow was obtained from the molecular mechanisms that control HSC self-renewal and femurs and tibias. For detection of SP cells, bone marrow cells differentiation. were stained with Hoechst 33342 dye (Sigma) as described (7). The paucity and heterogeneity of the HSC population has greatly hindered systematic efforts to identify HSC-specific Abbreviations: HSC, hematopoietic stem cell; SP, side population; LTR, long-term repopu- genes. A number of laboratories have used large-scale cDNA lating; NOD-SCID, nonobese diabetic͞severe combined immunodeficient; FACS, fluores- subtraction and microarray hybridization approaches to address cence-activated cell sorter; FDG, di-␤-galactopyranoside; CR, constant ratio; SCL, stem cell this question (9, 10). These technologies are extremely laborious leukemia; CRU, competitive repopulation units. and time-consuming. Thus far, no applicable markers that can be ††To whom correspondence should be addressed. E-mail: [email protected].

15468–15473 ͉ PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.202614899 Downloaded by guest on September 25, 2021 For antibody staining, primary antibodies were typically added at 1͞50 to 1͞100 dilutions. Secondary staining reagents including fluorescent conjugated secondary antibodies and streptavidin were titrated individually for minimal nonspecific signal. Anti- body staining was carried out for 15 min on ice (for endoglin staining) or at 4–8°C. When multiple antibody staining was required, primary and secondary staining for endoglin was carried out first. R-phycoerythrin- or allophycocyanin- conjugated goat anti-rat antibodies were used as the secondary staining reagent for the antiendoglin antibody (rat MJ7͞18). In case of multiple staining, the staining order was Hoechst 33342, FDG, antiendoglin͞secondary antibodies, lineage mixture, and other antibodies. Lineage antibodies included anti-CD11b (M1͞ 70), anti-Gr-1 (RB6-8C5), anti-B220 (RA3-6B2), anti-CD3␧ (145-2C11), and anti-Ter119 (Ter119). Other antibodies used in this study included anti-CD4 (GK1.5), anti-CD8a (53-6.7), anti- Cd45.1 (A20), anti-CD45.2 (104), anti-Sca-1 (E13–161.7), and anti-c-Kit. Fig. 1. Use of the SCL␤geo-3ЈEn transgene as a functional genetic marker to enrich HSCs from mouse bone marrow. (A and B) 5-Bromo-4-chloro-3-indolyl Constant Ratio (CR)-PCR and Chip Hybridization. For details see ␤-D-galactoside whole-mount staining of embryonic day 12.5 mice. Compared Supporting Materials and Methods, which is published as sup- with the wild-type embryo (A), an embryo carrying the 6E5-␤geo-3ЈEn trans- porting information on the PNAS web site, www.pnas.org. gene (SCL␤geo) shows predominant expression of lacZ in the fetal liver (B). Approximately 1–2% of mouse bone marrow cells are positive for the SCL␤geo Competitive Repopulation Assay. The competitive repopulation transgene (C). (D–F) Expression of the SCL␤geo transgene in mouse bone assay was performed by using the congenic Ly5.1͞Ly5.2 system marrow analyzed by staining with Hoechst 33342 and the ␤-galactosidase as described (18). Total bone marrow cells (2 ϫ 105 cells) from substrate FDG. SP cells account for Ϸ0.06% of bone marrow cells (D). Among ␤ Ϸ female C57͞BL6 (Ly5.2) mice were used as competitors. NOD- SCL geo transgene-positive cells (FDG-positive gated), 1.5% are SP cells (E). Ϸ (F) SCL␤geo and lineage-expression proﬁle of the bone marrow SP cells (SP SCID mice 8 weeks old were sublethally irradiated (350–400 cells gated); among all the gated SP cells, Ϸ30% are positive for the SCL␤geo Gy) and used to measure HSC activity of cells from stem cell transgene and approximately half of these are lineage-negative or -low. leukemia (SCL)-␤geo-3ЈEn transgenic mice (FVB). Trans- planted mice were analyzed for donor cell contribution by retroorbital bleeding at various times (4, 16, or 28 weeks) specifically to HSCs͞progenitor cells (17, 24). Therefore, we posttransplantation. Nucleated cells were stained with appro- generated transgenic mice using the reporter construct 6E5- priate conjugated Ly5.1 and Ly5.2 antibodies to determine the ␤geo-3ЈEn (SCL␤geo) in which ␤geo expression is controlled by percentage of donor contribution in the recipient mice. The cells the SCL promoter (6E5) and the 3ЈEn. As shown by whole- were stained simultaneously with R-phycoerythrin-conjugated mount 5-bromo-4-chloro-3-indolyl ␤-D-galactoside embryo anti-B220, a mixture of R-phycoerythrin-conjugated anti-Mac-1 staining (17), the selected founder line no. 336 showed predom- and anti-Gr-1 antibodies, or a mixture of R-phycoerythrin- inant expression of the SCL␤geo transgene in embryonic day conjugated anti-CD4 and anti-CD8 antibodies to determine 11.5–12.5 fetal liver (Fig. 1 A and B). Further analysis by FDG multilineage repopulation. Competitive repopulation units staining (17) showed Ϸ1–2% of bone marrow cells from the 5 ͞ Ϫ (CRU) per 10 were calculated by the formula [%Ly5.1 (100 transgenic mice was positive for the SCL␤geo transgene (Fig. ϫ ϫ 5 %Ly5.1)] (2 10 cells per number of cells injected). 1C). More than 30% of the FDG-positive bone marrow cells Results were lineage-negative or -low (data not shown), suggesting that SCL␤geo-positive cells were enriched for hematopoietic pro- Enrichment of HSCs from Bone Marrow SP Cells with a Transgenic Stem genitor or stem cells. Cell Marker. Although a combination of surface markers can be To examine whether the SCL␤geo reporter can enrich HSCs used to enrich LTR HSCs, such markers are rarely available for from bone marrow SP cells, we first stained bone marrow cells the purification of other adult stem cells. Nevertheless, many with Hoechst 33342 dye and FDG. The percentage of SP cells types of adult stem cells from a variety of tissues such as bone ␤ Ϸ marrow (7), muscle (19), neurosphere (20), and testis can be among the SCL geo-positive bone marrow cells ( 1.5%) is Ϸ25-fold higher than that in total bone marrow (Ϸ0.06%), enriched as SP cells based on the differential efflux of the ␤ fluorescent dye Hoechst 33342. This suggests that the conserved suggesting that SCL geo-positive bone marrow cells are en- SP phenotype may represent a functional feature shared by stem riched for HSCs (Fig. 1 D and E). Interestingly, we observed that Ϸ30% of the bone marrow SP cells expressed the SCL␤geo cells of different tissue origin (21). However, SP cells purified ␤ from bone marrow and other tissues are heterogeneous and may transgene (Fig. 1F), indicating that the SCL geo reporter can contain progenitors or differentiated cells or adult stem cells of fractionate SP cells into two distinctive populations. Because the Ј different tissue origins (22). We hypothesized that a transgenic 3 En was able to target reporter gene expression to both adult and embryonic LTR HSCs (17, 24), HSC activity is likely stem cell marker, a reporter transgene that recapitulates the in BIOLOGY ␤ pos ␤ neg vivo expression of a functionally defined critical regulator of stem enriched in the SCL geo SP cells but not in the SCL geo ␤ pos DEVELOPMENTAL cell formation and maintenance, may be used to enrich corre- SP cell population. Furthermore, we found that SCL geo SP spondent tissue-specific stem cells from SP cells. cells are heterogeneous for lineage marker expression (Fig. 1F). To test this hypothesis we selected the SCL gene as a func- Because LTR HSCs are lineage-negative or -low (25), the tional genetic marker to enrich HSCs from bone marrow SP cells. SCL␤geopos SP Linneg/low cells, which account for 0.01% nucle- SCL, a basic helix–loop–helix transcription factor, is essential for ated bone marrow cells, are likely more enriched for LTR HSCs. mouse primitive and definitive hematopoiesis and also plays an To estimate the frequency of HSCs within the SCL␤geopos SP important role in endothelial development (23). Detailed anal- Linneg/low cell population, we carried out competitive repopulat- ysis of the regulation of the mouse SCL gene identified one 3Ј ing assays using NOD-SCID mice as recipients. We injected 10 enhancer (3ЈEn) that directed expression of a linked transgene SCL␤geopos SP Linneg/low cells along with 2 ϫ 105 competitor

Chen et al. PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 ͉ 15469 Downloaded by guest on September 25, 2021 cells from NOD-SCID mice into each of 10 recipient mice. Because NOD-SCID mice express the Ly5.1 antigen and the transgenic mice (FVB strain) express the Ly5.2 antigen, donor white blood cell (WBC) contributions can be quantified by staining Ly5.1 and Ly5.2 antigens. Two months posttransplantation, we analyzed the donor WBC contribution of the seven surviving recipients. The percentages of donor (Ly5.2) WBCs in these recipients were 1.1%, 0%, 0.8%, 1.12%, 0%, 2.03%, and 25.83%, respectively. This result demonstrated that the SCL␤geopos SP Linneg/low cells indeed are highly enriched for LTR-HSC activity.

Identification of HSC-Specific Genes by CR-PCR and Oligonucleotide Microarray Analysis. To provide control cell populations for SCL␤geopos SP Linneg/low cells, we fractionated lineage-negative or -low bone marrow cells into four cell populations by using the SCL␤geo marker and the SP phenotype: (i) SCL␤geopos SP Linneg/low,(ii) SCL␤geoneg SP Linneg/low,(iii) SCL␤geopos non-SP neg/low neg neg/low Lin , and (iv) SCL␤geo non-SP Lin . These popu- Fig. 2. Schematic diagram of CR-PCR and its application in gene-expression lations represent Ϸ0.01, 0.01, 0.6, and 0.6% of total bone marrow profiling using oligonucleotide microarrays. The key feature of this protocol cells, respectively. Linneg/low cells, which comprise Ϸ2% of total is that sample and control cDNAs are amplified in the same PCR such that the bone marrow cells, are quite heterogeneous and are mostly early ratio of individual genes between the sample and control RNAs will not be myeloid or lymphoid progenitor cells. No LTR activity was found skewed during amplification. The poly(T) primers consist of oligo(dT), unique in SCL␤geoneg cell fractions (data not shown). The frequency of sequences (T-1, dark blue; T-2, dark orange), and universal forward sequences ␤ pos neg/low (black). These primers are designed to label an individual RNA sample with a HSCs in SCL geo non-SP Lin cells, as measured by the specific sequence tag (T1 or T2) in the RT step. A universal primer (SMART Ϸ ␤ pos CRU frequency, was 38 times lower than that in SCL geo oligo, dark green with GGG at the 3Ј end) was added to the end of the newly neg/low SP Lin cells (data not shown). Therefore, genes specifically synthesized cDNA by a template-switching mechanism (33). Thus the cDNA pos neg/low expressed in SCL␤geo SP Lin cells but not the other products contain common 5Ј and 3Ј flanking sequences (universal forward, three populations are likely to be HSC-specific. black͞gray; universal reverse, dark green͞light green) that allow PCR ampli- The frequency of SCL␤geopos SP Linneg/low cells is only fication. Equal amounts of RT products from each sample were mixed and Ϸ0.01% of nucleated bone marrow cells. Because of the low amplified with the universal forward and reverse PCR primers. Amplified abundance of the these cells and the toxic nature of the Hoechst samples were split into two (or more) equal fractions. A specific T7 promoter 33342- and FDG-staining procedures, only Ϸ1,000 SCL␤geopos containing either the T1 or T2 sequence tag (T7-1 or T7-2) matched to the neg/low corresponding poly(T) primer [poly(T-1) or poly(T-2)] was used to selectively SP Lin cells could be isolated by FACS from the bone add a T7 promoter to the corresponding fraction of cDNAs in the mixture by marrow of one transgenic mouse. Less than 1 ng of total RNA linear extension reaction using DNA polymerase. cRNA samples were gener- was obtained from 1,000 cells. Significant amplification is ated by in vitro transcription and subjected to oligonucleotide microarray needed to identify genes that are differentially expressed in analysis. Differentially expressed genes were identified based on the fluores- SCL␤geopos SP Linneg/low cells by oligonucleotide microarray cent intensity ratio of individual genes. Throughout, complementary se- analysis. To maintain the ratio of individual genes between quences are shown as a lighter color of the matching strand (e.g., light blue samples during PCR amplification, we developed an amplifica- versus dark blue). tion protocol, termed CR-PCR and depicted in Fig. 2. The key feature of CR-PCR is that sample and control cDNAs are ␤ pos neg/low amplified in the same PCR such that the ratio of individual genes SCL geo SP Lin cells but not in the other cell popula- between the sample and controls will not be skewed. tions (Table 1). To verify this, we used antibody staining and FACS analysis to examine surface endoglin expression on Two sets of CR-PCRs were established to identify genes that pos neg/low ␤ pos neg/low SCL␤geo SP Lin cells and the three control cell popu- are differentially expressed in SCL geo SP Lin cells, the ␤ enriched HSC population. cDNAs were synthesized by using 1 ng lations. To this end bone marrow cells from SCL geo transgenic of total RNA from each cell population. The first PCR contained mice were stained with Hoechst 33342, FDG, antiendoglin cDNAs from SCL␤geopos SP Linneg/low (HSC) and SCL␤geoneg antibody, and antibodies against lineage markers. Approxi- SP Linneg/low cells. The second PCR contained cDNAs from mately 0.4% of the total bone marrow cells are positive for ␤ SCL␤geopos SP Linneg/low, SCL␤geopos non-SP Linneg/low, and endoglin and SCL geo (Fig. 3A). As before, SP cells comprised Ϸ neg/low SCL␤geoneg non-SP Linneg/low cells. PCR amplifications were 0.06% of total bone marrow cells. The Lin gate was set Ϸ done in duplicates, and samples were amplified and subjected to such that these cells account for 2% of the total bone marrow oligonucleotide microarray hybridization. cells (Fig. 3B). ␤ To identify HSC-specific candidate genes, we focused on those We then examined the expression of endoglin and SCL geo abundantly expressed in SCL␤geopos SP Linneg/low cells but not in in gated SP and Linneg/low cells (Fig. 3C) or gated non-SP and the other three cell populations; a selected set is shown in Table Linneg/low cells (Fig. 3D). As shown in Fig. 3C, 66% (ϭ56 ϩ 10) 1. Among those genes, c-fos was identified previously as an of the SP Linneg/low cells were SCL␤geo-positive, and 85% HSC-specific gene in a cDNA subtraction and microarray screen (ϭ56͞66) of these SCL␤geopos SP Linneg/low cells also expressed (19) and may play an important role in maintaining stem cells in endoglin. In contrast, 34% (ϭ27 ϩ 7) of the SP Linneg/low cells a dormant state (26). Moreover, c-fos is also differentially were SCL␤geo-negative, and only 20% (ϭ7͞34) of these expressed in the population of human CD34pos CD38neg cells that SCL␤geoneg SP Linneg/low cells were positive for endoglin (Fig. are enriched in primitive HSCs but not in CD34pos CD38pos cells 3C). If all the endoglin-positive (Endopos) cells express equal that do not contain primitive HSCs (27, 28). amounts of endoglin protein and mRNA, these FACS data indicate that endoglin expression on SCL␤geopos SP Linneg/low Verification of Endoglin Expression on Highly Enriched HSCs by FACS cells is 4.3-fold that in SCL␤geoneg SP Linneg/low cells. The mRNA Analysis. We also found that endoglin, an ancillary transforming ratio determined by array analysis is 13-fold (Table 1). Of the growth factor ␤ receptor, was highly differentially expressed on non-SP Linneg/low cells (Fig. 3D), 35% (ϭ33 ϩ 2) were SCL␤geo-

15470 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.202614899 Chen et al. Downloaded by guest on September 25, 2021 Table 1. Genes differentially expressed in the enriched HSC population Ampliﬁcation set 1 Ampliﬁcation set 2

GenBank Gene (1) SCL␤geoϩ (2) SCL␤geoϪ Fold (3) SCL␤geoϩ (4) SCL␤geoϩ (5) SCL␤geoϪ Fold Fold accession no. description SP LinϪ SP LinϪ (1)͞(2) SP LinϪ non-SP LinϪ non-SP LinϪ (3)͞(4) (3)͞(5)

AI840975 EST 1,383.45 9.58 144 1,673.96 18.7 3.79 90 442 X77952 Endoglin 996.39 78.36 13 3,501.58 48.71 90.36 72 39 J05479 Calcineurin 746.15 36.58 20 1,016.78 18.84 2.48 54 410 V00727 c-fos 4,645.57 143.68 32 433.82 16.97 8.01 26 54 AW125272 EST 5,781.49 263.45 22 1,640.78 227.01 55.24 7 30 AI853703 EST 234.64 9.67 24 2,003.22 156.98 214.93 13 9 AV293396 EST 2,107.39 208.26 10 313.34 43.26 24.95 7 13

Columns (1)–(5) show the normalized ﬂuorescence intensity for the indicated genes in the indicated sample. ‘‘Fold’’ lists the ratios of ﬂuorescence intensity between the indicated samples and is a measure of enrichment of these mRNAs in the SCL␤geopos SP Linneg͞low cells relative to the indicated control cell population.

positive, and only 6% (ϭ2͞35) of these SCL␤geopos non-SP enriched for HSC activity, these results suggest that endoglin is Linneg/low cells also expressed endoglin. Similarly, 65% (ϭ63 ϩ likely to be a marker for HSCs within the SP cells. 2) of the non-SP Linneg/low cells were SCL␤geo-negative, and 3% (ϭ2͞65) of SCL␤geoneg non-SP Linneg/low cells were positive for Endopos SP Cells Contain Essentially All the LTR-HSC Activity Among SP endoglin (Fig. 3D). Applying the same calculation, endoglin Cells. Although endoglin is expressed specifically on the ␤ pos neg/low expression on SCL␤geopos SP Linneg/low cells should be 14-fold of SCL geo SP Lin cells, it marks a slightly smaller cell ␤ that of SCL␤geopos non-SP Linneg/low cells and 28-fold of that of population of the total SP cells than does SCL geo. Approxi- ␤ SCL␤geoneg non-SP Linneg/low cells, respectively. The mRNA mately 30% and 20% of SP cells were positive for the SCL geo ratios determined by array analysis were 72 and 39, respectively marker or endoglin expression, respectively (Fig. 4B). As indi- pos ␤ pos (Table 1). cated by the lineage profile of Endo SP and SCL geo SP Thus, the FACS analysis confirmed the ratio of endoglin expression in the enriched HSC population versus the other control population, suggesting that endoglin is highly differentially expressed on the surface of the enriched HSC population. This result further demonstrated that CR-PCR can maintain the ratio of differentially expressed genes when amplifying as little as 1 ng of total RNA. Because our competitive repopulation studies showed that SCL␤geopos SP Linneg/low cells are highly

Fig. 4. Endopos cells are enriched for LTR-HSC activity in bone marrow SP cells. (A and B) Correlation of endoglin and SCL␤geo expression in bone marrow SP cells. (A) Lineage and SCL␤geo (FDG) proﬁle of bone marrow SP cells. (B) Lineage and endoglin proﬁle of bone marrow SP cells. In both A and B Endopos SP cells are depicted as yellow dots and Endoneg SP cells are red dots. Arbitrary gates were set for lineage-marker expression: neg, low, and high. The blue

quadrant in B was the sorting gate used to divide bone marrow SP cells into BIOLOGY four cell populations: Endopos Linneg, Endopos Linlow, Endoneg Linneg, and En- doneg Linlow/hi cells. (C) Competitive repopulation analysis of Endopos SP cells DEVELOPMENTAL (solid orange line and error bar) and Endoneg SP cells (dotted green line and error bar). Shown here is the donor contribution (% Ly5.1) by Endopos SP cells Fig. 3. FACS analysis shows that endoglin is differentially expressed on the and Endoneg SP cells at 4, 16, and 26 weeks posttransplantation. One hundred enriched HSC SCL␤geopos SP Linneg/low population. Bone marrow cells from Endopos SP or 375 Endoneg SP cells were injected into each recipient. (D) SCL␤geo transgenic mice were stained with antiendoglin antibody, Hoechst Competitive repopulation analysis of Endopos SP Linneg (orange bar) and 33342, and lineage markers. (A) Endoglin and SCL␤geo expression in total Endopos SP Linlow (green bar) cells. Shown here is the CRU per 105 donor cells bone marrow cells. (B) Gate for Linneg/low cells. FSC, forward scatter. (C) in the Endopos SP Linneg (orange bar) and Endopos SP Linlow (green bar) groups Endoglin and SCL␤geo expression proﬁle of gated Linneg/low SP cells. (D) at 4, 14, and 28 weeks posttransplantation. Forty-two Endopos SP Linneg and Endoglin and SCL␤geo expression proﬁle of gated Linneg/low non-SP cells. 139 Endopos SP Linlow cells were injected into each recipient.

Chen et al. PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 ͉ 15471 Downloaded by guest on September 25, 2021 cells (Fig. 4 A and B), this is largely due to the difference in 400 bone marrow Ly5.1 SP cells from the primary recipient along lineage-marker expression between Endopos SP and SCL␤geopos with 2 ϫ 105 Ly5.2 competitor bone marrow cells. Four of the five SP cells. The majority of Endopos SP cells also expressed the secondary recipients were reconstituted with Ly5.1 SP cells SCL␤geo transgene (Endopos cells are shown as yellow dots in (1.8 Ϯ 0.6% of donor contribution). These results confirmed that Fig. 4 A and B). Endopos SP cells are mostly lineage-negative or Endopos SP cells are enriched for LTR HSCs that are capable of -low (Fig. 4B). In contrast, Ϸ33% of the SCL␤geopos SP cells are self-renewal. predominantly lineage-high and are endoglin-negative (Endoneg, red dots on right side of Fig. 4A), and the SCL␤geopos SP cells Discussion that are endoglin-positive (yellow dots in right side of Fig. 4A) Here we developed a strategy to isolate highly enriched HSCs are all Linneg/low. from the bone marrow SP cells using a transgenic stem cell To demonstrate that endoglin is indeed a marker for LTR reporter driven by the SCL 3ЈEn (17, 24). Furthermore, we HSCs, we examined the reconstitution potential of Endopos SP developed an amplification protocol, CR-PCR, that made it and Endoneg SP cells using a competitive repopulation assay (29). possible to analyze the gene-expression profile by oligonucleo- We injected 100 Endopos SP cells or 375 Endoneg SP cells isolated tide microarray analysis using as little as 1 ng of total RNA. With from a Ly5.1 donor along with 2 ϫ 105 Ly5.2 competitor cells into these technical advances we identified endoglin as a protein lethally irradiated congenic Ly5.2 mice. The number of Endopos differentially expressed on SCL␤geopos SP Linneg/low cells, a SP and Endoneg SP cells injected was proportional to their highly enriched HSC population. Most importantly, we demon- abundance in the bone marrow SP-cell population. At least five strated that endoglin is a functional marker for LTR HSCs and recipient mice were used for each cell sample. We observed can be used to enrich Ϸ5-fold LTR HSCs from SP cells. These distinct reconstitution kinetics in the recipients that received results validate our approach for enrichment of HSCs and Endopos SP or Endoneg SP cells, as indicated by the fraction of identification of stem cell-specific genes or markers and suggest white blood cells expressing the donor Ly5.1 antigen (Fig. 4C). that our overall strategy may be applicable for the identification At 4 weeks posttransplantation, the donor contribution by 100 of markers for other types of tissue-specific stem cells. Endopos SP and 375 Endoneg SP cells was comparable (Fig. 4C). Isolation of highly enriched LTR HSCs by fractionating bone However, the contribution by Endoneg SP cells decreased sig- marrow SP cells with a transgenic stem cell reporter represents nificantly after 4 weeks, whereas the contribution by Endopos SP an alternative HSC-purification approach that requires minimal cells increased dramatically (Fig. 4C). The distinct reconstitution knowledge about specific surface markers. This approach was kinetics indicated that the Endoneg SP cells contain mostly validated by the identification of endoglin as a marker for LTR short-term HSCs, which can only maintain transient reconstitu- HSCs. Endoglin was not found in two previous large-scale tion. Significant long-term multilineage reconstitution, as indi- expression analyses using HSCs enriched by conventional sur- cated by the percentage chimerism in T-lymphoid, B-lymphoid, face markers (9, 10). Moreover, because the SP phenotype is and myeloid cells, was seen only in recipient mice that received shared by many other adult stem cell types, our results also Endopos SP cells (not shown). Our results established that suggest that a similar strategy can be devised to enrich stem cells Endopos SP cells contain essentially all the LTR activity within of different tissue origins with surface markers that are largely bone marrow SP cells (Fig. 4C). Because Endopos SP cells unknown. account for Ϸ20% of total SP cells (Fig. 4B), a 5-fold enrichment The CR-PCR protocol, in which coamplification of cDNA of LTR HSCs from the SP cells was achieved by using endoglin samples was used to maintain the ratio of individual genes in as a marker. different RNA samples, was also key to the identification of Some of the Endopos SP cells are still expressing lineage endoglin as an HSC-specific marker. This protocol allows the use markers at a low level. We suspected that Endopos SP Linneg cells of DNA microarrays to identify differentially expressed genes may be enriched further for LTR HSCs and that the Endopos SP even when mRNA is available from only a minute number of Linlow cells may contain more short-term HSCs (25). We frac- cells, in our case Ϸ1,000 cells (Ϸ1 ng of total RNA). As evidence, tionated SP cells into Endoneg Linneg, Endoneg Linlow/hi, Endopos we were able to identify endoglin as a gene that is specifically Linlow, and Endopos Linneg fractions (Fig. 4B, blue lines); these expressed on the majority of SCL␤geopos SP Lin neg/low cells but represent Ϸ3%, 50%, 34%, and 13% of the SP cells, respectively. not in three control bone marrow cell populations using 1 ng of No LTR-HSC activity was found in the two endoglin-negative total RNA from each population. Compared with the two-round fractions (data not shown). We injected 139 Endopos Linlow SP in vitro-transcription protocol used in other studies (15, 16), the or 42 Endopos Linneg SP cells into lethally irradiated mice along CR-PCR amplification protocol requires at least 10–50 times with 2 ϫ 105 Ly5.2 competitor cells. The number of cells injected less RNA. Moreover, the relative abundance of endoglin expres- was proportional to the cell distribution within each quadrant. At sion in the HSC-enriched populations versus control cell groups 4 weeks posttransplantation, Endopos Linlow SP and Endopos were comparable at the mRNA level, as determined by array Linneg SP cells contained an equivalent amount of CRU activity analysis, and at the level of cell-surface protein expression (Table per 105 cells (Fig. 4D). However, at 14 weeks posttransplanta- 1 and Fig. 3). This result demonstrated that CR-PCR can tion, the competitive repopulating activity of the Endopos Linneg maintain the ratio of individual genes during amplification. In SP cells was Ͼ2-fold that of the Endopos Linlow SP cells. Notably, two recent studies endoglin was identified as one of many genes the CRU activity per 105 cells in the Endopos Linlow SP cells expressed in enriched HSC populations. In one, endoglin was decreased significantly from 4 to 28 weeks posttransplantation, found to be highly expressed on many bone marrow cell popu- whereas that of the Endopos Linneg SP cells increased (Fig. 4D). lations, as shown by quantitative PCR (15). In the other, gene The distinct reconstitution kinetics indicated that, as anticipated, expression in LTR HSCs was not compared with that of any the Endopos SP Linlow cells contained a significant number of differentiated hematopoietic progenitor cells (16). short-term HSCs, but the Endopos SP Linneg cells did not (Fig. As with all methodologies, the CR-PCR protocol has its 4D). Based on this study, one can further enrich for LTR HSCs limitations. We noticed that many genes were undetectable on by selecting the lineage-negative fraction of Endopos SP cells. the chips, presumably because of inefficient PCR amplification To evaluate the self-renewal potential of Endopos SP cells, we of these transcripts. We also observed relatively low replicate carried out secondary transplantations using Ly5.1 SP cells correlation coefficients (Ϸ0.65) that may be due to the quantum isolated from primary Ly5.2 recipients effectively reconstituted effects in amplifying low numbers of mRNA molecules isolated with Ly5.1 Endopos SP cells at 14 weeks posttransplantation. from very small numbers of cells. Variations in the early Each lethally irradiated secondary recipient was injected with manipulations are amplified during the later PCR steps. Despite

15472 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.202614899 Chen et al. Downloaded by guest on September 25, 2021 these limitations, we demonstrated that CR-PCR did provide a embryonic day 10.5, the time when adult repopulating cells simple protocol that can be used to discover differentially appear, it is unclear whether endoglin plays an essential role in expressed genes by oligonucleotide microarray analysis when the regulating HSC self-renewal and differentiation (31). To defin- number of cells available is extremely limited. itively establish a role of endoglin in regulating HSC function it Differential expression of endoglin in the enriched LTR-HSC may be necessary to examine the developmental potential of population but not the control populations (Fig. 3) suggested endoglin null embryonic stem cells in chimeric mice. Nonethe- that endoglin could be used as an effective surface marker to less, endoglin is a functional marker that defines and enables enrich LTR HSCs. Indeed, we demonstrated that endoglin can enrichment of LTR HSCs. be used to further enrich LTR HSCs from bone marrow SP cells. This extends earlier work, suggesting that endoglin may have Note Added in Proof. It was noted recently by another group that important functions in angiogenesis and hematopoiesis. Muta- endoglin may be differentially expressed in enriched HSCs (34). tions in the endoglin gene that lead to haploinsufficiency are We thank Glenn Paradis and Michael Doire (Flow Cytometry Core associated with the inherited human disorder hereditary hem- Facility, Center for Cancer Research, Massachusetts Institute of Tech- orrhagic telangiectasia type 1, a disease characterized by bleed- nology) for invaluable help with FACS sorting and C. Ladd (Whitehead ing caused by vascular malformations (30). Endoglin null em- Genome Center) and Dr. K. Chen (Transgenic Facility, Albert Einstein bryos die at embryonic day 10.0–10.5 because of defects in blood Medical School, New York) for excellent technical assistance. We also vessel and heart development and show severe defects in yolk-sac thank Drs. M. Xun, G. Wang, S. Ghaffari, C. Zhang, and B. Luo for hematopoiesis (31). A recent study of in vitro differentiation of helpful discussion and critical reading of the manuscript. Support to EndoϪ/Ϫ embryonic stem cells suggested that endoglin also may H.F.L. was from the Engineering Research Centers Program of the be important in definitive hematopoiesis, particularly myelopoi- National Science Foundation under Award EEC 9843342 through the esis and erythropoiesis (32). Together with our work showing Biotechnology Process Engineering Center (Massachusetts Institute of Technology). Work in the A.R.G. laboratory is supported by the specific expression of endoglin on LTR HSCs, this result indi- ␤ Wellcome Trust, Leukaemia Research Fund, and Medical Research cates that endoglin and transforming growth factor signaling Council. This work is supported in part by Affymetrix and Bristol–Myers may play important roles in regulating HSC self-renewal and͞or Squibb. C.-Z.C. holds a Cancer Research Institute͞Donaldson, Lufkin, differentiation. However, because endoglin null mice die around and Jenrette postdoctoral fellowship.

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