Primary Mesenchymal Stem and Progenitor Cells from Bone Marrow Lack Expression of CD44 Protein
Hong Qian, Katarina Le Blanc and Mikael Sigvardsson
Linköping University Post Print
N.B.: When citing this work, cite the original article.
Original Publication:
Hong Qian, Katarina Le Blanc and Mikael Sigvardsson, Primary Mesenchymal Stem and Progenitor Cells from Bone Marrow Lack Expression of CD44 Protein, 2012, Journal of Biological Chemistry, (287), 31, 25795-25807. http://dx.doi.org/10.1074/jbc.M112.339622 Copyright: American Society for Biochemistry and Molecular Biology http://www.asbmb.org/
Postprint available at: Linköping University Electronic Press http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-80787
Primary Mesenchymal Stem and Progenitor Cells from Bone Marrow Lack Expression of
CD44
Hong Qian1,2*, Katarina Le Blanc2, Mikael Sigvardsson1
1Department of Clinical and Experimental Medicine, Linköping University, SE-58185 Linköping, Sweden. 2Center for Hematology and Regenerative Medicine, Department of Medicine, Karolinska University Hospital Huddinge, S-141 86, Stockholm, Sweden.
Running title: Native mesenchymal stem cells do not express CD44
*To whom correspondence should be addressed: Hong Qian, HERM, Novum Floor 4, Karolinska University Hospital Huddinge, Hälsovagen 7, S-141 86, Stockholm, Sweden. E-mail: [email protected], Tel: +46-8-58583623, Fax: +46- 8-585 836 05.
Key words: Mesenchymal stem cells, CD44, microarray, Bone Marrow, Flow cytometry.
Background: Natural phenotype of mesenchymal stem cells (MSCs) has not been well- characterized. Results: MSCs from bone marrow naturally are CD44-, however, in vitro cultivation results in acquisition of CD44 expression on the cells. Conclusion: Native MSCs in bone marrow lack CD44 expression. Significance: Our findings highlight natural phenotype of MSCs and open new possibilities for prospective isolation of MSCs from bone marrow.
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SUMMARY the information has been obtained from in vitro studies on culture-expanded cells, which Despite significant progress in our may not represent the phenotype of MSCs in understanding of mesenchymal stem cell vivo (2-5). Multi-color fluorescence activated (MSC) biology during the last years, much of cell sorting (FACS) have been fundamental for the information is based on experiments using definition and prospective isolation of in vitro culture-selected stromal progenitor different cell populations of the hematopoietic cells. Therefore, the natural cellular identity of system over the last 20 years. Recent MSCs remains poorly defined. Numerous development of FACS-based protocols for the studies have reported that CD44 expression is isolation and characterization of MSCs one of the characteristics of MSCs in both directly from BM opens the possibility to human and mice, however, we here have better identify and characterize non- prospectively isolated bone marrow stromal hematopoietic cell compartments in the BM. cell subsets from both human and mouse bone In mice, platelet derived growth factor marrow by flow cytometry and characterized receptor α (PDGFRα), stem cell antigen-1 them by gene expression analysis and function (SCA1), CD51 and Nestin are expressed on assays. Our data provide functional and freshly isolated BM stromal cell populations molecular evidence suggesting that primary enriched with MSCs (6-8). In human, several mesenchymal stem and progenitor cells of - surface proteins including Stro-1, CD271 and bone marrow reside in the CD44 cell fraction CD146 may be used as markers for in both mice and humans. The finding that - mesenchymal stem and progenitor cells (9-14). these CD44 cells acquire CD44 expression In addition, expression of markers such as after in vitro culture provides an explanation to CD105, CD90 and CD49A have been the previous misconceptions concerning CD44 diversely reported to be characteristic of expression on MSCs. In addition, the other MSCs (15). Among those, CD44 has been previous reported MSC markers including reported to be highly expressed on in vitro CD73, CD146, CD271, CD106/VCAM1 are expanded MSCs from both humans and mice also differentially expressed on those two cell (16-22). CD44 is an adhesion molecule types. Our microarray data revealed distinct - existing in different isoforms that interact with gene expression profile of the freshly CD44 multiple ligands such as hyaluronan, selectins, cells and the cultured MSCs generated from collagen and fibronectin (23). It is widely these cells. Thus, we conclude that bone expressed in multiple cell types including marrow MSCs physiologically lack expression hematopoietic cells, cancer stem cells (24). of CD44, highlighting the natural phenotype of MSCs and open new possibilities to In the present study, by using multi- prospectively isolate MSCs from the bone color FACS, microarray analysis and CFU-F marrow. assay, we have found that while freshly isolated MSCs from human and mouse BM express the surface markers previously INTRODUCTION reported to mark early mesenchymal Mesenchymal stem cells (MSCs) was progenitors, they lack expression of CD44. originally isolated from bone marrow (BM) by Further characterization of the cells revealed their capacity to generate colony-forming unit- that the CD44+ cells displayed little or no fibroblast (CFU-F) in vitro (1). Although there CFU-F activity whereas the CD44- cells has been significant progress in understanding contain almost all the clonogenic cells with of the biological features of MSCs, much of multilineage differentiation potentials. 2
However, in vitro culture of the CD44- MSCs excluded by propidium iodide (PI) staining. and progenitor cells resulted in their The cells were analyzed and sorted on conversion to a CD44 positive phenotype, FACSAria II Sorp (BD). providing an explanation to the previous observations suggesting CD44 as a marker for FACS isolation and analysis of mouse MSCs. MSCs. Furthermore, the cultured MSCs The BM mononuclear cells from femurs, tibias derived from the fresh CD44- stromal cells and iliac crest of FVB/N mice were isolated display distinct gene expression profiles of cell using a standard protocol which was tested in adhesion molecules and growth factors as well our laboratory without affecting cell surface as cytokines. These findings highlight the marker expression. The bones were first importance of in vivo/ex vivo analysis of crushed in PBS+10%FBS (PAA) in order to mesenchymal cells for identifying their obtain maximal cells in BM endosteal region physiological properties and suggest that prior to enzyme treatment. The marrow cells CD44 expression can be used as a negative were collected and the bone fragments were rather than a positive marker for prospective then treated with 0.1% Collagenase II (CLS II isolation of MSCs from BM. Worthin gton Biochemical) and 0.05% trypsin-EDTA for 45 min at 37°C. The tubes EXPERIMENTAL PROCEDURES were shaken every 10min during incubation. Subject: BM aspirates were obtained from The treatment was stopped by adding ice cold iliac crest of normal young adult volunteers FBS to reach a final concentration of 20% following informed consent according to the FBS, subsequently wash the bones by procedures approved by local ethics committee PBS+10% FBS. The cells were collected and at Karolinska Institute (Stockholm, Sweden). filtered via 70 µm cell strainer (BD). The bone Mouse bones were obtained from adult (3-4 and marrow cells were pooled and spun down month old) normal FVB/N mice. Animal at 300g for 10 min and then resuspended in procedures were performed with approval PBS+10% FBS. The stromal cells were first from the ethics committee at Linköping enriched by depleting hematopoietic cells University (Linköping, Sweden). using purified rat anti-mouse antibodies against CD45 and LIN (TER119, B220, CD4, FACS isolation and analysis of human BM CD8, GR1 and MAC1) and subsequently MSCs. Mononuclear cells from BM aspirates using sheep anti-rat Dynal beads (Invitrogen). of healthy adult volunteers were isolated by The endothelial cells and the residual Ficoll-Hypaque (Lymphoprep, Axis-Shield hematopoietic cells were visualized by CD31 PoC AS) density centrifugation. The CD45- - and goat-anti-rat tricolor antibody and/or CD235 cells were enriched by negative CD45 and TER119. The dead cells were selection using CD45 and CD235 microbeads excluded by PI staining. The CD44+ and and magnetic-activated cell sorting (MACS, CD44- stromal cells were gated or analyzed Miltenyi Biotec). The cells were then stained based on fluorescent minus one (FMO) with anti-human CD271 CD146, CD105, controls for CD44 expression on FACS aria II CD106, CD73, STRO-1, CD29, CD45 and Sorp (BD). For information about the Glycophorin A/CD235. Anti-human CD19 antibodies used in the study, see Supplemental was included in the staining in order to information. exclude possible contamination of B cells in the sorted stromal cells. For information about the antibodies used in the study, see CFU-F assay. The stromal cells (CD45-LIN- supplemental information. Dead cells were CD31-CD44+ and CD45-LIN- CD31-CD44-) 3
from normal mouse and human BM were Assays-on-Demand probes, see below list of sorted and plated into 96-well plates or 12- probes used for Q-PCR. well plates containing complete Mesencult medium in MesenCult® Proliferation Kit for Cell Cycle Analysis. The analysis was mouse (#05511) and human (#05411), performed as described(25). BM mononuclear respectively (Stem cell Technologies, cells from wild-type FVB/N mice were Vancouver, Canada) in hypoxic (1% O2) for initially stained with antibodies against CD45, 10-12 days (mouse) or 12-14 days (human). lineage cells and CD44. After incubation with The mouse cells were seeded at a density of the cell surface antibodies, the cells underwent 10, 40, 100 and 200 cells/well for the CD44- fixation with a Cytofix/Cytoperm kit (BD cells and 100, 200, 400, 1000 and 2000 Biosciences) and staining of PE-anti-KI67 and cells/well and the human cells were plated at DAPI. Analysis was performed on a FACS 2, 5, 10 cells/well for the CD44- cells and 10, ARIA II SORP (BD Biosciences). 50, 100 and 1000 cells/well for the CD44+ cells. The complete MesenCult medium was In vitro differentiation assays. The assay was prepared by mixing one part of MSC performed on culture-expanded cells at stimulatory Supplements and 4 parts of passage 2-4. The cells were under stimulation Mesencult Basal Medium for mouse cells and with differentiation media for 3-4 weeks. The one part of MSC stimulatory Supplements and media was changed every 2-3 days. For 9 parts of Mesencult Basal Medium for human osteoblast differentiation, the cells were cells (Stem cell Technologies,Vancouver, cultured in complete alpha MEM medium or Canada). The colonies were stained with DMEM containing 10 % FBS, 10nM HEPES Giemsa (Sigma) and scored under inverted (1M), 100 U/mL of penicillin, 100 µg/mL microscope (Leica DMIL, Leica streptomycin, 50µg/ml ascorbic acid (Sigma), Microsystems, Germany). Cluster of more 1-5 x10-7 M dexamethasone (Sigma) and than 50 cells was counted as one colony and 10mM glycerol phosphate were used. The the images were taken using Leica Appication cells were fixed with 10 % formalin or ice- Suite software. cold methanol and the calcium deposit were verified by using 1% Alizarin Red S (Sigma) Quantitative RT-PCR. Cells were sorted (pH 4.1) or von Kossa staining (26). For the directly into buffer-RLT (Qiagen) and frozen von Kossa silver nitrate staining method, at –80°C. RNA extraction and DNase cultures were fixed in cold methanol for 15–20 treatment was performed with the RNeasy min. After rinsing, the fixed plates were Micro Kit (Qiagen) according to the incubated with 5% silver nitrate solution under manufacturer’s instructions for samples UV light using a UVllinker (UVitec, containing less than 105 cells. Eluted RNA Cambridge, UK). Mineralized nodules were samples were reverse transcribed using seen as dark brown to black spots. On the SuperScript III and random primers other hand, for alizarin red S (sodium alizarin (Invitrogen) according to protocol supplied by sulphonate) staining, 1 % alizarin red S the manufacturer. Real-time quantitative PCR (Sigma) was prepared in distilled water and (Q-PCR) reactions were performed by mixing the pH was adjusted to 4.1–4.3 using 0.5 % 2 x TaqMan universal PCR master mix, 20 x ammonium hydroxide. Cultures were stained with alizarin red S for 10–15 min after the TaqMan primer/probe mix, RNase-free H20 and 2.5 µl of cDNA for a final reaction fixation. After removal of unincorporated volume of 10 µl. For information about excess dye with distilled water, the 4
mineralized nodules were labeled as red spots. (www.dchip.org) for dCHIP analysis. For For adipogenesis, the cultures were incubated Gene set enrichment analysis (GSEA), the data in DMEM Glumax (Gibco) supplemented with are normalized by RMAExpress software 10 % FBS, 10nM HEPES, 100 U/mL (http://rmaexpress.bmbolstad.com). GSEA of penicillin, 100 µg/mL streptomycin, 5- the microarray data were performed according 10µg/mL insulin (Sigma), 0.5 mM to the instructions isobutylmethylxanthine (Sigma), 1-5 x10-6 M (http://www.broadinstitute.org/gsea/index.jsp). dexamethasone (Sigma). The cells were fixed Gene sets tested included gene ontology with 10 % formalin and stained with 0.3 % Oil (c5.all.v2.5.symbols.gmt), BioCarta Red O (Sigma) in methanol (Sigma). The (c2.biocarta.v2.5.symbols.gmt) and KEGG chondrocyte differentiation was induced in (c2.kegg.v2.5.symbols.gmt). After collapsing, monolayer culture, the cells were cultured in there are 20606 genes left from microarray 12-well plates in complete DMEM with high datasets and there are1164 remaining gene sets glucose 4,5 g/L containing 10-7 M after gene set size-filtering (min=15, dexamethsane, 1% ITS (Sigma), 2mM sodium max=500). Gene sets with a nominal p- pyruvate (Sigma), 0.35mM proline (Sigma) value < 0.05 and false discovery rate and 10ng/ml TGF-β3 (R&D System) or (FDR) < 0.25 were considered to be complete chondrocyte differentiation medium significantly enriched. Those genes occurring (mixed by StemXVivo Human/Mouse in the ranked list before the point at which a Chondrogenic Supplement (Catalog # maximal GSEA enrichment score is achieved CCM006) and StemXVivo Human/Mouse are referred to as the leading edge subset and Chondrogenic Base Media (Catalog # thus are responsible for the core enrichment CCM005) in 1/100 ration (R&D system). The observed for a given gene set. Within each chondrocyte differentiation was verified by gene set, the farther the position of a gene to staining of proteoglycan with both 0.1% the left (red) implies a higher correlation with Toluidine blue (Sigma) (pH 2.0 to 2.5) or 1% CD44 negative phenotype, and the farther to Alcian blue in 3% acetic acid solution (pH the right (blue) implies a higher correlation 2.5). Then removed the excess dye and washed with genes down-regulated upon CD44 three times with distilled water. Then plates expression. were mounted with Clear MountTM Mounting solution (Invitrogen, CA, USA). The images Biochemical pathways analysis of the were taken using bright field using Leica microarray data. The lists of 2-fold changed Appication Suite software. genes from the microarray data were applied to the Database for Annotation, Visualization and Integrated Discovery (DAVID) Microarray analysis. RNA is extracted from - - + - Bioinformatics resources v6.7 the sorted CD45 LIN CD44 and CD44 (http://david.abcc.ncifcrf.gov/tools.jsp) for for subsets or culture-expanded MSCs at passage mapping KEGG pathways(27). 1-3, labeled and amplified according to Affymetrix™ GeneChip Expression Analysis Statistical analysis. The unpaired t test or Technical Manual. Chips are scanned using Mann-Whitney test was used to compare the GeneChipTM Scanner 3000. Human Genome differences between the cell types based on the U133 plus 2.0 Chips are normalized using data distribution. All reported p-values were invariant set normalization and probe level obtained using the Graph Pad Prism 4.0 expression values are calculated using the PM- software and less than 0.05 was considered MM model provided by the dCHIP software statistically significant. The frequencies of 5
CFU-Fs were calculated by either Pearson in densities. The CFU-Fs generated from the Excel or L-Calc software (Stem cell CD44- cells were fibroblast-like consistent Technologies INC). with immature phenotype of mesenchymal progenitor cells (Figure 1E). In some of the RESULTS experiments, CD45+LIN+ cells were sorted for Freshly isolated mesenchymal stem and CFU-F assay as controls. However, no progenitor cells of mouse BM lack expression colonies were observed from 200,000 cells of CD44. plated. These data indicate that the CD44- cells Since accumulated evidence has suggested that contain almost all CFU-Fs in mouse BM CD44 is a common positive marker on whereas CD44 expression marks the cells expanded MSCs (28), we wanted to lacking colony-forming capacity. investigate if this protein could be used to Nestin and fibromodulin (Fmod) have purify and characterize primary MSCs ex vivo. been reported to mark primary MSCs in mouse We first analyzed CD44 expression in BM (31) and human (32). Consistent with the stromal cells (CD45-LIN-CD31-) of mouse finding of enrichment of CFU-Fs in the CD44- BM by FACS using an antibody recognizing cell fraction, Q-PCR analysis showed that all forms of CD44 (clone IM7) (Figure 1A). Nestin and FmoD mRNA were enriched in the This revealed that CD44 was expressed on a CD44- cells and almost undetectable in the significant subfraction (45 ± 7 %) of BM CD44+ cells (Figure 1F). In addition, the stromal cells. However, upon investigation of CD44- cells expressed higher levels of mRNA co-expression of CD44 with MSC-associated encoding matrix protein and growth factors cell surface markers including SCA1, including collagen type I (Col1a1), PDGFRa/CD140a, CD51/integrin αv (6,7,29), nephroblastoma overexpressed gene (Nov), we found that the cells expressing these MSC Angiopoietin like-1 (Angptl1) and insulin markers were exclusively detected in the growth factor 1 (Igf1), all reported to be CD44- cell fraction (Figure 1B). expressed in BM MSCs (7,33,34) (Figure 1F). VCAM1/CD106 and CD105 have been In all, these data provide molecular support for reported to be expressed on cultured MSCs the idea that the CD44- cell population is (30). However, analysis of the expression of enriched with MSCs and progenitors. these markers on freshly isolated BM cells Another feature commonly associated suggested that even though the CD44- stromal with stem cell populations is quiescence under cells display high expression of steady state conditions. In order to investigate VCAM1/CD106 and CD105, the expression of the cell cycle status of the CD44+/- cells in these surface molecules is also detected on the mouse BM, we performed cell cycle analysis CD44+ stromal cells (Figure 1B). These data by simultaneous staining of KI67 and DAPI suggest that the phenotypically defined MSCs revealing that while 0.6 % of the CD44+ cells are enriched in the CD44- cells of mouse BM. were in G0, around 15% of the CD44- cells To investigate the functional properties resided in a dormant state (G0) (Figure 2A- of the CD44+ and CD44- stromal cells, we 2B). Correspondingly, the absolute majority of sorted the cells (Figure 1A) and evaluated their the CD44+ cells were accumulated in cycling clonogenic potential by limiting dilution CFU- (G1 and S/G2/M) stages (Figure 2), indicating F assay (Figure 1C-1D). The frequency of that a proportion of the CD44- cells remained CFU-Fs in the CD44- cells was 1/167 whereas quiescent whereas most of the CD44+ cells no CFU-F could be detected in the CD44+ were in active cell cycle. This was supported cells when plated at any of the indicated cell by Q-PCR analysis showing upregulation of
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the cell cycle inhibitor genes p21Cip and p27 in much smaller than that of the CD44+ cells in the CD44- cells, compared to the CD44+ cells the BM, the higher recovery of CFU-Fs from (Figure 2C). These data suggest that the the CD44- cells suggest that they still contain physiologically quiescent cells reside in the the majority of the CFU-Fs (Figure 3F). CD44-, not the CD44+ cells. However, it is important to note that the most Taken together, the phenotypically, of the colonies formed from the CD44+ cells functionally and molecularly defined BM were observed when the cells were plated at mesenchymal stem and progenitor cells higher, nonclonal density (2000 cells/cm2) naturally do not express CD44. (Figure 3E). This could lead to an overestimation of the CFU-F frequency and Human BM MSCs are enriched in CD44- the possibility of that the CFU-F frequency mesenchymal cells. may not reflect the frequency of the To investigate whether our findings from clonogenic cells in this cell population mouse studies hold true in human, we according to previous observation (2). analyzed CD44 expression in normal human Moreover, while the freshly sorted CD44- cells BM stromal cells of healthy donors and were highly proliferative and generated characterized the CD44+/- cell subsets by fibroblast-like cells when plated in culture multi-color FACS, colony assay and global (Figure 3G), the colonies generated from the gene expression analysis. Within CD45- freshly sorted CD44+ cells displayed a CD235-CD31- cells, the majority of the cells dramatically reduced expansion capacity and express CD44 on the cell surface while less could not be replated after the secondary than 10 % of them are CD44- (Figure 3A-3B). culture. This together with growth Since CD146 and CD271 have been reported characteristics of the cells suggests that most to be expressed on freshly isolated human BM of the clonogenic cells in human BM are MSCs (10,11,14,35), we analyzed their naturally CD44-. expression in relation to that of CD44 in Multipotency is a key stem cell feature CD45-CD235-CD31- cells. Interestingly, while for MSCs. We next performed in vitro the majority of the CD44- cells are positive for differentiation assay on expanded cells both CD146 and CD271 only around 4% of generated from freshly sorted CD44- cells to the CD44+ cells express these markers (Figure test their multilineage differentiation potentials. 3A-3B). Unique co-expression of CD146 and This revealed that the CD44- cells could CD271 in the CD44- cells was also reflected in generate adipocytes, osteoblasts and similar frequencies of the CD44- cells to that chondrocytes in vitro (Figure 3H). The of the CD146+CD271+ cells in BM (Figure differentiation assay could not be performed 3B-3D). These data suggest that the absolute with the freshly sorted CD44+ cells since they majority of the phenotypically defined MSCs could not be sufficiently expanded in culture. reside in the CD44- cells in human BM. These data suggest that the CD44- cells To test their clonogenic capacity, we contain a major part of the MSCs in human sorted the CD44+/- subsets directly from BM. human BM by FACS (Figure 3A) and Furthermore, FACS analysis of the performed limiting dilution assay of CFU-Fs. reported MSC-associated martkers CD73, We found that the CFU-F frequency in the CD29, VCAM1 and STRO1 on BM cells CD44- cells reached 1/14, 94-fold higher than indicated that majority of the CD44- stromal that (1/1314) in the CD44+ cells (Figure 3E). cells from human BM expressed these surface Although the proportion of CD44- cells is antigens (Figure S1). Surprisingly, the CD44-
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cells displayed low expression CD105, include gene sets coding for extracellular presenting a discrepancy from what has been molecules that are significantly enriched in the reported for cultured MSCs(15). CD44- cells (Figure 4B-4C, Table S3). Furthermore, enrichment of cell cycle Human CD44- mesenchymal cells display progression genes in the CD44+ cells (Figure MSC-associated molecular phenotype. S3B-S3C) confirms relatively quiescent status To further investigate molecular properties of of the CD44- cells. These data provide the CD44- and CD44+ cells from human BM, comprehensive views into potential functions we performed microarray analysis on freshly of the CD44- and CD44+ subsets and important sorted human BM CD45-CD235-CD31- hints for further investigation of the role of CD44+/- subsets. dCHIP analysis revealed 929 human BM stromal cells in homeostasis and genes that are more than 4-fold differentially diseases. expressed in the CD44+ as compared to the - Acquisition of CD44 expression on CD44 cells. Among genes reported to be mesenchymal stem and progenitor cells related to MSC properties, we noted during in vitro culture. downregulation of cell cycle progression genes The finding that the CD44- stromal cells and upregulation of the cell cycle inhibitor contain essentially all MSCs is completely on genes including CDKN1A(p21), GAS1 and the contrary to what has been reported for GAS6 in the CD44- cells as compared to + culture-expanded MSCs. In order to test CD44 cells (Figure 4A and S2-S3), whether expression of CD44 was due to in suggesting a relatively quiescent status of the - - vitro manipulation of the cells, we cultured the CD44 cells. Importantly, the CD44 cells freshly isolated CD44- MSCs from mouse and expressed higher levels of a set of cytokines human BM and analyzed CD44 expression on and growth factors including KIT ligand the cells after culture at different passages. As (KITLG), vascular endothelial growth factor previously reported, these cells acquired CD44 (VEGFC), Jagget-1 (JAG1), Angiopoietin-like expression on their surface after in vitro 4 (ANGPTL4), ANGPT1 and CXCL12 as well expansion early at the first passage and as extracellular matrix proteins (ECM) such as remained at a high level during the later laminin α4 (LAMA4), fibronectin (FN1), passages (>98.7±1.5%) (Figure 5). We have fibromodulin (FMOD), necdin (NDN) and developed multiple cell clones from single collagen type I (COL1A1), all reported to be CFU-F generated from mouse CD44- cells in enriched in human BM MSCs (32-34,36) and the limiting dilution assays and all of the shown to be important for hematopoiesis clones were positive for CD44 in culture at (36,37). On the contrary, expression of these early and later passages (passage 5 to 14) genes is undetectable or low in the CD44+ - (Figure 5B). However, acquisition of CD44 cells (Figure 4A-4C). The CD44 cell expression on the MSCs upon culture did not population also displays relatively higher affect proliferation capacity of the cells expression of other MSC-related genes (Figure (Figure 5D). These data suggested that CD44 S1). In addition, Gene Sets Enrichment was dramatically up-regulated on Analysis (GSEA) revealed that multiple genes mesenchymal stem and progenitor cells during sets for known biological in vitro culture and CD44+ phenotype of functions/processes/pathways are significantly expanded MSCs does not reflect the true negatively or positively correlated with CD44- cellular identity of the primary MSCs and phenotype (p <0.001 and FDR <0.25) (Table progenitor cells. S1-S3, Figure 4B-4C, Figure S3). These
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In vitro cultivation induce extensive changes contrary, CXCL12, MPO and EGR1 are in gene expression in human MSCs. The significantly downregulated in the cultured induction of dramatic changes in CD44 MSCs. Hence, we conclude that in vitro expression and proliferation characteristics cultivation of human MSCs results in dramatic upon cultivation of the CD44- BM stromal and consistent changes in gene expression cells prompted us to search for molecular patterns. mechanisms underlying culture-related changes in the MSCs. To this end, we performed microarray experiments to compare DISCUSSION gene expression patterns in cultured MSCs at In the present study, we have prospectively + - passage 1-3 to that of the freshly sorted CD44- isolated the CD44 and CD44 stromal cells mesenchymal cells (CD45-CD235-CD31- from both human and mouse BM by multi- CD44-) from the same donors. dCHIP analysis color FACS and characterized them of the microarray data revealed that 2708 phenotypically, functionally and molecularly. genes were more than 2-fold differentially In striking contrast to the previous finding of expressed in the CD44+ cultured MSCs as high expression of CD44 on culture-expanded compared to the freshly sorted CD44- cells. MSCs, we have uncovered that native Several of the changes appeared to be mesenchymal stem and progenitor cells lack consistent between two different donors and CD44 expression. This finding is of large over three passages. Among the differentially importance for the development of methods expressed genes we identified a large number for isolation of BM MSCs both for of surface antigens in addition to CD44 experimental and clinical porposes. (Figure 6). These included cell adhesion SCA1, PDGFRa and Nestin have receptors such as integrins (ITGA3, ITGAE, recently been shown to be expressed on ITGB5 and ITGA6) and CD109, ADAM12, freshly isolated mouse BM MSCs (6,31,39). CD151, CD59, CD248, some of which have We here report that the BM stromal cells been reported to be expressed on cultured positive for those markers are enriched in the - MSCs (30,38). While MSC-associated CD44 cells in mice. Similarly, the majority of + + markers such as CD73 and CD146 are the previously defined CD271 CD146 MSCs upregulated, NGFR/CD271, VCAM1, CD36 (14) from human BM do not express CD44. and EPOR are downregulated in the cultured Furthermore, we also show that the commonly MSCs compared to the fresh ex vivo analysed used positive MSC markers CD73, CD106, CD44- cells. Importantly, in keeping with our CD29 and STRO1 are highly expressed on the - FACS data, CD44 is dramatically upregulated freshly sorted CD44 cells. These data together in the cultured MSCs. In addition, we with the finding of enrichment of CFU-F - observed dramatic upregulation of hyaluronan activities in the CD44 cells strongly support synthase (HAS1, HAS2), growth factor and CD44 negative phenotype of the MSCs. This matrix protein genes including VEGF, result can be further corroborated by the cell WNT5A,WNT5B, FN1, LAMAB1, BDGF cycle analysis and molecular data showing and Collagens in the cultured MSCs (Figure 7). enrichment of quiescent cells and the cells Microarray data-based signal pathway- with multilineage differentiation potential in - mapping illustrated upregulated (≥2-fold) the CD44 cells. Another interesting finding in - genes for the WNT signal, Focal adhesion and the present study is that the CD44 stromal MAPK signal pathways in the cultured MSCs cells express higher levels of hematopoiesis- expressing CD44 (Figure S4, S5). On the regulating growth factors and matrix proteins including CXCL12, KIT Ligand, Angiopoietin 9
1, VEGF, JAG1, LAMA4 and FN1, compared MSCs showed more efficient homing to BM to the CD44+ cell fraction. Thus, this cell compared to the cultured MSCs (40). Loss of population might play an important role for BM homing capacity of the cultured MSCs maintenance of normal hematopoiesis in vivo. might be due to culture-induced changes in Taken together, our data strongly suggest that expression of adhesion receptors including CD44- stromal cells are enriched with CD44 and CXCR4 on the cells, which might multipotent mesenchymal stem and progenitor lead to unwanted entrapment in other organs cells. Although MSCs are only a small (6,38). CD44 exists in multiple isoforms on fraction of the CD44- cells, given the fact that different type of the cells and CD44 CD44 is widely expressed in hematopoietic expression as well as its binding capapcity cells and endothelial cells, using this marker towards their ligands are regulated by different alone or in combination with other negative cytokine stimulation and other environmental markers including CD45 and CD235 in human changes (41). It has been shown that Early and in combination with a positive marker growth response 1(EGR1) regulate CD44 such as SCA1 in mice would significantly transcription via binding to CD44 promoter in facilitate prospective isolation of MSCs from B cells (42). However, our gene expression BM. data suggest that the level of EGR1 transcript Most importantly, our data provide is dramatically reduced (by 14 fold) in the clear evidence for alteration of MSC cultured MSCs while these cells acquire CD44 phenotype during in vitro manipulations of the expression. Hence, apparently the functional cells, emphasizing the importance of linkage between those two molecules in the prospective isolation of MSCs in order to MSCs require further investigations. uncover the nature and therapeutic potentials Comparing gene expression patterns of the cells. The functional consequence for and surface marker expression in the freshly acquisition of CD44 expression on the isolated and cultured MSCs revealed that in expanded MSCs during therapeutic use is vitro manipulation of MSCs resulted in unclear. However, since the acquisition of dramatic overall changes in gene expression CD44 expression already occurred at the first patterns. We here provided new evidence for passage and remained on the cells at the later changes in expression of adhesion receptors passages during in vitro expansion, it is likely and signalling molecules. While CD105, that stem cell growth characteristics such as integrin α1/CD49A, α3/CD49C, α4/CD49D, sustainable expansion capacity of the MSCs α5/CD49E, α6/CD49F, CD151 and CD109 are remained after acquiring CD44 expression. In upregulated on the cultured MSCs, expression addition, the cells expanded in culture from of other cell surface antigens such as VCAM1, the CD44- cells could give rise to adipocytes, ICAM4, CD36, EPOR and PTHGR are chondrocytes and osteobalsts, suggesting reduced in the culture-expanded MSCs, as maintenance of multilineage differentiation compared to the freshly sorted CD44- stromal potential of the cells after acquisition of CD44 cells. These changes could possibly result in expression on the surface. Hence, expression changes in differentiation potential and other of CD44 per se does not appear to reduce the cellular processes of the cultured MSCs since MSC potential of the cultured cells in vitro. those factors have been reported to be However, it was reported that the culture- important for regulating lineage differentiation expanded MSCs displayed reduced or loss of of human MSCs (reviewed in (43))(44-47). In homing capacity to BM although highly addition, the changes in expression cell surface expressed CD44 (6). On the contrary, primary receptors including CD44 could result in the
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upregulation of multiple signal molecules findings highlight importance of ex vivo related to cell adhesion, growth factor analysis of the cells and provide clear evidence pathways in the cultured MSCs, however, the for true cellular identity of MSCs and latter could in turn lead to further changes of progenitor cells. the cell surface receptor expression through inside-out signal transduction. CD44 function is controlled by its ACKNOWLEDGMENTS posttranslational modifications such as The authors thank Liselotte Lenner in sialofucosylations. It has been shown that the Linköping University for the valuable advice CD44 glycoform bearing alpha-2,3-sialyl and technical assistance and thank Professor modifications on the cultured MSCs is not Eva Hellström Lindberg at Karolinska reactive with BM vascular E-selectins, which University hospitial, Karolinska Institute for resulted in poor osteotropism after systemic providing us bone marrow samples from transplantation of the cells. However, this can healthy voluteers. The work was generously be rescued by converting the glycoform to supported by grants from the Swedish Cancer selectin-binding glycoform of CD44 (48). On Society, the Swedish Research Council, the the other hand, it was reported that CD44 on Swedish Childhood Cancer Foundation and the cultured MSCs contributed to migration of the faculty of Medicine at Linköping the cells into injured kidney via interaction University. with hyaluronic acid at sites of injury (17,49). It is important to note that hyaluronic acid is FOOTNOTES: also upregulated in many solid cancers (24). Author Contributions: HQ designed and Positive contribution of cultured MSCs to performed research, collected, analyzed and breast cancer development has been reported interpreted data, wrote the manuscript and did (50). Therefore, although MSCs hold great final approval of the manuscript. KLB promise for cancer therapy(51), safety issue performed research, analyzed data and did should be seriously concerned before clinical final approval of the manuscript. MS designed use of culture-expanded MSCs for gene or research, analyzed and interpreted data, wrote drug delivery. the manuscript and did final approval of the In summary, we have here provided manuscript. phenotypic, functional and molecular evidence Disclosure of potential Conflict of Interest: that BM mesenchymal stem and progenitor All authors have no conflicts of interest to cells physiologically do not express CD44 in disclose. both humans and mice. However, in vitro Abbreviations: MSCs, Mesenchymal stem culture could result in acquisition of CD44 cells; BM, Bone marrow; FACS, fluorescent expression on their surface and changes in activated cell sorting; CFU-F, colony-forming expression of cytokine, growth factor, matrix unit-fibroblast. protein and other signalling molecules. These
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Figure Legends
Figure 1. Clonogenic mesenchymal stem and progenitor cells lack expression of CD44 in mouse BM. (A) One representative FACS profile shows analysis of CD44 expression in CD45- LIN-CD31- cells. The numbers in the panels are mean % of the CD44+/- cells, from 10 experiments. (B) FACS analysis of expressions of SCA1, CD51, CD90.1, CD105, VCAM1/CD106 and PDGFRa/CD140a in CD45-LIN-CD44+/- cells. The numbers in the panels indicated mean percentages of the gated cells within CD45-LIN-CD31- cells. The data were from 3-10 experiments. (C) Limiting dilution of CFU-Fs in the CD44+/- cells. The cells were plated at densities of 10, 50, 100, 200 cells for the CD44- cells and 200, 500, 1000, 2000 cells per well for the CD44+ cells in 96-well plates The cell dose yielding 37.5% negative wells for CD44- cells was 167, indicated by dashed line. The 95% confidence interval (CI) bands are shown in dotted lines. There was no CFU-Fs observed from the CD44+ cells at any of the doses. (D) The frequencies of CFU-Fs in the CD44+ and CD44- cells calculated by L-Calc (Stem Cell Technologies). Data were mean ± 95% CI, from 3 experiments. nd, Not detectable. (E) Morphology of Giemsa stained CFU-Fs derived from the CD44- cells. (F) Q-PCR analysis of expressions of MSC-associated genes. Data were 3 independent sorting experiments. Each dot represents mean of triplicate measurements in each experiment. MSC associated genes include Fmod, Igf1, Nov, Nes, Col1a1 and Angptl1. The differences between the two cell types were compared by unpaired one-tailed t test.
Figure 2. Quiescent mesenchymal cells reside in the CD44- stromal cell fraction. (A) One representative FACS profile of cell cycle analysis of the CD45-LIN-CD44+ and CD44- cells by KI67 and DNA staining. Cell-cycle status within the defined the CD45-LIN-CD44+ and CD44- cells was determined by simultaneous two-parameter analysis with DNA content versus KI67 expression. Numbers in quadrants show the percentages of the gated CD44+ and CD44- cells in each of the cell-cycle phases (G0, G1, and S/G2/M). (B) Mean cell-cycle distribution of total CD44+ and CD44- cells. Data were from 2 experiments. (C) Q-PCR analysis of cell cycle regulator genes p21, p27 and Cdk6 in the CD44+ and CD44- cells. The data were normalized to endogenous Hprt expression, from 2 independent sorting experiments. Every dot represents mean of triplicate measurements for each gene. The differences between the two cell populations are indicated in the panels.
Figure 3. Human BM mesenchymal stem and progenitor cells primarily reside in CD44- stromal cell fraction. The data were from 5 analysis experiments with 4 healthy donors. (A) FACS profiles show CD44, CD271, CD146 expression in human BM CD45-CD235-CD31- cells and sorting of the CD44+/- cells. The stromal cells were first enriched by MACS prior to staining of the antibodies. (B) Percentages of the CD44+ and CD44- cells within stromal cells in BM. (C) Expressions of CD146 and CD271 in the CD44+ and CD44- cells. (D) The frequencies of CD146+CD271+cells, CD44+ and CD44- cells in BM. (E) Limiting dilution of CFU-Fs in the CD44+/- cells. The cell dose yielding 37.5% negative wells were indicated by dashed lines on X- axis. (F) CFU-F recovery from the CD44+/- cells of BM. (G) Morphology of Giemsa stained CFU-Fs. (H) In vitro differentiation of the CD44- cells. The adipocytes were identified by oil red stainings. The osteoblasts were confirmed by both Alizarin red and von konssa stainings. The chondrocytes were identified by both Toluidine blue and Alcian blue.
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Figure 4. Microarray data provide molecular evidence for immature phenotype of the CD44- cells from human BM. (A) dCHIP analysis of gene expressions of cytokines, growth factors and ECM proteins in the CD44+ and CD44- cells from normal human BM. Clustering shows the genes being up-regulated more than 4-fold in the CD44- cells compared to the CD44+ cells. Red represents high and blue low expression. The numbers in the heatmap are expression values of each indicated genes. (B-C). GSEA analysis of significantly up-regulated gene sets in the CD44- cells. The green curves plot the ES (enrichment score). Black vertical dashed lines specify the maximum ES score. Significantly enriched data sets are defined according to GSEA default settings (p < 0.001 and FDR < 0.25). The heatmap shows expressions of the genes in the leading edge subsets (only top 40 genes were shown for clarity if more than 40 genes in the leading edge subsets). (B) Enrichment of genes in the gene sets of extracellular matrix region in the CD44- cells. (C) Enrichment of the gene set of extracellular matrix structural constituents in the CD44- cells. The data were from 8 microarray platforms and 2 sorting experiments, normalized by RMAExpress software. See also in Figure S1-S2 and Table S1-S3.
Figure 5. Acquisition of CD44 expression in CD45-LIN-CD44- MSCs and progenitor cells after culture. The freshly sorted CD44- cells from human and mouse BM were cultured and the expanded cells were analysed by FACS for CD44 expression. (A) CD44 expression in the expanded mouse stromal cells after 8-10 days culture. (B) CD44 expressions in the cultured cells at later passages (passage 5-15) derived a single CD44- mouse cell clone. The red lines indicated expression of CD44 and the blue line indicated the isotype control stainings. (C) CD44 expression in the cultured human BM stromal cells. The freshly sorted CD45-CD235-CD31-CD44- cells from BM of healthy humans were cultured for 14 days. A15, 16 and A17 indicated different donors. The red lines indicated expression of CD44 and the blue line indicated the isotype control stainings. (D) Fold expansion of the cells generated from the freshly sorted CD44- cells during culture. Limited number (10-50 cells) of the sorted CD44- cells were plated in culture and the cells generated from the culture were counted and calculated for fold expansion. The data were mean ± SEM, from 3 independent experiments on BM from 3 healthy volunteers. X-axis indicates the number of the passages (p).
Figure 6. Culture-induced alteration of cell adhesion molecule expression in MSCs from human BM. The microarray data on the freshly sorted CD44- mesenchymal cells and on the culture-expanded MSCs (acquired CD44 expression) derived from the fresh CD44- cells of the same donors were analysed by dCHIP software. The microarray experiments on the culture- expanded MSCs were performed at passage (p) 1-3. The ‘a’ and ‘b’ indicated replicate samples at each passage (p1-p3) from the indicated donors (a15 or a17). Clustering shows the genes being up-regulated more than 3-fold in the cultured cells compared to the freshly sorted CD44- cells. Red represents high and blue low expression. The numbers in the heatmap are expression values of each indicated genes and the fold changes in expression of the receptors are caculated based on the mean expression values from each cell type. The data were from 10 microarray platforms (6 arrays of the cultured cells and 4 arrays of the fresh cells) and 2 sorting experiments on 2 healthy volunteers, normalized by RMAExpress software.
15
Figure 7. Differential expression of growth factor, matrix protein and signaling molecules in freshly sorted MSCs and the cultured MSCs of human BM. Gene expressions of cytokines, growth factors and ECM proteins in the freshly sorted CD44- mesenchymal cells and the culture- expanded MSCs (acquired CD44 expression) derived from the freshly sorted CD44- cells of the same donors were analysed by dCHIP software. The microarray experiments on the culture- expanded MSCs were performed at passage (p) 1-3. The ‘a’ and ‘b’ indicated replicate samples at each passage (p1-p3) from the indicated donors (a15 or a17). Clustering shows selected genes being up-regulated more than 8-fold in the cultured cells compared to the freshly sorted CD44- cells. For clarity, only part of the growth factors and signalling molecules are shown in the panel. Red represents high and blue low expression. The numbers in the heatmap are expression values of each indicated genes and the fold changes are caculated based on the mean expression values from each cell type. The data were from 10 microarray experiments (6 arrays of the cultured cells and 4 arrays of the fresh cells) and 2 sorting experiments on 2 healthy volunteers, normalized by RMAExpress software. See also in Figure S4 and Figure S5.
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Fig. 1 (Qian, et al.) A PI- CD45-LIN-CD31-
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o t o o r t t t o t o
2.0 t t
t
t
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n n n n
150 3 o p n o n n n n i
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i i s s 1.0 o s s s s s s s 1.5 s t s o s s s 60
s s e s e s s 0.20 s t 1.5
s e e s r r n e e e e e e r r n e p r r e p r r 2 r r 100 0.1 o p p r i r x p p o p x p p p i x x p p 1.0s e x x x e
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s 0.5 0.2 e e 0.15 x x s e e e
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e a a e e e 5 e l l R e
R e 0.0 0.0 e e R R R R R e e e
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i CD44+ CD44- v R
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0 R 0.00 R Angptl1 Igf1 CD44+ CD44- CD44+ CD44- Nov t
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0 R 0.00 R CD44+ CD44- CD44+ CD44- Fig. 2 (Qian, et al.)
A CD44+ CD44- 46 54 36 49 G1 S/G2/M KI67
G0 KI67
DAPI 0.6 15
DAPI
B P < 0.0001 P=0.008 ns, P=0.065
CD44+ CD44- CD44+ CD44- CD44+ CD44- G0 G1 S/G2/M
C p27 p21 t Cdk6 t t r r r
0.15 p
1.5 p p P = 0.008! 6 ns
P = 0.016! H H H
o o o t t t
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18
19
20
Fig. 5 (Qian, et al.)
A Mouse 96.7
Culture
Freshly sorted CD44 CD44 CD44- Max %of
CD44 B #1 #2 #3 #4
Max of #2 #3 #4 %
CD44 C Human From CD45-CD235-CD31-CD44- cells A15 A16 A17
Max of %
CD44 D
21
Figure 6. (Qian et al.) fresh fresh fresh fresh ,_a15_P1b ,_a17_P3a _a17_P3b _a15_P1a _a15_P2a _a15_P2b , , , , ------
Gene name Fold change CD44 CD44 CD44 CD44 CD44 CD44 CD44 CD44 CD44 CD44
1494 300 242 919 767 951 15 49 24 21 ITGA6 28 1245 402 325 772 661 772 11 26 12 18 CD9 41 1735 1172 1152 1331 1016 1071 27 34 69 35 ADAM12 30 3567 2345 2378 3001 2528 2702 41 32 15 43 MET 85 152 92 72 125 93 108 15 24 7 22 PTPN13 6 3063 1528 1431 2255 2083 2218 36 49 28 26 CD109 61 3372 1608 1437 2109 2291 2559 24 30 19 27 NT5E/CD73 90 224 171 155 327 291 276 48 39 39 28 ITGA3 6 4803 3191 3761 4957 5004 4909 73 570 342 948 CD59 9 423 296 283 382 439 436 50 45 60 47 CD33L3 7 392 329 284 321 382 413 33 16 37 43 PROCR 11 586 403 420 473 552 540 10 20 16 53 ASAM 20 567 712 714 786 815 790 172 95 82 59 CEECAM1 7 394 337 345 436 378 436 57 14 17 23 CD44 14 721 667 637 896 767 769 62 141 31 46 CD99 11 2497 2278 2462 3006 2857 2582 399 244 156 110 CD248 12 2312 2132 2308 2636 3048 2967 144 195 66 49 ANPEP/CD13 23 1101 1112 1092 1423 1476 1376 30 267 73 176 CD151 9 1494 1226 1218 977 1025 970 248 233 259 373 ITGAE 4 408 425 423 395 324 356 110 91 103 62 MCAM/CD146 4 1525 1974 2172 1910 1879 1820 190 100 96 42 ITGA5 18 200 195 214 215 198 185 38 14 28 53 BMPR2 6 599 979 981 704 683 671 53 70 104 16 FZD2 13 276 338 302 229 239 252 18 72 27 52 ITGB5 6 92 77 75 94 102 93 480 1902 230 947 GHR -10 31 39 38 40 41 40 175 424 47 157 CD53 -5 6 5 4 5 6 5 2006 2141 37 136 CEACAM8 -214 19 18 24 25 23 23 69 113 46 64 CD300A -3 13 17 17 15 15 14 58 81 49 40 NGFR/CD271 -4 55 41 46 60 45 55 181 150 138 102 PTHR1 -3 20 11 10 14 15 12 408 393 147 79 FRZB -19 835 21 19 653 242 298 5212 6080 3194 1890 VCAM1 -12 194 137 112 196 200 211 353 598 794 589 CD82 -3 29 29 27 31 29 27 135 70 114 205 EPOR -5 10 7 7 9 10 11 19 108 172 208 ICAM4 -14 12 13 13 12 11 13 102 247 789 1482 CD36 -53 32 30 34 29 31 25 151 78 543 598 SPN -11 1365 1350 1312 1093 1231 1271 1095 2333 5846 6866 TFRC -3
22
Figure 7. (Qian et al.) fresh fresh fresh fresh ,_a15_P1a ,_a15_P1b _a15_P2a _a15_P2b _a17_P3a _a17_P3b , , , , ------Gene name Fold change CD44 CD44 CD44 CD44 CD44 CD44 CD44 CD44 CD44 CD44 500 282 417 497 10 10 10 10 9 9 SPARCL1 -44 18656 9132 13347 6944 16 16 20 17 17 15 DEFA1 /// DEFA3 -713 13047 6707 12141 6393 1479 1339 991 963 1083 1153 CXCL12 -8 1764 97 1078 127 14 16 15 15 14 14 MPO -53 2411 381 3476 939 112 128 143 137 146 130 EGR1 -14 20 9 42 23 454 308 107 152 510 358 TNFRSF11B 13 27 9 25 19 1001 359 456 359 293 831 FN1 27 24 30 66 48 167 466 436 415 404 428 COL6A1 9 445 56 224 89 1658 4517 3510 3771 3334 3883 COL6A2 17 20 28 28 19 1314 859 767 756 617 581 BDNF 34 31 16 48 18 2457 2079 1723 1872 1044 1006 WNT5A 60 61 28 18 26 4819 4111 2569 2623 3217 3119 COL4A1 102 20 13 31 8 256 214 96 112 136 95 COL8A2 8 365 35 59 47 4533 3568 2251 2395 2298 2142 COL5A2 23 164 45 30 46 8233 6298 3190 3337 3440 3379 COL12A1 65 11 37 64 40 402 446 583 520 407 465 LIF 12 77 72 62 32 1726 2433 2393 2167 2047 2012 HAS1 35 20 27 10 8 2154 2677 3058 3016 2646 2824 SPOCK1 167 241 105 37 30 2044 1576 1672 1759 1699 1590 LAMB1 17 15 16 7 6 3730 2973 3073 3248 3273 3273 DKK1 305 14 23 57 17 394 436 378 436 337 345 CD44 14 73 111 91 68 1517 1422 1481 1384 1056 1203 LAMB2 16 99 4 39 20 1701 1741 1529 1526 1247 1288 WNT5B 37 293 66 417 175 6389 7301 5559 5070 5143 5669 COL4A2 25 713 94 414 87 15859 14483 11633 12164 12246 12408 TGFBI 40 285 65 209 53 8758 7844 6123 6502 5682 5788 COL6A3 44 8 53 28 40 395 231 231 265 293 234 OSTM1 8 164 146 68 196 1375 1456 1318 1518 1835 1730 VEGF 11 9 25 24 14 814 573 416 463 917 744 POSTN 37 14 13 43 21 1696 1341 1027 1071 1596 1392 HAS2 60
23
Supplemental Information
Primary Mesenchymal Stem and Progenitor Cells from Bone Marrow Lack Expression of
CD44
Hong Qian1,2*, Katarina Le Blanc2, Mikael Sigvardsson1
Supplemental data:
Figure S1: related to Figure 3; Figure S2: related to Figure 4A; Figure S3: related to Figure 4B; Figure S4: related to Figure 7; Figure S5: related to Figure 7; Table S1-S3, related to Figure 4B. Supplemental materials:
Antibodies used for flow cytometry.
Probes used for Q-PCR.
Supplemental Data:
Fig. S1. (Qian, et al) Fig. S1 (Qian, et al.) CD44- CD44+ 6 70 8 1 CD106
15 70 99 0.4 CD29
12 71 2 0.6 CD73
7 48 4 .1.8 STRO-1
1 23 98 0.2 CD105
!"#$%&
Supplemental Figure 1. FACS analysis of STRO1, CD73, CD29, CD105 and CD106 expression on the CD44- mesenchymal cells of human bone marrow. The stromal cells were first enriched by MACS using CD45 and CD235 microbeads. The enriched cells were stained with fluorochrome conjugated antibodies against CD45, CD235, CD31, CD44, CD271 and the respective cell surface markers. The CD44+ and CD44- cells were gated within CD45-CD235- CD31- live cells. The data were from 3 independent experiments with 3 healthy young adult donors. The numbers in the panels are mean % of the CD44+ or CD44- cells.
Fig. S3.(QianFig., Set2 al). (Qian, et al) a1 a b a1 b1 a b b1 + + + + - - - - CD44+BMa.CEL CD44+BMb.CEL CD44+a_(HG-U133_Plus_2).CEL CD44+b_(HG-U133_Plus_2).CEL BMa.CELCD44- CD44-a_(HG-U133_Plus_2).CEL BMb.CELCD44- CD44-b_(HG-U133_Plus_2).CEL CD44 CD44 CD44 CD44 CD44 CD44 CD44 CD44 Genes Foldchanges 8 6 6 7 202 442 38 35 AMOTL2AMOTL2: 27 angiomotin like 2 5 6 5 5 103 275 22 11 PGF PGF:21 placental growth factor, vascular endothelial growth ... 23 27 24 24 963 2643 53 26 PRG2PRG2: 38 proteoglycan 2, bone marrow (natural killer cell acti... 6 7 7 7 804 1312 50 67 S100PS100P: 82 S100 calcium binding protein P 3 5 4 4 237 413 48 93 CSPG2CSPG2: 48 chondroitin sulfate proteoglycan 2 (versican) 3 3 3 4 209 614 47 205 GHRGHR: 77 growth hormone receptor 15 15 24 23 536 1798 154 314 BGLAPBGLAP: 37 bone gamma-carboxyglutamate (gla) protein (oste... 78 79 77 76 267 1420 160 231 STAT2STAT2: 7 signal transducer and activator of transcription 2, ... 7 13 9 15 357 1611 137 213 MXRA5MXRA5: 53 matrix-remodelling associated 5 5 5 5 5 38 464 31 81 ECM2ECM2: 30 extracellular matrix protein 2, female organ and adi... 4 3 3 4 69 741 41 24 BCL6BCL6: 60 B-cell CLL/lymphoma 6 (zinc finger protein 51) /// B... 24 29 24 25 515 112 41 72 ALDH2ALDH2: 7 aldehyde dehydrogenase 2 family (mitochondrial) 18 17 15 12 525 26 74 10 EGFREGFR: 10 epidermal growth factor receptor (erythroblastic leu... 11 11 8 12 720 45 29 24 SVEP1SVEP1: 20 sushi, von Willebrand factor type A, EGF and pen... 21 14 16 15 204 44 49 17 ITGB7ITGB7: 5 integrin, beta 7 17 16 17 14 149 28 50 27 HLXB9HLXB9: 4 homeobox HB9 3 4 4 5 102 128 29 41 COL14A1COL14A1: 18 collagen, type XIV, alpha 1 (undulin) 8 6 6 8 3153 2943 482 541 SPARCSPARC: 252 secreted protein, acidic, cysteine-rich (osteonecti... 18 21 7 24 4003 3623 610 580 CEBPDCEBPD: 125 CCAAT/enhancer binding protein (C/EBP), delta 25 20 23 25 1966 2191 330 504 LTBP2LTBP2: 54 latent transforming growth factor beta binding prot... 6 6 3 25 4714 4729 805 1202 FOXC1FOXC1: 285 forkhead box C1 15 25 21 29 2946 2970 617 672 JUN JUN:81 v-jun sarcoma virus 17 oncogene homolog (avian) 5 4 5 6 5329 6443 236 384 S100A8S100A8: 623 S100 calcium binding protein A8 (calgranulin A) 23 22 27 28 583 721 29 41 IER3IER3: 14 immediate early response 3 16 16 13 23 877 1158 63 65 SLPI SLPI:32 secretory leukocyte peptidase inhibitor 14 16 10 20 223 281 33 32 FZD1FZD1: 10 frizzled homolog 1 (Drosophila) 7 9 11 12 520 648 60 58 EFEMP2EFEMP2: 33 EGF-containing fibulin-like extracellular matrix pr... 12 10 11 12 118 133 23 17 MMP2MMP2: 7 matrix metallopeptidase 2 (gelatinase A, 72kDa ge... 6 7 8 6 639 749 97 72 IGFBP3IGFBP3: 59 insulin-like growth factor binding protein 3 33 29 26 24 3159 3661 758 434 RASD1RASD1: 71 RAS, dexamethasone-induced 1 10 12 10 9 92 99 25 18 NAV1NAV1: 6 neuron navigator 1 17 18 19 13 479 348 42 230 LGALS1LGALS1: 16 lectin, galactoside-binding, soluble, 1 (galectin 1) 4 3 4 5 189 100 78 43 SPARCL1SPARCL1: 26 SPARC-like 1 (mast9, hevin) 53 55 37 53 6417 5142 1365 903 FOSBFOSB: 70 FBJ murine osteosarcoma viral oncogene homolog B 17 16 14 14 533 444 145 59 HNMTHNMT: 20 histamine N-methyltransferase 10 10 17 21 879 694 55 151 NFIL3NFIL3: 30 nuclear factor, interleukin 3 regulated 126 157 208 228 4712 3498 590 719 MCL1MCL1: 13 myeloid cell leukemia sequence 1 (BCL2-related) 7 7 5 6 179 145 17 18 PDGFRLPDGFRL: 15 platelet-derived growth factor receptor-like 41 35 35 35 1736 1369 158 179 IGFBP7IGFBP7: 23 insulin-like growth factor binding protein 7 23 23 20 24 840 632 158 181 ANXA2ANXA2: 20 annexin A2 3 3 3 3 479 327 75 58 APP APP:82 amyloid beta (A4) precursor protein (peptidase nexin-... 10 14 11 20 2915 1830 426 322 FOS FOS:100 v-fos FBJ murine osteosarcoma viral oncogene hom... 296 378 241 245 5595 3801 1127 748 JUNDJUND: 10 jun D proto-oncogene 10 14 10 13 4470 2850 747 342 VCAM1VCAM1: 177 vascular cell adhesion molecule 1 95 111 111 116 3050 1815 157 208 IER2IER2: 12 immediate early response 2 8 7 7 8 1273 603 34 25 MPOMPO: 65 myeloperoxidase 72 107 112 94 3136 1584 505 281 CEBPBCEBPB: 14 CCAAT/enhancer binding protein (C/EBP), beta 6 9 8 8 1599 646 230 269 FMO2FMO2: 87 flavin containing monooxygenase 2 (non-functional... 9 10 9 8 1818 641 141 71 EGR1EGR1: 75 early growth response 1 13 9 11 8 192 79 40 23 PDGFRBPDGFRB: 8 platelet-derived growth factor receptor, beta pol... -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0 0.5 1.0 1.5 2.0 2.5 3.0
Supplemental Figure 2. Up-regulation of MSC-related genes, oncogenes and anti-apoptotic genes in the CD44- cells. dCHIP analysis of global gene expression in the freshly sorted CD44+ cells and CD44- cells from normal human BM. Clustering shows some of the genes being up- regulated more than 4-fold in the CD44- cells compared to the CD44+ cells. Red represents high and blue low expression. Data were from 2 independent sorting experiments, 4 microarray experiments. The numbers in the heat map are expression values of each indicated genes, normalized by dCHIP software. Related to Figure 4.
Fig. S3 (Qian, et al,.) Fig. S4.(Qian, et al) A B
P <0.001 FDR=0.15
Leading edge subsets TRIAP1 NBN BUB1 ERCC3 KNTC1 CDC45L ZW10 MAD2L2 BIRC5 CCNA2 RINT1 RBBP8 CCNE2 ZAK ATM HUS1 TIPIN CHEK1 BUB1B RB1 ATR SMC1A CHEK2 RAD17 MAD2L1 TTK + CD44 - CD44 C P =0.02 FDR=0.12
Leading edge subsets TRIAP1 NBN DCTN3 UBE2C ANAPC5 BUB1 SMC3 KIF22 TARDBP KNTC1 RAN NUSAP1 KIF2C NOLC1 ZW10 PRMT5 MAD2L2 ANAPC4 BIRC5 CDC25C CCNA2 RINT1 CDCA5 NEK2 MPHOSPH6 ATM SMC4 ESPL1 CENPE BUB1B CDC23 KIF11 DLG7 KIF15 SMC1A RAD17 AURKA MAD2L1 ANLN TTK + CD44 - CD44
Supplemental Figure 3. GSEA analysis of cell-cycle regulator genes in the CD44- cells. (A) Plot of p values versus normalized enrichment scores (NES) shows the number of enriched gene sets that are significantly different between the two cell populations. False discovery rate (FDR)-q value is the estimated probability that the normalized enrichment score represents a false positive finding. NES is the enrichment score for the gene set after it has been normalized across analyzed gene sets. (B-C) Enrichment of cell cycle genes in the CD44+ cells. (B) Enrichment of the genes in the gene set for M phase mitotic cell cycle. (C) Enrichment of genes in the gene set of cell cycle checkpoint in the CD44+ cells. The green curves plot the ES. Black vertical dashed lines specify the maximum ES scores. Within each gene set, the farther the position of a gene to the left (red) implies a higher correlation with CD44 negative phenotype, and the farther to the right (blue) implies a higher correlation with genes down-regulated upon CD44 expression. Significantly enriched data sets are defined according to GSEA default settings (p < 0.001 and FDR < 0.25). The heatmap shows expressions of the genes in the leading edge subsets (only the top 40 genes were shown for clarity if more than 40 genes in the leading edge subsets). Data were from 8 microarray platforms and 2 independent experiments. The data were normalized by RMAExpress software. Related to Figure 4.
Pathway 4/10/12 4:00 PM
Pathway:WntFig. signalingS4 (Qian, pathway et al,.) PathwayFigure information generated S4. ( byQian KEGG. et Stop al.) Blinking A
Pathway 4/14/12 11:46 PM
Pathway:Focal adhesion Pathway information generated by KEGG. Stop Blinking
B
List genes are shown in red DAVID Gene Name
C-terminal binding protein 1 C-terminal binding protein 2 CREB binding protein CXXC finger 4 E1A binding protein p300 F-box and WD repeat domain containing 11 FOS-like antigen 1 Rho-associated, coiled-coil containing protein kinase 2 RuvB-like 1 (E. coli) S-phase kinase-associated protein 1 SMAD family member 2 SMAD family member 3 SMAD family member 4 SRY (sex determining region Y)-box 17 SUMO1/sentrin/SMT3 specific peptidase 2 WNT inhibitory factor 1 adenomatosis polyposis coli 2 adenomatous polyposis coli axin 1 axin 2 beta-transducin repeat containing calcineurin B homologous protein 2 calcium binding protein P22 calcium/calmodulin-dependent protein kinase II alpha calcium/calmodulin-dependent protein kinase II beta calcium/calmodulin-dependent protein kinase II delta calcium/calmodulin-dependent protein kinase II gamma casein kinase 1, alpha 1 casein kinase 1, alpha 1-like casein kinase 1, epsilon casein kinase 2, alpha 1 polypeptide pseudogene; casein kinase 2, alpha 1 polypeptide casein kinase 2, alpha prime polypeptide catenin (cadherin-associated protein), beta 1, 88kDa catenin, beta interacting protein 1 cerberus 1, cysteine knot superfamily, homolog (Xenopus laevis)
http://david.abcc.ncifcrf.gov/kegg.jsp?path=hsa04310$Wnt%20signaling%20pathway&termId=470038846&source=kegg Page 1 of 3
List genes are shown in red http://david.abcc.ncifcrf.gov/kegg.jsp?path=hsa04510$Focal%20adhesion&termId=470038853&source=kegg Page 1 of 6 Supplemental Figure 4. The biological pathways analysis of the genes in culture-expanded human BM MSCs. The genes from microarray data that are more than 5-fold upregulated in the culture-expanded MSCs compared to the freshly sorted CD44- cells were used for mapping KEGG pathways using the Database for Annotation, Visualization and Integrated Discovery (DAVID ) Bioinformatics resources v6.7 (http://david.abcc.ncifcrf.gov/tools.jsp). (A), WNT signal pathway. (B) Focal adhesion pathway. Red stars indicate the genes are more than 5 fold increase in the cultured MSCs compared to the freshly sorted CD44- cells.
Pathway 4/10/12 8:03 PM
Pathway:MAPK signaling pathway Pathway informationFigure generated S5. by KEGG (Qian. Stop Blinkinget al.) Fig. S5 (Qian, et al,.)
List genes are shown in red DAVIDSupplemental Gene Name Figure 5. Upregulation of the genes in MAPK pathway in culture-expanded CD14 molecule DNA-damage-induciblehuman transcriptBM 3 MSCs. The genes from microarray data that are more than 5-fold upregulated in the ECSIT homolog (Drosophila) - ELK1,culture member of ETS -oncogeneexpanded family MSCs compared to the freshly sorted CD44 cells were used for mapping ELK4, ETS-domain protein (SRF accessory protein 1) Fas (TNFMAPK receptor superfamily, signal member pathway6) analyzed using the DAVID Bioinformatics resources v6.7. Red stars Fas ligand (TNF superfamily, member 6) JMJD7-PLA2G4Bindicate readthrough the transcript; genes phospholipase are A2, moregroup IVB (cytosolic); than jumonji 5 fold domain containing increase 7 in the cultured MSCs compared to the freshly MAP kinase interacting serine/threonine- kinase 1 MAP kinasesorted interacting CD44 serine/threonine cells. kinase 2 MAPK scaffold protein 1 MYC associated factor X RAP1A, member of RAS oncogene family RAP1B, member of RAS oncogene family RAS guanyl releasing protein 1 (calcium and DAG-regulated) RAS guanyl releasing protein 2 (calcium and DAG-regulated) RAS guanyl releasing protein 3 (calcium and DAG-regulated) RAS guanyl releasing protein 4 RAS p21 protein activator (GTPase activating protein) 1 RAS p21 protein activator 2 Rap guanine nucleotide exchange factor (GEF) 2; similar to RAPGEF2 protein Ras protein-specific guanine nucleotide-releasing factor 1 Ras protein-specific guanine nucleotide-releasing factor 2 TAO kinase 1 TAO kinase 2 TAO kinase 3 TNF receptor-associated factor 2 TNF receptor-associated factor 6 activating transcription factor 2 activating transcription factor 4 (tax-responsive enhancer element B67); activating transcription factor 4C activin A receptor, type IB activin A receptor, type IC arrestin, beta 1 arrestin, beta 2 brain-derived neurotrophic factor calcineurin B homologous protein 2 calcium binding protein P22 calcium channel, voltage-dependent, L type, alpha 1D subunit calcium channel, voltage-dependent, L type, alpha 1F subunit http://david.abcc.ncifcrf.gov/kegg.jsp?path=hsa04010$MAPK%20signaling%20pathway&termId=470038827&source=kegg Page 1 of 4 Supplemental Table 1. GSEA analysis of enrichment of gene sets that are positively correlated to CD44- phenotype. Related to Figure 4.
NAME SIZE ES NES NOM p-value FDR q-value RESPONSE_TO_WOUNDING 180 0.28 1.74 0.00 0.15 HSA04510_FOCAL_ADHESION 194 0.20 1.72 0.00 0.15 EXTRACELLULAR_REGION 425 0.30 1.77 0.00 0.15 EXTRACELLULAR_MATRIX_STRUCTURAL_CONSTITUENT 25 0.49 1.74 0.00 0.15 POSITIVE_REGULATION_OF_METABOLIC_PROCESS 213 0.11 1.75 0.00 0.16 CATION_HOMEOSTASIS 103 0.23 1.78 0.00 0.16 HSA00641_3_CHLOROACRYLIC_ACID_DEGRADATION 15 0.54 2.02 0.00 0.16 AMINE_RECEPTOR_ACTIVITY 34 0.46 1.74 0.00 0.16 TRANSMEMBRANE_RECEPTOR_PROTEIN_KINASE_ACTIVITY 51 0.38 1.72 0.00 0.16 POSITIVE_REGULATION_OF_CYTOKINE_BIOSYNTHETIC_PROCESS 25 0.37 1.77 0.00 0.16 EXTRACELLULAR_REGION_PART 321 0.30 1.74 0.00 0.17 CENTRAL_NERVOUS_SYSTEM_DEVELOPMENT 112 0.21 1.61 0.00 0.17 CELL_JUNCTION 76 0.30 1.61 0.00 0.17 CHEMICAL_HOMEOSTASIS 146 0.19 1.67 0.00 0.17 NERVOUS_SYSTEM_DEVELOPMENT 360 0.21 1.62 0.00 0.17 ANATOMICAL_STRUCTURE_MORPHOGENESIS 359 0.19 1.79 0.00 0.17 WOUND_HEALING 53 0.31 1.77 0.00 0.17 POSITIVE_REGULATION_OF_CELLULAR_PROTEIN_METABOLIC_PROCESS 69 0.18 1.62 0.00 0.17 REGULATION_OF_CYTOKINE_BIOSYNTHETIC_PROCESS 37 0.35 1.67 0.00 0.17 RESPONSE_TO_BACTERIUM 27 0.38 1.62 0.00 0.17 TRANSMEMBRANE_RECEPTOR_ACTIVITY 402 0.30 1.67 0.00 0.17 HSA04742_TASTE_TRANSDUCTION 45 0.27 1.66 0.00 0.18 NEURITE_DEVELOPMENT 53 0.30 1.62 0.00 0.18 HOMEOSTATIC_PROCESS 196 0.14 1.67 0.00 0.18 EXTRACELLULAR_SPACE 234 0.29 1.62 0.00 0.18 POSITIVE_REGULATION_OF_CELLULAR_METABOLIC_PROCESS 206 0.11 1.70 0.00 0.18 BASOLATERAL_PLASMA_MEMBRANE 31 0.32 1.60 0.00 0.18 HSA04940_TYPE_I_DIABETES_MELLITUS 41 0.36 1.79 0.00 0.18 CELLULAR_MORPHOGENESIS_DURING_DIFFERENTIATION 49 0.32 1.60 0.00 0.18 RHODOPSIN_LIKE_RECEPTOR_ACTIVITY 129 0.31 1.60 0.00 0.18 SECOND_MESSENGER_MEDIATED_SIGNALING 151 0.27 1.67 0.00 0.18 ENDOPEPTIDASE_ACTIVITY 110 0.19 1.63 0.00 0.18 TRANSMEMBRANE_RECEPTOR_PROTEIN_TYROSINE_KINASE_ACTIVITY 43 0.37 1.59 0.00 0.19 HSA04080_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION 250 0.35 1.69 0.00 0.19 ADHERENS_JUNCTION 22 0.46 1.67 0.00 0.19 NEGATIVE_REGULATION_OF_CELL_DIFFERENTIATION 27 0.34 1.59 0.00 0.19 G_PROTEIN_SIGNALING__COUPLED_TO_CYCLIC_NUCLEOTIDE_SECOND_MESSENGER 100 0.32 1.65 0.00 0.19 ION_HOMEOSTASIS 121 0.22 1.80 0.00 0.19 CELL_MATRIX_JUNCTION 17 0.43 1.59 0.00 0.19 PHOSPHOINOSITIDE_MEDIATED_SIGNALING 46 0.30 1.59 0.00 0.19 NEURON_DIFFERENTIATION 73 0.30 1.59 0.00 0.19 G_PROTEIN_COUPLED_RECEPTOR_ACTIVITY 183 0.30 1.68 0.00 0.19 CYTOKINE_METABOLIC_PROCESS 40 0.32 1.64 0.00 0.19 G_PROTEIN_SIGNALING__COUPLED_TO_IP3_SECOND_MESSENGER__PHOSPHOLIPASE_C_ACTIVATING43 0.29 1.58 0.00 0.19 CYTOKINE_BIOSYNTHETIC_PROCESS 39 0.34 1.68 0.00 0.19 HSA01430_CELL_COMMUNICATION 123 0.32 1.67 0.00 0.19 AXONOGENESIS 43 0.38 1.64 0.00 0.19 CYCLIC_NUCLEOTIDE_MEDIATED_SIGNALING 102 0.31 1.64 0.00 0.19 POSITIVE_REGULATION_OF_PROTEIN_METABOLIC_PROCESS 71 0.17 1.58 0.00 0.19 HSA04610_COMPLEMENT_AND_COAGULATION_CASCADES 66 0.34 1.58 0.00 0.19 CELL_MIGRATION 93 0.26 1.81 0.00 0.19 FDR-q value: the estimated probability that the normalized enrichment score represents a false positive finding; NES: Normalized enrichment score; the enrichment score for the gene set after it has been normalized across analyzed gene sets; Size: Number of genes in the gene set after filtering out those genes not in the expression dataset; The more interesting gene sets achieve the maximum enrichment score near the top or bottom of the ranked list; Supplemental Table 2. Enrichment of gene sets that are negatively correlated to CD44- phenotype. Related to Figure 4. NAME SIZE ES NES NOM p-value FDR q-value CELL_CYCLE_ARREST_GO_0007050 55 -0.22 -1.66 0.00 0.23 NUCLEOBASE__NUCLEOSIDE_AND_NUCLEOTIDE_METABOLIC_PROCESS 49 -0.35 -1.49 0.00 0.23 CELLULAR_CATABOLIC_PROCESS 198 -0.27 -1.46 0.00 0.23 REGULATION_OF_CYCLIN_DEPENDENT_PROTEIN_KINASE_ACTIVITY 42 -0.32 -1.66 0.00 0.23 CATABOLIC_PROCESS 210 -0.26 -1.46 0.00 0.23 MICROTUBULE_CYTOSKELETON_ORGANIZATION_AND_BIOGENESIS 34 -0.46 -1.49 0.00 0.23 CELLULAR_CARBOHYDRATE_METABOLIC_PROCESS 123 -0.12 -1.44 0.00 0.23 HSA00240_PYRIMIDINE_METABOLISM 85 -0.43 -1.51 0.00 0.23 NUCLEOTIDYLTRANSFERASE_ACTIVITY 45 -0.46 -1.48 0.00 0.23 MICROTUBULE_BINDING 29 -0.37 -1.45 0.00 0.23 CELLULAR_PROTEIN_COMPLEX_ASSEMBLY 31 -0.34 -1.51 0.00 0.23 BIOSYNTHETIC_PROCESS 433 -0.21 -1.45 0.00 0.23 UNFOLDED_PROTEIN_BINDING 41 -0.39 -1.49 0.00 0.23 ESTABLISHMENT_OF_ORGANELLE_LOCALIZATION 17 -0.51 -1.46 0.00 0.23 NUCLEAR_CHROMOSOME_PART 32 -0.35 -1.52 0.00 0.23 RNA_BINDING 236 -0.46 -1.48 0.00 0.23 RNA_SPLICING__VIA_TRANSESTERIFICATION_REACTIONS 32 -0.57 -1.52 0.00 0.23 ATPASE_ACTIVITY__COUPLED 87 -0.30 -1.66 0.00 0.23 SECRETION_BY_CELL 103 -0.16 -1.53 0.00 0.23 HSA00450_SELENOAMINO_ACID_METABOLISM 28 -0.37 -1.53 0.00 0.23 SPINDLE_MICROTUBULE 16 -0.70 -1.53 0.00 0.23 RNA_SPLICING_FACTOR_ACTIVITY__TRANSESTERIFICATION_MECHANISM 18 -0.72 -1.53 0.00 0.22 RESPONSE_TO_DNA_DAMAGE_STIMULUS 151 -0.39 -1.66 0.00 0.22 UBIQUITIN_CYCLE 46 -0.32 -1.54 0.00 0.22 DNA_DIRECTED_RNA_POLYMERASE_II__HOLOENZYME 55 -0.53 -1.54 0.00 0.22 PHOSPHOINOSITIDE_METABOLIC_PROCESS 27 -0.41 -1.66 0.00 0.22 GLYCEROPHOSPHOLIPID_BIOSYNTHETIC_PROCESS 26 -0.44 -1.65 0.00 0.22 RESPONSE_TO_ENDOGENOUS_STIMULUS 187 -0.34 -1.65 0.00 0.22 NUCLEOSIDE_TRIPHOSPHATASE_ACTIVITY 195 -0.22 -1.55 0.00 0.21 SMALL_NUCLEAR_RIBONUCLEOPROTEIN_COMPLEX 21 -0.73 -1.55 0.00 0.21 PHOSPHOINOSITIDE_BIOSYNTHETIC_PROCESS 20 -0.47 -1.63 0.00 0.21 CELL_CYCLE_PHASE 158 -0.34 -1.57 0.00 0.21 HSA04110_CELL_CYCLE 110 -0.45 -1.56 0.00 0.21 PURINE_NUCLEOTIDE_BINDING 199 -0.15 -1.57 0.00 0.21 MRNA_PROCESSING_GO_0006397 68 -0.57 -1.56 0.00 0.21 HSA00564_GLYCEROPHOSPHOLIPID_METABOLISM 67 -0.18 -1.63 0.00 0.21 LIPID_BIOSYNTHETIC_PROCESS 89 -0.23 -1.55 0.00 0.21 HYDROLASE_ACTIVITY__ACTING_ON_ACID_ANHYDRIDES 211 -0.22 -1.61 0.00 0.21 NUCLEAR_LUMEN 342 -0.42 -1.64 0.00 0.21 RNA_PROCESSING 161 -0.58 -1.63 0.00 0.21 NUCLEOTIDE_BIOSYNTHETIC_PROCESS 19 -0.38 -1.64 0.00 0.20 DNA_DEPENDENT_DNA_REPLICATION 53 -0.49 -1.65 0.00 0.20 DNA_INTEGRITY_CHECKPOINT 21 -0.48 -1.58 0.00 0.20 SPLICEOSOME 46 -0.65 -1.61 0.00 0.20 COENZYME_METABOLIC_PROCESS 35 -0.53 -1.63 0.00 0.20 HSA00440_AMINOPHOSPHONATE_METABOLISM 15 -0.62 -1.63 0.00 0.20 DAMAGED_DNA_BINDING 20 -0.59 -1.64 0.00 0.20 MRNA_METABOLIC_PROCESS 79 -0.54 -1.57 0.00 0.20 HSA00563_GLYCOSYLPHOSPHATIDYLINOSITOL_ANCHOR_BIOSYNTHESIS 22 -0.44 -1.65 0.00 0.20 CHROMOSOMAL_PART 89 -0.46 -1.62 0.00 0.20 ENDOPLASMIC_RETICULUM 271 -0.22 -1.64 0.00 0.20 ORGANELLE_LUMEN 410 -0.42 -1.61 0.00 0.20 DNA_DEPENDENT_ATPASE_ACTIVITY 19 -0.61 -1.62 0.00 0.20 CELL_CYCLE_PROCESS 181 -0.36 -1.62 0.00 0.20 MEMBRANE_ENCLOSED_LUMEN 410 -0.42 -1.61 0.00 0.20 CHROMATIN 34 -0.37 -1.60 0.00 0.19 PYROPHOSPHATASE_ACTIVITY 209 -0.21 -1.59 0.00 0.19 GENERAL_RNA_POLYMERASE_II_TRANSCRIPTION_FACTOR_ACTIVITY 24 -0.51 -1.59 0.00 0.19 DNA_REPLICATION_INITIATION 16 -0.60 -1.59 0.00 0.19 NUCLEOTIDE_BINDING 212 -0.16 -1.60 0.00 0.19 HSA00100_BIOSYNTHESIS_OF_STEROIDS 24 -0.43 -1.60 0.00 0.19 CHROMOSOME 115 -0.45 -1.60 0.00 0.19 DOUBLE_STRAND_BREAK_REPAIR 23 -0.49 -1.59 0.00 0.19 COFACTOR_METABOLIC_PROCESS 49 -0.51 -1.60 0.00 0.19 PURINE_RIBONUCLEOTIDE_BINDING 194 -0.15 -1.60 0.00 0.19 Supplemental Table 3. A full list of the ECM and growth factor genes in the leading edge subsets enriched in the CD44- cells. FDR-q value: the estimated probability that the normalized enrichment score represents a false positive finding; NES: Normalized enrichment score; the enrichment score for the gene set after it has been normalized across analyzed gene sets; Size: Number of genes in the gene set after filtering out those genes not in the expression dataset; The more interesting gene sets achieve the maximum enrichment score. The ranking metric measures a gene’s correlation with a phenotype. A positive value indicates correlation with the CD44 negative phenotype. Related to Fig.4B.
Supplementary Table 3. A full list of ECM and growth factor genes of leading edge subsets enriched in the CD44- cells. Related to Fig. 4B. FDR-q value: the estimated probability that the normalized enrichment score represents a false positive finding; NES: Normalized enrichment score; The ranking metric measures a gene’s correlation with a phenotype.
GENE SYMBOLGENE_TITLE RANK IN GENE LIST RANK METRIC SCORE RUNNING ES CORE ENRICHMENT LUM lumican 5 2.976 0.002 Yes HTRA1 HtrA serine peptidase 1 10 2.544 0.004 Yes EFEMP1 EGF-containing fibulin-like extracellular matrix protein 1 12 2.415 0.007 Yes GSN gelsolin (amyloidosis, Finnish type) 14 2.358 0.009 Yes ERBB2 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) 25 2.086 0.011 Yes SFRP5 secreted frizzled-related protein 5 26 2.056 0.013 Yes COL3A1 collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant) 29 2.027 0.015 Yes INHBB inhibin, beta B (activin AB beta polypeptide) 73 1.723 0.016 Yes PRELP proline/arginine-rich end leucine-rich repeat protein 127 1.584 0.015 Yes FMOD fibromodulin 180 1.496 0.015 Yes LTBP2 latent transforming growth factor beta binding protein 2 194 1.468 0.017 Yes PCSK2 proprotein convertase subtilisin/kexin type 2 209 1.455 0.018 Yes MUC5AC mucin 5AC, oligomeric mucus/gel-forming 252 1.382 0.019 Yes LPL lipoprotein lipase 257 1.377 0.021 Yes IL16 interleukin 16 (lymphocyte chemoattractant factor) 264 1.372 0.023 Yes PDZD2 PDZ domain containing 2 273 1.363 0.025 Yes CTGF connective tissue growth factor 279 1.361 0.027 Yes FBLN1 fibulin 1 283 1.355 0.029 Yes PTX3 pentraxin-related gene, rapidly induced by IL-1 beta 290 1.351 0.031 Yes COL1A2 collagen, type I, alpha 2 303 1.338 0.033 Yes ANGPTL2 angiopoietin-like 2 311 1.333 0.035 Yes LAMA4 laminin, alpha 4 320 1.328 0.037 Yes FRZB frizzled-related protein 332 1.317 0.039 Yes PVR poliovirus receptor 362 1.290 0.040 Yes LOXL1 lysyl oxidase-like 1 363 1.289 0.042 Yes ANGPTL4 angiopoietin-like 4 374 1.283 0.044 Yes ADM adrenomedullin 392 1.274 0.045 Yes CD248 CD248 molecule, endosialin 394 1.273 0.048 Yes MATN3 matrilin 3 419 1.259 0.049 Yes SERPINF1 serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1 429 1.255 0.051 Yes IL1RN interleukin 1 receptor antagonist 449 1.246 0.052 Yes CCL22 chemokine (C-C motif) ligand 22 460 1.238 0.054 Yes MMP11 matrix metallopeptidase 11 (stromelysin 3) 469 1.235 0.056 Yes ANGPTL3 angiopoietin-like 3 471 1.235 0.058 Yes FBN1 fibrillin 1 499 1.223 0.059 Yes GPX3 glutathione peroxidase 3 (plasma) 522 1.211 0.061 Yes COL6A3 collagen, type VI, alpha 3 526 1.208 0.063 Yes SGCB sarcoglycan, beta (43kDa dystrophin-associated glycoprotein) 530 1.207 0.065 Yes PAPPA pregnancy-associated plasma protein A, pappalysin 1 543 1.198 0.067 Yes LAMC1 laminin, gamma 1 (formerly LAMB2) 546 1.196 0.069 Yes TFPI2 tissue factor pathway inhibitor 2 560 1.191 0.071 Yes PTN pleiotrophin (heparin binding growth factor 8, neurite growth-promoting factor 1) 565 1.187 0.073 Yes FAM12A family with sequence similarity 12, member A 618 1.165 0.073 Yes PCSK5 proprotein convertase subtilisin/kexin type 5 622 1.163 0.075 Yes PLA2G3 phospholipase A2, group III 643 1.158 0.076 Yes FSTL1 follistatin-like 1 647 1.157 0.078 Yes PLA2G5 phospholipase A2, group V 656 1.156 0.080 Yes PTGDS prostaglandin D2 synthase 21kDa (brain) 661 1.155 0.083 Yes LAMB2 laminin, beta 2 (laminin S) 667 1.153 0.085 Yes PLA2G2E phospholipase A2, group IIE 685 1.147 0.086 Yes LOXL2 lysyl oxidase-like 2 687 1.147 0.088 Yes CDH13 cadherin 13, H-cadherin (heart) 720 1.136 0.089 Yes IL1B interleukin 1, beta 735 1.131 0.091 Yes CHI3L1 chitinase 3-like 1 (cartilage glycoprotein-39) 740 1.129 0.093 Yes CCL2 chemokine (C-C motif) ligand 2 800 1.110 0.092 Yes AREG amphiregulin (schwannoma-derived growth factor) 835 1.101 0.093 Yes PRSS3 protease, serine, 3 (mesotrypsin) 867 1.092 0.094 Yes MYT2 myelin transcription factor 2 875 1.091 0.096 Yes APOA5 apolipoprotein A-V 909 1.082 0.097 Yes FXYD6 FXYD domain containing ion transport regulator 6 915 1.081 0.099 Yes APOE apolipoprotein E 1013 1.057 0.096 Yes PLA2R1 phospholipase A2 receptor 1, 180kDa 1022 1.056 0.098 Yes PTHLH parathyroid hormone-like hormone 1034 1.054 0.100 Yes SPINT1 serine peptidase inhibitor, Kunitz type 1 1094 1.041 0.100 Yes LIPE lipase, hormone-sensitive 1104 1.037 0.101 Yes TINAG tubulointerstitial nephritis antigen 1110 1.035 0.104 Yes COL7A1 collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive) 1130 1.030 0.105 Yes TGFBI transforming growth factor, beta-induced, 68kDa 1221 1.017 0.103 Yes SPN sialophorin (leukosialin, CD43) 1256 1.011 0.103 Yes CTF1 cardiotrophin 1 1311 0.999 0.103 Yes OPTC opticin 1312 0.999 0.106 Yes IL17A interleukin 17A 1366 0.983 0.105 Yes FGB fibrinogen beta chain 1398 0.979 0.106 Yes COL4A5 collagen, type IV, alpha 5 (Alport syndrome) 1426 0.973 0.107 Yes KLK13 kallikrein 13 1466 0.967 0.107 Yes ORM1 orosomucoid 1 1479 0.964 0.109 Yes CP ceruloplasmin (ferroxidase) 1497 0.962 0.111 Yes COL15A1 collagen, type XV, alpha 1 1512 0.960 0.112 Yes SFRP1 secreted frizzled-related protein 1 1531 0.957 0.114 Yes GPHA2 glycoprotein hormone alpha 2 1533 0.957 0.116 Yes MMP8 matrix metallopeptidase 8 (neutrophil collagenase) 1595 0.948 0.116 Yes COL4A2 collagen, type IV, alpha 2 1599 0.947 0.118 Yes CABP4 calcium binding protein 4 1631 0.943 0.119 Yes CEACAM8 carcinoembryonic antigen-related cell adhesion molecule 8 1677 0.936 0.119 Yes LEP leptin (obesity homolog, mouse) 1712 0.932 0.119 Yes COL5A1 collagen, type V, alpha 1 1731 0.930 0.121 Yes SEMG1 semenogelin I 1763 0.925 0.122 Yes APP amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease) 1807 0.920 0.122 Yes SEMA3E sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3E 1849 0.913 0.122 Yes VWF von Willebrand factor 1923 0.904 0.121 Yes SMR3B submaxillary gland androgen regulated protein 3 homolog B (mouse) 1933 0.903 0.123 Yes MMP2 matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase) 1937 0.902 0.125 Yes EFEMP2 EGF-containing fibulin-like extracellular matrix protein 2 1990 0.895 0.125 Yes CXCL3 chemokine (C-X-C motif) ligand 3 1992 0.894 0.127 Yes COL4A3 collagen, type IV, alpha 3 (Goodpasture antigen) 2090 0.881 0.125 Yes IL17C interleukin 17C 2092 0.881 0.127 Yes DCD dermcidin 2095 0.881 0.129 Yes COL8A1 collagen, type VIII, alpha 1 2150 0.874 0.129 Yes FBN2 fibrillin 2 (congenital contractural arachnodactyly) 2173 0.872 0.130 Yes PNOC prepronociceptin 2182 0.870 0.132 Yes APOD apolipoprotein D 2244 0.864 0.131 Yes NID2 nidogen 2 (osteonidogen) 2253 0.863 0.133 Yes ADAMTS9 ADAM metallopeptidase with thrombospondin type 1 motif, 9 2255 0.863 0.136 Yes IMPG2 interphotoreceptor matrix proteoglycan 2 2409 0.845 0.130 Yes CCL20 chemokine (C-C motif) ligand 20 2444 0.842 0.131 Yes IFNAR2 interferon (alpha, beta and omega) receptor 2 2546 0.830 0.128 Yes LTBP4 latent transforming growth factor beta binding protein 4 2582 0.826 0.129 Yes IL6 interleukin 6 (interferon, beta 2) 2601 0.825 0.131 Yes HYAL1 hyaluronoglucosaminidase 1 2609 0.824 0.133 Yes LBP lipopolysaccharide binding protein 2657 0.819 0.133 Yes TGFB2 transforming growth factor, beta 2 2666 0.819 0.135 Yes PNLIPRP2 pancreatic lipase-related protein 2 2681 0.817 0.136 Yes FGF2 fibroblast growth factor 2 (basic) 2708 0.814 0.137 Yes RS1 retinoschisis (X-linked, juvenile) 1 2714 0.813 0.139 Yes RNASE2 ribonuclease, RNase A family, 2 (liver, eosinophil-derived neurotoxin) 2719 0.813 0.142 Yes IL4 interleukin 4 2734 0.812 0.143 Yes APOA1 apolipoprotein A-I 2778 0.806 0.143 Yes RNASE3 ribonuclease, RNase A family, 3 (eosinophil cationic protein) 2783 0.806 0.146 Yes CCL13 chemokine (C-C motif) ligand 13 2790 0.805 0.148 Yes FGG fibrinogen gamma chain 2891 0.796 0.145 Yes CHAD chondroadherin 2917 0.793 0.146 Yes GPC1 glypican 1 2923 0.793 0.148 Yes CFH complement factor H 2929 0.792 0.150 Yes SGCE sarcoglycan, epsilon 2934 0.792 0.152 Yes CHI3L2 chitinase 3-like 2 2942 0.791 0.154 Yes PNLIPRP1 pancreatic lipase-related protein 1 2943 0.791 0.157 Yes FGF5 fibroblast growth factor 5 3019 0.785 0.155 Yes CPA1 carboxypeptidase A1 (pancreatic) 3042 0.783 0.157 Yes CLEC11A C-type lectin domain family 11, member A 3090 0.777 0.157 Yes HBEGF heparin-binding EGF-like growth factor 3116 0.775 0.158 Yes IL1F7 interleukin 1 family, member 7 (zeta) 3131 0.773 0.160 Yes MMP20 matrix metallopeptidase 20 (enamelysin) 3158 0.771 0.161 Yes IGFALS insulin-like growth factor binding protein, acid labile subunit 3209 0.767 0.160 Yes NDP Norrie disease (pseudoglioma) 3242 0.764 0.161 Yes GREM1 gremlin 1, cysteine knot superfamily, homolog (Xenopus laevis) 3252 0.763 0.163 Yes ECM2 extracellular matrix protein 2, female organ and adipocyte specific 3283 0.760 0.164 Yes DST dystonin 3292 0.760 0.166 Yes CETP cholesteryl ester transfer protein, plasma 3294 0.760 0.168 Yes IL18BP interleukin 18 binding protein 3329 0.756 0.169 Yes NPPB natriuretic peptide precursor B 3331 0.756 0.171 Yes SLIT2 slit homolog 2 (Drosophila) 3335 0.756 0.173 Yes PLA2G7 phospholipase A2, group VII (platelet-activating factor acetylhydrolase, plasma) 3361 0.754 0.175 Yes C2 complement component 2 3390 0.751 0.176 Yes SSPN sarcospan (Kras oncogene-associated gene) 3407 0.750 0.177 Yes IL29 interleukin 29 (interferon, lambda 1) 3450 0.746 0.177 Yes ACHE acetylcholinesterase (Yt blood group) 3457 0.746 0.179 Yes ADAMTS5 ADAM metallopeptidase with thrombospondin type 1 motif, 5 (aggrecanase-2) 3518 0.740 0.179 Yes DMD dystrophin (muscular dystrophy, Duchenne and Becker types) 3547 0.737 0.180 Yes CCL7 chemokine (C-C motif) ligand 7 3563 0.736 0.181 Yes MMP3 matrix metallopeptidase 3 (stromelysin 1, progelatinase) 3575 0.735 0.183 Yes CXCL2 chemokine (C-X-C motif) ligand 2 3581 0.734 0.185 Yes FGF18 fibroblast growth factor 18 3681 0.725 0.183 Yes SERPINA1 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 3778 0.717 0.180 Yes IMPG1 interphotoreceptor matrix proteoglycan 1 3804 0.715 0.181 Yes MMP13 matrix metallopeptidase 13 (collagenase 3) 3810 0.715 0.184 Yes SFTPD surfactant, pulmonary-associated protein D 3927 0.706 0.180 Yes FGA fibrinogen alpha chain 3942 0.705 0.182 Yes KLK6 kallikrein 6 (neurosin, zyme) 3978 0.702 0.182 Yes CLCA3 chloride channel, calcium activated, family member 3 3979 0.702 0.185 Yes IL1F8 interleukin 1 family, member 8 (eta) 4006 0.699 0.186 Yes FGF17 fibroblast growth factor 17 4056 0.696 0.186 Yes DMP1 dentin matrix acidic phosphoprotein 4077 0.695 0.187 Yes C5 complement component 5 4080 0.694 0.189 Yes IFI30 interferon, gamma-inducible protein 30 4154 0.689 0.188 Yes MGP matrix Gla protein 4211 0.684 0.188 Yes EFNA5 ephrin-A5 4232 0.683 0.189 Yes SEMA3F sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3F 4235 0.683 0.191 Yes IL5RA interleukin 5 receptor, alpha 4294 0.679 0.191 Yes LAMA2 laminin, alpha 2 (merosin, congenital muscular dystrophy) 4347 0.676 0.191 Yes FSTL3 follistatin-like 3 (secreted glycoprotein) 4383 0.673 0.191 Yes IL13RA2 interleukin 13 receptor, alpha 2 4395 0.672 0.193 Yes INHBA inhibin, beta A (activin A, activin AB alpha polypeptide) 4401 0.672 0.195 Yes ANGPT2 angiopoietin 2 4441 0.668 0.196 Yes CX3CL1 chemokine (C-X3-C motif) ligand 1 4506 0.662 0.195 Yes LGALS3BP lectin, galactoside-binding, soluble, 3 binding protein 4569 0.658 0.194 Yes FGF10 fibroblast growth factor 10 4605 0.656 0.195 Yes F13A1 coagulation factor XIII, A1 polypeptide 4640 0.653 0.195 Yes COL16A1 collagen, type XVI, alpha 1 4700 0.650 0.195 Yes MMP9 matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase) 4730 0.648 0.196 Yes PCSK1N proprotein convertase subtilisin/kexin type 1 inhibitor 4761 0.646 0.196 Yes TNFRSF11Btumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) 4769 0.645 0.198 Yes PLA2G2A phospholipase A2, group IIA (platelets, synovial fluid) 4790 0.644 0.200 Yes ASIP agouti signaling protein, nonagouti homolog (mouse) 4804 0.643 0.202 Yes RBP4 retinol binding protein 4, plasma 4826 0.641 0.203 Yes ELA2B - 4833 0.640 0.205 Yes SLIT3 slit homolog 3 (Drosophila) 4861 0.638 0.206 Yes CARTPT CART prepropeptide 4872 0.637 0.208 Yes APCS amyloid P component, serum 4876 0.637 0.210 Yes LAMA3 laminin, alpha 3 4916 0.634 0.210 Yes COL5A3 collagen, type V, alpha 3 4967 0.631 0.210 Yes CRISP1 cysteine-rich secretory protein 1 4988 0.630 0.212 Yes NOG noggin 4997 0.629 0.214 Yes PRG2 proteoglycan 2, bone marrow (natural killer cell activator, eosinophil granule major basic protein) 5065 0.625 0.213 Yes F2 coagulation factor II (thrombin) 5249 0.613 0.206 Yes IL8 interleukin 8 5285 0.610 0.207 Yes FCN3 ficolin (collagen/fibrinogen domain containing) 3 (Hakata antigen) 5351 0.605 0.206 Yes FGF21 fibroblast growth factor 21 5421 0.600 0.205 Yes USH2A Usher syndrome 2A (autosomal recessive, mild) 5455 0.598 0.205 Yes IL26 interleukin 26 5550 0.591 0.203 Yes CXCL9 chemokine (C-X-C motif) ligand 9 5584 0.589 0.204 Yes S100A7 S100 calcium binding protein A7 5633 0.586 0.204 Yes KL klotho 5642 0.586 0.206 Yes CFP complement factor properdin 5645 0.585 0.208 Yes SCUBE1 signal peptide, CUB domain, EGF-like 1 5660 0.584 0.210 Yes ACE2 angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 5675 0.583 0.211 Yes COL10A1 collagen, type X, alpha 1(Schmid metaphyseal chondrodysplasia) 5678 0.583 0.214 Yes DKK3 dickkopf homolog 3 (Xenopus laevis) 5704 0.581 0.215 Yes COL5A2 collagen, type V, alpha 2 5709 0.581 0.217 Yes HPX hemopexin 5787 0.576 0.215 Yes CDA cytidine deaminase 5802 0.575 0.217 Yes IL6R interleukin 6 receptor 5815 0.574 0.219 Yes MATN1 matrilin 1, cartilage matrix protein 5860 0.571 0.219 Yes POSTN periostin, osteoblast specific factor 5912 0.567 0.219 Yes FGF20 fibroblast growth factor 20 5915 0.567 0.221 Yes REG3A regenerating islet-derived 3 alpha 5927 0.566 0.223 Yes KLK5 kallikrein 5 5938 0.565 0.225 Yes WNT2B wingless-type MMTV integration site family, member 2B 6041 0.558 0.222 Yes COL9A3 collagen, type IX, alpha 3 6044 0.557 0.224 Yes ODZ1 odz, odd Oz/ten-m homolog 1(Drosophila) 6058 0.556 0.226 Yes CRISP3 cysteine-rich secretory protein 3 6081 0.555 0.227 Yes COMP cartilage oligomeric matrix protein 6129 0.551 0.227 Yes APOA2 apolipoprotein A-II 6178 0.548 0.227 Yes EBI3 Epstein-Barr virus induced gene 3 6275 0.541 0.225 Yes TNFAIP2 tumor necrosis factor, alpha-induced protein 2 6286 0.541 0.227 Yes APOL1 apolipoprotein L, 1 6307 0.539 0.228 Yes SGCA sarcoglycan, alpha (50kDa dystrophin-associated glycoprotein) 6346 0.537 0.228 Yes VTN vitronectin 6393 0.535 0.229 Yes TAC1 tachykinin, precursor 1 (substance K, substance P, neurokinin 1, neurokinin 2, neuromedin L, neurokinin alpha, neuropeptide K, neuropeptide6408 gamma) 0.534 0.230 Yes F7 coagulation factor VII (serum prothrombin conversion accelerator) 6421 0.533 0.232 Yes FBLN2 fibulin 2 6447 0.532 0.233 Yes PRR4 proline rich 4 (lacrimal) 6544 0.525 0.231 Yes CFHR1 complement factor H-related 1 6637 0.519 0.228 Yes TGFB1 transforming growth factor, beta 1 (Camurati-Engelmann disease) 6652 0.517 0.230 Yes FGL2 fibrinogen-like 2 6669 0.516 0.232 Yes FGFBP1 fibroblast growth factor binding protein 1 6682 0.516 0.233 Yes MEP1B meprin A, beta 6739 0.511 0.233 Yes GHRL ghrelin/obestatin preprohormone 6844 0.505 0.230 Yes THBS4 thrombospondin 4 6888 0.503 0.230 Yes EREG epiregulin 6908 0.501 0.232 Yes GNLY granulysin 6937 0.500 0.233 Yes EGFL6 EGF-like-domain, multiple 6 6955 0.498 0.234 Yes SHH sonic hedgehog homolog (Drosophila) 6980 0.496 0.235 Yes MMP10 matrix metallopeptidase 10 (stromelysin 2) 6994 0.496 0.237 Yes IL20 interleukin 20 6995 0.496 0.240 Yes MYOC myocilin, trabecular meshwork inducible glucocorticoid response 7009 0.495 0.241 Yes A1BG alpha-1-B glycoprotein 7161 0.485 0.236 Yes CALCA calcitonin/calcitonin-related polypeptide, alpha 7187 0.483 0.237 Yes PI3 peptidase inhibitor 3, skin-derived (SKALP) 7256 0.478 0.236 Yes IL12B interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p40) 7258 0.478 0.239 Yes INSL4 insulin-like 4 (placenta) 7299 0.474 0.239 Yes SGCD sarcoglycan, delta (35kDa dystrophin-associated glycoprotein) 7347 0.471 0.239 Yes COL13A1 collagen, type XIII, alpha 1 7363 0.470 0.241 Yes SERPINA7 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 7 7386 0.468 0.242 Yes LOXL3 lysyl oxidase-like 3 7398 0.468 0.244 Yes SNTB2 syntrophin, beta 2 (dystrophin-associated protein A1, 59kDa, basic component 2) 7459 0.462 0.243 Yes SCGB1D2 secretoglobin, family 1D, member 2 7462 0.462 0.245 Yes SST somatostatin 7463 0.462 0.248 Yes FGF12 fibroblast growth factor 12 7493 0.460 0.249 Yes IL5 interleukin 5 (colony-stimulating factor, eosinophil) 7578 0.454 0.247 Yes PLG plasminogen 7582 0.454 0.249 Yes FGF9 fibroblast growth factor 9 (glia-activating factor) 7647 0.450 0.248 Yes CXCL1 chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) 7656 0.449 0.250 Yes EPO erythropoietin 7664 0.449 0.252 Yes PSAP prosaposin (variant Gaucher disease and variant metachromatic leukodystrophy) 7725 0.444 0.251 Yes LYZ lysozyme (renal amyloidosis) 7751 0.442 0.253 Yes LECT2 leukocyte cell-derived chemotaxin 2 7833 0.436 0.251 Yes IGFBP1 insulin-like growth factor binding protein 1 7847 0.435 0.253 Yes KLK8 kallikrein 8 (neuropsin/ovasin) 7848 0.435 0.255 Yes GRP gastrin-releasing peptide 7894 0.432 0.255 Yes REN renin 7904 0.431 0.257 Yes DKKL1 dickkopf-like 1 (soggy) 7909 0.431 0.259 Yes PLA2G2D phospholipase A2, group IID 8033 0.421 0.255 Yes MATN4 matrilin 4 8039 0.420 0.257 Yes APLP1 amyloid beta (A4) precursor-like protein 1 8053 0.419 0.259 Yes IL2 interleukin 2 8081 0.418 0.260 Yes MEP1A meprin A, alpha (PABA peptide hydrolase) 8107 0.416 0.261 Yes FIGF c-fos induced growth factor (vascular endothelial growth factor D) 8128 0.414 0.263 Yes CFHR3 complement factor H-related 3 8141 0.413 0.264 Yes IL27 interleukin 27 8182 0.409 0.265 Yes CD5L CD5 molecule-like 8191 0.409 0.267 Yes SPACA3 sperm acrosome associated 3 8216 0.407 0.268 Yes GDF15 growth differentiation factor 15 8274 0.403 0.267 Yes GLA galactosidase, alpha 8321 0.398 0.268 Yes SCG2 secretogranin II (chromogranin C) 8332 0.398 0.269 Yes ADAMTS13 ADAM metallopeptidase with thrombospondin type 1 motif, 13 8356 0.396 0.271 Yes CRLF1 cytokine receptor-like factor 1 8386 0.394 0.272 Yes SNTG2 syntrophin, gamma 2 8409 0.392 0.273 Yes LGALS7 lectin, galactoside-binding, soluble, 7 (galectin 7) 8508 0.383 0.270 Yes SEMA3A sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A 8552 0.379 0.271 Yes SEMG2 semenogelin II 8568 0.378 0.272 Yes PRSS8 protease, serine, 8 (prostasin) 8603 0.375 0.273 Yes DNASE1 deoxyribonuclease I 8621 0.374 0.274 Yes COL25A1 collagen, type XXV, alpha 1 8625 0.373 0.276 Yes MMP7 matrix metallopeptidase 7 (matrilysin, uterine) 8729 0.364 0.274 Yes INHA inhibin, alpha 8753 0.362 0.275 Yes FGF16 fibroblast growth factor 16 8774 0.360 0.276 Yes SGCG sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein) 8851 0.352 0.275 Yes CPB2 carboxypeptidase B2 (plasma, carboxypeptidase U) 8855 0.352 0.277 Yes FGF6 fibroblast growth factor 6 8887 0.349 0.278 Yes PLUNC palate, lung and nasal epithelium carcinoma associated 8898 0.348 0.280 Yes COL11A1 collagen, type XI, alpha 1 8919 0.346 0.281 Yes MAGEE1 melanoma antigen family E, 1 8920 0.346 0.284 Yes ANGPTL1 angiopoietin-like 1 8956 0.343 0.284 Yes MEPE matrix, extracellular phosphoglycoprotein with ASARM motif (bone) 8957 0.343 0.286 Yes PLA2G10 phospholipase A2, group X 9011 0.338 0.286 Yes GDNF glial cell derived neurotrophic factor 9018 0.338 0.288 Yes P11 - 9052 0.334 0.289 Yes PCSK9 proprotein convertase subtilisin/kexin type 9 9061 0.334 0.291 Yes WNT5A wingless-type MMTV integration site family, member 5A 9068 0.333 0.293 Yes SMR3A submaxillary gland androgen regulated protein 3 homolog A (mouse) 9185 0.321 0.290 Yes INSL3 insulin-like 3 (Leydig cell) 9208 0.318 0.291 Yes MBL2 mannose-binding lectin (protein C) 2, soluble (opsonic defect) 9233 0.316 0.292 Yes ECM1 extracellular matrix protein 1 9257 0.313 0.293 Yes FJX1 four jointed box 1 (Drosophila) 9272 0.311 0.295 Yes SPINK5 serine peptidase inhibitor, Kazal type 5 9299 0.307 0.296 Yes Supplemental materials:
Antibodies used for flow cytometry. Antigen Conjugates Clone Isotype Sources
Rat TER-119 Purified TER-119 Biolegend
Rat CD45 Purified 30-F11 Biolegend
Rat B220 Purified RA3-6B2 Biolegend
Rat CD4 Purified GK1.5 Biolegend
Rat CD8 Purified 53-6.7 Biolegend
Rat CD11B Purified M1/70 Biolegend
Rat GR1 Purified RB6-8C5 Biolegend
Rat CD19 APC 1D3 Imgenex
Rat B220 PECY7 RA3-6B2 Biolegend
Rat CD45 PECy5 30-F11 eBioscience
Rat CD31 PECY7 390 eBioscience
Rat CD44 APCCY7 IM7 Rat IgG2b, k eBioscience
Rat TER-119 PECY5 TER-119 eBioscience
Rat CD51 PE RMV-7 Rat IgG1,k Biolegend
Rat SCA1 PE D7 Rat IgG2a, k BD
Rat SCA1 Pacific blue D7 Rat IgG2a, k eBioscience
Rat KI67 PE B56 Mouse IgG1 BD
Rat CD32/16 Purified 93 eBioscience
Goat anti-rat IgG Cedar Lane (H+L) polyclonal PECY5 CLCC40106 laboratory
Antibodies used for flow cytometry (continued). Antigen Conjugates Clone Isotype Sources
Rat CD140/PDGFRa APC APA5 Rat IgG2a, k eBioscience
Rat CD19 PECY5 1D3 Biolegend
Rat B220 PECY5 RA3-6B2 Biolegend
Mouse anti-rat/mouse CD90.1 Alexa Fluor® 647 OX-7 Mouse IgG1, κ Ebioscience
Mouse CD45 eFluor® 450 2D1 Mouse IgG1, κ Ebioscience
Mouse CD235 eFluor® 450 6A7M Mouse IgG1, κ Ebioscience
Mouse CD31 APC WM59 Mouse IgG1, κ Ebioscience
Mouse CD146 PE SHM-57 Mouse IgG2a, κ Biolegend
Mouse CD271 FITC ME20.4 Mouse IgG1, κ Biolegend
Mouse CD19 eFluor® 450 HIB19 Mouse IgG1, κ Ebioscience
Rat CD105 PE MJ7/18 Rat IgG2a, k eBioscience
Rat CD106 APC 429 Rat IgG2a, k Biolegend
Mouse CD31 PE-CY7 WM-59 Mouse IgG1, k eBioscience
Mouse STRO-1 Alexa Fluor® 647 STRO-1 Mouse IgM, λ Biolegend
Mouse CD105 Alexa Fluor® 647 43A3 Rat IgG1, k Biolegend
Mouse CD106 PECY5 STA Rat IgG1, k Biolegend
Mouse CD29 APC TS2/16 Rat IgG1, k Biolegend
Mouse CD73 APC AD2 Rat IgG1, k Biolegend
List of the probes used for Q- RT-PCR. Gene name Assay ID Full name
Fmod Mm00491215_m1 Fibromodulin
Angptl1 Mm00472259_m1 Angiopoietin-Like 1
Col1a1 Mm00801666_g1 Collagen, Type I, alpha 1
Igf1 Mm00439560_m1 Insulin-Like Growth Factor 1 p27 Mm00438168_m1 p27/cdkn1b p21 CIP1 Mm00432448_m1 p21/cdkn1a
Nov Mm00456855_m1 Nephroblastoma overexpressed gene
Nes Mm00450205_m1 nestin
Cdk6 Mm00438163_m1 Cyclin-dependent kinase 6
Hprt Mm00446968_ml hypoxanthine guanine phosphoribosyl transferase