Table SI. Genes overexpressed in CD34 + CD7 + prothymocytes.

Transcription

1 Table SI. Genes overexpressed in CD34 + CD7 + prothymocytes _at UMODL1 uromodulin-like 1 NM_ _s_at CEL carboxyl ester lipase (bile salt-stimulated lipase) BC _at CD96 CD96 molecule BC _at ZDHHC14 zinc finger, DHHC-type containing 14 BC _at LOC hypothetical protein LOC BC _s_at THEMIS thymocyte selection associated BC _at LOC similar to hcg AI _a_at SCML4 sex comb on midleg-like 4 (Drosophila) BC _s_at SLC7A5 solute carrier family 7 (cationic amino acid transporter, y+ system) member 5 AB _s_at TOX thymocyte selection-associated high mobility group box AI _at AOX1 aldehyde oxidase 1 NM_ _at IL2RB interleukin 2 receptor, beta NM_ _at MN1 meningioma (disrupted in balanced translocation) 1 NM_ _at GZMA granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase 3) NM_ _at KLRK1 killer cell lectin-like receptor subfamily K, member 1 NM_ _s_at CEL carboxyl ester lipase (bile salt-stimulated lipase) NM_ _s_at WT1 Wilms tumor 1 NM_ _at CCR7 chemokine (C-C motif) receptor 7 NM_ _x_at XCL1 chemokine (C motif) ligand 1 U _s_at LDB2 LIM domain binding 2 NM_ _at TNFRSF17 tumor necrosis factor receptor superfamily, member 17 NM_ _at CD96 CD96 molecule NM_ _s_at KLRC1 /// KLRC2 killer cell lectin-like receptor subfamily C, member 1 /// killer cell lectin-like receptor subfamily C, member 2 NM_ _s_at KLRC3 killer cell lectin-like receptor subfamily C, member 3 NM_ _at CD160 CD160 molecule NM_ _at AGR2 anterior gradient homolog 2 (Xenopus laevis) AF _s_at GATA3 GATA binding protein 3 BC _s_at SMAGP small cell adhesion glycoprotein BC _s_at NRXN2 neurexin 2 AA _s_at NRXN2 neurexin 2 AB _s_at IGFBP3 insulin-like growth factor binding protein 3 M

2 Table SI. Continued _at SH2D1A SH2 domain containing 1A AF _s_at LY9 lymphocyte antigen 9 AF _s_at SCN3A sodium channel, voltage-gated, type III, alpha subunit AF _x_at TRBC1 T cell receptor beta constant 1 M _s_at TRBC1 T cell receptor beta constant 1 AF _x_at TRBC1 T cell receptor beta constant 1 AL _at TRD@ T cell receptor delta locus AW _s_at IFITM1 interferon induced transmembrane protein 1 (9-27) AA _x_at CD7 CD7 molecule AI _at KLRB1 killer cell lectin-like receptor subfamily B, member 1 NM_ _s_at CD7 CD7 molecule NM_ _s_at XCL1 /// XCL2 chemokine (C motif) ligand 1 /// chemokine (C motif) ligand 2 NM_ _at PRF1 perforin 1 (pore forming protein) AI _s_at TRA@ /// TRD@ T cell receptor alpha locus /// T cell receptor delta locus X _s_at TRA@ /// TRD@ T cell receptor alpha locus /// T cell receptor delta locus X _at MYLK3 myosin light chain kinase 3 BF _s_at KLRF1 killer cell lectin-like receptor subfamily F, member 1 NM_ _s_at DNMT3B DNA (cytosine-5-)-methyltransferase 3 beta NM_ _s_at NCR2 natural cytotoxicity triggering receptor 2 NM_ _at ATL1 atlastin GTPase 1 AF _at SUCNR1 succinate receptor 1 AF _x_at H19 H19, imprinted maternally expressed transcript (non-protein coding) BF _at BF _at RORA RAR-related orphan receptor A AW _at ELL2 elongation factor, RNA polymerase II, 2 AI _at SFMBT2 Scm-like with four mbt domains 2 T _at EPHA4 EPH receptor A4 AI _at KLHL13 kelch-like 13 (Drosophila) AB _at MS4A1 membrane-spanning 4-domains, subfamily A, member 1 AI _at TOX2 TOX high mobility group box family member 2 AA _at AI

3 Table SI. Continued _at BF _at ACY3 aspartoacylase (aminocyclase) 3 AI _at NCRNA00158 non-protein coding RNA 158 BE _at TMEM200A transmembrane protein 200A AA _at AI _at VASH2 vasohibin 2 AI _s_at RGL4 ral guanine nucleotide dissociation stimulator-like 4 AI _at AA _at GFI1B growth factor independent 1B transcription repressor AI _at BF _at AA

4 Table SII. Genes overexpressed in CD34 + CD19 + pro-b cells _at FLJ39653 hypothetical FLJ39653 BC _at WDR78 WD repeat domain 78 BC _at AL _at ARPP21 cyclic AMP-regulated phosphoprotein, 21 kd AI _s_at ARPP21 cyclic AMP-regulated phosphoprotein, 21 kd AI _at GAB1 GRB2-associated binding protein 1 BC _at ZHX2 zinc fingers and homeoboxes 2 BM _at BC _s_at LOC hypothetical protein LOC AK _at LOC hypothetical protein LOC BQ _at LCN6 lipocalin 6 BC _at ARPP21 cyclic AMP-regulated phosphoprotein, 21 kd H _at BU _a_at AI _a_at MACROD2 MACRO domain containing 2 BC _at SH2D4B SH2 domain containing 4B AK _at DNTT Deoxynucleotidyltransferase, terminal AA _at DNTT deoxynucleotidyltransferase, terminal AA _at AL _at CA _at CD9 CD9 molecule NM_ _s_at AK1 adenylate kinase 1 BC _s_at FOXO1 forkhead box O1 AW _s_at FOXO1 forkhead box O1 NM_ _at P4HA2 prolyl 4-hydroxylase, alpha polypeptide II NM_ _s_at SYNE2 spectrin repeat containing, nuclear envelope 2 NM_ _at CHST15 carbohydrate (N-acetylgalactosamine 4-sulfate 6-O) sulfotransferase 15 NM_ _s_at COL5A1 collagen, type V, alpha 1 AI _s_at PSD3 pleckstrin and Sec7 domain containing 3 AW _s_at PSD3 pleckstrin and Sec7 domain containing 3 NM_ _s_at SOCS2 suppressor of cytokine signaling 2 AB

5 Table SII. Continued _s_at MME membrane metallo-endopeptidase AI _s_at MME membrane metallo-endopeptidase NM_ _s_at ABCA1 ATP-binding cassette, sub-family A (ABC1), member 1 NM_ _at ABCA1 ATP-binding cassette, sub-family A (ABC1), member 1 AF _at ZHX2 zinc fingers and homeoboxes 2 NM_ _s_at SCHIP1 schwannomin interacting protein 1 NM_ _at NID2 nidogen 2 (osteonidogen) NM_ _at NPTX1 neuronal pentraxin I NM_ _at RIMS3 regulating synaptic membrane exocytosis 3 NM_ _at POU2AF1 POU class 2 associating factor 1 NM_ _s_at CD79B CD79b molecule, immunoglobulin-associated beta NM_ _at SERPINI1 serpin peptidase inhibitor, clade I (neuroserpin), member 1 NM_ _at NPY neuropeptide Y NM_ _at HIST1H3H histone cluster 1, H3h NM_ _at CD27 CD27 molecule NM_ _at FHIT fragile histidine triad gene NM_ _at RAG1 recombination activating gene 1 NM_ _at SPTA1 spectrin, alpha, erythrocytic 1 (elliptocytosis 2) NM_ _s_at ID3 inhibitor of DNA binding 3, dominant negative helix-loop-helix protein NM_ _at SMAD1 SMAD family member 1 NM_ _at HMHB1 histocompatibility (minor) HB-1 NM_ _s_at CD24 CD24 molecule BG _x_at CD24 CD24 molecule M _at CTGF connective tissue growth factor M _s_at C10orf10 chromosome 10 open reading frame 10 AL _at HIST1H1C histone cluster 1, H1c BC _s_at HIP1R /// LOC huntingtin interacting protein 1 related /// similar to KIAA0655 protein AB _s_at DOK4 docking protein 4 BC _x_at CD24 CD24 molecule AA _s_at CD24 CD24 molecule X

6 Table SII. Continued _at CORO2B coronin, actin binding protein, 2B BF _x_at HIST1H2BD histone cluster 1, H2bd BC _at HIST1H2BG histone cluster 1, H2bg BC _at LOC90925 hypothetical protein LOC90925 BC _at DNTT deoxynucleotidyltransferase, terminal M _s_at AKAP12 A kinase (PRKA) anchor protein 12 AB _s_at LEF1 lymphoid enhancer-binding factor 1 AF _s_at SMAD1 SMAD family member 1 U _s_at LRIG1 leucine-rich repeats and immunoglobulin-like domains 1 AB _at PXDN peroxidasin homolog (Drosophila) BF _at PXDN peroxidasin homolog (Drosophila) D _at COL5A1 collagen, type V, alpha 1 N _at MYBL1 v-myb myeloblastosis viral oncogene homolog (avian)-like 1 AW _at AI _at HIST1H2AD /// HIST1H3D histone cluster 1, H2ad /// histone cluster 1, H3d NM_ _at SEMA6A sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6A AB _s_at HIST1H2AC histone cluster 1, H2ac AL _at RAG2 recombination activating gene 2 AW _at IGHG1 immunoglobulin heavy constant gamma 1 (G1m marker) X _s_at HIST1H2BG histone cluster 1, H2bg BE _s_at CD72 CD72 molecule AF _s_at FADS3 fatty acid desaturase 3 AC _x_at CD24 CD24 molecule AK _at AL _s_at KANK2 KN motif and ankyrin repeat domains 2 NM_ _at PSD3 pleckstrin and Sec7 domain containing 3 NM_ _at NRN1 neuritin 1 NM_ _s_at CHD7 chromodomain helicase DNA binding protein 7 NM_ _at PBK PDZ binding kinase NM_ _s_at NEIL1 nei endonuclease VIII-like 1 (E. coli) NM_ _at FJX1 four jointed box 1 (Drosophila) NM_

7 Table SII. Continued _s_at TCL6 T-cell leukemia/lymphoma 6 AF _at VPREB3 pre-b lymphocyte 3 NM_ _s_at ARPP21 cyclic AMP-regulated phosphoprotein, 21 kd NM_ _at NM_ _at SPANXB1 /// SPANXB2 /// SPANX family, member B1 /// SPANX family, member B2 /// SPANX SPANXF1 family, member F1 NM_ _s_at sperm protein associated with the nucleus, X-linked, family member SPANXA1 /// SPANXA2 /// A1 /// SPANX family, member A2 /// SPANX family, member B1 /// SPANXB1 /// SPANXB2 /// SPANX family, member B2 /// SPANX family, member C /// SPANX SPANXC /// SPANXF1 family, member F1 NM_ _s_at TCL6 T-cell leukemia/lymphoma 6 NM_ _s_at BACH2 BTB and CNC homology 1, basic leucine zipper transcription factor 2 NM_ _x_at MAGED4 /// MAGED4B melanoma antigen family D, 4 /// melanoma antigen family D, 4B NM_ _at VPREB1 pre-b lymphocyte 1 NM_ _s_at LEF1 lymphoid enhancer-binding factor 1 AF _s_at FAIM3 Fas apoptotic inhibitory molecule 3 AI _at RNFT2 ring finger protein, transmembrane 2 AW _at PAX5 paired box 5 BF _s_at MAGED4 /// MAGED4B melanoma antigen family D, 4 /// melanoma antigen family D, 4B BC _at SEMA6A sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6A AF _at SLC23A1 solute carrier family 23 (nucleobase transporters), member 1 AF _at NPCDR1 nasopharyngeal carcinoma, down-regulated 1 AF _at SCD5 stearoyl-coa desaturase 5 AL _at SLC41A1 solute carrier family 41, member 1 AW _at TP53INP1 tumor protein p53 inducible nuclear protein 1 AW _at GAB1 GRB2-associated binding protein 1 AK _at GAB1 GRB2-associated binding protein 1 AK _s_at BACH2 BTB and CNC homology 1, basic leucine zipper transcription factor 2 AW _at DTX1 deltex homolog 1 (Drosophila) AW _s_at AKAP12 A kinase (PRKA) anchor protein 12 BF _at AKAP12 A kinase (PRKA) anchor protein 12 BF _at SMAD1 SMAD family member 1 AU

8 Table SII. Continued _at C6orf105 chromosome 6 open reading frame 105 AA _at GAB1 GRB2-associated binding protein 1 AW _at ZNF608 zinc finger protein 608 AI _at LOC Hypothetical protein LOC BF _at AI _s_at AKAP12 A kinase (PRKA) anchor protein 12 BF _at BE _at ARPP21 cyclic AMP-regulated phosphoprotein, 21 kd AL _at AU _at AA _at AV _at CMTM8 CKLF-like MARVEL transmembrane domain containing 8 AW _at GBP4 guanylate binding protein 4 BG _at MACROD2 MACRO domain containing 2 BF _at AI _at HIST1H2BC histone cluster 1, H2bc AA _at AA _at BACH2 BTB and CNC homology 1, basic leucine zipper transcription factor 2 AI _at BE _at LCN10 /// LCN6 lipocalin 10 /// lipocalin 6 AI _at AI _at LOC Hypothetical LOC AA _at AW _at R _at AI _s_at LOC hypothetical LOC AA _at LOC hypothetical LOC AA _x_at FCRL1 Fc receptor-like 1 AI _s_at H _at W _at C6orf105 chromosome 6 open reading frame 105 AA

9 Table SII. Continued _at AA _s_at CD24 CD24 molecule L _at HIP1R /// LOC huntingtin interacting protein 1 related /// similar to KIAA0655 protein AB Footnote: Results in Tables SI and SII are expressed as fluorescence intensity Log2 values. Comparative analysis of the gene expression profiles of CD34 + CD7 + (pro-t), CD34 + CD19 + (pro-b) and CD34 + Lin - (Imm) HPCs populations was performed with the R package "locfdr" (lfdr < 5%). CD34 + CD7 + prothymocytes differentially expressed 73 probe sets corresponding to 64 annotated genes (Table SI). CD34 + CD19 + pro-b cells differentially expressed 153 probe sets corresponding to 116 annotated genes (table SII) 10

10 Supplemental Figure Legends FIGURE S1. Sequential analysis of BM cells and thymocytes from humanized mice. BM, spleen and thymus cells of irradiated NSG mice injected with UCB CD34 + cells at 2 mo of age were harvested 1M (n=10), 2M (n=10), 3M (n=10) or 5M (n=10) post-graft, and FACS analyzed after labeling with the indicated mabs. Gates are set on hucd45 + cells. Evolution of (A) BM CD34 ++ CD38 low cells or (B) CD4 + CD8 + thymocytes. Numbers in quadrant indicate percentages of labeled cells. Data are from one representative mouse out of 10. FIGURE S2. Effect of BM aging on prothymocyte development and thymus colonization. UCB CD34 + HPCs were injected into irradiated NSG mice aged 2 (2m), 4 (4m) or 8 (8m) mo (n = 5 of each group). BM or thymus cells were analyzed at 3M post-graft. (A, B). FACS analysis of (A) BM cells and (B) thymocytes. Cells were labeled with the indicated mabs; data are shown as dot-plots; numbers in quadrants indicate labeled cell percentages. (C). Quantification of hucd45 + cell populations in the thymus; data are shown as absolute cell numbers (x10-6 ) or percentages relative to total cells (%); values, and presented as Box and Whiskers plots, thick lines indicating the medians; statistically significant differences (p < 0.05) are marked by asterisks. (D). Cultures on OP9-DL1 cells. Pro-T2 and CD7 lo prothymocytes from the cultures were FACS analyzed at culture d 21. Data are shown as dot-plots or histograms; numbers in quadrants indicate percentages of labeled cells. 11

11 A. 1M 2M 3M 5M Figure S1 0.9% 0.3% 0.2% 0.1% bone marrow CD34 CD38 B. 1M 2M 3M 5M 15% 4% 5% 90% 15% 77% 88% thymus 71% 5% 3% 2% 3% 0.1% 8% CD4 CD8

12 A. 2m mouse 4m mouse 8m mouse Figure S2 0.15% 0.1% < 0.05% CD34 CD7 CD34 CD38 1.9% 1.2% 0.4% 0.3% 0.8% 2.0% 21% 17% 19% BONE MARROW 3M B. 2m mouse 4m mouse 8m mouse 1.0% 0.4% 0.2% CD34 CD7 6% 88% 8% 86% 4% 91% THYMUS 3M CD4 CD8 2% 4% 3% 3% 1% 4% C. cell number (x10 6 ) * DP cells (%) D. CD7 lo 68% 6% OP9 DL1 (day 21) 1% 7% 25% 2m 4m 8m 2m 4m 8m pro-t 87% 2% 7% 8% 3% CD5 CD56 CD3