© 2012 Nature America, Inc. All rights reserved. Transplantation, Children’s Hospital Los Angeles, Los Angeles, California, USA. USA. California, Angeles, Los Correspondence should be addressed to Angeles, G.M.C. ([email protected]). Los California, USA. Hospital Children’s Los Angeles, Los Angeles, California, USA. Transplantation, 1 been has marrow bone human in (HSCs) cells stem hematopoietic marrow bone human postnatal from progenitors and study stem of direct cells requires adult hematopoietic hematopoiesis steady-state during regulated is lymphopoiesis how understanding However, cells. hematopoietic of source the as (UCB) blood cord cal umbili neonatal have used of human hematopoiesis stages progenitor study under of cells the profiles tional functionalabilities,the immunophenotypessurface transcripand influence can hematopoiesis of human ontogeny in stage and source hematopoiesis human of specifics the to studies mouse from derived hierarchies cellular about knowledge of lation’ been called ‘lymphoid-primed multipotent progenitors’ (LMPPs) of expression of the cell-surface receptor Flt3, and cells at this stage havebefore complete loss of myeloid potential has been defined on the basis A stage at which mouse bone marrow progenitors are ‘lymphoid tial demonstrated in certain primed’ tivepathways, althoughphysiologicalthe relevance lineageofpoten of gradual loss of lineage potential that can occur via multiple alterna pathways of lineage commitment dichotomous strictly assumed studies mouse from developed models Early lymphoidhumanofhematopoieticcommitment cells. ofstages mouse lymphopoiesis, considerably in stages less progenitor is of understood identity about the theabout critical known is much Although thymus. Our studies identify the earliest stage of lymphoid priming in human bone marrow. expressed at the junction cortico-medullary of the thymus, which suggested a possible role for this molecule in homing to the HSCs and lineage-negative (Lin potential. Gene-expression profiling placed the CD10 homing molecule L-selectin (CD62L). CD10 before onset of the expression of CD10 and commitment to the B cell lineage. We identified this subset by high expression of the report a unique population lymphoid-primed in human bone marrow that was generated from stem hematopoietic cells (HSCs) Expression of the cell-surface antigen CD10 has long been used to define the lymphoid commitment of human cells. Here we Mikkola A K Hanna A Lisa Kohn of L-selectin before expression of CD10 with upregulation Lymphoid priming in human bone marrow begins NATURE NATURE IMMUNOLOGY Received 21 February; accepted 24 July; published online 2 September 2012; Department of Pathology & Laboratory Medicine, University of California, Los Angeles, California, USA. The The stepwise process of the lymphoid differentiation of multipotent differences create challenges for species-specific Critical the ‘trans 5 Eli Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California, USA. 1 , Qian-Lin , HaoQian-Lin

3 ADV , 5 & Gay M Crooks M Gay & in vitro A 1 NCE ONLINE PUBLIC . Those ideas have evolved into models − assays continues to be debated ) CD34 2 4 , , Rajkumar Sasidharan Department Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, 6– 8 . Most studies of the earliest of earliest the . Most studies + CD10 − CD62L 1 6 + , . In addition, the the addition, In . 4 progenitors. CD62L was expressed on immature thymocytes, and its ligands were A , 5 TION 8 , 9 . hi −

progenitors had full lymphoid and monocytic potential but lacked erythroid CD62L 2 . 3 2– , Chintan Parekh hi doi:10.1038/ni.240 3 5 - - - - - population at an intermediate stage of between differentiation Department Department of Molecular, Cell and Biology,Developmental University of California, .

able to generate B cells, NK cells and T cells, as well as monocytic monocytic as well LMPPs to the marrow similar mousebone in cells, and that dendritic as cells, T and cells NK cells, B generate to able were cells these cultures, stromal In potential. erythroid or myeloid that had expressionhigh of CD62L and that was devoid of clonogenic CD34 engraftment thymic efficient cells correlates with loss of erythroid and megakaryocyte potential and lation of CD62L expression on c-Kit ing to peripheral lymphoid organs expression. CD10 of independently or before to the either commitment B that lymphoid lineage, precedes priming commitment of human cells, we sought to identify lymphoid a stage the of of lymphoid hierarchy progenitor the understand to Therefore, to the B cell lineage, with expression of genes B several cell–specific population but nonetheless show of evidence molecular commitment CD34 the of precursors are CD24 of expression or cell T little natural relatively killer (NK) cell potential with potential cell B toward bias strong a age markers (Lin shown that CD34 to generate all lymphoid lineages CD10 CD34 on MME) or (CALLA CD10 antigen surface cell the of expression with begin to assumed L-selectin (CD62L) is expressed on lymphocytes and mediates hom + + Lin progenitors lack myeloid and erythroid potential but are able potential and myeloid erythroid lack progenitors − CD10 5 4 2 , Shundi Ge − Division Division of Research Immunology & Bone Marrow − + : CD3 progenitor subpopulation humanin bone marrow CD10 − + CD14 cells, even those without expression of line + 14– progenitors, based on the finding that that finding the on based progenitors, − 1 CD15 11 1 , Yuhua Zhu 1 6 1 0 , . In this study we have identified a identified have we study this In . 3 1 . . However, subsequent studies have . Studies have reported that upregu 2 + . CD34 Lin − CD19 − Sca-1 + Lin − CD56 + − 1

mouse bone marrow CD10 , +

Lin ARTICLES − CD235a − + CD10 cells that lack + − CD24 ), ), show 1 2 1 + - - - .

© 2012 Nature America, Inc. All rights reserved. five ( Numbers in quadrants indicate percent cells in each throughout. * expression on subsets of the CD34 common myeloid progenitor; IL-3R ( full gating strategy, cells by flow cytometry ( CD34 of CD34 CD10 n total, number: Sample monocyte-macrophage. macrophage; GEMM, granulocyte-erythrocyte- forming unit, erythroid; GM, granulocyte- various CD34 of output clonogenic erythroid and myeloid ( throughout. each in cells percent indicate areas outlined to) adjacent (or in Numbers and CD7 on CD34 clonogenic potential. ( erythroid and myeloid lack that progenitors Figure 1 CD34 the of restriction lymphoid define to insufficient was alone expression CD7 that demonstrated assays of population small a donors), most of which did not coexpress CD10 on ( CD7 of CD34 expression low detected we lymphoidofstagesearly tocommitment UCB CD7 in marker lymphoid the of expression linking reports published Given The lymphoid marker CD7 does not define lymphoid commitment RESULTS hematopoiesis. human aberrant and normal during HSCs of ment commit lymphoid the of regulation the of understanding complete a more facilitate will population of progenitor this The identification primed. lymphoid become progenitors human adult which at stage the that CD10 propose We thymus. human the to homing in role a have may CD62L that possibility the suggested which CD62L, for ligands of human expressed thymus junction lature at cortico-medullary the vascu the that and CD62L expressed thymus human in progenitors CD34 multipotent the diate between CD34 the CD10 transferase. Genome-wide expression and functional analysis identified of expression mal component recombinase DNArecombination, of expressionlack by their of the determined as cells not only lacked B cell–specific transcripts but also had CD10 not that showedinitiated analysis comprehensivemolecular ing, of skew lymphoid evidence Despite B and cells. myeloid cells producing ‘CD10 before reported been have ARTICLES 2 experiments with three independent samples ( investigated whether expression of CD7 was sufficient to identify lym bone marrow ( marrow bone UCB in before identified population age marker–expressing cells showed that the CD34 of CD34 Examination expression. CD10 of independently marrow bone human in commitment phoid d b = 4; CD10 4; =

) Myeloid and erythroid clonogenic capacity of various CD34 ) Methylcellulose assay (for CFU-C) of the the of CFU-C) (for assay ) Methylcellulose e lo on ta piiie ypodrsrce CD34 lymphoid-restricted primitive that found also We b + + ) or nine ( + − + Lin , − Lin CD62L Lin CD62L n +

Lin = 5. ( 5. = Identification of bone marrow marrow bone of Identification − − CD10 − CD62L − CD10 − − CD38 + CD7 CD10 Lin c hi d hi Supplementary Fig. 1a Fig. Supplementary − ) Strategy for the isolation isolation the for ) Strategy ) independent experiments (error bars ( progenitor in bone marrow represents the earliest earliest the represents marrow bone in progenitor cells’ here) rapidly mice, immunodeficient engrafted CD62L − + − + and CD10 and hi Lin + + subsets. BFU-E, burst- BFU-E, subsets. DNTT Supplementary Fig. 2 lymphoid progenitor population. progenitor lymphoid (CD10 human bone marrow cells (2.8% progenitor population as a developmental interme developmental a as population progenitor − a bone marrow cells. cells. marrow bone ) Expression of CD10 CD10 of ) Expression n hi > 30 samples; samples; > 30 (CD10 – , which encodes terminal deoxynucleotidyl terminal encodes which , + encoding genes ) cells and and ) cells 2 − . CD34 . CD7 − CD62L + − + Lin CD45RA , + n + cell populations depleted of line depleted populations cell = 2; − Lin +

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– 50 – BFU-E GEMM 0 Supplementary Table 1 Supplementary myelo clonogenic of devoid were CD34 of CD34 marrow had high expression of CD62L ( donors) of CD34 ing of lymphocytes to peripheral lymph nodes lymph peripheral to lymphocytes of ing noted above, receptor is Asthat CD62L the mediates a hom cell-surface potential. myeloid clonogenic lacked that cells those identify to ( tors progenitors and common myeloid megakarocytic-erythroid progeni CD34 ( granulocytes- the CFU of in assay by macrophages, detectable, readily still were tors progeni myeloid clonogenic but absent, was potential Erythroid tions; in contrast, all CD34 increasing CD62L expression. CD62L increasing which suggested that progressive loss of multipotency correlated with but low had CD62L of detectable CFU-C potential (population B, expression intermediate with population here) and the CD34 subpopulation(9% distinct a CD62L, ofexpression CD45RA potential or megakaryocytic erythroid lack that progenitors marrow bone mouse certain on CD45RA CD45RA both of presence the demonstrated progenitors progenitorslymphoid various on expressed be to shown been has CD45RA marker naive-cell The CD62L ( progenitors erythroid CD34 of studies with published Consistent bone marrow and UCB populationby assay ofcolony-forming unit cells (CFU-C; CD34 the in detectable readily were progenitors, erythroid particularly cells, clonogenic nonlymphoid marrow; bone P

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CD45RA + < 0.001 ( 0.001 < c + − Lin progenitors were of devoid myeloid or clonogenic CD34 CD62L 10 10 10 10 + + , granulocyte-macrophage progenitor. ( Lin 0

+ 2 3 4 5 − + Li 0 CD10 Lin n Fig. 1 Fig. − 10 ). Of note,CD34 Of the ). CD10 e 2 − hi 10 Events (%) + CD10 66.4 t Lin 1 100 Supplementary Table 1a population (called ‘CD10 (called population -test). Data are representative of seven ( 3 20 40 60 80 4 10 − 0 Aayi o te CD34 the of Analysis . b CD45RA 4 CD62L ). + − 10 10 0 + population (called ‘CD10 (called population CD10 − 10 5 , Lin cells, assessed by cytometry.flow assessed cells, 17– 2 − 10 - CD45RA CD10 ( potential erythroid 3 10 10 10 10 1 A − 10 9 0 3 2 4 5 CD10 TION + and granulocyte 4 CD45RA 10 cells were CD45RA + 0 Fig. Fig. 1 + 5 cells in normal human bone in normal cells 10 Lin 2 Supplementary Fig. 1b Supplementary

10 Unstained CD62L CD10 CD10 CD38 − c 3 − + NATURE NATURE IMMUNOLOGY CD45RA c , 10 CD10 population was needed + e ). ). Functional screening − Lin 4 ) or three independent ) or independent three 82.7 13.5 and CD45RA – + – 10 hi 1 5 , b 3 + − and is expressed expressed is and ): IL-3R Lin CD10 − f

CD62L CD62L subpopulation − o 10 10 10 10 10 10 10 10 + CD62L − 0 1.5%; 2 3 4 5 0 - population population 2 3 4 5 + CD10 e CD45RA macrophage macrophage CD7 Fig. 1 Fig. ) CD62L ) CD62L + Lin 0 84. 12.0 − 0 lo cells’ here) 10 + 10 CD45RA 8 CD45RA ( 2 2 7 Fig. 1 Fig. 10 10 − Fig. , 10– CD10 − 3 hi 3 + n 10 10 d CD7 frac = 14 14 = cells’ a 4 12 and and 4

10 7.11 2 0. 1 ), ), 10 .7 , 5 , b 5 c c 2 5 − 1 ). ). ), ), ). + − + , - - - - ,

© 2012 Nature America, Inc. All rights reserved. erate low numbers of myeloid cells when cultured on stromal layers; layers; stromal on cultured when cells myeloid of numbers low erate CFU-C, both the CD10 CD10 with ated T in higher significantly was output Cell CD8). coexpressed which of and genes T the cell–associated and that expressed CD4, CD8, CD3 and TCR immunophenotype typical of (expression thymocytes of CD1A, CD7, conditions,CD10 cell T those initiated with CD10 CD10 with initiated cultures in higher be to tended conditions lymphoid cell cell–NK B under output Cell cells. NK into develop to potential weak relatively with B cells mostly ated CD19 Consistent with published studies population( cellsrobustlyNK and cellsgenerated B both CD10 of Culture in potential lymphoid conditions demonstrated that monocyte the CD10 and Lymphoid CD10 of presence the showed consistently adulthood to infancy from ranged ages whose donors different 20 from marrow bone of Analysis tors. that have been relied on for the isolation of human lymphoid progeni highest CD62L expression expressed neither CD10 nor CD7, markers ( CD7 express not did cells CD62L ( (MPPs) progenitors multipotent CD34the ( for CD38 CD10 and Flt3 ligand) initiated with HSCs (CD34 erythropoietin ligand, c-Kit thrombopoietin, IL-3, with stroma (OP9 marrow CD34 (one-way analysis of variance (ANOVA)). ( and cultured in T cell conditions (presented as in populations initiated with CD10 ( with CD10 initiated IL-7), and ligand Flt3 ligand, c-Kit of presence the in DLL1, ligand Notch the expressing cells stromal OP9 (on conditions T cell ( or CD10 in B cell–NK cell lymphoid cultures initiated with CD10 assessed by flow cytometry. ( initiated with CD10 thrombopoietin), and ligand Flt3 ligand, c-Kit with cells stromal OP9 (on conditions lymphoid cell B cell–NK in 4 weeks for cultured cells ( Figure 2 NATURE NATURE IMMUNOLOGY significantly was types progenitor both from output cell however, c a a c a

, ) Expression of CD19 (B cells) and CD56 (NK cells) by CD34 , ( row)). (bottom cell cultures initiated with CD10 with initiated cultures cell b d

Although we did not detect clonogenic myeloid cells by assay of of assay by cells myeloid clonogenic detect not did we Although CD10 CD19 10 10 10 10 10 ), six ( ) Flow cytometry of CD34 RAG1 0 1 2 3 4 10 − CD56 − 20.5 48. CD10 CD62L 0 through depletion of lineage marker–expressing cells) gener cells) marker–expressing of lineage depletion through + 10

2 − Fig. Fig. 1 + − cells (key), presented relative to cell number at day 0. day at number cell to relative presented (key), cells Lympho-myeloid potential of bone marrow progenitors. progenitors. marrow bone of potential Lympho-myeloid 1 versus CD10 versus c cells had low or undetectable expression of CD62L, and and CD62L, of expression undetectable or low had cells CD62L − + ( – ) or three ( 10 CD62L Lin CD62 Supplementary Fig. 3 Fig. Supplementary 2 + hi Lin 10 e cells or CD10 or cells − 31.1 and and 3 L CD38 0 + hi .1 − 10 e cells ( cells hi hi populations in myelo-erythroid cocultures cocultures myelo-erythroid in populations ) Cell output of bulk cultures of CD34 4 − cells ( cells cells ( cells CD62L 27. 69. Supplementary Fig. 1b Supplementary d 6 5 ) experiments or are from six experiments ( − − CD62L CD10 population, which is enriched for HSCs and and HSCs for enriched is which population, + Fig. 2 Fig.

and CD10 − c Supplementary Fig. 2a Fig. Supplementary hi A + CD62L (top row) and and row) (top ADV b cells ( cells cells (left) or CD10 or (left) cells + Fig. 1 Fig. + B + ) Cell output of CD34 Lin cells (presented as in in as (presented cells ) hi 2. 0 e 2 − .1 8 A 3 or CD10 or ). CD62L − ( NCE ONLINE PUBLIC cells cultured for 4–8 weeks in weeks 4–8 for cultured cells Fig. Fig. 2c f Fig. Fig. 2 hi 2 ). Thus, the progenitor subset with with subset progenitor the Thus, ). 1 ), as well as CD56 − 1 cells generated cells that had the the had that cells generated cells − , had intermediate expression of of expression intermediate had , 1 CD62L CD62L , CD10 , b hi

f Cell output (fold) or CD10 or b + ) Cell growth of bone bone of growth ) Cell 10 10 10 d , , or * d + ). ). After ) or CD10 0 1 2 Lin and and 07 ). Notably, CD10 hi hi + − subsets were able to gen P cells than in those initi those in than cells cells (all of which were were which of (all cells CD38 + − = 0.49 for CD10 for 0.49 = CD10 CD10 Supplementary Supplementary Fig. 3 b cells (right), (right), cells + CD62L TCF7 ). * − cells (key) (key) cells in in vitro + CD62L Lin – b + 10 + – c ). * P cells cells − CD62 ). < 0.038 < 0.038 − − A ), ), + Time (d) CD62L populations populations , + TION NK cells (some (some NK cells P

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hi versus CD10 versus CD62L CD10 differentiation of the CD10 the of differentiation some potential, myelo-erythroid clonogenic lacked population CD10 the did than cells T into develop to tial CD10 the was than lineage B the toward skewed less was potential, lymphoid full had population model. mouse adult xenogeneic this in expected not be would Differentiation of non–self-renewing progenitors into T lymphoid cells ( cells lymphoid B and myeloid 2 receptor interleukin (IL-2)IL-2R the in deficient strain, immunodeficiency combined diabetic–severe (nonobese strain NSG the of mice immunodeficient potential, intratibial transplantation of CD10 cultures( cell T B cell–NK cell conditions ( in lineage a assigned be could that clones the of 86% in myeloidcells ( progenitors cell B unipotent predominantly progenitors, cell cell–NK B CD10 the whereas demonstrated that the CD10 CD10 (95% confidence interval, 1 in 4.6–6.9); cultures cell T for cells 5.6 in cell 1 and 4.4–6.4), in 1 interval, dence confi (95% cells cells–NK B for cells 5.3 in cell 1 efficiency, (cloning analysis limiting-dilution by and (~11%) cell single a with initiated CD38 from cultures in robust was cells in CD10 differentiation We erythroid uncommon. noted were rarely ulocytes CD209 CD14 were coculture stromal CD10 the from generated ( HSCs-MPPs of that than lower CD10 d e c Cell output (fold) CD4 10 10 10 10 In summary, our assays functional showed that the CD10 The cloning efficiency of CD10 10 10 10 10 – 0 2 3 4 5 0 1 2 3 hi + CD8 40.2 14.9 07 0 + CD7 cells (~12% from single cells). However, lineage analysis of clones 10 10 10 10 10 + CD1a 0 + 2 2 3 4 5 10 or CD10 or CD10 CD10 CD1A 75. 13. 57. 11.3 0 + 3 (one-way ANOVA). Data are from one representative of ten ten of representative one from are Data ANOVA). (one-way 10 10 6 6 3 Week 2 4 21.2 23.7 + – + 10 10 CD62L dendritic cells ( cells dendritic 14 Fig. 3 Fig. 5 3 Time (d) 10 4 − 4 CD4 22. 10 10 10 10 CD62L 4. 6 9. hi 10 .3 21 5 2 2 0 2 3 4 5 5 e G TCR ) produced rapid marrow engraftment of both both of engraftment marrowrapid produced ) 35.1 15.2 ). Consistent with the the with Consistent ). 0 f 10 ; error bars ( Fig. Fig. 3
 2 28

10 CD8 hi 10 10 10 10 − − 3 0 + cultures, but the production of erythroid 2 3 4 5 CD62L 10 CD62L and CD10 CD4 84. 10. 89. 3.0 + 0 4 Supplementary Fig. 4 Supplementary + d 35 26. 23. Fig. population, and had greater poten 10 10 9 1 8 CD33 ) and in 97% of all clones assayed from 2 5 W * 5 2 − Fig. 2 Fig. 10 CD62L eek

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f Cell output (fold) HSCs-MPPs. 4 9 10 10 10 10 10 5 ). Most nonlymphoid cells cells nonlymphoid Most ). 10 hi − 1 2 3 0 4 2 CD62L 94.4 0. 70.9 2. cells in lymphoid cultures − 07 10 2 7 CD62L + in vitro in Supplementary Fig. 5 Fig. Supplementary 3 , population. Although Although population. + b 10 population contained Fig. eek ) was similar to that of 4 25. 24. CD10 CD10 CD38 10 3 3 5 hi 7W ARTICLES

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© 2012 Nature America, Inc. All rights reserved. CD10 with CD38 presented as mean florescence intensity (MFI). * markers on various CD34 Figure 4 was absent. potential and erythroid its myeloid potential was lower significantly than that of HSCs-MPPs, However, assays. engraftment short-term in and cocultures stromal in induced was cells) and dendritic monocytes-macrophages (mostly experiments ( three from are Data mean. the indicate lines horizontal small mouse; individual an represents symbol scatter. ( forward FSC, scatter; side SSC, row). (bottom above plots from cells myeloid and B cells of back-gating and CD45), B cells (CD19 CD34 carrier cells only (negative control; left) or 3 × 10 NSG strain, analyzed 2 weeks after transplantation of 1 × 10 (CD4 T cell conditions showing T cell potential (CD4 single-cell clone (far right). ( a from markers cell dendritic and myeloid of coexpression or right); middle and middle left); B cell potential (CD19 potential (CD14 from one to three CD10 (B & NK). ( both or (B) B cells (NK), cells NK containing growth clonal with wells of frequency CD10 cells (B-NK B cells–NK lacking wells of frequency T cell conditions ( in B cell–NK cell conditions ( analysis of CD10 studies. ( analysis and CD10 Figure 3 ARTICLES 4 (or fourteen samples for CD38; a

a Events (%) Events (%) B-NK– wells (%) 10 10 100 20 40 60 80 20 40 60 80 dim 10 37 − + − − 0 0 0 0 CD62L Lin CD62L CD62L c-Kit CD38 CD14

01 g 01 a Lineage potential of potential Lineage CD10 − ) Frequency of human myeloid cells and human B cells among the total human cells in 5 0 , − 0 cells (right), showing human engraftment (top row; cells positive for HLA class 1 and human human 1 and class HLA for positive cells row; (top engraftment human showing (right), cells b 2 d − 2 , CD10 Cells plated 10 10 10 ) Limiting-dilution ) Limiting-dilution ) Flow cytometry of clones generated in B cell–NK cell conditions conditions cell B cell–NK in generated clones of cytometry ) Flow wells) or T cells (T cells T or wells) in vivo a + hi hi 3 3 hi CD15 – 10 15 or CD10 or CD10 or − 10 cells by by cells e CD38 Unstained + CD62L 4 4 − ; error bars ( CD15 20 10 CD62L b 10 − ), presented as presented ), transplantation transplantation 5 CD62L 5 + 25 – 10 10 ). ( 20 40 60 80 20 40 60 80 + 0 0 0 0 + ) and myeloid cells (CD14, CD15 & CD33 hi 30 − ) and dendritic cell potential (CD1a + + Flt3 f HLA-DR CD62L cells represent an intermediate stage of differentiation between HSCs and CD10 and HSCs between differentiation of stage intermediate an represent cells in vitro hi + ) Flow cytometry of bone marrow from a mouse of the the of a mouse from marrow bone of cytometry ) Flow cells in B cell–NK cell lymphoid coculture, presented as presented coculture, lymphoid cell B cell–NK in cells populations from human bone marrow. Data are from eighteen experiments with two or three independent samples per marker marker per samples independent three or two with experiments eighteen from are Data marrow. bone human from populations 01 01 Lin cells grown grown cells hi b 0 0 2 2 – or CD10 or e 10 CD10 CD62L 10 T wells (%) − a 100 a ) Flow cytometry of a single clone generated in generated clone a single of cytometry ) Flow

3 populations (key), assessed by flow cytometry (top row, left; bottom row), and summary of those results (top right), right), (top results those of summary and row), bottom row, left; (top cytometry flow by assessed (key), populations 10 37 – 3 ) ) or

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CD38 populations compared with those of the the of those with compared populations 3.0 0.1 10 10 ). 10 10 5 3 5 5 10 – CD19 4 10 10 10 10 1. 0. 10 0 2 3 4 5 2 03 g 5 A CD1A 57.3 39.4 CD10 64 0.03 10 10 10 10 0 TION 10 0 2 3 4 5 Human cells (%) 100 a 2 20 40 60 80 34.7 CD10 ) Expression of cell-surface cell-surface of ) Expression 0 10 – 0 6. CD10 CD10 CD62L 23 10 CD62L c 3 6

). 2 10 10 – NATURE NATURE IMMUNOLOGY 4 CD62L + – 3 3.3 0.0 Lin 10 hi hi CD38 10 5 – 4 21.6 7. 10 hi 0 – CD10 CD14 & CD15 5 10 10 10 10 10 10 10 10 0 2 3 4 5 0 CD1A 2 3 4 5 64 26.6 47.1 2.2 0 – CD62L 16. 1. 10 0 − 9 10 2 3 , 10 2 0.4 Lin CD10 10 3 hi – 3 10 B cells Myeloid 10 24.2 4 + 4 74. 2.1 10 7 10 .6 5 3 5

© 2012 Nature America, Inc. All rights reserved. between the CD38 also samples marrow CD10 the placed bone different three of analysis microarray that CD10 did than able to CD34 generate faster CD34 molecule CD10 adhesion the of expression lost CD10 culture, lymphoid in week 1 After presented as the frequency of single cells expressing the gene. Data are representative of three independent experiments ( CD10 CD10 > CD62L upregulated only in CD38 of more than twofold by pairwise comparison ( lymphoid cells. ( Figure 5 CD10 in both upregulated Thy-1 (CD90) had its highest expression on CD38 CD10 integrin 4 Fig. population and was maximal in the CD10 CD34 from the progressively increased CD38 marker differentiation the of Expression cells’ here). most primitive CD34 NATURE NATURE IMMUNOLOGY three biological replicates ( Cluster a Cluster Cluster Cluster Cluster Cluster Principal-component analysis of global gene-expression data from of analysis gene-expression global Principal-component − + a − − CD62L ) > CD38 2 1 6 5 4 3 CD62L CD62L ). Expression of the stem cell–associated receptors c-Kit, Flt3, c-Kit, receptors cell–associated stem the of Expression ).

hi A CD10 > CD10 > CD38 6 − (CD49F) and (CD49F) Prom-1 CD38 in wassimilar (CD133) CD62L hi – cells. * cells. − hi hi − CD62L a ; cluster 6, CD10 cells ( cells populations but was downregulated in CD10 CD62L ) Expression of genes encoding transcription factors ( − + − ; cluster 3, (CD38 CD62L hi HSCs-MPPs and the CD10 the and HSCs-MPPs cells were precursors of the CD10 the of precursors were cells + P Lin hi hi + Fig. 4 Fig.

cells represent a distinct progenitor population with a unique expression profile that combines genes of HSCs and early early and HSCs of genes combines that profile expression a unique with population progenitor a distinct represent cells CD10 a −

CD10 0.050, ** 0.050, − cells relative to expression in the two other equivalent populations (CD38 populations equivalent other two the in expression to relative cells c CD38 − hi ADV ; mean and s.e.m.) or one experiment with 13 cells assayed per gene ( CD62L progenitors in an intermediate position position intermediate an in progenitors b + + + CD38 ). In addition, CD10 cells cells A EBF1 MTF2 PAX5 FOXO1 ZHX2 LEF1 TCF3 MEF2A CBX4 EED SUZ12 EZH2 PHF19 HDAC9 HDAC4 HDAC3 HDAC1 RUNX3 RUNX2 RUNX1 BCL2 BCL6 CEBPD CEBPA HOXA10 HOXA9 HOXA7 HOXA5 FOS FOSB FOSL2 FOSL1 HMGA2 PBX1 CEBPB MEIS1 HOXA3 ZBTB16 SCML4 PHC2 PCGF2 HOXB6 HOXB5 HOXB4 HOXB3 TAL1 PRDM16 GATA3 GATA2 MYCT1 − NCE ONLINE PUBLIC + HSC-MPP population (called ‘CD38 (called population HSC-MPP > (CD10 > P hi − − in in vitro < 0.010 and *** and 0.010 < and CD10 and population to the CD10 the to population = CD10 = + − population ( population CD62L Cluster Cluster Cluster Cluster Cluster b Cluster ( P + Fig. 4 Fig. − < 0.05) and defined as follows based on statistical analysis (not heat-map appearance): cluster 1, cluster appearance): heat-map (not analysis statistical on based follows as defined and < 0.05) cells differentiated and and differentiated cells CD62L + + progenitors ( progenitors progenitors ( progenitors 5 4 3 2 1 6 hi − − b = CD38 = cells. HLA-DR was CD62L CD38 ), ), which suggested hi P A ) > CD10 < 0.001 (one-way ANOVA). ( ANOVA). (one-way 0.001 < TION + n – population. = 14 donors; hi CD62L −

). ( cells were − CD62L Fig. Fig. 4 Fig. Fig. 4 + c ; cluster 4, CD10 + ) Quantitative PCR analysis of gene expression, presented relative to expression in expression to relative presented expression, gene of analysis PCR ) Quantitative hi cells; cells; − and CD10 a a c ) or cytoplasmic and cell-surface molecules ( hi ). ). ). ). −

+ ROBO4 CD27 CD24 CD22 CD19 CD79B CD79A VPREB3 RAG RAG CD10 VPREB1 DNTT DCLRE1C CD38 CSF2RB CSF2RA CSF1R MP TLR2 ITGB2 ITGB7 IL1RN IL13RA1 IL10RB IL17RA IL10RA IFNGR1 CD62L GZM IL2RG CD2 CD33 FLT3 KIT PROM1 ITGA9 EPHB4 MP ADA ALDH2 hematopoietic stages of differentiation to delineate the molecular molecular the delineate to differentiation of stages hematopoietic critical to known regulate molecules of encoding genes patterns sion expres the PCR quantitative and microarray by analyzed Wethen Downregulation of HSC-associated genes in CD10 the CD10 ways, but additional transcriptional modulation seemed to occur after differentiation of HSCs-MPPs involved many shared molecular path CD10 downregulatedin tion, and most downregulated genes in CD10 CD10 in the were downregulated than population CD10 upregulatedin were genes ofnumbers similar ( withcallyCD38 Gene expression of CD10 More than twice as many genes were downregulated in the CD10 the in downregulated were genes many as twice than More ( types progenitor both to common were CD10 Supplementary Fig. 6a Supplementary O L 2 1 B + − CD62L populations; approximately half of those upregulated genes genes upregulated those of half approximately populations; d ) Quantitative PCR analysis of gene expression in single cells, cells, single in expression gene of analysis PCR ) Quantitative − d c Gene-expressing EBF1 (fold) TCF3 (fold) KIT (fold) TAL1 (fold) CD62L 150 100 0.5 1.0 1.5 10 20 30 40 50 50 hi cells (%) 0 0 2 4 6 0 0 100 > (CD38 > 20 40 60 80 0 − HSCs-MPPs rather than with CD10 hi d ** stage during the generation of CD10 of generation the during stage TAL1 ). − *** > (CD10 > * *** * − = CD10 = ). By pairwise ). comparison By with HSCs-MPPs, pairwise ** + cells ( − CD62L MPL − CD62L

+ PAX5 (fold) DNTT (fold) CD3E (fold) MPL (fold) Supplementary Fig. 6b Fig. Supplementary ); cluster 5, (CD10 0.5 1.0 1.5 30 10 20 30 40 50 10 15 10 20 FLT3 0 0 0 5 0 hi progenitors clustered hierarchi clustered progenitors b ) with a difference in expression expression in a difference ) with hi *** = CD10 = ** TCF3 a *** , * *** b Supplementary Fig. 6b Fig. Supplementary ), three experiments with with experiments three ), *** * − CD62L + RAG1 ); cluster 2, CD38 − − ARTICLES CD62L IL7R (fold) RAG1 (fold) CD62L CD10 CD38 CD10 − hi 10 15 20 0 2 4 6 8 0 5 CD62L PAX5 − cells were also + CD62L CD10 CD10 CD38 progenitors progenitors + – – ). Thus, the the Thus, ). CD62L + hi cells. hi = + – – CD62L popula hi hi hi cells and and −

hi ). ). 5 + - - - -

© 2012 Nature America, Inc. All rights reserved. vessels ( showing costaining of MECA-79 ( merged image ( positive for vascular endothelial cadherin (VE-cadherin (CD144); of the region in of the thymus. Scale bars, 200 junction cortico-medullary the at staining MECA-79 showing flow cytometry. ( or CD34 CD34 (one-way ANOVA). ( expression in CD10 CD38 ( assessed by flow cytometry (gated as CD34 molecules on CD10 progenitors to human thymus. ( Figure 6 CD10 the of 5 Fig. HSCs-MPPs and CD10 Flt3, of expression surface cell the with cell–associated genes ( IL10RB ( receptors cytokine lymphoid encoding genes and ( lineages cell NK and cell T with associated genes Specifically, population. this of potential monocyte and phoid 4; (cluster CD10-CD62L of stages of Analysis genes only upregulated in the CD10 Lymphoid-differentiation committed to B lymphopoiesis. then and primed lymphoid became multipotency, lost HSCs as regulation transcriptional of program coordinated highly (clusters and1 respectively; 5, PHC2 by (encoded PRC1 complexes repressive polycomb the of 5 Fig. CD10 the differentiationat in of the of genes expression In contrast, no significant further change at the CD10 the CD38 HOXB in CD38 the expression lated in both CD10 ( tors 1–6; (clusters 5a Fig. patterns expression different six of one to belonged in the other two populations ( expression to their relative population one in twofold of at sion least tions. All genes included in the heat maps had a difference in expres among relationships the CD38 ARTICLES 6 with three biological replicates ( b a

) Quantitative PCR analysis of Events (%) 100 20 40 60 80 0 + − b TAL1 , , and a CXCR4 or CD34 or CD10 and family were also downregulated during the transition from from transition the during downregulated also were family 01 ). Consistent with the B cell–skewed differentiation potential potential differentiation cell–skewed B the with Consistent ). , ). We noted reciprocal patterns of expression for members members for expression of patterns reciprocal noted We ). + ,

i b ) at the cortico-medullary junction of the thymus, and a merged image ( CD1A High CD62L expression and recruitment of recruitment and expression CD62L High 0 IL17RA ). Genes encoding known HSC-related transcription fac transcriptionHSC-related known encoding Genes ). 2 − HSC-MPP stage to the CD10 the to stage HSC-MPP 10 Fig. 5 Fig. SCML4 , 3 + GATA2 population, genes known to be expressed specifically specifically expressed be to known genes population, 10 − g − d thymocytes (bottom), assessed by assessed (bottom), thymocytes Unstained d ). Scale bars, 100 4 thymocytes (top) and CD34 and (top) thymocytes showing costaining of MECA-79 ( MECA-79 of costaining showing + ) Chromagen immunohistochemistry immunohistochemistry ) Chromagen 10 and cells, presented relative to relative presented cells, b − − c 5 ) showed a profile consistent with the dual lym dual the with consistentprofile a showed ) ) ) and PRC2 (encoded by CD62L CD62L ) CD62L expression in human human in expression ) CD62L − 100 and CD62L 20 40 60 80 0 IFNGR1 − MPO PSGL − CD62L 01 population (cluster 1; (cluster population hi hi 0 CD62L PRDM16 cells. * cells. cells and CD10 and cells 2 , 10 hi a CSF1R M a 3 cells and CD10 and cells CCR7 ) Expression of homing homing of ) Expression m. ( − P – h hi 10 ) were upregulated, as were myeloid myeloid were as upregulated, were ) + hi , CD10 Fig. 5 Fig. c ) in a subset of P-selectin–positive blood blood P-selectin–positive of a subset ) in < 0.05 (moderated (moderated 0.05 < cells but not in CD10 in not but cells progenitor stage (clusters 2 and 3; 3; and 2 (clusters stage progenitor 4 M ; mean and s.e.m. in 10 m. ( P e

– expression in expression 5 a ) were significantly downregu significantly were ) and and g CD10 0.05 0.05 100 ) Fluorescence immunohistochemistry immunohistochemistry ) Fluorescence h a 20 40 60 80 − 0 – ). These analyses identified a identified analyses These ). HOXA CD62L + j CD44 ) Fluorescence immunohistochemistry immunohistochemistry ) Fluorescence FLT3 Lin + CSF2R − 01 + + CD62L CD1A + cells, cells,

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CD34 CD34 Unstained CD34 CD34 Unstained – To investigate further the degree of heterogeneity of the three popu three the of heterogeneity of degree the further investigate To although that showed PCR quantitative by analysis Detailed j ). Scale bars, 100 j ). ).

in a similar percentage of CD10 − − − − + + + – cell to CD10 to cell CD62L 1A 1A CD62L CD62L , VPREB1 − – + CD62L − d − ADV CD62L FLT3 CD62L h e MECA-79 MECA-79 hi hi hi A − cell to CD10 to cell stage. Expression of of Expression stage. cells. NCE ONLINE PUBLIC expression in single cells was limited almost exclu almost limited was cells single in expression CD62L hi , RAG1 VPREB3 hi − cell and increased 12.0-fold in the transition from in the transition 12.0-fold and cell increased − hi + + CD62L − population. CD62L population was committed to B lymphopoiesis, lymphopoiesis, B to committed was population cells (cluster 6; 6; (cluster cells M CD62L population included cells with evidence of of evidence with cells included population m. Data are representative of three experiments experiments three of representative are Data m. hi and and cell to CD10 to cell + hi , cells ( hi hi CD19 + and CD10 population ( population PAX5 cell and increased 4.4-fold in the transi the in 4.4-fold increased and cell cell ( cell + f i P-selectin VE-cadherin cells, upregulation of certain genes genes certain of upregulation cells, Fig. 5 Fig. , + Fig. Fig. 5 (which encodes the transcription transcription the encodes (which CD22 cells. We detected expression of of expression Wedetected cells. Fig. Fig. 5a A TCF3 TION + cell ( cell c − c + , ), which demonstrated that that demonstrated which ), CD62L ). In contrast, EBF1 populations had distinct distinct had populations CD24 Fig. 5 Fig. –

(which encodes the tran the encodes (which MPL c Fig. Fig. 5 NATURE NATURE IMMUNOLOGY ). ). Notably, none of those TAL1 , and and (which encodes the the encodes (which PAX5 d hi ). Thus, CD38 the Merge Merge j g c and CD10 ). Similarly, (which encodes encodes (which CD27 + , CD38 IL7R RAG1 ) had high high ) had , − Fig. MECA-79 CD79A cells or cells in in vitro expres + − DNTT cells. cells. cells, cells, −

5 cell cell d ). ). − ------, .

© 2012 Nature America, Inc. All rights reserved. ated with the onset of CD10 expression and further upregulation upregulation further expression. CD38 of and expression CD10 of onset the with ated tive to its expression in HSCs) and that B cell commitment is initi (rela CD38propose Weupregulationof with begins priminglymphoidfraction. that HSC primitive most the in that than higher CD38, similar to expression in the CD10 of expression intermediate with cells included progenitors ymphoid gene CD10 potential erythroid no but potential cell dritic CD34 expression. CD10 ent with a model that positions the CD10 of the expression of antigens were genes and consist also cell-surface CD10 of cultures in generated that to equivalent least at was culture in generated cells B of number the potential, cell CD10 the although In addition, generate generate CD10 tor stage, and cultures initiated with CD10 that all human B cell differentiation passes through a CD10 CD10 restricted theCD10 that suggested evidence of pieces Several CD62L. molecule homing CD34 of subset a in sion expres CD10 of onset the before began marrow bone human in ing’ prim ‘lymphoid that have here demonstrated presented studies Our DISCUSSION thymus. human the to homing the into precursors marrow-derived ( thymus of entry of the site of the junction thymus, cortico-medullary the at cells endothelial positive P-selectin- of subset a in specifically vasculature thymic the in 79 addressins’ node ‘peripheral as known ligands CD62L of family the on found epitope carbohydrate CD34 mature more the were CD34 in the cells CD62L-expressing tionationCD34 theof (which represent >95% of all thymocytes; CD34 mature more the in than progenitors thymic CD34 in higher was expression CD62L thymus. human from tions shown). not (data CCR9 chemokine the of expression their in populations the between Wedifferences consistent no noted its expression in either CD10 significantly upregulated in the CD10 progenitors and mediates the migration of early thymocytes chemokine receptor CCR7, which is expressed on mouse early thymic CD10 in than CD10 in expression higher had both CD44 marker memory and activation the and P-selectin) for ligand (the PSGL-1 similar in CD10 the thymus ies as in important the being homing of to cells of and in cells settling stud in mouse before reported that have pairs been of receptor-ligand We analyzed by and flow gene cytometry expression the coexpression CD62L and ligand expression in human thymus ( lineage cell B the to commitment with associated genes of expression no but priming lymphoid early NATURE NATURE IMMUNOLOGY pbihd td hs ecie a CD10 a described has study published A We next examined the expression We of popula in progenitor CD62L expression the examined next PAX5 + + ‘multilymphoid progenitor’ also expressed the B cell–specific B the cell–specific expressed also progenitor’ ‘multilymphoid CD38 i. 6d Fig. − CD62L . Notably, the strategy for the isolation of those multil those ofisolationNotably, the . for strategy the 28– lo − neg 3 + + 0 cells ( − progenitors before differentiating into CD19 into differentiating before progenitors . Expression of the chemokine receptor CXCR4 was was CXCR4 receptor chemokine the of Expression . + CD62L – stage of lymphopoiesis. First, it is widely assumed assumed widely is it First, lymphopoiesis. of stage hi j populationwith lymphoid, and monocytic den ); this suggested a possible role for CD62L in in CD62L for role possible a suggested this ); population was a precursor of the more B cell– B more the of precursor a was population Fig. 6 Fig. + +

thymocyte population, we found that most most that found we population, thymocyte hi CD1a and CD10 + ADV progenitors with high expression of the the of expression high with progenitors + a cells or the CD38 ). In addition, the gene encoding the the encoding gene the addition, In ). A + − NCE ONLINE PUBLIC subset ( subset 3 CD62L 1 . We detected staining of MECA- of staining We detected . Supplementary Fig. 7 Fig. Supplementary + populations ( − CD62L hi Fig. 6 Fig. − population had population greater NK CD62L Fig. Fig. 6 − + − CD62L CD1a CD62L hi c − ). MECA-79 detects a detects MECA-79 ). population relative to c population ( ). After further frac + − hi cells. The patterns patterns The cells. subset rather than rather subset hi 2 population before Fig. Fig. 6 hi 1 A cells were able to population and and population . However, this this However, . + TION − ust n a in subset CD62L − thymocytes thymocytes a ). ). ). However,

+ 28– progeni Fig. + B hi 3 0

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the postnatal circulation and that are those life circulation throughout generated the postnatal in transiently detected are that cells between expected be would that differences UCB and marrow,bone functional as as well intrinsic the in immunophenotypes similar with cells of potentials lineage the in differences The data). unpublished G.M.C., and (Q.-L.H. CFU-C of numbers detectable readily but small included UCB in populations to ficult in detect UCB, and CD34 CD62L distinct a marrow; bone in than UCB in population lymphoid-primed pure a of identification the for able of CD10 the those to similar are CD7) CD34 express not do (which progenitors the functional and molecular profiles of CD10 mouse CLPs in the c-Kit the in CLPs mouse marrow bone mouse c-Kit of subpopulation a from LMPPs isolate to used been has Flt3 of expression surface Cell population. progenitor lymphoid-restricted mature, more a represented (CLPs) progenitors lymphoid common whereas potential, erythro-megakaryocytic lost had but potential myeloid some and lymphoid full retained marrow pro stage multipotent genitor a from separately emerge pathways erythroid myelo- and lymphoid the that idea classic the challenge and support marrow. bone human on focus that studies in the bone marrow microenvironment, highlight the critical need for knownto exist for both immunophenotype and function from progenitors UCB and bone marrow between are differences tial does not represent steady-state postnatal hematopoiesis, and substan UCB However, unfeasible. sometimes and difficult populations tor in vivo UCB in homologs immunophenotypic their of that than lower much is tors than progeni marrow bone ofoutput proliferativemarrow. accessible The bone is readily more is cells human of source this because complete lack of either CFU activity or activity CFU either of lack complete IL-7R mouse that noted has paper published progenitors lymphoid-committed more even for reported been has potential dendritic and myeloid residual of type This cells. dendritic and of monocytes-macrophages numbers limited to generate able are and expression CD10 precede that progenitors primed’ CD10 the that proposal the favor we Instead, progenitors. granulocyte-macrophage and progenitors loid mye common by initiated pathways myelo-erythroid main the of thatCD10the clonogenic myeloid-erythroid potential in assays of CFU-C suggested of absence The cells. and dendritic of monocyte-macrophage mostly LMPPs and progenitors multipotent mouse between discriminating for marker alternative as be might used an expression that CD62L suggests which potential, with high expression of Flt3 and loss and of megakaryocyte erythroid CD62L c-Kit in of expression upregulation that reported have studies Notably, here). LMPPs for and HSCs marker human a between discriminating as useful be to found been not has Flt3 of expression of upregulation despite the and LMPPs, CD10 mouse the to similar most seems progenitor row CD10 the data, molecular and tional Considerable data have been generated in mouse studies to both both to studies mouse in generated been have data Considerable Most human hematopoietic studies have used UCB, largely largely UCB, used have studies hematopoietic human Most The myeloid output of the CD10 the outputmyeloidof The + + assessment of rare, non–self-renewing bone marrow progeni marrow bone non–self-renewing rare, of assessment progenitor is more analogous to the mouse CLPs. However, CLPs. mouse the to analogous more is progenitor CD38 7 , 17 − CD62L , 32– − 1– 3 CD7 − 4 4 CD62L or that of HSCs from either source either from HSCs of that or , 3 6 hi . The ‘lymphoid-primed’ LMPPs in mouse bone bone mouse in LMPPs ‘lymphoid-primed’ The . + + immunophenotype described here was less reli less was here described immunophenotype UCB progenitors Lin 2 , and the IL-7 receptor , IL-7R and receptor IL-7 the hi FLT3 − 1 population does not represent a precursor precursor a represent not does population 4 Sca-1 − . Lin at the transcriptional level, cell-surface cell-surface level, at transcriptional the − + Sca-1 mouse bone marrow cells correlates correlates cells marrow bone mouse + Lin − lo CD62L − − population − CD62L 2 CD10 CD62L 0 . Moreover, we found that that found we Moreover, . in vivo in hi − − A CD45RA hi CD62L hi population consisted consisted population hi + cells are ‘lymphoid- population was dif + CLPs, despite their their despite CLPs, human bone mar myeloid potential, potential, myeloid Lin 1 A ARTICLES . Given our func our Given . 2 is used to define to is define used 3 1 − 5 hi (and reported reported (and , which makes makes which , Sca-1 bone marrow + CD62L 6 . Notably, . 10 + cells in in cells , 17 , + 2 1 cell . A . 7 ------

© 2012 Nature America, Inc. All rights reserved. tute the mouse thymus after tute transplantation thymus after mouse the mouse bone marrow progenitors that and efficiently rapidly reconsti of a population to identify mouse used been has to expression CD62L thymus, homing in described been not have CD62L with actions inter although note, Of unclear. yet as is population mobilized this of potential lineage and relevance physiological the but shown), not CD34 cells of (but not CD34 subset a in CD62L of expression noted have We thymus. human to progenitors of homing the in role a have may in the homing of to lymphocytes lymphoid peripheral organs, CD62L medullary region, raise the cortico- possibilitythe in that in specifically addition thymus, to human being the involvedin ligands CD62L of CD34 However, of mechanism. in expression CD62L the primitive the high same the involves thymus human to homing that possible is it cells, CD10 and HSCs-MPPs both on expression thymus mouse to homing of mediators thymic microenvironment. PSGL-1–P-selectin interactions are critical CD7 when upregulated rapidly is CD34 into differentiate CD34 as upregulated becomes CD62L whether assays genic clono in potential erythroid and myeloid high have thymocytes (CD7 itive CD10 although CD62L expression that was noted highest be on CD34 should It population. precursor thymic alternative or have provided evidence that CD10 set among human thymocytes sub immunophenotypic of a similar finding the given thymus, human bone marrow possibly represents a lymphoid progenitor that seeds the CD10 The thymus.human the seed normally that precursors marrow bone of the identity prove the definitively to impossible it make restrictions Experimental thymopoiesis. initiate to able be may progenitor marrow bone of type one than more that the mouse thymus has continued for over a decade, and it seed seems likely that precursors of identity the about Controversy ferentiation. CD10 CD10 the of precursor a be in CD10 and studies, human in LMPPs mouse to have expression seem bination high recom DNA for required machinery molecular the of components as such genes specific CD10 the of LMPPs mouse of opment Expression of the gene encoding E2A, which is foressential the devel HSCs, whereas expression of the gene encoding c-Kit in is retained expression their to relative downregulated receptor substantially are cytokine MPL the and Tal-1 factor CD10 transcription the human encoding and LMPPs mouse both In CD10 monocytic mostly lost. is potential residual, erythroid after retained is potential this that and proceeds commitment differentiation myeloid certain in pathway vitro in alternative an be may differentiation myeloid that suggests which cocultures, in stromal cells myeloid can generate ARTICLES 8

The identification of a lymphoid-primed progenitor that may may that progenitor lymphoid-primed a of identification The We noted both differences and similarities between the the between similarities and differences both noted We DNTT − − + − CD62L CD62L CD1a CD62L conditions − CD62L − expression was significantly higher in CD10 in higher significantly was expression ) ) subset of CD34 3 − 9 − hi thymocyte population and the endothelial expression expression endothelial the and population thymocyte and do not express CD62L. It is not clear at this time time this at clear not is It CD62L. express not do and hi CD62L hi bone marrow cells are not precursors of the most prim cells are recruited to the thymus to initiate T cell dif cell T initiate to thymus the to recruited are cells cells and mouse LMPPs in their gene expression gene their in LMPPs mouse and cells RAG1 hi cells. + 5 CD10 . Nonetheless, it is clear that the capacity for for capacity the that clear is it Nonetheless, . hi and and population from HSCs-MPPs, but B cell– + n vitro in CD1a EBF1 3 + 8 RAG2 cells) in mobilized blood (data peripheral , was also upregulated during generation generation during upregulated also was , + + CD1a ‘CLP’ 1 and 2 − . The thymocyte data presented here here presented data thymocyte The . CD7 progressively wanes as lymphoid lymphoid as wanes progressively were expressed at the CD10 the at expressed were − − CD62L − CD62L 1 thymocytes. CD34 thymocytes. 0 + raises the question of whether whether of question the raises PAX5 thymocytes or whether CD7 CD7 whether or thymocytes 4 0 . As PSGL-1 had abundant abundant had PSGL-1 As . hi hi cells may be an additional were not. In contrast, contrast, In not. were LMPPs engage with the the with engage LMPPs 15 − CD62L − , + 1 CD62L CD24 6 , and a population , of and a population + + CD1a CD1a hi − hi bone marrow marrow bone population in in population + − − cells, genes genes cells, 3 + CD1a CD7 progenitors, 6 Lin + , , but in our cells than than cells − − CD10 − + MPPs MPPs CD7 stage stage 36 2 , , 3 3 6 7 − − ------. .

Supported by the US National Institutes of Health (P01 HL073104 and RO1 D. Kohn, G. Dravid, S. Sandoval and M. forCorselli advice on the manuscript. We thank J. Scholes, F. X. Codrea, Li and S. Dandekar for technical assistance; and the in available is information Supplementary Note: codes. Accession the in available are pape the of version references associated any and Methods M are during aberrant states. hematopoiesis affected in disease hematopoiesis. It will also allow understanding of how these processes human in commitment lymphoid of stages first the of regulation lar before B cell commitment, will now permit delineation of the stage molecuat a and lymphoid differentiation, forfull marrowprimed bone mitted to the B cell lineage. The identification of a progenitor in human compared only between be multipotent could progenitors and differentiation progenitors of com states that meant now until has marrow ment in previous studies of hematopoietic progenitors in human bone mouse bone marrow and mouse thymus c-Kit 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.html. R Published online at The authors declare no competing financial interests. experiments and wrote the paper. bioinformatics H.K.A.M. supervised analysis; and G.M.C. anddesigned analyzed microarray data; S.G. and Y.Z. assisted in experiments; C.P. did experiments; designed, did and analyzed experiments, did R.S. bioinformatics analysis of L.A.K. designed, did and analyzed experiments and wrote Q.-L.H.the paper; Angeles. Los California, of University and the Jonsson Comprehensive Cancer Center Genomics Shared Resource of the the Broad Stem Research Cell Center of the University of Los Angeles,California, RM1-01717 to G.M.C.; and to RN1-00557-1 FlowH.K.A.M.), Cytometry Core of the HL077912), InstituteCalifornia of Regenerative Medicine (RC1-00108 and A C AU eprints and permissions information is available online at http://www.nature.com/at online available is information permissions and eprints CK O

ETHODS The The reliance on CD10 expression as a marker of lymphoid commit

T MP Rosen, S.D. Ligands for L-selectin: homing, inflammation, and beyond. and inflammation, homing, L-selectin: for Ligands S.D. Rosen, E.M. Six, M. Ichii, dendritic and killer, natural B, T, Human B. Chen, & D. Cen, M., Travis, A., Galy, tolerance. baby Haematopoiesis: O. Leavy, J.E. Mold, M.I. Rossi, Payne, K.J. & Crooks, G.M. Immune-cell lineage commitment: translation from mice RichieEhrlich, L.I.,Serwold, T. of Weissman, & vitroI.L.Inassays pitfalls misrepresent vivoin the and development cell T Early H.R. Rodewald, & S.M. Schlenner, Forsberg, E.C., Serwold, T., Kogan, S., Weissman, I.L. & Passegue, E. New evidence common clonogenic Adolfsson, J. of Identification K. Akashi, & I.L. Weissman, M., Kondo, Immunol. thymus. the seeding lineage. lymphoid B human the in subset. cell progenitor marrow bone (1995). common 459–473 a from arise cells humans. in lineages cell T changes. age-related developmental humans. to progenitors. lymphoid murine of potentials lineage flk2/flt3 potential. of potential progenitors. hematopoietic megakaryocyte-erythrocyte supporting Cell commitment. lineage blood adult for map road revised a potential megakaryocytic marrow. bone mouse in progenitors lymphoid H NOW + O ET

Lin 121 R C I N L − , 295–306 (2005). 295–306 , ED ONT

ADV et al. et Sca-1 G FI et al. et TrendsImmunol. 22 t al. et t al. et Immunity GM et al. , 129–156 (2004). 129–156 , N A The density of CD10 corresponds to commitment and progression and commitment to corresponds CD10 of density The RIBU A human postnatal lymphoid progenitor capable of circulating and circulating of capable progenitor lymphoid postnatal human A http://www.nature.com/doifinder/10.1038/ni.240 NCE ONLINE PUBLIC A + ea ad dl hmtpitc tm el gv rs t distinct to rise give cells stem hematopoietic adult and Fetal ENT lmhpiss s cie hogot ua lf, u tee are there but life, human throughout active is lymphopoiesis B Identification of Flt3+ lympho-myeloid stem cells lacking erythro- GEO: microarray data, data, microarray GEO: CD62L N r CIAL I J. Exp. Med. Exp. J. . T

S 26 I ON , 674–677 (2007). 674–677 ,

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29 26 9 , ,

© 2012 Nature America, Inc. All rights reserved. software. T LSR II for and FlowJo with was (BD) used data cytometry, were flow analyzed or Fortessa A cells). granulocytic for (G10F5; anti-CD66B or cells); myeloid for (WM53); anti-CD33 and (W6D3) anti-CD15 (M5E2), anti-CD14 cells); cells); anti-CD14 (M5E2) and erythroid anti-CD11B for(ICRF44); monocytic (RPA-T8) anti-TCR and anti-CD8 anti-CD7(M-T701), (RPA-T4), anti-CD4 (SK7), CD3 anti- (HI149), NK anti-CD1A cells); for dendritic for (MY31; (DCN46; anti-CD209 cells); anti-CD56 cells); lymphoid B for SJ25C1; and (4G7 CD19 hematopoietic human anti-HLA-A,cells); all humananti-HLA-Bcells); HLA-Candforall (G46-2.6; for (HI30; anti-CD45 BD): from (alls antibodies monoclonal human-specific following the with stained were then cultured, Lineage-specificanalysis. described as done were erythropoietin assays CFU Systems). and R&D U/ml; (4 ng/ml) (50 thrombopoietin ng/ml), (5 factor stem-cell ng/ml), (5 ligand Flt3 ng/ml), (5 IL-3 with FBS, 10% with DMEM in stroma assay. CFU and cultures Myelo-erythroid factor stem-cell and ng/ml) (5 ligand (1 ng/ml; R&D Flt3 Systems) ng/ml), (5 IL-7 with medium unit) on established OP9 stroma expressing cell-deposition the Notch automated ligand DL1 an in lymphoid (with dilution limiting by or cells single as or T cell lymphoid cultures. information, plating Table(limiting-dilution 2 Supplementary 100 × wells) total / wells (positive follows: as defined was cells single of efficiency Cloning cells. 100 than more included they if positive being as recorded were Clones Systems). R&D from thrombopoietin and ng/ml) d 3–5 for (cytokines of first culture (5 or IL-3 ng/ml), with (5 without (5 ng/ml), ligand Flt3 ng/ml), (5 IL-7 of presence the penicillin-streptomycin, (Sigma), 50 (Biowhittaker), FCS 5% with Scientific) (Irvine deposition unit. Cells were cultured cell- automatedin an lymphoid with mediumplates (RPMI-164096-well mediumin stroma MS5 or OP9 on dilution or by cells or limiting plates as single on in in bulk 48-well OP9 stroma plated cultures. lymphoid cell cell–NK B Systems). Immunocytometry BD BD). lasers; 633-nm and 561- from 488-, 405-, on a FACSAria (355-, all isolated were Cells (G44-26), (L234; phycoerythrin–anti-HLA-DR and (5E10) anti-CD90 allophycocyanin–anti-CD44 4G8), phycoerythrin– allophycocyanin–anti-CD62L (DREG-56), KPL-1), (anti-CD135; phycoerythrin-indodicarbocyanine– anti-SELPLG; anti-Flt3 or (anti-CD162 anti-PSGL-1 allophycocyanin– 12G5), (anti-CXCR4;allophycocyanin–anti-CD184 B8), (HIL-7R-M21),allophycocyanin–anti-CD117647–anti-CD127(YB5. Fluor defined negative gates. Additional analyses used the following antibodies: Alexa ino-2-phenylindole) was added for analysis of viability. A ‘no-antibody’ control (4 DAPI dye DNA-intercalating The Dickinson). Becton from all CD14 (M2E2), anti-CD19 (4G7), anti-CD56 (MY31) and anti-CD235aanti- (SK7), (GA-R2; anti-CD3 antibodies lineage-depletion isothiocyanate–labeled or − phycoerythrin (DREG-56), phycoerythrin–anti-CD62L (HI10a), anti-CD10 phycoerythrin-indotricarbocyanine– allo (HIT2), (HI100), phycocyanin–anti-CD38 phycoerythrin-indodicarbocyanine–anti-CD45RA and Biolegend); 581; (allophycocyanin- (anti-CD34; to antibody molecules human indotricarbocyanine–conjugated for specific antibodies monoclonal (Miltenyi Biotec). Review Institutional CD34 by enriched for were Samples approved Angeles. Los California, of guidelines University the of to Board according Hospital, AllCells Children’s or Cincinnati Angeles, Los of California, Core of Tissue University Pathology the the via donors healthy from obtained were cells cells. marrow bone of Isolation ONLINE METHODS NATURE NATURE IMMUNOLOGY CD29 mouse and

Samples enriched for CD34 for enriched Samples phycoerythrin-indodicarbocyanine–anti-CD7 (M-T701), and fluorescein fluorescein and (M-T701), phycoerythrin-indodicarbocyanine–anti-CD7 +

cells by the magnetic-activated cell-sorting (MACS) system system (MACS) cell-sorting magnetic-activated the by cells cell cell differentiation was assessed by RT-PCR of human CD45 A B (WT31); for T lymphoid cells); anti-CD235a (GA-R2; for for (GA-R2; anti-CD235a cells); lymphoid T for (WT31); − cells isolated at 4–5 weeks from T cell lymphoid cultures. lymphoid cell T from weeks at 4–5 isolated cells ). 4 1 Cells were Cells plated in bulk on 6-well or plates 96-well . Cells were collected from transplanted mice and and mice transplanted from collected were Cells + cells were incubated with combinations of of combinations with incubated were cells Normal human bone marrow and thymic thymic and marrow bone human Normal " -glutamine (Gemini Bio Products)) in in Products)) Bio (Gemini -glutamine Cells isolated by flow cytometry were were cytometry flow by isolated Cells Populations were plated on OP9 OP9 on plated were Populations M M 2-mercaptoethanol 2-mercaptoethanol M 3 9 . ` ,6-diamid + cells - -

control for presence of cDNA. of presence for control encodinggene TheAngeles. Los Software v3.0.2 (Fluidigm) at the GeneSeq Core of the University of California, expression chip with Taqman probes and results were analyzed with Real-Time ( methods change-in-cycling-threshold the of use means of results obtained for the reference genes genes reference candidate reference genes were analyzed with geNorm 3 Table probe– TaqMan and Mastermix Taqman based gene-expression with analysis (probes (Applied PCR Biosystems), real-time for used was Viia7 with Omniscript RT, OLIGO DT, and RNAguard (Pharmacia Biotech). An ABI with a and RNAEsay Qiagen reverse-transcribed (Qiagen) Microkit extracted Quantitative PCR analysis. cells. carrier irradiated only received mice control Negative CD45. human and HLA-B,HLA-A,HLA-Cfor positive cells as defined was engraftment human Totalcytometry. flow by analysis for later weeks 2 killed were and cells) UCB cells ( CD10 10 × 3 of injection intratibial before cGy) (375 Angeles. irradiated were Los mice NSG Adult California of University of Committee Use and Care Animal for vivo In Plus 2.0 Array (Affymetrix). Plus The 2.0 Array robust (Affymetrix). multichip average method U133 Affymetrix onto hybridized and (Qiagen) Microkit with extracted was analysis. Microarray of populations, mean fluorescence intensity and quantitative PCR, and the the and PCR, quantitative and intensity fluorescence mean populations, of of growth The potential. mean and s.e.m. were for calculated total CFU output of was The for analysis used two-way variance analysis. comparison statistical analysis. Statistical or an MRm camera HRc (fluorescence) camera (chromagen;Axiocam Zeiss). with captured were images and ), × (10 System Imagining Apotome with hematoxylin (Jackson Immunoresearch). Sections were viewed with Axioimager ing, DAB tetrahydrochloride) was (3,3-diaminobenzidine applied, followed by tion; T20925 and T20912; Invitrogen Molecular Probes). For chromagen stain amplifica signal applied was (for tyramide tyramide Fluor 488–labeled Alexa Invitrogen). For fluorescence immunohistochemistry, Alexa Fluor 594– and/or (anti-rat (MP-7444; antibody Vector secondary or (T20912; anti-mouse Labs) International)), followed by incubation with horseradish peroxidase–conjugated and/or Cruz Biotechnology) (1:83 dilution; anti-VE-cadherin BV6; Chemicon were incubated then with primaryformalin, antibody buffered (anti-MECA-79 neutral 10% (1:83 in dilution; fixed sc-19602; were Santa sections chemistry, 5 sections and (Tissue-Tek) compound temperature cutting optimum in ded Immunohistochemistry. maps) heat and (dendrograms Java TreeView software as described done was analysis enrichment set Gene (Limma). condition one least at in cells of cant at a they had a if difference only inmap expressionheat the ofin inclusion for considered were Genes excluded. were P of ware in expression in Venn diagrams was calculated with the R/Bioconductor soft difference a with genes of number The clustering. hierarchical for used were in MAS5 method all in replicates any of probes) (24,067 the populations three ‘present’ by the considered sets probe Only method. (agglomeration) linkage and average metric) (distance correlation rank Spearman with clustered cally hierarchi were populations three the from arrays Replicate replicates. all for algorithm (MAS5) 5 Suite Microarray The populations. three the from expression normalized to obtain

value was chosen. Probe sets not mapped to a gene with an official symbol symbol official an with gene a to mapped not sets Probe chosen. was value M o Single-cell quantitative PCR was done on a Fluidigm BioMark 48.48 gene- 48.48 BioMark on a Fluidigm done was PCR quantitative Single-cell m in thickness were stained with hematoxylin and eosin. For immunohisto in vivo in twofold. For genes with multiple probe sets, the probe set with the lowest the with set probe the sets, probe multiple For with twofold. genes 4 n 5 − package Limma package CD62L = 3 mice), each with 1 × 10 studies. studies. ). Reactions were done in technical and biological triplicates. Nine Nine triplicates. biological and technical in done were Reactions ). P value of less than 0.05 relative to expression by the other population experiments according to protocols approved by the Institutional the by approved protocols to according experiments hi cells ( cells 4 Adult mice of the NSG strain (Jackson Laboratories) were used 2 . Quantitative PCR results were normalized to . PCR the were results geometric Quantitative normalized 4 7 . For presentation, Cluster 3.0 software (clustering) software 3.0 Cluster . For presentation, Prism version 5 (GraphPad Software Inc) was used for for used was Inc) Software (GraphPad 5 version Prism n 10 × 2 or mice) 3 = RNA from bone marrow from three different donors donors different three from marrow bone from RNA 4 6 at a a at Human thymuses were frozen at 80 − °C and embed 4 4 After After isolation of cells with a FACSAria, RNA was was used for present, marginal or absent ‘calls’ absent or marginal present, for used was P value of value less than 0.01 and change in threshold 5 ‘carrier’ cells (irradiated (3,000 cGy) CD34 B 2 o -microglobulin was used as a positive positive a as used was -microglobulin twofold and the was difference signifi 4 10 × 15 to ACTB $ 4 $ CD34 C plus 4 T and and 9 ) were used. were software for optimal . doi:10.1038/ni.2405 + Lin B2M Supplementary Supplementary − bone marrow through the through 4 3 was used 4 8 and − 4 ------

© 2012 Nature America, Inc. All rights reserved. 44. 43. 42. 41. software ELDA analysis. statistical for used was post-test Tukey a with variance of analysis one-way doi:10.1038/ni.2405

i, W.M. Liu, Bolstad, B.M., Irizarry, R.A., Astrand, M. & Speed, T.P. A comparison of normalization of generation system: J. Vandesompele, OP9–DL1 The J.C. Zuniga-Pflucker, & R. Holmes, call algorithms. call bias. and variance on Bioinformatics based data array oligonucleotide density high for methods (2002). genes. research0034–research0034.11 control internal multiple of averaging geometric by data vitro. Protoc. Harb. in cells stem hematopoietic or embryonic from T-lymphocytes t al. et doi:10.1101/pdb.prot5156 (2009). doi:10.1101/pdb.prot5156

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