Leukemia (2007) 21, 311–319 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu ORIGINAL ARTICLE T-, B- and NK-lymphoid, but not myeloid cells arise from human CD34 þ CD38ÀCD7 þ common lymphoid progenitors expressing lymphoid-specific genes I Hoebeke1,3, M De Smedt1, F Stolz1,4, K Pike-Overzet2, FJT Staal2, J Plum1 and G Leclercq1 1Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium and 2Department of Immunology, Erasmus University Medical Center, Rotterdam, The Netherlands Hematopoietic stem cells in the bone marrow (BM) give rise to share a direct common progenitor either, as CLPs were not all blood cells. According to the classic model of hematopoi- found in the fetal liver.5 Instead, fetal B and T cells would esis, the differentiation paths leading to the myeloid and develop through B/myeloid and T/myeloid intermediates. lymphoid lineages segregate early. A candidate ‘common 6 lymphoid progenitor’ (CLP) has been isolated from The first report of a human CLP came from Galy et al. who À þ CD34 þ CD38À human cord blood cells based on CD7 expres- showed that a subpopulation of adult and fetal BM Lin CD34 sion. Here, we confirm the B- and NK-differentiation potential of cells expressing the early B- and T-cell marker CD10 is not þ À þ CD34 CD38 CD7 cells and show in addition that this capable of generating monocytic, granulocytic, erythroid or population has strong capacity to differentiate into T cells. As megakaryocytic cells, but can differentiate into dendritic cells, CD34 þ CD38ÀCD7 þ cells are virtually devoid of myeloid B, T and NK cells. These LinÀCD34 þ CD10 þ cells homo- differentiation potential, these cells represent true CLPs. To 7 unravel the molecular mechanisms underlying lymphoid com- genously expressed CD38. According to Ishii et al. expression þ mitment, we performed genome-wide gene expression profiling of the chemokine receptor CXCR4 on BM CD34 cells would on sorted CD34 þ CD38ÀCD7 þ and CD34 þ CD38ÀCD7À cells. be sufficient to restrict their differentiation potential to the Interestingly, lymphoid-affiliated genes were mainly upregu- lymphoid lineage. A human CMP was recently also identified in þ lated in the CD7 population, while myeloid-specific genes the LinÀCD34 þ CD38 þ fraction of BM and cord blood. These were downregulated. This supports the hypothesis that lineage CMPs are CD45RAÀ and express low levels of IL-3Ra.8 commitment is accompanied by the shutdown of inappropriate þ gene expression and the upregulation of lineage-specific In cord blood, expression of CD10 on CD34 cells does not genes. In addition, we identified several highly expressed discriminate progenitor cells with lymphoid-restricted potential genes that have not been described in hematopoiesis before. from multipotent cells.9 However, Hao et al.9 detected in the Leukemia (2007) 21, 311–319. doi:10.1038/sj.leu.2404488; most primitive CD34 þ CD38À cord blood fraction a subpopula- published online 14 December 2006 tion expressing CD7, an antigen that was previously identified Keywords: human; hematopoiesis; stem cells; cord blood; lymphoid progenitor on early human T-lymphoid progenitors, and they showed that single CD34 þ CD38ÀCD7 þ cord blood cells can generate B cells, NK cells and dendritic cells, but are devoid of myeloid or Introduction erythroid differentiation potential. T-cell potential was not addressed by these investigators. All blood cells ultimately derive from a rare population of In a recent study, Haddad et al.10 compared the differentiation hematopoietic stem cells in the bone marrow (BM) that are potential of cord blood CD34 þ CD45RAhiLinÀCD10 þ cells, multipotent and have the ability to self-renew. According to the which correspond to the BM CLP, with that of cord blood classic model of hematopoiesis, all lymphoid cells (T, B and CD34 þ CD45RAhiCD7 þ cells, which comprise the natural killer (NK) cells) develop through a common precursor CD34 þ CD38ÀCD7 þ CLP, as these uniformly express CD45RA. stage, the so-called ‘common lymphoid progenitor’ (CLP), and The authors showed that the differentiation potential of accordingly, cells from the myeloid lineages share a ‘common CD34 þ CD45RAhiCD7 þ cells is skewed toward the T/NK myeloid progenitor’ (CMP). This model was supported by the lineages, while CD34 þ CD45RAhiLinÀCD10 þ cells predomi- prospective isolation of cell populations with CLP and CMP nantly possess B-cell differentiation potential. Additionally, both 1,2 function from the murine BM. Recent evidence, however, populations retain some degree of myeloid differentiation indicated that BM CLPs are not physiological T-cell progenitors, capacity. Gene expression data from microarray analyses as early thymic progenitors (ETPs) do not have the CLP supported their conclusions. 3 4 phenotype and CLPs are not present in the peripheral blood. In the present study, we confirm that the CD34 þ CD38ÀCD7 þ Instead, the thymus is most likely seeded by a multipotent cord blood population is lymphoid-committed and we show that progenitor. During fetal hematopoiesis, B and T cells do not it also has strong T-lymphoid differentiation potential in fetal thymus organ culture (FTOC). To investigate the molecular Correspondence: Professor G Leclercq, Department of Clinical mechanisms driving lymphoid commitment, we studied the Chemistry, Microbiology and Immunology, Ghent University Hospital, differential gene expression between CD34 þ CD38ÀCD7À multi- 4 Blok A, De Pintelaan 185, B-9000 Ghent, Belgium. þ À þ E-mail: [email protected] potent cells and CD34 CD38 CD7 CLP cells using Affymetrix 3Current address: I Hoebeke, Diabetes Research Center, Brussels Free GeneChip technology. University (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium. 4 Current address: Laboratory of Molecular Cell Biology, Institute of Materials and methods Botany and Microbiology, Katholieke Universiteit Leuven and Department of Molecular Microbiology, Flanders Interuniversity Institute of Biotechnology (VIB), Leuven, Belgium Cell sorting Received 31 August 2006; accepted 19 October 2006; published Cord blood was obtained and used following the guidelines of online 14 December 2006 the Medical Ethical Commission of the Ghent University Molecular characterization of human cord blood CLP I Hoebeke et al 312 Hospital. Within 12 h after collection of human umbilical cord Flow cytometry blood samples, mononuclear cells were isolated over a Before labelling with antibodies, cells were pre-incubated Lymphoprep density-gradient (Axis-Shield PoC AS, Oslo, 15 min with anti-mouse FcRgII/III (clone 2.4.G2, a kind gift of Norway) and CD34 þ cells were isolated by positive selection with Dr J Unkeless, Mount Sinai School of Medicine, New York, NY, MACS magnetic beads (Miltenyi Biotec, Bergisch Gladbach, USA) and human IgG (Miltenyi Biotec) to block murine and Germany). Cells were labelled with anti-CD34-allophycocyanin human Fc receptors, respectively. Cells were incubated with (APC), anti-CD38-phycoerythrin (PE) and anti-CD7-fluorescein appropriate amounts of combinations of the following mouse isothiocyanate (FITC) monoclonal antibodies (BD Biosciences, anti-human monoclonal antibodies: CD19-PE, CD34-APC, San Jose, CA, USA) and CD34 þ CD38ÀCD7 þ and CD4-APC, CD33-FITC, CD14-FITC (all from BD Biosciences), CD34 þ CD38ÀCD7À cells were sorted with a FACSVantage CD56-APC and CD8b-PE (both from Immunotech, Beckman Cell sorter (Becton and Dickinson Immunocytometry Systems Coulter, Fullerton, CA, USA). Cell populations containing (BDIS), San Jose, CA, USA). The purity of the sorted cells was mouse leukocytes (from FTOC) were simultaneously stained checked on a FACSCalibur (BDIS) and was always 495%. with anti-mouse CD45-CyChrome (BD Pharmingen, San Diego, Sorted cells were either directly used in MS-5 co-culture or CA, USA). After 45 min, cells were washed with ice-cold FTOC, or either stored in 200 ml TRIZOL (Invitrogen, Carlsbad, PBS þ 1% BSA þ 0.1% NaN3, propidium iodide (4 mg/ml) was CA,USA)atÀ701C for later RNA isolation and use in added and cells were analyzed on a FACSCalibur. Propidium microarray experiments or real-time PCR. iodide positive and mouse CD45 þ cells, representing dead cells and mouse leukocytes, respectively, were excluded from analysis, which was performed with CellQuest software (BDIS). Co-culture on MS-5 stromal cells The differentiation of stem cells to most lymphoid (except RNA isolation and amplification The TRIZOL lysates of different sorts, corresponding to a total of T cells) and myeloid cell types can be accomplished in vitro by culture in the presence of the appropriate human recombinant 100 000 sorted cells, were pooled and total RNA was extracted and purified on an RNeasy column (Qiagen, Venlo, The Nether- cytokines on a feeder layer of the murine stromal cell line MS-5.11 Four days before their use in co-culture experiments, MS-5 lands) according to the instructions of the manufacturers. The RNA was concentrated to 10 ml with Microcon YM-50 columns cells (kindly provided by L Coulombel, Institut Gustave Roussy, (Millipore, Billerica, MA, USA) and subjected to Degenerative Villejuif, France) were seeded in 96-well plates at a density of 3 Oligonucleotide Primer- (DOP) mediated amplification. The 5 Â 10 cells per well. Co-cultures were initiated by incubating detailed protocol, which was developed in our lab, is available human sorted cells in 200 ml Iscove’s Modified Dulbecco’s upon request. Briefly, mRNA was first reverse transcribed using a Medium (IMDM)