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Notch Receptor LigationProgress to DN2/3 Stage Thymocytes with Propensity of Adult Lymphoid Progenitors to

Zúñiga-Pflücker, Howard T. Petrie and Paul W. KincadeJiaxue Huang, Karla P. Garrett, Rosana Pelayo, Juan Carlos

http://www.jimmunol.org/content/175/8/4858doi: 10.4049/jimmunol.175.8.4858

2005; 175:4858-4865; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Propensity of Adult Lymphoid Progenitors to Progress to DN2/3 Stage Thymocytes with Notch Receptor Ligation1

Jiaxue Huang,*† Karla P. Garrett,* Rosana Pelayo,* Juan Carlos Zuniga-Pflucker,‡

Howard T. Petrie,§ and Paul W. Kincade2*

Notch family receptors control critical events in the production and replenishment of specialized cells in the immune system.However, it is unclear whether Notch signaling regulates abrupt binary lineage choices in homogeneous progenitors or has moregradual influence over multiple aspects of the process. A recently developed coculture system with Delta 1-transduced stromal cellsis being extensively used to address such fundamental questions. Different from fetal progenitors, multiple types of adult marrowcells expanded indefinitely in murine Delta-like 1-transduced OP9 cell cocultures, progressed to a DN2/DN3 thymocyte stage, andslowly produced TCR� and NK cells. Long-term cultured cells of this kind retained some potential for T lymphopoiesis in vivo.Adult marrow progressed through double-positive and single-positive stages only when IL-7 concentrations were low and passageswere infrequent. Lin�c-KitlowGFP�IL-7R��/� prolymphocytes were the most efficient of adult bone marrow cells in short-termcultures, but the assay does not necessarily reflect cells normally responsible for replenishing the adult thymus. Although marrow-derived progenitors with Ig DH-JH rearrangements acquired T lineage characteristics in this model, that was not the case for moreB committed cells with VH-DHJH rearrangement products. The Journal of Immunology, 2005, 175: 4858–4865.

T he Notch family of receptor/transcription factors has at-tracted great interest because of its potential importancein establishment of the immune system, stem cell self-

renewal, and lineage fate decisions (1). Knockout and overexpres-sion experiments have generated an impressive body of data indi-cating that Notch determines whether lymphoid progenitorsgenerate B or T lineage lymphocytes (2, 3). Moreover, two labo-ratories demonstrated that the behavior of normal progenitorscould be modified by contact with ligand modified stromal cells (4,5). Indeed, experimental models were described that support allstages of T lymphopoiesis and even when embryonic stem celllines were used to initiate the cultures (6).

However, the range of target cells that can respond to Notchreceptor ligation with a lineage choice decision and the extent towhich partially committed progenitors can be reprogrammed re-main unclear. Indeed, we need to rigorously test whether there aresingle branch points where progenitors at a discrete stage are ir-reversibly diverted into a particular pathway. Moreover, it is notknown if B lineage inhibition and T lineage induction representcompletely separate or related events (7, 8). Controversy remainsabout the role of Notch in determining the relative production ofTCR�� vs TCR�� lymphocytes and biasing the formation of CD4

and CD8 subsets (9–11). Additionally, most of the studies to datehave used fetal lymphoid progenitors and direct comparisons withtheir counterparts in adult bone marrow have not been reported.

One of the most unique and important features of hemopoieticstem cells (HSCs)3 is their ability to extensively self-renew whileretaining the ability to give rise to multiple specialized types ofblood cells. Understanding how this process is regulated maysomeday facilitate expansion of stem cells in culture, opening pos-sibilities for genetic engineering and other therapeutic applica-tions. In that context, it is interesting that human stem cells wereexpanded 10-fold in culture by ligation of Notch receptors (12),and stem cell lines were established by introduction of constitu-tively active Notch 1 (13). Therefore, it is possible that Notchreceptor ligation might trap cells in a proliferative mode and blocktheir differentiation. If so, lymphohematopoietic cells might be ex-panded and maintained for prolonged periods on Delta 1-trans-duced stromal cells.

We have now used RAG-1/GFP-knockin mice, cell sorting, andcoculture with Notch ligand-transduced stromal cells to addressthese questions. Unlike their fetal counterparts, multiple subsets ofadult bone marrow cells were able to generate long-term, Notchligand, and IL-7-dependent cell lines paused at theCD4�D8�thymocyte (DN)2/3 thymocyte stage. Progression ofadult progenitors in the T lineage was blocked in the presence of5 ng/ml IL-7. These findings provide a basis for further investiga-tion of adult lymphoid progenitors in short- and long-term cultures.

Materials and MethodsMice and embryos

RAG-1/GFP-knockin mice have been described previously (14). FemaleB6.PL-Thy1a/CyJ (Thy-1/CD90.1) congenic, and C57BL/6 mice werebought from The Jackson Laboratory. Mating homozygous male RAG-1/GFP-knockin mice with wild-type C57BL/6 strain female mice generated

*Immunobiology and Cancer Program, Oklahoma Medical Research Foundation,Oklahoma City, OK 73104; †Department of Microbiology and Immunology, Univer-sity of Oklahoma Health Sciences Center, Oklahoma City, OK 73190; ‡Departmentof Immunology, University of Toronto, Sunnybrook and Women’s Research Institute,Toronto, Ontario, Canada; and §The Scripps/Florida Research Institute, Jupiter, FL33458

Received for publication May 18, 2005. Accepted for publication July 25, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grants AI20069, AI58162,AI33940, and AI53739 and National Center for Research Resources Grant P20RR15577. J.C.Z.-P. is supported by an Investigator Award from the Canadian Insti-tutes of Health Research.2 Address correspondence and reprint requests to Dr. Paul W. Kincade, Immunobi-ology and Cancer Program, Oklahoma Medical Research Foundation, 825 N.E. 13thStreet, Oklahoma City, OK 73104. E-mail address: kincade@omrf.ouhsc.edu

3 Abbreviations used in this paper: HSC, hemopoietic stem cell; CLP, common lym-phoid progenitor; DN, CD4�D8� thymocyte; DP, CD4�CD8� thymocyte; ELP,early lymphoid progenitor; LSP, L-selectin-positive progenitor; OP9-DL1, murineDelta-like 1-transduced OP9 cell.

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heterozygous RAG-1/GFP embryos, as well as mice that were studied asadults. Mating homozygous male RAG-1/GFP-knockin mice with femaleB6.PL-Thy1a/CyJ mice yielded heterozygous RAG-1/GFP animals bearingthe Thy-1/CD90.1 Ag. Timed pregnancies were determined by designatingthe day of vaginal plug observation as day 0.5 postcoitus (E0.5). For ex-periments with adult mice, 5- to 10-wk-old male mice were used. All of thestudies were reviewed and approved by an institutional review committee.

Antibodies

FITC-conjugated CD45R/B220 (RA3-6B2), CD3� (145-2C11), CD8�(53-6.7), CD43, CD25, and Mac-1 Abs, as well as PE-conjugated CD3�(145-2C11), TCR��, NK1.1(PK136), CD45R/B220 (RA3/6B2), CD19 (ID3),and IL-7R�(SB/199) Abs were all purchased from BD Pharmingen. Thesame source was used for biotinylated-CD106/VCAM-1, biotinylated-CD19 (clone 1D3), CD90.1 (OX-7) and allophycocyanin-conjugatedCD11b/Mac-1 (M1/70), DX-5, c-Kit (2B8), CD44 (1M7), TCR�, CD4,and CD45R/B220. Allophycocyanin-Cy-7-CD62L (MEL-14) and PE-Cy5-Sca-1 (D7) were obtained from eBioscience. A PE-Texas red tandem-con-jugated streptavidin (PE-TR-streptavidin) was purchased from Caltag Lab-oratories. Purified rabbit anti-TdT polyclonal Ab and a FITC-conjugatedF(ab�)2 fraction of goat anti-rabbit IgG Ab were purchased from Super-techs. FITC-conjugated goat anti-mouse IgM Ab was purchased fromZymed Laboratories.

OP9 cocultures

Transduced OP9-MigR1 (OP9-vector) and murine Delta-like 1-transducedOP9 cells (OP9-DL1) stromal cell lines have been described previously (5).They were maintained in modified MEM (�-MEM; Invitrogen Life Tech-nologies) supplemented with 20% FCS (HyClone). Flt3-L, stem cell factor,and IL-7 were all purchased from R&D Systems and used at concentrationsspecified in the text. Coculture of hemopoietic progenitors was modified toallow calculation and comparisons of proliferation and differentiation fromstem cells/progenitors (5, 15). Briefly, 1 day before plating of progenitors,2 � 104 stromal cells were plated into each well of 24-well plates. Pro-genitors were then added to the stromal cells at different densities to de-termine the numbers of input cells that gave optimal lymphocyte yields. Ona weekly basis, stromal cells and hemopoietic cells were harvested using0.53 mM EDTA/PBS (pH 7.4). Then, 3,000–10,000 hemopoietic cells to-gether with stromal cells were subcultured onto freshly prepared stromalcells. For weekly monitoring of differentiation, stromal cells were excludedby staining with an Ab to VCAM-1, and dead cells were excluded bystaining with 7-aminoactinomycin D. Culture conditions and cell densitieswere considered acceptable when �95% of the recovered hemopoieticcells were viable.

Cell sorting and flow cytometry

E13.5-15.5 fetal livers were harvested and subjected to cell sorting as de-scribed previously (16). Bone marrow cells were harvested and enrichedfor lineage-negative cells by incubation with a mixture of Abs to lineagemarkers, CD45RA (supernatant from clone 14.8), Gr-1 (Ly-6G; RB6-8C5),CD11b/Mac-1 (M1/70), CD19 (1D3), and Ter-119, followed by negative

selection with the MACS cell separation system (Miltenyi Biotec). Thesepartially lineage-depleted cells were further stained with rat anti-mouselineage markers (biotin-Gr-1, Mac-1, CD2, CD3�, CD8, Ter-119, CD45R,DX-5, and CD19), as well as allophycocyanin-labeled c-Kit and PE-Sca-1.Lineage-positive cells were electronically gated out, and lineage negativefractions were sorted on the basis of RAG-1/GFP, c-Kit, and Sca-1 ex-pression with a MoFlo (DakoCytomation). Similar separations were doneto isolate the HSC (Lin�Sca-1�c-KithighCD90.1low), L-selectin-positiveprogenitors (LSPs, Lin�CD90.1�Sca-1�c-KithighCD62L�), early lym-phoid progenitors (ELPs, Lin�Sca-1�c-KithighRAG-1�), and commonlymphoid progenitors (CLPs, Lin�Sca-1lowc-KitlowIL-7R��), as describedin Fig. 6. These previously described fractions (17–20) were sorted on aFACSAria (BD Biosciences) after thoroughly lineage depleting bone mar-row cells and then staining with biotin-CD90.1 revealed by PE-TR, allo-phycocyanin-Cy7-CD62L, PE-IL-7R�, PE-Cy-5-Sca-1, and allophycocya-nin-c-Kit. Postsort evaluation of sorted preparations revealed purities of�97%. All analyses were performed on a FACSCalibur (BD Biosciences).

Transplantation

Congenic Ly 5.1 mice were lethally irradiated with a single dose of 960 rad4 h before transplantation. Then, 105 recipient whole bone marrow cellswere mixed with 105 sorted Lin�c-Kit�Sca-1� cultured cells and injectedi.v. into recipients. Cells were harvested from bone marrows, thymuses,and spleens 21–28 days after injection and stained to check lineage differ-entiation. Additional transplanted animals were observed for 2 mo andautopsied to see whether tumors developed.

RT-PCR analysis of gene expression

The mRNAs were isolated from sorted cells using MicroPoly(A) Pure(Ambion) and converted to cDNA with Moloney murine leukemia virusreverse transcriptase (Invitrogen Life Technologies). cDNA amounts werenormalized by comparing the PCR products using probes specific for �-ac-tin. The PCR was conducted by using combination with ampli-TaqDNApolymerase (Takara) and TaqStart Ab (BD Clontech) at three differentcycles to confirm the reaction is in the exponential phase. To compare therelative expression level of relevant genes, serial 5-fold dilutions of cDNAwere amplified at appropriate cycles. In the case of no amplification, sec-ondary PCR was performed by using 2 �l for 20-�l reaction volume fromfirst reaction as a template to confirm the lack of transcripts. Anti-Taq Abwas inactivated by heating at 95°C for 7 min before amplification con-ducted as follows: 15 s at 94°C, 15 s at 57°C, and 30 s at 72°C. Gene-specific primer sequences are listed in Table I.

ResultsFetal and adult lymphoid progenitors can respond quitedifferently to Notch receptor ligation

Primitive cells in the aorta/gonad/mesonephros and fetal liver canefficiently give rise to T lineage lymphocytes (defined asTCR���CD4�CD8� or TCR���) when cultured on OP9-DL1 in�-MEM supplemented with 5 ng/ml IL-7 and 5 ng/ml Flt3-L

TABLE I. Primers used for RT-PCR analyses

Notch 1 5�-CGGTGTGAGGGTGATGTCAATG-3� 5�-GAATGTCCGGGCCAGCGCCACC-3�Notch 2 5�-CTACAACTGTATCTGCCGCAGC-3� 5�-CCTTGTCGGCGCAGTACTGACTC-3�IL-7R� 5�-CCCCATAACGATTACTTCAAAGGC-3� 5�-AGAGTTTGGCAGCAAGTCTTGATA-3�Bcl-2 5�-TCGCTACCGTCGTGACTTC-3� 5�-AAACAGAGGTCGCATGCTG-3�EBF 5�-GAGATTTTTCCACAAGAAAAGGTTG-3� 5�-GGAAGAACCTGTCAATTATCACTGG-3��5 5�-CTTGAGGGTCAATGAAGCTCAGAAGA-3� 5�-CTTGGGCTGACCTAGGATTG-3�mb1 5�-GCCAGGGGGTCTAGAAGC-3� 5�-TCACTTGGCACCCAGTACAA-3�PU.1 5�-CGGATGACTTGGTTACTTACG-3� 5�-GTAGGAAACCTGGTGACTGAG-3�GATA1 5�-TCATACCACTAAGGTGGCTGAATC-3� 5�-ACACAGTTGAGGCAGGGTAGAG-3�GATA2 5�-CGGAATTCGACACACCACCCGATACC-3� 5�-CGGAATTCGCCTACGCCATGGCAGT-3�GATA3 5�-GGCCAGGCAAGATGAGAAAGA-3� 5�-TCTGACAGTTCGCGCAGGATG-3�Rag 1 5�-TGCAGACATTCTAGCACTCTGG-3� 5�-ACATCTGCCTTCACGTCGAT-3�pT� 5�-CATGCTTCTCCACGAGTG-3� 5�-CTATGTCCAAATTCTGTGGGTG-3�Id1 5�-TCCAACTTCTTGTTCTCTCCC-3� 5�-CACAAGATGCGATCGTCG-3�E47 5�-CGCACTGACCACGAGCTTCAC-3� 5�-TCCAGGGACAGCACCTCATCTG-3�Hes-1 5�-GCCAGTGTCAACACGACACCGG-3� 5�-TCACCTCGTTCATGCACTCG-3�Irf5 5�-TTCCAGAAGGGCCAGACTAAT-3� 5�-TGACATCAGGCCATTCTTCTC-3�Bh11hB2 5�-AGCCGTGGACTTGAAAGAGA-3� 5�-TGGATGACTGGCACACAGTT-3�c-Fos 5�-ATCCTTGGAGCCAGTCAAGA-3� 5�-TCCCAGTCTGCTGCATAGAA-3�IL-2R� 5�-AACGGCACCATCCTAAACTG-3� 5�-CTGTGTTGGCTTCTGCATGT-3�

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(T. Yokota et al., submitted for publication), and we assumed thesame would be true for adult bone marrow. However, this was not thecase, and only very small yields of ��� T lymphocytes were obtained(Fig. 1). In the example shown, RAG-1/GFPlow progenitors from day14 fetal liver were directly compared with Lin�c-KitlowRAG-1/GFP�IL-7R�� cells isolated from adult bone marrow and found togenerate at least 1000 times more T cells (Fig. 1B).

It seemed possible that this might not have represented an ap-propriate Notch ligand responding subset of marrow progenitors,so subsequent experiments used a series of Lin� fractions. Manydifferent subsets of marrow were capable of B lymphopoiesis whenplaced on OP9-vector stromal cells, but this differentiation optionwas completely blocked in OP9-DL1 cocultures (Fig. 2 and data

not shown). The production of CD11b/Mac-1� myeloid cells wasalso inhibited by Notch receptor ligation. Percentages and totalnumbers of recovered TCR� cells were always extremely low inOP9-DL1 cocultures initiated with marrow fractions. In addition,the cultures usually contained approximately the same numbers ofNK1.1� and an unidentified population of DX5�NK1.1� cells(data not shown). Similar observations were obtained in more than10 experiments where different parameters were exploited for sep-aration of a total of 9 marrow cell populations. This was also thecase when fetal and adult progenitors were isolated at the sametime using the same sorting strategy and cultured side by side.Therefore, it is highly unlikely that any subset in adult bone mar-row has comparable activity to fetal progenitors, suggesting thatadult progenitors have requirements not recapitulated by these par-ticular culture conditions. This issue is addressed in more detailbelow.

Homogeneous populations of early T lineage lymphocytesexpand in OP9-DL1 cocultures initiated with adult bone marrow

It seemed possible that adult bone marrow progenitors of T cellsmight benefit from extended culture intervals. However, this wasnot the case, and surface TCR� cells represented a minor fractionof recovered populations even after 5 wk of coculture (data notshown). Note that stromal cell overgrowth was detrimental to totalcell yields, and we subcultured lymphoid cells at weekly intervalsto freshly prepared stromal cell layers. Lymphocyte growth couldnot be sustained when long-term OP9-DL1-cultured cells weretransferred to OP9-vector stromal cells or when IL-7 was with-drawn, indicating that survival and/or proliferation required sus-tained Notch and IL-7 signaling (data not shown). In contrast,OP9-DL1 cocultures with added cytokines continued to thrive forat least 3 mo (Fig. 3A and data not shown). Although culturescould be established from six discrete fractions of bone marrow,the efficiency of growth during the first week was not uniform. Themost efficient Lin�c-KitlowRAG-1/GFP�IL-7R��/� prolympho-cytes/CLP produced 5000 cells/input progenitor during this inter-val, while the yield from other populations was 1 log less. Fromthe second week, each cell that was subcultured produced an av-erage of 65 cells, regardless of the starting population. After be-coming established, the cultures could be propagated indefinitely,and we estimate that one progenitor produced at least 1016 lym-phocytes within 2 mo (Fig. 3A).

Weekly examination of cells by flow cytometry revealed that thelong-term propagated cells were nearly homogeneous. Indeed, re-covered cells had very similar characteristics even when very dif-ferent subsets of marrow were used to initiate the cultures. That is,most cells propagated for extended periods on OP9-DL1 were me-dium to large in size and lacked markers of erythroid (TER119),

FIGURE 1. Fetal/adult disparity in T lymphopoi-etic potential in OP9-DL1 cocultures. A, E14 Fetalliver progenitors and adult bone marrow Lin�RAG-1/GFP�c-KitlowIL-7R�� cells were cultured with 5ng/ml IL-7 and 5 ng/Flt3-L on OP9-DL1 for 14days. Representative plots are shown for culturedcells stained for TCR� and TCR��. Similar resultswere obtained in experiments with eight other frac-tions of adult bone marrow. B, Percentages ofTCR�� and TCR��� cells present at the end of cul-tures are shown along with yields per input progen-itor. The values are means of duplicate cultures �SD in this typical experiment.

FIGURE 2. Delta 1-mediated Notch signals inhibit B and myeloid lin-eage differentiation from adult progenitors in short-term (9 days) cultures.A, Six subsets of adult progenitors were sorted according to expression ofRAG1/GFP, c-Kit, and Sca-1 after lineage depletion before culture onOP9-vector (top row) and OP9-DL1 (bottom row) for 9 days (5 ng/ml IL-7and 5 ng/ml Flt-L). Recovered cells were then stained for CD11c/Mac-1and CD19, and results from four of the major populations are illustratedhere. B, Yields of B lineage cells (left panel) and myeloid cells (rightpanel) per input progenitor were calculated for all six groups of cultures.�, Cells recovered from OP9-vector cultures; p, corresponds to OP9-DL1cultures. The values are means of duplicate cultures � SD in this repre-sentative of three similar experiments.

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myeloid (CD11b/Mac-1 and Gr-1), NK (DX-5 and NK1.1), den-dritic (CD11c), and B lymphocyte (CD45R/B220, BP-1 and

CD19) lineages (Fig. 3, B and C, and data not shown). Markersassociated with mature lymphoid cells (CD40, CD23, CD21,CD28, and B7) were also undetectable (data not shown). Otherhemopoietic markers that were detectable include CD43, Ly6C,Fas, and CD26, while the AA4.1 Ag associated with fetal stemcells and developing B lineage lymphocytes was absent.

Long-term cultured cells were CD45�Sca-1�c-Kit�CD43�CD44�/�CD25�CD5� and thus resembled thymo-cytes at the DN2/DN3 stage (Fig. 3C). Flow cytometry revealedlow to high levels of RAG-1/GFP and RAG-1 transcripts weredetected by RT-PCR (data not shown). TdT is a valuable markerof lymphopoiesis that is expressed from the ELP stage in bonemarrow and by thymocytes. Long-term cultured cells wereTdTlow/� (Fig. 3D). Components of the TCR complex were alsoevaluated. Small numbers of cells with cytoplasmic CD3� orTCR� were detectable by flow cytometry (Fig. 3D). RT-PCR re-vealed mRNA for pT� (data not shown). We conclude that whileonly prolymphocytes expand rapidly, many other discrete fractionsof adult bone marrow progenitors can be extensively propagated inresponse to Notch receptor-mediated signals and acquire indistin-guishable properties. Our subsequent experiments focused on ex-tensive characterization of these long-term cultured cells.

Continued, low-level T and NK lineage differentiation fromlong-term propagated lymphocytes arrested at the DN 2/3 stage

It has been reported that Notch can promote self-renewal of HSCs(21) and the above results show that bone marrow cells could beexpanded for months on the Delta 1 ligand. Thus, it seemed pos-sible that long-term propagated cells generated from the variousmarrow fractions were reprogrammed and assumed stem cell prop-erties. This was addressed with a more extensive RT-PCR andfunctional analysis of the cultured cells.

Semiquantitative RT-PCR analyses revealed that the GATA-3and PU.1 and E47 transcription factors were expressed, and theId-1 repressor of the bHLH family of transcription factors was justdetectable. Consistent with the growth selection conditions, thecells expressed Bcl-2, Notch 1, Hes-1, and IL-7R� transcripts.Levels of the IL-7R were beneath that detectable by flow cytom-etry, even though their survival required the continuous presenceof this cytokine. In contrast, the �c (CD132) cytokine receptorcomponent was easily seen by staining. Although the cells ex-pressed the IL-2R�-chain (CD25), the IL-2R� (CD122) compo-nent was absent (data not shown).

A recent study identified genes whose expression changessharply as cells progress through early differentiation stages in thethymus (22). Therefore, we evaluated four of these by RT-PCRanalysis, comparing long-term cultured lymphocytes to freshlysorted CD4�CD8�CD3�CD19�CD11c�DX-5� thymocytes(Fig. 4). This revealed that cells propagated in Notch-dependentcultures resembled CD44�CD25� (DN2) and CD44�CD25�

(DN3) cells within a normal thymus. Additional PCR analyseswere performed to assess the status of receptor gene rearrange-ments. As expected, evidence was obtained for rearrangement ofall of four TCR gene segments (V�3, V�6, V�8, and V�17) thatwere evaluated (data not shown). Although long-term cultures al-ways contained very small percentages (�3%) of surface TCR� orNK1.1� lymphocytes, it seemed possible that these were self-re-newing. Therefore, we rigorously depleted these differentiatedcells by cell sorting and returned the remaining cells to culture.Although the majority population remained immature Sca-1�c-Kit� cells 1 wk later, T and NK lineage cells were again detectable(Fig. 5A).

Ly-5.2 marked long-term cultured lymphocytes (1 � 105 cells)were then transferred with an equal number of Ly-5.1 competitor

FIGURE 3. A range of adult bone marrow progenitors representing var-ious stages of differentiation expand under the influence of Delta 1. A, Sixfractions of bone marrow progenitors were sorted on the basis of RAG-1/GFP expression and cultured for 5 wk on stromal cells plus 5 ng/ml IL-7 and5 ng/ml FL. The yields per input were calculated for each weekly expansion.B, Light scatter properties of cultured cells are compared with freshly isolatedlymphocytes (left panel). May-Grunwald-Giemsa staining of a cytocentrifugepreparation is also shown (right panel). C, Representative flow cytometryresults are shown for Lin�c-KitlowIL-7R�� marrow cells expanded on OP9-DL1 for 4 wk and then stained for progenitor and lineage-specific markers.Identical results were obtained when eight other fractions of bone marrowwere cultured in this way. D, Expanded cells were first stained for surfacelineage markers (VCAM-1 for stromal cells, DX-5 for NK cells, CD3� for Tcells, and Mac-1 for myeloid cells), along with CD25 and CD44. They werethen permeabilized and stained for cytoplasmic T lineage stage-specific pro-teins. Quadrants were set based on isotype-matched controls (ISO) andpositive controls using bone marrow and thymocytes (data not shown).Cultured cells were gated for VCAM-1�DX-5�CD3��Mac-1� cells, isotypecontrol stainings for cytoplasmic (cyt) TdT (rabbit IgG and FITC goatanti-rabbit IgG), cyt CD3� (FITC IgG1), and cyt TCR� (PE IgG2) were alsoprepared with cultured cells and are shown in the upper panel. CytoplasmicTdT, CD3�, and TCR� are shown in the lower panel.

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bone marrow cells to irradiated (9.6 Gy) Ly-5.1-recipient mice toassess their differentiation potential (Fig. 5B). In two experimentsof this kind, a total of 3.9(�0.4) � 105 donor-type cells was re-covered from the thymus 21–28 days later, where they expressedRAG-1/GFP and had progressed through CD4�CD8� toCD4�CD8� thymocyte stages. However, cultured cells competedpoorly with Ly5.1 bone marrow cells, and no donor-type cellswere detected in either the spleen or bone marrow. The fact thatthere was no evidence of malignant transformation in transplantedanimals suggests that long-term cultured lymphocytes retainednormal properties.

Adult lymphoid progenitors have unique requirements for Tlineage differentiation in culture

Recent studies have demonstrated remarkable differences in cyto-kine requirements for fetal and adult lymphopoiesis (23–25). Inparticular, IL-7 is only essential for formation of T and B cellsbeyond the early neonatal period (26). Therefore, we consideredthat T cell progenitors within adult bone marrow might requirespecial conditions. Therefore, cultures were initiated with prolym-phocytes and maintained for the first 2 wk in the presence of 5ng/ml IL-7. They were then subcultured weekly with a range ofIL-7 concentrations for an additional 2 wk (Fig. 6A). This revealedthat low doses of IL-7 dramatically increased percentages ofCD4�CD8� thymocyte (DP) T lineage cells. However, total yieldsof TCR��� cells were much less, and slightly fewer TCR�� cellswere produced when IL-7 was low during the last 2 wk (data notshown).

Four subsets of adult marrow were then isolated and placed onOP9-DL1 stromal cells with 1 ng/ml IL-7. They were passaged tofresh OP9-DL1 cells just once after 8 days. The Lin�Sca-1lowc-Kitlow IL-7R�� (CLP) fraction generated lymphocytes on day 18,and they emerged in other cultures somewhat later. Impressivepercentages of CD4�CD8� DP cells, and even CD4� cells werepresent in all of the cultures when they were evaluated after a totalof 24 days (Fig. 6B). Similar results were obtained in parallel cul-tures that were held with 0.2 ng/ml IL-7. Total yields of DP Tlineage cells were highest in cultures initiated with CLP and whenthe higher concentration of IL-7 was used (Fig. 6C).

The frequency of subculture is another variable that might in-fluence T lineage differentiation, and we sought conditions thatmight favor T cell production from stem cells. Therefore,

Lin�Sca-1�c-KithighThy-1/CD90.1low HSC were isolated fromadult bone marrow and placed in OP9-DL1 cocultures for 8 dayswith 1 ng/ml IL-7. A subset of those cultures was passaged andheld for an additional 19 days (Fig. 6D). An additional group wassubcultured a second time after 16 days, and all were evaluated atthe same time. Although absolute yields of DP cells were not dra-matically changed by the additional passage, numbers of DN cellssubstantially increased.

Unlike fetal progenitors, which produced large numbers of DPcells in 2 wk, adult progenitors required more time, and somesubculture was required to maintain optimal yields. Although adultprogenitors progressed to the DN2/3 stage and proliferated indef-initely in high IL-7 concentrations, further differentiation was fa-vored by much lower concentrations of this cytokine and a mini-mal number of passages. In addition, long-term Notch-dependentcell cultures could be easily established from adult but not fetalcells.

Long-term OP9-DL1 cocultures expand marrow progenitorsbefore VHDHJH rearrangement

There are at least two possible explanations why long-term cul-tures initiated with a wide variety of bone marrow cells are indis-tinguishable. Rare cells with uniform properties might have beenselected for expansion, or alternatively, progenitors dedicated toalternate fates could have been redirected toward the T lineage.Therefore, we prepared cultures with three types of relatively dif-ferentiated CD45R/B220� marrow cells. We know from otherstudies that several types of stromal cells are suppressive for com-mitted B lineage progenitors (T. Yokota et al., submitted for pub-lication) and that was the case in these experiments. That is, incontrast to prolymphocytes, the three CD45R/B220� fractionsgenerated very small numbers of cells in 1-wk OP9-DL1 cocul-tures (data not shown). It is also important to note that even when

FIGURE 4. Expanded cells have properties similar to DN2/DN3 stagethymocytes. Lin�Sca-1�c-Kit� cells were prepared by sorting cells recoveredfrom 28 day cultures of Lin�c-KitlowIL-7R�� bone marrow progenitors. Thiseliminated the small numbers of stromal, T, and NK cells present at that time.Highly enriched lymphocytes from the long-term cultures were then comparedwith freshly sorted CD3��CD4�CD8�NK1.1�Mac-1�CD19� adult thymo-cytes that were further resolved into DN subsets according to expressionpatterns of CD44 and CD25. RT-PCR analyses were performed to detecttranscripts that vary in a stage-dependent fashion (22). The plots depict valuesobtained with freshly isolated thymocytes, and the stars show results obtainedwith cultured cells.

FIGURE 5. Expanded cells retain the potential to become T and NKcells. A, Long-term (28 days) cultured lymphocytes derived from theLin�c-KitlowIL-7R�� subset of marrow were purified by sorting forLin�c-Kit�Sca-1� cells. They were then recultured on OP9-DL1 for an-other week before staining for NK1.1, Mac-1, TCR�, TCR��, c-Kit, andSca-1 Ags. B, A total of 105 sorted c-Kit�Sca-1� cells was mixed with thesame amount of host type, whole bone marrow cells and i.v. injected intolethally irradiated Ly5.1 mice. Twenty-eight days after transplantation, thy-mocytes from nontransplanted host and transplanted animals were col-lected and assayed for thymus reconstitution. The left panels show gatingfor donor Ly5.2� lymphocytes, while the middle and right panels illustratetheir properties.

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cultures were initiated with CD19� cells, numbers of CD19� cellspresent in OP9-DL1 cultures 1 wk later were only a fraction ofthose present in OP9 stromal cell cocultures. However, these couldbe expanded with three subsequent passages, and sufficient cellswere harvested at 4 wk for analysis. At that time, no CD19� cellswere present, and the lymphocytes resembled DN2/DN3 stage thy-mocytes as described above. That is, the cultures contained 3–8%TCR���, 2–15% NK�, and 75–90% CD44�/�CD25� cells. Wereasoned that CD45R/B220�CD19�CD43�IgM� and CD45R/B220�CD19�CD43�IgM� fractions would be highly enrichedfor B lineage cells, and genomic PCR analyses confirmed that atleast some in the starting populations had undergone VH to DHJH

recombination (Fig. 7). However, DHJH, but not VH-DHJH rear-rangement, products were found in long-term cultured lympho-cytes. We conclude that long-term OP9-DL1 cocultures can sup-press expansion of cells that would normally become B cells and

provide a very powerful selective pressure to cells not fully com-mitted to the B lymphocyte lineage.

DiscussionThis study demonstrates that numerous types of progenitors de-rived from adult marrow can be used to establish long-term Tlineage cell cultures on DL1-transduced stromal cells However,while fetal cells progressed efficiently to CD4�CD8� and evensingle-positive T lineage cells, progenitors from adult bone mar-row expanded continuously but failed to progress past a stageequivalent to DN2/DN3 thymocytes in the presence of 5 ng/mlIL-7. Reduced cytokine concentrations and a minimum of subcul-tures supported the production of TCR�� lymphocytes from adultprogenitors. This information extends previous indications that fe-tal and adult progenitors differ in differentiation requirements. Aprolymphocyte fraction, including cells designated CLP, grewmost efficiently during the first week on OP9-DL1, but establishedcultures initiated with a wide range of marrow subsets were indis-tinguishable. This resulted at least in part from the selective ex-pansion of rare progenitors that were not fully committed to the Blymphocyte lineage.

Adult marrow progenitors were dramatically different from theirfetal counterparts in several respects. Two Lin�c-Kitlow RAG-1/GFP�IL-7R��/� prolymphocyte fractions of bone marrow ex-panded as efficiently as fetal liver in 1-wk OP9-DL1 cocultures(Fig. 3A and data not shown). However, the yield of TCR� lym-phocytes 1 wk later was at least 1000-fold less than that obtainedfrom cultures initiated at the same time from fetal liver progenitors(Fig. 1). Although cocultures initiated with fetal cells usuallylasted no longer than 4 wk, we found that adult marrow progenitorscould be serially transferred to fresh stromal cells an indefinitenumber of times. These adult-derived cells retained dependence onIL-7 and Notch receptor ligation while acquiring homogeneouscharacteristics. Indeed, cultures initiated with six distinctly differ-ent subsets of bone marrow were remarkably similar by the secondpassage and subsequently expanded at approximately the samerate. Other fetal/adult differences in T lymphopoiesis have been

FIGURE 6. T lineage differentiation from marrow progenitors is influ-enced by IL-7 concentration and subculture. A, Lin�c-KitlowIL-7Ra�

prolymphocytes were cultured first on OP9-DL1 with 5 ng/ml IL-7 and 5ng/ml Flt3-L for 2 wk. They were then subcultured to fresh stromal cellswith the indicated concentrations of IL-7 plus Flt3-L and subcultured again1 wk later. After a total of 4 wk of culture, cells were harvested and stainedfor flow cytometry analysis. B, HSC, LSP, ELP, and CLP fractions werecultured on OP9-DL1 with 1 ng/ml IL-7 and 5 ng/ml Flt3-L for 8 days.They were then subcultured under the same conditions for another 16 daysbefore harvest and staining for CD4 and CD8. C, Total yields ofCD4�CD8� (DP) cells were calculated per input HSC, LSP, ELP, or CLPin cultures that were maintained for the whole time on OP9-DL1 withFlt3-L and either 1 ng/ml IL-7 or 0.2 ng/ml IL-7. After the first 8 days, allgroups were subcultured with the original conditions for another 16 daysbefore harvest and analysis. The absolute numbers of DP cells per sortedprogenitor were calculated by multiplying yields of the first culture intervalwith that of the second interval and the frequency of DP cells. D, HSCswere cultured on OP9-DL1 with Flt3-L and 1 ng/ml IL-7 for 8 days andthen subcultured in two groups. One group (shown by �) was harvestedand stained on day 27. The parallel group (f) was maintained in the sameway but passaged one additional time on day 24. Numbers of total cells,CD4�CD8� (DN) and DP cells are shown.

FIGURE 7. Long-term OP9-DL1 cocultures selectively expand bonemarrow cells that lack Ig VH-DHJH rearrangements. B220�IgM� B pro-genitors from adult bone marrow were sorted into CD43�CD19�,CD43�CD19�, and CD43�CD19� fractions before culture for 4 wk.CD19�c-Kit�Sca-1� cells were then sorted to exclude differentiated andstromal cells. Genomic DNA was extracted, and DH-JH (upper panel) andVH-DHJH Ig gene rearrangement were tested and compared with thoseprepared from initiating progenitors. Germline and rearrangement productsare labeled, while two artifact bands are designated with asterisks.

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reported previously (27, 28). In addition, a very recent study in-dicates that IL-7 blocks adult but not fetal thymocyte differentia-tion (29, 30).

The density and complexity of Notch ligands, APC, and cyto-kines found in the normal thymus is unlikely to be fully replicatedin this coculture model. Despite that, progression of T lymphopoi-esis and production of TCR� cells from fetal progenitors are sur-prisingly robust. All three of our labs routinely obtain lower cellyields and slower expansion from adult marrow progenitors, butonly one routinely observes efficient T cell production from thatsource (6). We show here that low cytokine concentrations and aminimal number of passages favor lineage progression from adultcells. Subsequent analysis may reveal important technical vari-ables such as serum batches, cytokine source, passage number ofstromal cells, and mouse strains may represent other importanttechnical variables.

There is growing evidence that many discrete cell types in bonemarrow have the potential to become T lymphocytes, and it isdifficult to discern which ones normally replenish the thymus (31,32). The most primitive Lin�Sca-1�c-Kithigh fraction of bonemarrow contains stem cells, LSPs and ELPs, with the potential forsustained T cell production (20, 33). Moreover, cells with thesegeneral properties are detectable within the bloodstream and re-semble canonical prothymocytes in some respects (7, 34). On theother hand, cells with at least short-term T lymphopoietic potentialcan be found within Lin�c-Kitlow and even CD45R/B220� frac-tions, and it is possible they contribute under some circumstances(18, 20, 35). Although direct comparisons under physiologicalconditions could be informative, it seems likely that the T vs B fatedecision is not as abrupt as generally believed. There are likely tobe two factors that contribute to this misconception. One is thatmultiple types of progenitors can be diverted into the T cell lineageunder the conditions found in OP9-DL1 cocultures (present dataand Ref. 7), although this is not recapitulated under normal phys-iologic conditions (7). The other is that Notch signaling can, tosome extent, redirect the lineage fate of cells that would normallynot become T cells.

In any event, the Lin�c-KitlowRAG-1/GFP�IL-7R��/� cate-gory of bone marrow gave the most impressive short-term expan-sion in OP9-DL1 cocultures. Although this includes cells origi-nally described as CLPs, there are reasons to believe they are notthe most efficient for replenishing the adult thymus (20, 32). It isalso important to note that at least two stromal cell lines can ac-tually suppress the activity of fully committed B lineage progen-itors (16). We do not understand the basis for this inhibition butcould speculate that there is constitutive low level expression ofNotch ligands. Any functional assessment of differentiation poten-tial is subject to bias, and caution is advised when interpretingculture results in terms of in vivo relevance.

Long-term propagated adult lymphocytes closely resembledprimitive DN2/DN3 stage thymocytes with respect to expressionof four genes (22). Although largely arrested at that stage, thecultures continued to contain small numbers of cytoplasmicTCR�� cells and slowly generated NK and T cells. In addition,they successfully competed with normal bone marrow and pro-gressed to CD4� and CD8� lymphocytes in the thymuses of ir-radiated recipients. Furthermore, we found evidence of polyclonalrearrangement of four TCR� genes (data not shown). It is curiousthat fetal progenitors, such as those freshly isolated from the thy-mus (5, 7), efficiently complete maturation in the OP9-DL1 culturesystem, while adult bone marrow progenitors transiently arrest atthe DN2/DN3 stage when 5 ng/ml IL-7 is added. When the purifiedLin�c-Kit�Sca-1� cells from long-term cultures were trans-planted with equal number of total recipient bone marrow cells,

only 0.3% of thymic cells were derived from the cultured cells.This indicates that thymic homing and/or T cell differentiation ofbone marrow progenitors cultured on OP9-DL1 may decrease overtime. It has been shown previously that T lineage progression fromfetal progenitors in a fetal thymic organ culture system is inhibitedby high cytokine concentrations (36). However, in the OP9-DL1coculture system, this is only the case for adult progenitors (thisreport and Ref. 30). These findings again highlight fetal/adult dif-ferences and indicate that the cultures do not optimally provide allenvironmental cues required by adult marrow-derived progenitors.Further study might implicate other Notch ligands (37) or addi-tional components of the normal adult thymus.

The bone marrow, rather than the thymus, is likely tobe the source of NK cells during adult life (38). Indeed, theLin�c-KitlowFlk-2� prolymphocyte fraction of marrow is the bestsource of NK progenitors, and before acquisition of the CD122receptor for IL-15, many of them still retain B lineage potential(39, 40). We did not intentionally promote development of NKcells by addition of IL-15 in the present study. However, smallnumbers of DX-5�NK1.1� NK lineage cells were produced in allof our OP9-DL1 cultures, regardless of whether they were initiatedwith fetal liver or adult bone marrow. These emerged during thefirst week on OP9-vector control stromal cells and from the secondweek in OP9-DL1 cocultures (data not shown). Other than thisapparent difference in kinetics, we saw no strong evidence that theNK lineage choice was either positively or negatively influencedby Notch receptor ligation. A recent analysis of fetal thymocytesdemonstrated that residual NK lineage potential is normally down-regulated at the DN2 stage and suppressed by Notch receptor li-gation (8).

Notch is thought to influence a variety of events in hematopoi-esis, including the self-renewal of stem cells (12, 13, 21, 41). How-ever, recombinant forms of Delta 1 have been shown to suppressmyelopoiesis (42, 43). We consistently observed a reduction inproduction of myeloid cells in OP9-DL1 cocultures, regardless ofwhether fetal or adult hemopoietic cells were used. It is unclearwhether strong Notch signals killed myeloid progenitors, heldthem in an undifferentiated, cytokine unresponsive state, or di-rected them to alternative fates.

It is critical to know if long-term cultured marrow cells weresimilar because of reprogramming, or alternatively, if rare cellscontaminating all of the sorted fractions were selected over time.This is a difficult determination when the starting populations to becompared are all very primitive. Therefore, we started some cul-tures with highly enriched (98% by postsort analysis) and rela-tively late CD45R/B220�CD19�CD43�sIgM� (Hardy FractionE) cells. Although there is almost no proliferative activity in thestarting population (44) and we saw no expansion during the firstweek of coculture, long-term lines were established within 3 wk ofserial passage. Remarkably, there was no evidence for Ig VH toDHJH rearrangements and no cytoplasmic � H chain staining in theexpanded lymphocytes, even though small pre-B cells made upalmost all of the starting population. We interpret these results asshowing that the OP9-DL1 coculture system has tremendous po-tential for selection and expansion of rare, undifferentiated lym-phocytes that might represent contaminants in sorted populations.Although cells can progress substantially in the B lineage and remainNotch ligation sensitive, late-stage progenitors with productivelyrearranged Ig genes are not reprogrammed to adopt a T cell fate. Wedemonstrate that an impressive range of different progenitors in adultmarrow could be propagated indefinitely in OP9-DL1 cocultures.Regardless of starting population, the cells assumed properties ofDN2/DN3 stage thymocytes and retained some potential for terminal

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T lineage differentiation. Although a degree of lineage reprogram-ming may occur in this culture model, it can preferentially expandrare, relatively undifferentiated cells. The findings are compatible witha model where Notch regulates multiple events and differentiationoptions become gradually restricted (45).

AcknowledgmentsWe thank Rob Welner and Scott Perry for help in sorting some of the adultmarrow cells. We also thank Shelli Wasson for assistance with preparingthe manuscript.

DisclosuresThe authors have no financial conflict of interest.

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