July 10, 2003 originally published onlinedoi:10.1182/blood-2003-04-1249

2003 102: 3120-3128  

Kim Klarmann, Mariaestela Ortiz, Meghan Davies and Jonathan R. Keller hematopoietic stem cells

negative−Identification of in vitro growth conditions for c-Kit 

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HEMATOPOIESIS

Identification of in vitro growth conditions for c-Kit–negativehematopoietic stem cellsKim Klarmann, Mariaestela Ortiz, Meghan Davies, and Jonathan R. Keller

Our laboratory recently identified a quies-cent class of pluripotent hematopoieticstem cells (PHSCs) that are lineage nega-tive (Linneg), lack c-Kit, and are able togive rise to c-Kit–positive (c-Kitpos) PHSCsin vivo. This population fails to proliferatein vitro but has delayed reconstitutingactivity in vivo. In this study, we purifiedthese cells to enrich for the PHSCs andwe identified in vitro conditions capableof supporting their maturation. The c-Kit–negative (c-Kitneg) cells exhibited differen-

tial expression of Sca-1, CD34, CD43,CD45, and Thy 1.2. We purified the cellsbased on Sca-1, as it is expressed onactive PHSCs. We detected pre–colony-forming unit spleen (pre–CFU-s) activityin both the Sca-1neg and Sca-1pos popula-tions, indicating the presence of primitivePHSCs in both populations. However, ourin vitro studies suggest that the Sca-1pos

population is enriched for PHSCs. The invitro systems that support the growth ofthese dormant cells include a modified

long-term marrow culture and variousstromal cell lines. In modified long-termbone marrow cultures, c-Kitneg cells gaverise to c-Kitpos PHSCs, with long-termreconstitution activity in vivo. Thus wehave established an in vitro system toexamine PHSC maturation that will allowus to study the mediators of the c-Kitneg toc-Kitpos transition. (Blood. 2003;102:3120-3128)

© 2003 by The American Society of Hematology

Introduction

Pluripotent hematopoietic stem cells (PHSCs) are rare cells thathave the capacity to self-renew and give rise to all lineages ofhematopoietic cells. The current cell surface phenotype of thePHSCs is considered to be c-KitposSca-1posLinneg-low.1-3 Whentransplanted, these cells provide both short-term (30-90 days) andlong-term (8-10 months) reconstitution of all hematopoietic lin-eages. In addition, they give rise to colonies on the spleens oflethally irradiated mice within 12 days of transplantation (colony-forming unit spleen [CFU-s]). They also form colonies in soft agar(CFU-culture [CFU-c]) in response to combinations of hematopoi-etic growth factors (HGFs), including stem cell factor (SCF) andinterleukin-3 (IL-3).4 Induction of c-Kit expression is essential forproper hematopoietic development, as illustrated by knock-outanimal systems in which deletion of c-Kit, or its ligand, SCF,results in prenatal lethality due to the disruption of hematopoi-esis.5,6 Similarly, antibodies that prevent c-Kit from interactingwith SCF block hematopoietic development both in vitro and invivo.7-9 However, while c-Kit expression is known to be crucial forhematopoiesis, the conditions that support the development of thec-Kit–negative (c-Kitneg) cell to a c-Kit–positive (c-Kitpos) cellremain poorly understood.

Previously, our laboratory identified and characterized a novelc-Kitneg PHSC population that does not have short-term reconstitu-tion activity, does not form CFU-c’s or CFU-s’s, yet gives rise toc-Kitpos PHSCs when transplanted into lethally irradiated recipientsafter 8 to 10 months.10 This delayed reconstitution suggestedc-Kitpos cells develop over time. These dormant c-Kitneg cells are

purified from mouse bone marrow by counter current elutriation toobtain blast-sized cells (counter current elutriation at 25 mL/min � CCE-25). Elutriation is followed by fluorescence-activatedcell sorting (FACS) to remove lineage-committed (B, T, anderythroid) and c-Kitpos cells. CCE-25–c-Kitneg cells also lackantigens expressed on mature myeloid cells.10 Additionally, unlikec-Kitpos cells, CCE-25–c-Kitneg cells are unique in that they do notform colonies in soft agar in the presence of multiple growthfactors and do not radioprotect lethally irradiated mice or give riseto CFU-s’s. Identification of the PHSC c-Kitlow cells by ourlaboratory and others provides further support for the model ofc-Kitneg to c-Kitpos PHSC development.11,12 Although the c-Kitneg

cell is thought to be the ancestor of the c-Kitpos PHSC, it has beentechnically difficult to study the transition of this cell from thec-Kitneg state to the c-Kitpos state, because of its delayed engraft-ment kinetics (8-10 months) and its apparent inability to growin vitro.

To gain an understanding of the mechanisms controlling thegrowth of c-Kitneg cells, we had 2 goals. First, we needed to furtherpurify the PHSC activity in the CCE-25–c-Kitneg cell population.Previously, we discovered the CCE-25–c-Kitneg population washeterogeneous. In this study we increased the stringency of theCCE-25–c-Kitneg isolation and then examined this population forthe expression of other PHSC surface antigens, including Sca-1,CD34, CD43, and Thy 1.2.10 Second, we identified in vitroconditions that would support the growth of these cells. To do this,we used a recently developed, modified long-term marrow culture

From the Laboratory of Molecular Immunoregulation, Basic Research Program,Science Applications International Corporation-Frederick, Center for CancerResearch, National Cancer Institute at Frederick; Frederick, MD.

Submitted April 22, 2003; accepted June 23, 2003. Prepublished online asBlood First Edition Paper, July 10, 2003; DOI 10.1182/blood-2003-04-1249.

Supported in whole or in part with federal funds from the National Cancer Institute(NCI), National Institutes of Health, under contract number NO1-CO-12400.

The publisher or recipient acknowledges the right of the US government to retain anonexclusive, royalty-free license in and to any copyright covering this article.

Reprints: Kim D. Klarmann, Laboratory of Molecular Immunoregulation,Bldg 560, Rm 12-03, NCI-Frederick, Frederick, MD 21702; e-mail:kklarmann@ncifcrf.gov; or Jonathan R. Keller, Hematopoiesis and GeneTherapy Section, SAIC-IRSP, Bldg 560, Rm 12-03, SAIC-Frederick, Frederick,MD 21702; e-mail: kellerj@mail.ncifcrf.gov.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.

© 2003 by The American Society of Hematology

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system, as well as multiple stromal cell lines, both known tosupport PHSC growth, for their ability to support the growth anddifferentiation of c-Kitneg cells in vitro. Here we show the firstevidence for in vitro growth of these dormant CCE-25–c-Kitneg

PHSCs. These in vitro conditions can be used in future studies toidentify the factors controlling the proliferation and maturation ofthe c-Kitneg PHSCs.

Materials and methods

Mice

C57Bl/6 (Ly-5.1) and congenic C57Bl/6 (Ly-5.2) mice 6 to 12 weeks of agewere obtained from the animal production area at NCI-Frederick (Freder-ick, MD). Animal care was provided in accordance with the proceduresoutlined in the “Guide for Care and Use of Laboratory Animals” (NationalInstitutes of Health, Bethesda, MD, 1996).

CCE-25–c-Kitneg purification

Bone marrow was flushed from femurs of C57Bl/6 mice with Iscovesmodified Dulbecco medium (IMDM; GibcoBRL, Grand Island, NY)supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), andpenicillin/streptomycin (pen/strep; GibcoBRL). Cells were filtered througha 40-�m nylon mesh (BD Falcon, Bedford, MA). To obtain blast-sizedcells, the bone marrow cells (BMCs) were elutriated using a Beckman J-6Mcentrifuge with a JE-6B elutriator rotor and a standard chamber (BeckmanCoulter, Hialeah, FL). Bone marrow cells were loaded into the chamber at aflow rate of 15 mL/min, a centrifuge speed of 3000 rpm, and a maximumcell density of 1 � 109 cells per elutriation. The flow rate was adjusted to 25mL/min and 300 mL was collected (CCE-25). Red cells were lysed usingACK buffer (40 seconds at 4°C). The cells were allowed to recover inmedium for 10 minutes, then washed and resuspended in phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA), followed bytreatment with FcR (2.4 G2)–blocking antibodies for 10 minutes at 4°C.The cells were stained for lineage markers (lineage antigens: B220[RA3-6B2], CD3� [500A2], TER-119, and Gr-1 [RB6-8C5]), c-Kit (2B8),and other stem cell and progenitor markers including CD34 (RAM34),CD38 (90), CD43 (S7), CD45 (104), and Thy 1.2 (30 H-12). All antibodieswere from Pharmingen (San Diego, CA) and were added at 1 �g/106 cellswith the cells at a density of 107/mL. Cell-antibody mixtures were kept onice for 20 minutes followed by 2 washes with PBS/0.1% BSA. The cellswere sorted using a MoFlo high-speed cell sorter (Dako Cytomation, FortCollins, CO). Cells were gated to exclude large, granular, and lineage-positive (Linpos) cells. Viability after sorting was determined by trypanblue exclusion.

Pre–CFU-s assay

Animals were pretreated for 7 days with antibiotics prior to lethalirradiation (9.5 Gy). Purified cells were injected via tail vein. At 12 daysafter transplantation, the animals were killed. Their bone marrow washarvested and transplanted into secondary, lethally irradiated recipients at1 � 106 to 5 � 105 cells per recipient. Spleens of both the primary andsecondary recipients were harvested and placed in Tellesniczky solution(375 mL 70% ethanol, 18.75 mL glacial acetic acid, 37 mL formalin) tovisualize colonies on the spleens of the recipients (CFU-s).

RAG-2�/� transplant assay

RAG-2�/� animals were obtained from Jackson Laboratory and weremaintained in pathogen-free conditions. Animals were treated with acidwater and antibiotics for 7 days prior to transplantation with CCE-25–c-Kitneg purified cells. On the day of transplantation, the animals weresublethally irradiated with 4.0 Gy. Peripheral blood was assayed for thepresence of donor B220� and CD3�� cells at weeks 2, 6, and 14 aftertransplantation. At 14 weeks the animals were killed and the thymuses and

spleens were analyzed for the presence of donor CD3�� or B220� cells byflow cytometry.

CFU-c

Purified CCE-25–c-Kitneg cells were plated in 35-mm tissue culture dishesin IMDM with 20% horse serum (HS), pen/strep, 0.3% Seaplaque Agarose,and the following cytokines: human erythropoietin (hEPO, 20 ng/mL),human Flt-3 (huFlt-3) ligand (200 ng/mL), human granulocyte colony-stimulating factor (hG-CSF, 50 ng/mL), human interleukin-1� (hIL-1�, 20ng/mL), hIL-6 (50 ng/mL), hIL-11 (50 ng/mL), human leukemia inhibitoryfactor (hLIF, 50 ng/mL), human macrophage colony-stimulating factor(hM-CSF, 300 ng/mL), murine granulocyte-macrophage colony-stimulat-ing factor (mGM-CSF, 20 ng/mL), mIL-3 (30 ng/mL), mSCF (100 ng/mL),murine stromal cell–derived factor 1� (mSDF-1�, 100 ng/mL), and murinethrombopoietin (mTpo, 20 ng/mL) (Peprotech, Rocky Hill, NJ). Cultureswere kept at 37°C, 5% CO2. Colonies were counted after 12 days.

Liquid culture with HGFs, anti-TGF�, or immobilized DeltaExtIgG

Purified CCE-25–c-Kitpos, CCE-25–c-KitnegSca-1pos, or CCE-25–c-KitnegSca-1neg purified cells were cultured in IMDM with 20% horse serum, andpen/strep, supplemented with 100 ng/mL mSCF and 30 ng/mL mIL-3, incombination with murine vascular endothelial growth factor (mVEGF, 100ng/mL), murine basic fibroblast growth factor (mbFGF, 10 ng/mL), andmurine endothelial growth factor (mEGF, 100 ng/mL). Alternatively,anti–transforming growth factor � (TGF�), (clone 1D11), or an isotype-matched control, was added at a maximum of 26 �g/mL and a minimum of0.9 �g/mL (1:3 serial dilutions) in the presence of SCF and IL-3. To activatethe Notch signaling pathway, we coated plates with 10 �g/mL of eitherDeltaExtIgG or human immunoglobulin G (IgG; as a negative control)overnight at 4°C; the plates were then washed 5 times with PBS andblocked with culture medium (IMDM, 20% HS) in the presence of 100ng/mL mSCF, 100 ng/mL hTpo, 50 ng/mL IL-6, and 100 ng/mL Flt-3ligand. Cells were incubated at 37°C, 5% CO2 for 15 days and weremonitored for growth throughout this time.

Tpo-LTMCs

Tpo–long-term bone marrow cultures (LTMCs) were prepared as describedby Yagi et al.13 Briefly, bone marrow from C57Bl/6 animals was flushedwith Fischer medium supplemented with 20% horse serum (Hyclone lot no.AHA7635), 1 �M hydrocortisone (Sigma, St Louis, MO), and pen/strep(GibcoBRL). Bone marrow cells were cultured in T-25 flasks at a density of1 femur per flask. Cultures were supplemented with 10 ng/mL murine Tpo(R&D Systems, Minneapolis, MN). Cultures were maintained by biweeklydemirepopulation.

Stromal cell lines

Cells (14F1.1) were cultured in Dulbecco modified Eagle medium (DMEM),10% fetal calf serum (FCS). M2-10B4 cells were cultured in RPMI-1640,5% FCS, with 500 �g/mL G418. D4T cells were cultured in IMDM with20% FCS, supplemented with 100 �g/mL endothelial cell growth supple-ment (ECGS; Collaborative Biomedical Products, Bedford, MA). OMA-ADcells were cultured in minimum essential medium � (MEM�), 12% HS,12% FCS, 10 �M �-ME. OP9 cells were cultured in MEM�, 20% FCS, 50�M beta-mercaptoethanol (�-ME), supplemented with nonessential aminoacids. S17 cells were cultured in RPMI-1640 supplemented with 10% FCS,25 �M �-ME. All media contained pen/strep. The CCE-25–c-Kitneg–purified cell populations (Sca-1pos or Sca-1neg) were seeded onto 70%confluent stromal cell line monolayers and thereafter maintained in IMDM,20% horse serum, and pen/strep supplemented with 100 ng/mL murine SCFand 30 ng/mL murine IL-3 (Peprotech). Transwell experiments wereperformed in either 12- or 6-well tissue culture plates. (0.4-�m pores;Corning, Acton, MA). Cytospins were stained with PROTOCOL Hema3stain set (Biochemical Sciences, Swedesboro, NJ).

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Results

PHSC antigen expression by CCE-25–c-Kitneg cells

Our previous experiments10 demonstrated that CCE-25–c-Kitneg

cells were heterogeneous with respect to the expression of othercell surface antigens. Therefore, we assayed the cells for theexpression of antigens known to be expressed on PHSCs includingSca-1, CD34, CD38, CD43, CD45, and Thy 1.2. To purify theCCE-25–c-Kitneg cell population, we isolated mouse bone marrow(Figure 1A) and obtained small, blast-sized cells by counter currentelutriation (CCE) at 25 mL/min (CCE-25) (Figure 1B). We stainedthe CCE-25 cells with antibodies that recognize the Lin-specificmarkers B220, CD3�, Gr-1, TER119, as well as c-Kit (Figure 1C).We gated the population for blast-sized cells and sorted forLinnegc-Kitneg (CCE-25–c-Kitneg) cells, which we found comprise1% of the CCE-25 population. These cells were analyzed for theexpression of other progenitor markers (Figure 1C).

As shown in Figure 1, we observed that 19% of the cellsexpressed Sca-1 (Ly6-A/E) (Figure 1D), 10% were CD34pos

(Figure 1E), and 22% were CD38pos cells (Figure 1F). The lownumber of CD34pos cells was anticipated, as c-KitposSca-1pos

PHSCs also express low levels of CD34.14,15 CD43 (Ly-48,Leukosialin), which has been detected on PHSCs, has a role incell adhesion16 and was expressed by 38% of the CCE-25–c-Kitneg population (Figure 1G). In addition, a majority of theCCE-25–c-Kitneg population (62%) was CD45pos, as anticipated,being of hematopoietic origin (Figure 1H). Surprisingly, therewas a significant number of CD45neg cells as well (38%).Finally, Thy 1.2 (CD90), known to be present on PHSCs,17,18

was expressed on 19% of the c-Kitneg population (Figure 1I).We chose to purify the CCE-25–c-Kitneg cells based on Sca-1

expression, because of the essential role that Sca-1 has in hemato-poietic development.2,3,19 The CCE-25–c-KitnegSca-1pos and Sca-1neg populations were isolated by FACS and used in subsequentexperiments (Figure 1J-K). The purity of these sorted cells wasverified by postsort analysis (Figure 1L-M).

Pre–CFU-s activity of CCE-25–c-KitnegSca-1pos

and Sca-1neg purified cells

To determine whether the CCE-25–c-KitnegSca-1pos or Sca-1neg

population contained PHSCs, we used the pre–CFU-s, an in vivoassay that detects primitive progenitors.4,20 For the pre–CFU-sassay, the CCE-25–c-KitnegSca-1pos or Sca-1neg cells were trans-planted into lethally irradiated mice. After 12 days, the spleens

were removed and placed in Tellesniczky solution to determine thenumber of primary CFU-s’s. Bone marrow cells from these primaryrecipient animals were then transplanted into secondary recipients.Then 12 days later, the CFU-s of the secondary recipients wasdetermined. As expected, the unsorted CCE-25 cells had primaryCFU-s activity 12 days after transplantation, while the saline-injected controls and those animals that received CCE-25–c-Kitneg

cells (with or without Sca-1) did not (Figure 2; Table 1). In thesecondary recipients, the saline control, again, had no CFU-s’s,while the CCE-25 recipients had spleen colonies indicating thepresence of secondary pre–CFU-s, as anticipated. The averagepre–CFU-s activity for the CCE-25–c-KitnegSca-1pos populationswas 10.8 pre–CFU-s per 1 � 105 cells, while the average activityfor the Sca-1neg population was 8.6 pre–CFU-s per 1 � 105 cells.Therefore, both the CCE-25–c-KitnegSca-1pos cells and the Sca-1neg

cells had roughly equivalent pre–CFU-s activity in this assay,indicating the presence of stem cells in both populations.

CCE-25–c-Kitneg transplantation into RAG-2�/� mice

We also compared an alternative in vivo environment that sub-jected the CCE-25–c-Kitneg cells with a different selective pressurethan the pre–CFU-s assay. In particular, we examined the ability ofthe CCE-25–c-Kitneg cells to reconstitute the lymphoid deficiencyin RAG-2�/� (recombination activating gene–2 knock-out) mice.Based on our previous studies, we knew the CCE-25–c-Kitneg

population can give rise to lymphoid cells in vivo, 10 to 12 monthsafter transplantation.10 We hypothesized that the lack of maturelymphoid cells in these animals would create an environment that

Figure 1. Isolation and purification of the CCE-25–c-Kitneg cells. Representative forward and side scatter ofmouse bone marrow (A). Forward scatter (FSC) and sidescatter (SSC) profile of CCE-25–c-Kitneg cells isolated byelutriation according to the procedures outlined in “Mate-rials and methods” (B). FACS analysis of CCE-25 cellsincubated with lineage- and c-Kit–specific antibodies(Lin: B220, CD3�, TER-119, Gr-1) (C). The CCE-25–Linnegc-Kitneg cells were gated and examined for theexpression of Sca-1 (D), CD34 (E), CD38 (F), CD43 (G),CD45 (H), and Thy 1.2 (I). CCE-25–c-Kitneg cells (J) weresorted for Sca-1pos and Sca-1neg (K) populations and theirpurity was verified by postsort reanalysis (L-M). Isotype-matched control background staining was subtractedfrom the percentages shown. These results are represen-tative of 2 experiments.

Figure 2. Pre–CFU-s activity of the purified CCE-25–c-Kitneg cell populations.CCE-25–c-KitnegSca-1pos and Sca-1neg populations were isolated as described in“Materials and methods.” The purified cell populations were transplanted into lethallyirradiated mice. After 12 days, the animals were killed. Their spleens were fixed inTellesniczky solution to visualize spleen colonies (Primary CFU-s). Whole bonemarrow from these primary recipients was transplanted into lethally irradiatedsecondary recipients. After 12 days, spleens from secondary recipients were fixed inTellesniczky solution (Secondary CFU-s). These results are representative of 3experiments.

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would selectively pressure the CCE-25–c-Kitneg cells to developquickly. To test this, whole bone marrow cells (wBMCs), used as apositive control, or purified CCE-25–c-KitnegSca-1pos or Sca-1neg

cells were injected into sublethally irradiated (4.0 Gy) RAG-2�/�

mice. The peripheral blood of the mice was tested for the presenceof donor (Ly 5.2pos) T cells (CD3�pos) and B cells (B220pos)beginning 2 weeks after transplantation and subsequently moni-tored through 14 weeks. Animals that received whole bone marrowas a positive control showed both T- and B-cell reconstitution;however, no donor cells were detected in the peripheral blood ofanimals that received transplants of purified CCE-25–c-KitnegSca-1pos or Sca-1neg cells (data not shown). After 3.5 months, thymusesand spleens were harvested and analyzed for the presence of donorcells (Ly 5.2). Animals that received whole bone marrow hadthymic reconstitution by donor cells (23%) (Figure 3B), butanimals that received CCE-25–c-KitnegSca-1pos or Sca-1neg cells didnot show any thymic engraftment (Figure 3C and 3D, respec-tively). Analysis of the spleens gave the same result (data notshown). These data indicate that the selective pressure to correctthe lymphoid deficiency in these animals was insufficient to inducethe proliferation and maturation of the CCE-25–c-Kitneg cells.

CCE-25–c-Kitneg CFU-c’s with multiple HGFs

Although we and others have shown that CCE-25–c-Kitneg cellsdo not respond to multiple combinations of HGFs in vitro,10 weasked whether other HGFs not previously tested could promotethe growth and survival of FACS-purified CCE-25–c-KitnegSca-1pos or Sca-1neg cells. To examine the growth of the CCE-25–c-KitnegSca-1pos and Sca-1neg populations in vitro, we purified thecells as previously described, and then plated them in soft agar(CFU-c) with multiple growth factors, including the following:EPO, Flt-3 ligand, G-CSF, IL-1�, IL-6, IL-11, LIF, M-CSF,GM-CSF, IL-3, and SCF. CFU-c’s were counted 12 days afterplating. The novel growth factors we had not previously tested

were Tpo and SDF-1�. The data show that the CCE-25–c-KitnegSca-1pos cells give rise to an occasional, small colony,while the Sca-1neg cells do not grow in response to these factors(Table 2). This observation supports the following conclusions.First, that c-Kitpos cells did not contaminate the sorted c-KitnegSca-1neg or Sca-1pos populations, because they would have formedlarge, hyperproliferative colonies in the presence of SCF andIL-3 and multiple HGFs,10 as observed in the positive control.Second, the expanded HGF repertoire tested in these experi-ments was not sufficient to induce significant CFU-c’s by theCCE-25–c-Kitneg purified cells. We also found that EGF, bFGF,and VEGF in combination with SCF and IL-3 were insufficientto promote in vitro growth of the CCE-25–c-KitnegSca-1pos orSca-1neg cells in liquid cultures (data not shown).

Because TGF� is known to act as an autocrine inhibitor ofPHSC proliferation,21,22 we tested the ability of anti-TGF� antibod-ies to promote the growth of the CCE-25–c-KitnegSca-1pos andSca-1neg purified cells. To interrupt TGF� autocrine signaling andto determine if this interruption was sufficient to induce c-Kitexpression and subsequent proliferation of these cells, the purifiedCCE-25–c-KitnegSca-1pos or Sca-1neg cells were incubated withanti-TGF� (clone 1D11) antibodies along with SCF and IL-3 inliquid cultures for 15 days. However, while the CCE-25–c-Kitpos

cells, used as a positive control, grew well under these conditions,the CCE-25–c-KitnegSca-1pos and Sca-1neg cells did not (data notshown). Similarly, activation of the Notch signaling pathway wasshown to promote the growth of PHSCs,23-25 yet treatment of theCCE-25–c-KitnegSca-1pos or Sca-1neg cells with an activator ofNotch signaling, DeltaExtIgG (Varnum-Finney et al26,27) (in thepresence of SCF, Tpo, Flt-3 ligand, and IL-6), did not induceproliferation of these purified cells. Thus, the multi-HGFs, anti-TGF� and DeltaExtIgG failed to stimulate in vitro growth of theCCE-25–c-Kitneg cell populations.

Figure 3. Purified CCE-25–c-Kitneg cell growth in RAG-2�/� mice. CCE-25–c-KitnegSca-1pos and Sca-1neg cells were purified by elutriation and cell sorting asdescribed in “Materials and methods.” These cells were transplanted into sublethallyirradiated RAG-2�/� mice (4.0 Gy). At 14 weeks after transplantation, the thymuseswere harvested and assayed for the presence of donor (Ly 5.2) thymocytes (CD3�).Animals received transplants of saline (A), 1 � 106 whole bone marrow (wBMC,positive control) (B), 1 � 103 CCE-25–c-KitnegSca-1pos (C), or 1 � 103 CCE-25–c-KitnegSca-1neg cells (D). These results are representative of 5 animals per group and 2separate experiments.

Table 1. Pre–CFU-s activity of purified CCE-25 c-Kitneg cell populations

Cell fraction

Primary recipients Secondary recipientsPre-CFU-s per1 � 105 cellsNo. cells transplanted CFU-s No. cells transplanted CFU-s

Saline None 0 1 � 106 1 1 0

CCE-25 2 � 105 16 3 1 � 106 5 3 6

c-Kitneg 1 � 105 1 1 1 � 106 12 2 19

c-KitnegSca-1pos 1 � 105 1 1 1 � 106 4 3 8.3

5 � 105 3 3 13.3

c-KitnegSca-1neg 5 � 104 1 1 1 � 106 2 1 7.7

5 � 105 1 1 9.6

Spleen colonies were counted from 5 mice (per group, per experiment). Pre-CFU-s activity is determined by the following formula: [(No. of secondary CFU-s/number ofcells transplanted into the secondary recipient) � no. of BMCs per femur in the primary recipient after 12 days]/no. of cells transplanted into the primary recipient. These resultsare representative of 3 experiments.

Table 2. CCE-25-c-KitnegSca-1pos and Sca-1neg growth in responseto multi-HGFs in vitro

Cell type CFU-c/1 � 103 cells

c-Kitpos 176 85

c-Kitneg Sca-1pos 8 14

c-Kitneg Sca-1neg 1 0

The CCE-25-c-Kitneg Sca-1 were purified and plated in duplicate, in soft agarcolony assays and were scored for colony formation (CFU-c) after 10 days asdescribed in “Materials and methods.” The cultures were supplemented with thefollowing growth factors: EPO (20 ng/mL), Flt-3 ligand (200 ng/mL), G-CSF (50ng/mL), IL-1� (20 ng/mL), IL-6 (50 ng/mL), IL-11 (50 ng/mL), LIF (50 ng/mL), M-CSF(300 ng/mL), GM-CSF (20 ng/mL), IL-3 (30 ng/mL), SCF (100 ng/mL), SDF-1� (100ng/mL), and Tpo (20 ng/mL). Data are representative of 2 experiments.

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CCE-25–c-KitnegSca-1pos and Sca-1neg growth and maturationin modified long-term bone marrow cultures

To define the conditions that regulate the maturation of theCCE-25–c-Kitneg cells to c-Kitpos cells in vitro, we tracked donorcell development in primary long-term bone marrow cultures(LTMCs). We used a modified long-term bone marrow culture, thethrombopoietin (Tpo)–LTMC, instead of the traditional (Dexter)LTMC, because it has been shown to support high levels of c-Kitpos

PHSC production.13 These cultures differ from the traditionallong-term bone marrow culture in that they are maintained at 37°C,and their media contain 10 ng/mL Tpo, which is replenished duringbiweekly demirepopulation.

We evaluated the growth and maturation of purified CCE-25–c-Kitpos, –c-KitnegSca-1pos, or –c-KitnegSca-1neg (Ly 5.2) on Tpo-LTMC stromal layers established from the bone marrow of Ly 5.1mice. The Tpo-LTMCs were allowed to develop for 4 weeks toestablish stromal layers, after which they were irradiated to stopcell division, and nonadherent cells were removed. We addedpurified CCE-25–c-KitnegSca-1pos or Sca-1neg cells to the Tpo-LTMC stromal layers and the cultures were monitored for growth.After 25 days the entire contents of the cultures were harvested andexamined for the presence of donor (Ly 5.2) c-Kitpos cells, as wellas myeloid (Mac-1, Gr-1, and F4/80) and lymphoid (B220, CD3�)cells by flow cytometry.

CCE-25–c-KitnegSca-1pos cells (4 � 103) cultured on Tpo-LTMCs generated c-Kitpos (Ly 5.2pos) cells that remained presentover the 25-day culture period (29%) (Figure 4A). In addition, theCCE-25–c-KitnegSca-1pos produced lymphoid (11% B220pos, 7%CD3�pos) (Figure 4B, middle panel) and myeloid (3% Gr-1pos, 23%Mac-1pos) cells (Figure 4B, middle panel). We observed variation inthe final numbers of donor cells per flask. This may be due to thefact that the Tpo-LTMCs were primary, nonclonal cultures thatwere maintained for an extended period and supported differentlevels of donor cell proliferation. However, the capacity of theCCE-25–c-Kitneg cells to develop in to both lymphoid and myeloidcells was observed in multiple flasks in replicate experiments. TheCCE-25–c-KitnegSca-1pos cells expanded approximately 80-fold(data not shown). In contrast, when the same number (4 � 103) ofCCE-25–c-KitnegSca-1neg cells was added to Tpo-LTMC stroma, nogrowth was observed. However, when 100-fold more Sca-1neg cellswere added (4 � 105), 14% of the donor (Ly 5.2)–derived cellswere c-Kitpos after 35 days in culture; Figure 4A (right panels)shows lymphoid cells (5% B220pos, 5% CD3�pos) and Figure 4B(right panel), myeloid cells (4% Gr-1pos, 15% Mac-1pos). Therefore,these data yield 2 conclusive results: (1) the CCE-25–c-KitnegSca-1pos compartment is enriched for cells that grow on the Tpo-LTMCstroma, and (2) the Tpo-LTMC supports the transition of thec-Kitneg cells to c-Kitpos cells.

To determine if the CCE-25–c-KitnegSca-1pos cells generatedcells capable of multilineage reconstitution, a characteristic of thec-Kitpos PHSCs, we cultured the CCE-25–c-KitnegSca-1pos cells onTpo-LTMC stroma for 25 days and then transplanted them intolethally irradiated Ly 5.1 recipients, along with host supportmarrow (the CCE-25–c-KitnegSca-1neg cells grown on Tpo-LTMCdid not generate enough cells to transplant). Peripheral blood of therecipients was assayed by flow cytometry for the presence of donorlymphoid and myeloid cells 3.5 months after transplantation. Theanimals that received whole bone marrow, a positive control fortransplantation, showed an average of 50% donor reconstitution, asexpected for this assay (Figure 4C, top panels). Specifically, 36% ofthe T (CD3�pos) cells and 65% of the B (B220pos) cells were of

donor (Ly 5.2) origin, while in the myeloid lineages, 33% of theGr-1pos cells and 49% of the Mac-1pos cells were donor derived. Incomparison, when CCE-25–c-KitnegSca-1pos cells grown for 25days on Tpo-LTMC stroma were transplanted, they gave rise tomultilineage reconstitution. As shown in Figure 4 (bottom panels),11% of the CD3�pos cells and 21% of the B220pos cells were ofdonor origin, while 37% of the Gr-1pos and 46% of the Mac-1pos

cells were of donor origin. These data illustrate the ability of theTpo-LTMCs to support the CCE-25–c-KitnegSca-1pos cells to gener-ate PHSCs capable of producing multiple lineages, in vitro and invivo. Taken together, these data indicate the Sca-1pos populationcontains more cells with PHSC activity in vitro. Furthermore, thisis the first identification of in vitro culture conditions capable ofsupporting the maturation of CCE-25–c-KitnegSca-1pos cells toc-Kitpos PHSCs.

Figure 4. Purified CCE-25–c-KitnegSca-1pos and Sca-1neg cell growth on Tpo-LTMC stroma. Tpo-LTMCs were established according to procedures outlined in“Materials and methods.” Tpo-LTMCs (4-week-old; Ly 5.1 marked) were irradiated(13.0 Gy), nonadherent cells were removed, and the remaining stromal layers wereseeded with purified, Ly 5.2pos, whole bone marrow (positive control), CCE-25–c-KitnegSca-1pos, or Sca-1neg cells. (A) Flow cytometry for c-Kit and “donor” (Ly 5.2) cellsafter 25 days of culture. Panels are representative of 2 flasks per group in 2 separateexperiments. (B) Lineage composition of CCE-25–c-Kitneg (donor, Ly 5.2) cells grownin the Tpo-LTMCs. After 25 days of culture, the contents of the Tpo-LTMCs purifiedCCE-25–c-Kitneg (Ly 5.2) cells were analyzed by flow cytometry for Ly 5.2– andlineage-specific markers. Left panel: Tpo-LTMCs alone; middle panel: 4 � 103

CCE-25–c-KitnegSca-1pos cells; and right panel: 4 � 105 CCE-25–c-KitnegSca-1neg

cells. Data shown are from 1 flask, which represent 2 flasks per group in 2 separateexperiments. (C) Cells from the Tpo-LTMC stromal layers were harvested 25 daysafter CCE-25–c-KitnegSca-1pos (Ly 5.2) seeding and then transplanted into lethallyirradiated (9.5 Gy) Ly 5.1 recipients with 2 � 105 wBMC competitive host marrow.Peripheral blood was assayed for the presence of donor lymphoid (CD3� and B220)and myeloid (Gr-1 and Mac-1) cells by flow cytometry as described in “Materials andmethods.” The top panels show the distribution of donor-derived cells from animalsthat received transplants of 2 � 105 whole bone marrow cells (positive control). Thepercentages in the upper right corners of the dot plots represent the number of donorcells in the Linpos cell population. For these analyses, background staining, indicatedby staining with isotype-matched controls, was subtracted to yield the percentage ofcells shown on the dot plots.

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CCE-25–c-KitnegSca-1pos and Sca-1neg growthon stromal cell lines

To determine if stromal cell lines could support the growth of theCCE-25–c-KitnegSca-1pos and Sca-1neg cells, we cocultured thepurified populations on D4T,28 OP9,29 14F1.1,30 S17,31 M2-10B4,32

or OMA-AD cell lines known to support hematopoietic growth invitro. We consistently observed that 10- to 100-fold less Sca-1pos

cells than Sca-1neg cells were required to observe growth (data notshown), similar to the difference observed in the Tpo-LTMCs. TheCCE-25–c-KitnegSca-1pos or Sca-1neg cells were cocultured in directcontact with stromal cell lines, and were maintained by biweeklyfeeding over a 25-day period. We observed significant differencesin the ability of these stromal cell lines to support the growth of theCCE-25–c-KitnegSca-1pos or Sca-1neg cells. For example, we foundthat it was necessary to add a minimum of 1 � 105 CCE-25–c-KitnegSca-1pos or Sca-1neg cells to detect growth in OP9 cocultures,while the S17 and 14F1.1 lines required only 1 � 104 CCE-25–c-KitnegSca-1pos or Sca-1neg cells (data not shown). In contrast, farfewer c-KitnegSca-1pos or Sca-1neg cells were required for growth onthe OMA-AD or M2-10B4 cell lines (500 to 1000 cells). Thisshowed that stromal cell lines varied significantly in their capacityto support CCE-25–c-KitnegSca-1pos and Sca-1neg cell growth andsuggests that the OMA-AD and M2-10B4 stromal lines have thecapacity to support the growth of dormant PHSCs better than theother lines tested.

For this reason, we chose the M2-10B4 stromal cell line forfurther study. This cell line has been engineered to express humanIL-3 and human G-CSF.32 While human IL-3 does not cross-reactwith the mouse IL-3 receptor, human G-CSF does cross-react andcould promote the growth of developing PHSCs. The CCE-25–c-KitnegSca-1pos and Sca-1neg cells were cocultured with the M2-10B4cells for 25 days in the presence of SCF and IL-3. The data shownin Figure 5 are an average of 2 separate experiments. CCE-25–c-Kitpos cells were used as a positive control for development, andmatured into monocytic cells (50% Mac-1pos, 31% F4/80pos) and

maintained a population of c-Kitpos cells (47.5%) as shown inFigure 5 (left panel). The CCE-25–c-KitnegSca-1pos gave rise to68% monocytic cells (Mac-1pos), 40% macrophages (F4/80pos), andvery few c-Kitpos cells (7%) (Figure 5, middle panel). These cellsexpanded approximately 100-fold, from 1.4 � 104 cells to 1.6 � 106

cells. In comparison, the CCE-25–c-KitnegSca-1neg cells generated alower percentage of monocytic cells (37%) but produced a similarproportion of macrophages (41% F4/80pos). The CCE-25–c-KitnegSca-1neg cells expanded approximately 70-fold, from 1.4 � 104

cells to 1.0 � 106 cells. These data show that the M2-10B4 stromalcell line supports the maturation of the CCE-25–c-Kitneg cells togrow into heterogeneous populations of myeloid lineage cells.

To determine if the soluble growth factors produced by theM2-10B4 cells alone were sufficient to support the growth of theCCE-25–c-KitnegSca-1pos or Sca-1neg cells, we separated the puri-fied c-Kitneg cells from direct contact with the stromal cells usingtranswell inserts. Under these “noncontact” conditions, the CCE-25–c-Kitpos population, again used as a positive control for cell growth,generated both monocytic cells (Mac-1pos) (Figure 6A, left panel)and c-KitposSca-1pos cells (Figure 6B, left panel). The CCE-25–c-KitnegSca-1pos cells gave rise to exclusively Mac-1pos cells in these“noncontact” cultures (97%; Figure 6A, middle panel). A startingpopulation of 1.4 � 104 cells expanded to 2.2 � 106 cells, a150-fold expansion, similar to that seen in the direct contact

Figure 5. CCE-25–c-KitnegSca-1pos and Sca-1neg cell growth in “contact” M2-10B4 cocultures. Purified CCE-25–c-KitnegSca-1pos and Sca-1neg cells were addedto M2-10B4 monolayers and were cocultured for 25 days with biweekly feeding in thepresence of 100 ng/mL SCF and 30 ng/mL IL-3. Cells were harvested and stained asdescribed in “Materials and methods.” The bars represent the percentages ofgranulocytes (Gr-1), monocytic cells (Mac-1), and/or macrophages (F4/80), as wellas c-Kitpos cells as indicated, minus the background contributed by the isotype-matched controls. Left panel: CCE-25–c-Kitpos cells were used as a positive controlfor growth. Middle panel: CCE-25–c-KitnegSca-1pos cells. Left panel: CCE-25–c-KitnegSca-1neg cells. For these analyses, background staining, indicated by stainingwith isotype-matched controls, was subtracted to yield the percentages shown.Values were calculated by averaging the data from 2 separate experiments. Errorbars indicate the standard error between experiments. These data are representativeof 3 wells per group per experiment in 3 experiments.

Figure 6. CCE-25–c-KitnegSca-1pos and Sca-1neg cell growth in “noncontact”M2-10B4 cocultures. Purified CCE-25–c-KitnegSca-1pos and Sca-1neg cells wereadded to M2-10B4 monolayers and were cocultured in transwells over M2-10B4monolayers for 25 days with biweekly feeding in the presence of 100 ng/mL SCF and30 ng/mL IL-3. Cells were harvested and stained as described in “Materials andmethods.” (A) Flow cytometry for myeloid lineage markers Gr-1 and Mac-1. (B) Flowcytometry for PHSC markers c-Kit and Sca-1. These data are representative of 3points per group in 3 separate experiments. For these analyses, background staining,indicated by staining with isotype-matched controls, was subtracted to yield thepercentages shown on the dot plots. (C) The morphology of CCE-25–c-KitnegSca-1pos

and Sca-1neg cells grown in “noncontact” M2-10B4 cultures over time. Photomicro-graphs of purified CCE-25–c-KitnegSca-1pos and Sca-1neg cells that were cocultured intranswells over M2-10B4 monolayers for 25 days with biweekly feeding in thepresence of 100 ng/mL SCF and 30 ng/mL IL-3. Cells were harvested and stained asdescribed in “Materials and methods.” At 18 days after seeding, cells were harvestedand cytospun. M indicates macrophage morphology; mast, mast cell morphology.

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cultures. In contrast, the CCE-25–c-KitnegSca-1neg cells developedinto a homogeneous population of c-KitposSca-1pos cells (Figure 6B,right panel). These cells also expanded significantly, as 4.5 � 106

cells grew from a starting population of 1.4 � 104 cells. Todetermine if c-KitposSca-1pos cells produced by noncontact culturewith the M2-10B4 cells were PHSCs, we cytospun the cells andfound they had become predominantly mast cells, not c-Kitpos

PHSCs (Figure 6C, right panel). Transplantation and CFU-c assaysconfirmed that the c-KitposSca-1pos cells grown from the CCE-25–c-KitnegSca-1neg cells do not contain c-Kitpos PHSC activity (data notshown). Altogether these data show that we identified severalstromal cell lines that support c-Kitneg cell growth and that weobserved the Sca-1pos cells consistently grew at lower cell densitiesthan the Sca-1neg cells. We showed that soluble factors produced bythe M2-10B4 cells were sufficient to promote the maturation of theCCE-25–c-KitnegSca-1pos cells into macrophages and the Sca-1neg

cells to mast cells. In addition, the data show the maturation of c-Kitneg

cells can be mediated by cell-associated factors, as shown by theheterogeneity of the cells that matured in direct contact cultures.

Discussion

The requirements for the growth and maturation of the CCE-25–c-Kitneg cell have been difficult to identify because of its slow growthin vivo and an apparent inability to grow in vitro. Studies by ourlaboratory and others have shown this population does not formCFU-c’s in the presence of multi-HGF combinations, does not haveprimary CFU-s activity, but, importantly, does have the capacity fordelayed multilineage reconstitution, demonstrating PHSC poten-tial.10,33-35 In this report, we identified in vitro conditions thatsupport the growth and maturation of CCE-25–c-KitnegSca-1pos

PHSCs to c-Kitpos PHSCs and multilineage maturation (Tpo-LTMC) and conditions that support rapid myeloid differentiation(M2-10B4 stromal cell line). In addition, we found that theCCE-25–c-KitnegSca-1pos cells had greater growth potential thanthe Sca-1neg cells in vitro. The purification of the CCE-25–c-Kitneg

cells and identification of alternative in vitro growth conditionsenabled us to document the development of these primitive PHSCsand provides the foundation for future study of the factors thatcontrol their maturation.

In this study, we examined the heterogeneity of the CCE-25–c-Kitneg population using known stem and progenitor cell markers.Another technique used to purify stem and progenitor cells isisolation of the Hoechst side population (SP). The Hoechst SP frommurine bone marrow has been characterized as predominantlyLinneg, c-Kitpos, Sca-1pos, and CD45pos.36,37 Cohler et al havereported that LinnegSca-1posc-Kitneg cells do not lie in the HoechstSP.33 Based on these observations we speculate that most of theCCE-25–c-Kitneg population is not in the SP. However, it is possiblethat a small percentage of the CCE-25–c-Kitneg population could befound within the Hoechst SP.

We used Sca-1 as the basis for further purification of the c-Kitneg

population, because c-Kitpos PHSCs are known to express Sca-1,2,3

and a recent study using Sca-1�/� mice clearly shows the essentialnature of Sca-1 in hematopoietic development.19 Although bothpopulations have stem cell activity as indicated by the pre–CFU-sassay, because the Sca-1pos cells proliferated at lower seedingdensities in vitro than the Sca-1neg cells, we propose the CCE-25–c-KitnegSca-1neg cells are more dormant than the CCE-25–c-KitnegSca-1pos cells and precede them in the PHSC maturation pathway(Figure 7A). If the CCE-25–c-KitnegSca-1neg cells precede the

Sca-1pos cells in the maturation pathway, the requirement for morecells could be due to the CCE-25–c-KitnegSca-1neg cells being moredormant, or there may simply be fewer cells capable of respondingto the growth signals in these cultures. It is also possible that theSca-1neg cells may be a separate PHSC population that is develop-mentally independent of the Sca-1pos cells. Regardless, the fact thatSca-1neg cells can give rise to c-Kitpos cells in the Tpo-LTMCsuggests that these cells have PHSC potential like the Sca-1pos cells.

In the process of determining whether the CCE-25–c-KitnegSca-1pos or the Sca-1neg cells were enriched for PHSC activity, wecompared 2 transplantation models for their ability to support therapid maturation of CCE-25–c-Kitneg cells in vivo. The pre–CFU-sassay indicated the presence of PHSCs in both the c-KitnegSca-1pos

and Sca-1neg populations. However, this assay depends upon serialtransplantation of the cells through 2 lethally irradiated recipients,which should put extreme pressure on all PHSCs present todevelop. This level of developmental pressure may explain the factthat both the CCE-25–c-KitnegSca-1pos and Sca-1neg populationsgave rise to similar numbers of secondary CFU-s per 1 � 105 cellstransplanted. Therefore, we wanted to determine if the CCE-25–c-KitnegSca-1pos or Sca-1neg cells would respond to a more selectivehematopoietic pressure, generated by the absence of maturelymphoid cells, as in RAG-2�/� mice. Because there was no donorreconstitution in the RAG-2�/� animals that received transplants ofthe CCE-25–c-KitnegSca-1pos or Sca-1neg cells, the pressure for thetransplanted cells to develop was insufficient to induce prolifera-tion by these cells, or the CCE-25-c-Kitneg cells are predisposed tobecome myeloid cells in the RAG-2�/� model. Alternatively, it isalso possible that the reconstitution kinetics in RAG-2�/� mice aresimilar to the traditional transplantation assay,10 making it ineffec-tive to rapidly assay CCE-25–c-Kitneg cell growth.

Therefore, to better understand the conditions responsible forthe growth of these dormant c-Kitneg cells, we sought in vitromodels. The novel HGFs tested in this study, Tpo,38 SDF-1�,39-41

TGF�-blocking antibodies,21,22,42-44 and DeltaExtIgG, an activator ofthe Notch signaling pathway,23-27 did not promote significantgrowth of the CCE-25–c-KitnegSca-1pos or Sca-1neg cells. Jagged,another ligand known to activate the Notch signaling pathway, hasnot been tested with the CCE-25–c-Kitneg cells. In addition, thereare other mediators that have been reported to promote the

Figure 7. Proposed models for the c-Kitneg PHSC maturation pathway andmaturation in the in vitro growth conditions tested in this study. (A) Suggestedmodel of c-Kitneg to c-Kitpos maturation. (B) Model of Tpo-LTMCs–supported c-Kitneg

maturation. (C) Model of M2-10B4–driven c-Kitneg maturation.

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development of primitive cells, specifically, the bone morphogenicproteins,45 which remain to be evaluated. Therefore, we concludethat an unidentified growth factor or combination of multiplegrowth factors is required to induce CCE-25–c-KitnegSca-1pos andSca-1neg cell growth.

When cultured on Tpo-LTMC stroma, the CCE-25–c-KitnegSca-1pos population gave rise to cells capable of multilineage reconstitu-tion, a property of the c-Kitpos PHSCs46 (Figure 7B). These are thefirst in vitro conditions identified that support growth of these cells,and as such is an important first step toward understanding thenature of the environment that is required for these cells toproliferate. In the context of c-Kitneg development, the Tpo-LTMCdata suggest that the CCE-25–c-KitnegSca-1neg cell is the ancestorof the c-KitposSca-1pos PHSC. We believe that the c-Kitneg celltransitions through a c-Kitlow stage8,11,12,47 as it matures to a c-Kitpos

cell (Figure 7A). In addition, the Tpo-LTMC provides an in vitroenvironment that contains a complex milieu of uncharacterizedsecreted, membrane-bound, and extracellular matrix factors similarto those that are present in an irradiated recipient in vivo. However,it is due to the complexity of these cultures that the factorsresponsible for c-Kitneg growth are difficult to identify using thissystem. Future studies may depend upon establishment of a stromalcell line from the Tpo-LTMCs, which could support the expansionof primitive cells without promoting differentiation.

In addition to our experiments with the primary stroma from theTpo-LTMCs, we compared 6 previously cloned stromal cell linesfor their ability to support the growth of the CCE-25–c-KitnegSca-1pos

and Sca-1neg cells. While all of the cell lines tested have been shown tosupport in vitro hematopoiesis, our results indicate the M2-10B4and OMA-AD lines are superior in their ability to produce thefactors necessary to induce development of the CCE-25–c-Kitneg

cells. Specifically, factors secreted by the M2-10B4 cells promotegrowth of these cells, but do so without supporting a self-renewingc-Kitpos PHSC population, unlike the Tpo-LTMCs (Figure 7C). TheM2-10B4 stromal cells may produce maturation signals at concen-trations that promote the terminal differentiation of the CCE-25–c-Kitneg cells. For example, the presence of the hG-CSF, produced bythe M2-10B4, in combination with mSCF and mIL-3 may force thePHSCs that develop from the CCE-25–c-KitnegSca-1pos and Sca-1neg cells to mature completely. Yet, we know that G-CSF alone or

in combination with a multiple growth factor cocktail is insufficientto induce growth of the CCE-25–c-KitnegSca-1pos or Sca-1neg cells.A comparison of the genes expressed by the Tpo-LTMC stroma, orby a Tpo-LTMC stromal cell line, and the M2-10B4 stromal cellline could identify the factors that promote the self-renewal ofprimitive PHSCs in vitro, which would be a valuable tool for exvivo expansion of primitive hematopoietic stem cells.

Taken together, our findings and those of others suggest that theCCE-25–c-KitnegSca-1pos cell is a dormant stem cell that prolifer-ates in vivo in response to the extreme pressure present after toxichematopoietic insult (irradiation). However, because of their dor-mancy these cells may be a valuable reserve of primitive stem cellsin patients with leukemia that may be used for transplantation ifthey could be expanded ex vivo. Also, because of the primitivenature of the CCE-25–c-KitnegSca-1pos and Sca-1neg cells, we intendto test the ability of these cells to mature into nonhematopoietic celltypes. In addition, our analysis of this CCE-25–c-Kitneg compart-ment identified a unique, distinct CD45neg population. It is possiblethat these cells developmentally precede the hematopoietic-committed CD45pos cells and have the ability to develop intononhematopoietic tissue, a possibility we have not yet tested.

Acknowledgments

The content of this publication does not necessarily reflect theviews or policies of the Department of Health and Human Services,nor does the mention of trade names, commercial products, ororganizations imply endorsement by the US government.

We are grateful for the outstanding technical assistance of SteveStull, Mehrnoosh Abshari, Kathleen Noer, Roberta Matthai, andJohn Wine. The authors thank Sally Spence, Christine McCauslin,and Joost Oppenheim for their critical review of this manuscript. Inaddition, we are grateful to Scott Duram and Kathrin Muegge foradvice regarding the RAG-2�/� model and analysis, as well asDonna Hogge, Liz Welniak, Kyunghee Choi, Dov Zipori, and ToruNakano for stromal cell lines. In addition we thank Barbara Varnum-Finney and Irwin Bernstein for the generous gift of the DeltaExtIgG.Finally, we are especially grateful to Steve Bartelmez for his adviceon the Tpo-LTMC and for the generous gift of horse serum.

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