rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture...

Experimental Hematology 27 (1999) 904–915

0301-472X/99 $–see front matter. Copyright © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc.PII S0301-472X(99)00012-0

Rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture system

Hiroshi Kawada

a,b

, Kiyoshi Ando

a,b

, Takashi Tsuji

e

, Yasuhito Shimakura

a,b

, Yoshihiko Nakamura

a

, Jamel Chargui

a,b

, Masao Hagihara

a,b

, Hiroyuki Itagaki

d

, Takashi Shimizu

a

, Sadaki Inokuchi

a

, Shunichi Kato

a,c

, and Tomomitsu Hotta

a,b

a

Research Center for Genetic Engineering and Cell Transplantation,

b

Department of Internal Medicine, and

c

Department of Pediatrics, Tokai University School of Medicine,

d

The Cell Transfusion Center of Tokai University Hospital, and

e

Division of Hematology, Pharmaceutical Frontier Research Laboratory, JT Inc., Kanagawa, Japan

(Received 24 June 1998; revised 20 November 1998; accepted 14 December 1998)

Cell numbers limit the widespread clinical use of cord blood(CB) for gene therapy and marrow replacement in adults; asimple and effective method for ex vivo expansion of CB primi-tive progenitor cells (PPC) is required. Recently, the combina-tion of thrombopoietin (TPO) and Flk-2/Flt-3 ligand (FL-2)was reported to support slow proliferation of CB-PPC instroma-free liquid culture. We established a novel culture sys-tem in which the murine stromal cell line HESS-5 dramaticallysupports the rapid expansion of cryopreserved CB-PPC in syn-ergy with TPO/FL-2. Furthermore, while HESS-5 cells directlyadhered to human progenitors during culture, the cultured hu-man cells could easily be harvested without contamination byHESS-5 cells. Within 7 days of culture, a 100-fold increase inCD34

bright

/CD38

dim

cells was obtained in serum-containing cul-ture. When HESS-5 cells were physically separated from hu-man progenitor cells in the presence of TPO/FL-2, synergy wasblocked, suggesting that HESS-5 cells support proliferation ofPPC by direct cell-to-cell interaction. The hematopoietic-sup-portive effects of this xenogeneic coculture system were thenassessed in a very short-term (5 days) serum-free culture. Ex-pansion was further enhanced by addition of stem cell factor(SCF) or interleukin-3 (IL-3). As a result, a 50- to 100-fold in-crease in CD34

bright

/CD38

dim

cells was noted. Colony-formingunits in culture (CFU-C) and mixed colonies (CFU-GEMM)were enhanced by 10- to 30-fold and 10- to 20-fold, respec-tively. Moreover, generation of long-term-culture–initiatingcells (LTC-IC) from CD34

bright

/CD38

dim

cells was amplified by25-fold. The severe-combined immunodeficient (SCID) mouse-repopulating cell (SRC) assay confirmed extensive ability ofthe expanded cells to reconstitute long-term hematopoiesis.These results indicate that this xenogeneic coculture system, incombination with human cytokines, can rapidly generate PPC

from cryopreserved CB. © 1999 International Society for Ex-perimental Hematology. Published by Elsevier Science Inc.

Keywords:

Ex vivo expansion—Progenitor cells—Stromal cell

line—Thrombopoietin—Flk-2/Flt-3 ligand

Introduction

Human umbilical cord blood (CB) is an attractive alterna-tive to bone marrow or growth factor mobilized peripheralblood as a source of hematopoietic progenitors because CBcontains a high number of primitive progenitor cells (PPC)[1,2]. To date, over 500 transplants have been performedworldwide using unfractionated CB from related and unre-lated donors, and the results over the past 9 years of CBtransplantation for children with malignant and nonmalig-nant diseases have been promising [3]. However, there arepotential limitations to the widespread use of CB; there maybe enough hematopoietic stem cells to reconstitute children,but the ability to engraft an adult from CB may require exvivo manipulations.

Many studies to identify culture conditions able to sup-port expansion of PPC have been attempted [4–7]. Recentlyit was reported that the combination of thrombopoietin(TPO) and Flk-2/Flt-3 ligand (FL-2) maintained productionof CB-PPC for more than 6 months in stroma-free liquidculture [8]. Both early-acting cytokines were reported tosustain cell viability and promote proliferation preferen-tially of a minor subpopulation of CD34

bright

cells, i.e.,CD34

bright

/CD38

dim

cells [9–12]; PPC are reported to be in-cluded in this subset [13].

On the other hand, contact with murine stromal cells wasreported to preserve human PPC quality during ex vivo ma-nipulation [14,15]. We have established several murine stro-mal cell lines from bone marrow and spleen [16]. Among

Offprint requests to: Tomomitsu Hotta, M.D., Division of Hematology,Department of Internal Medicine, Tokai University School of Medicine,Bohseidai, Isehara, Kanagawa, 259-1193 Japan; E-mail: [email protected]

H. Kawada et al./Experimental Hematology 27 (1999) 904–915

905

these, we discovered a novel hematopoietic-supportive cellline, HESS-5, which effectively supports not only formationof murine granulocyte and macrophage colonies but alsoproliferation of human CB CD34

bright

/CD38

dim

cells in thepresence of human cytokines [17,18]. Progenitors expandedby this xenogeneic coculture system generate a number ofhigh proliferative potential colony-forming cells and mixedcolony-forming units and differentiate to CD10

1

/CD19

1

B-lymphoid cells in the presence of FL-2.Although it requires a relatively long process to obtain a

sufficient number of PPC by ex vivo manipulation [19–21],prolonged cultures often result in the exhaustion of the pro-liferative and lineage potential of most PPC [22,23]. An ef-fective culture system in which PPC can be rapidly ex-panded is more suitable for clinical settings because thisprevents infections or transformation. Rapid expansion alsois economical and timely for transplantation. Here, we dem-onstrate marked supportive effects of HESS-5 cells on pro-liferation of PPC isolated from cryopreserved CB in syn-ergy with TPO/FL-2 using a novel culture system by whichcontamination with murine stromal cells can be avoided.The number of CD34

bright

/CD38

dim

cells was dramaticallyincreased in a very short time. The long-term-culture–initi-ating cell (LTC-IC) assay and the severe-combined immu-nodeficient (SCID) mouse-repopulating cell (SRC) assayindicated extensive abilities of the expanded cells to sustainand reconstitute long-term hematopoiesis. This novel cul-ture system will be a good ex vivo model that provides valu-able information for regulatory mechanisms of rapid exvivo expansion of PPC.

Materials and methods

CD34

bright

cell purification

CB, collected according to institutional guidelines, was obtainedfrom normal full-term deliveries. Mononuclear cells (MNC) wereisolated from CB using Ficoll-Hypaque (Lymphoprep, 1.077

6

0.001 g/mL; Nycomed, Oslo, Norway) density gradient centrifuga-tion. CD34

bright

cell purification utilized positive selection using theMACS immunomagnetic separation system (Miltenyi Biotec,Glodbach, Germany) according to the manufacturer’s instructions.Briefly, MNC were suspended in buffer containing phosphate-buffered saline (PBS), 0.5% bovine serum albumin (BSA), and2 mM EDTA (BSA-EDTA-PBS), and incubated for 15 minuteswith monoclonal hapten-conjugated anti-CD34 antibody (clone:QBEND/10) and human Ig to prevent nonspecific binding. Washedcells were resuspended in BSA-EDTA-PBS and incubated for 15minutes with colloidal super-paramagnetic microbeads conjugatedto an anti-hapten antibody. After labeling, the cell suspension waspassed through a column (VS

1

separation column) held within amagnetic field causing CD34

bright

cells to be retained in the col-umn. CD34

bright

cells were collected by removal of the columnfrom the magnet and washing with BSA-EDTA-PBS. The cellswere then applied to a second column (RS

1

separation column)and the purification step repeated. Ninety-five percent or more ofthe enriched cells were CD34

bright

by flow cytometric analysis.

CB cryopreservation and thawing

CD34

bright

cells purified from CB were frozen in a medium supple-mented with dimethylsulfoxide and FCS (Cell-Banker; Nihon Zen-yaku Kohgyo, Fukushima, Japan) using a step-down freezing pro-cedure and placed in liquid nitrogen. Aliquots of frozen sampleswere thawed before use. The thawed cells were washed twice andtrypan blue viability determination was performed. When cell via-bility was more than 98%, the samples were subjected to furtherstudies.

Murine stromal cell line

The murine hematopoietic-supportive stromal cell line HESS-5was previously established from murine bone marrow [16]. HESS-5cells were maintained in minimal essential medium

a

(MEM-

a

;Nikken Bio Medical Laboratory, Kyoto, Japan) supplemented with10% (v/v) horse serum (GibcoBRL, Grand Island, NY) at 37

8

C un-der 5% CO

2

in humidified air.

Human cytokines

Recombinant human TPO, interleukin-3 (IL-3), granulocyte-mac-rophage colony-stimulating factor (GM-CSF), and erythropoietin(EPO) were generously provided by Kirin Brewery (Tokyo, Ja-pan). Recombinant human stem cell factor (SCF) was a gift fromAmgen Biologicals (Thousand Oaks, CA). Recombinant humanFL-2 was purchased from R&D Systems (Minneapolis, MN). Thefinal concentrations of cytokines were as follows: TPO, 50 ng/mL;FL-2, 50 ng/mL; IL-3, 20 ng/mL; SCF, 50 ng/mL; GM-CSF, 10ng/mL; and EPO, 3 U/mL.

Culture systems

Diagrams of several culture systems used in this study are dis-played in Fig. 1. Stroma-free culture and classical coculture withstromal cells were performed in culture media in 24-well micro-plates (Iwaki Glass, Chiba, Japan). In the classical coculture, whencontamination of stromal cells in the harvested cells was negligible(

,

2%) by microscopic visualization, cells were subjected to fur-ther examination. In some experiments, human hematopoietic cellswere physically separated from the stromal layer by a polyethyleneterephthalate track-etched membrane in cell culture inserts (BectonDickinson Labware, Franklin Lakes, NJ) (noncontact culture), asdescribed by Verfaillie et al. [24,25]. In this study, a novel culturesystem was devised by modifying the cell culture insert system.First, HESS-5 cells were cultured on the reverse (back) side of thetrack-etched membrane of the insert in MEM-

a

supplemented with10% horse serum. After obtaining a confluent feeder layer, stromalcells were washed five times and the medium was changed for co-culture. CD34

bright

cells isolated from CB were seeded on the upperside of the membrane of the insert where the cytoplasmic villi ofHESS-5 cells passed through the etched 0.45

m

m pores. Therefore,while HESS-5 cells directly adhered to human hematopoietic cellsduring culture, expanded cells could easily be harvested withoutcontamination with HESS-5 cells. Low pore density (LPD, 1.6

3

10

6

/cm

2

) and high pore density membranes (HPD, 1.0

3

10

8

/cm

2

)were used in the present study.

Short-term ex vivo expansion of hematopoietic progenitors

Serum-containing liquid culture was carried out using a mediumcontaining 12.5% horse serum, 12.5% fetal bovine serum, 10

2

4

M2-mercaptoethanol, 2 mM L-glutamine, 0.2 mM i-inositol, 20

m

M

906


folic acid, and MEM-

a

(MyeloCult; StemCell Technologies, Van-couver, Canada) supplemented with 10

2

6

M hydrocortisone (Sigma,St. Louis, MO) and penicillin/streptomycin (GibcoBRL) with orwithout designated cytokines. Three thousand and 100,000CD34

bright

cells were subjected to cytokine-containing and cyto-kine-free culture, respectively. Culture plates were incubated at 37

8

Cin a humidified atmosphere consisting of air enriched with 5% CO

2

.On Day 7 of culture, the medium in each well was removed and re-placed with fresh medium. On Days 7 and/or 14 of culture, aliquotsof cultured cells were harvested and subjected to cell count, clonalcell culture, and flow cytometric analysis. Serum-free liquid culturewas carried out using StemPro™-34SFM (GibcoBRL) supple-mented with StemPro™-34 Nutrient Supplement (GibcoBRL), 2 mML-glutamine (GibcoBRL), and penicillin/streptomycin with orwithout designated cytokines. Five thousand to 100,000 CD34

bright

cells were cultured under the same conditions as serum-containingculture. On Day 5 of culture, cells were harvested and counted. Al-iquots were applied to studies including the LTC-IC assay.

Immunophenotyping by flow cytometry

Aliquots of cells were suspended in EDTA-BSA-PBS and incu-bated with mouse IgG (Inter-Cell Technologies, Hopewell, NJ) toblock nonspecific binding. Cells were then reacted for 15 minutes

with FITC- and PE-conjugated several monoclonal antibodies at4

8

C. Unbound antibodies were removed by two washes, and cellswere resuspended in EDTA-BSA-PBS. Stained cells were thenpassed though a nylon mesh filter and subjected to two-color flowcytometric analysis. Cells labeled with FITC- and PE-conjugatedmouse isotype-matched antibodies were used as negative controls.The analysis was performed using a FACScan flow cytometer(Becton Dickinson, San Jose, CA) with LYSIS II software (BectonDickinson). At least 10,000 events were acquired for each analysis.Antibodies used were as follows; FITC-conjugated CD2, CD3,CD14, CD15, CD19, CD33, CD34, CD41, glycophorin A, andHLA-DR antibodies; PE-conjugated CD38 and CD45 antibodies.CD41 antibody was purchased from Nichirei (Tokyo, Japan). CD3and glycophorin A antibodies were from Immunotech (Marseille,France). CD14, CD33, and CD45 antibodies were from Pharmingen(San Diego, CA) and all others were from Becton Dickinson. Fur-thermore, in some experiments, aliquots of cultured cells were sub-jected to three-color flow cytometric analysis to assess the lineagecommitment of progenitors. Samples were incubated for 15 minuteswith biotin-conjugated anti-CD34 (Immunotech, Marseille, France).Cells labelled with a biotin-conjugated mouse isotype-matched anti-body were used as a negative control. After washing, cells were la-beled with streptavidin PerCP (Becton Dickinson), PE-conjugated

Figure 1. Diagrams of several culture systems used in the study. Low pore density (LPD) culture 5 novel culture using the LPD membrane; high pore den-sity (HPD) culture 5 novel culture using the HPD membrane.


907

anti-CD38, and various FITC-conjugated monoclonal antibodies.Three-color flow cytometry was performed using a FACSCalibur(Becton Dickinson) with CellQuest software (Becton Dickinson).

Clonal cell culture

Aliquots from initial CB samples or cultured cells were incubatedin methylcellulose media at concentrations of 1–2

3

10

2

cells/mLfor purified CD34

bright

cells and 5–10

3

10

2

cells/mL for culturedcells in 35-mm tissue culture dishes (Iwaki Glass). One milliliter ofculture mixture contained 1.2% 1500 cp methylcellulose (Sigma),MEM-

a

, 1% deionized fraction

∨

BSA (Sigma), 10

2

4

M/L 2-mer-captoethanol (Wako Pure Chemical Industries, Osaka, Japan), 30%fetal calf serum (JRH Biosciences, Lenexa, KS), EPO, IL-3, SCF,GM-CSF, and cells. Dishes were incubated at 37

8

C in a humidifiedatmosphere with 5% CO

2

in air. All cultures were done in tripli-cate. Total colony-forming units in culture (CFU-C) and mixedcolonies containing erythroid and myeloid cells and megakaryo-cytes (CFU-GEMM) consisting of 50 or more cells were scored onan inverted microscope at 14 days of culture. To assess the accu-racy of in situ identification, individual colonies were lifted with anEppendorf micropipette under direct microscopic visualization,

spread on glass slides using a cytocentrifuge and studied withMay-Grunwald-Giemsa staining.

LTC-IC assay

LTC-IC assay was performed as described by Sutherland et al. [26]with slight modifications. Briefly, bone marrow stromal cells de-rived from hematologically normal donors were seeded at 10

5

cellsper well in 96-well flat-bottomed plates with MEM-

a

supple-mented with 10% FCS. After obtaining semiconfluent feeder lay-ers, stromal cells were irradiated with 15-Gy using a

137

Cs

g

-irradi-ator. CD34

bright

cell subpopulations (i.e., CD34

bright

/CD38

bright

andCD34

bright

/CD38

dim

cells) purified from CB or those isolated fromcultured cells by sorting with a FACSVantage (Becton Dickinson)were seeded at limiting dilution on the feeder layer in serum-con-taining media. For each evaluation, at least three cell concentra-tions were used with 24 replicates per concentration. Culture plateswere incubated at 37

8

C with 5% CO

2

for 3 to 5 days and then incu-bated at 34

8

C with weekly changes of medium. After 5 weeks ofculture, cells were assayed for CFU-C in methylcellulose medium.Colonies were scored 2 weeks later. The frequency of wells inwhich there were no clonogenic progenitors was determined ac-

Figure 2. Electron microscopic view of the novel culture system. HESS-5 cells were cultured at the reverse (back) side of the polyethylene terephthalatetrack-etched membrane of the cell culture insert. After obtaining a confluent feeder layer, the novel coculture was initiated by seeding cord blood (CB)CD34bright cells on the upper side of the membrane of the cell culture insert. On Day 5 of coculture, the membrane to which both cultured cells were adherentwas removed from the cell culture insert and subjected to electron microscopy. Photos represent views of the upper side of the membrane, demonstrating directcontact between cultured human hematopoietic cells and cytoplasmic villi of HESS-5 cells, which passed through the etched 0.45-mm pores of membrane.

Table 1.

Evaluation of synergistic effects between HESS-5 cells and TPO/FL-2 on ex vivo expansion of hematopoietic progenitors

Cell population or colony None HESS-5 TPO/FL-2 HESS-5 & TPO/FL-2

Total number of cells 0.7

6

0.1 2.9

6

0.5 98.7

6

20.7 414.7

6

10.1CD34

bright

cells 0.1

6

0.0 0.4

6

0.1 4.3

6

0.4 16.0

6

2.5CD34

bright

/CD38

bright

cells 0.1

6

0.0 0.4

6

0.1 4.3

6

0.4 14.7

6

2.1CD34

bright

/CD38

dim

cells 0.1

6

0.0 0.4

6

0.3 4.2

6

2.0 150.3

6

84.3*CFU-C ND 1.7

6

0.0

#

13.3

6

2.3 74.3

6

11.2**CFU-GEMM ND 2.4

6

0.3

#

7.3

6

5.3 35.7

6

7.6*

Cord blood (CB) CD34

bright

cells were cultured in the presence or absence of TPO/FL-2 with or without HESS-5 cells. In coculture, HESS-5 cells directly ad-hered to the CB cells through 0.45-

m

m pores of low pore-density membranes in the cell culture insert system. Each number for the above-mentioned cell pop-ulations indicates the mean fold increase

6

SEM of three different experiments on Day 14 of culture. Suitable aliquots of cultured cells were assayed forCFU-C and CFU-GEMM. After 2 weeks of clonal cell culture, colonies were scored and the results represent the fold increase (mean 6 SEM of three differ-ent experiments). #Only one experiment was performed in triplicate; *p , 0.05; **p , 0.01 vs those without HESS-5; ND 5 not determined.

908 H. Kawada et al./Experimental Hematology 27 (1999) 904–915

cording to the number of the initial input population, and the fre-quency of LTC-IC was calculated.

SRC assaySRC assay was performed as previously described [13], with slightmodifications. Briefly, 8-week-old male NOD/Shi-scid (NOD/SCID) mice were obtained from the Central Institute for Experi-mental Animals (Kawasaki, Japan). All animals were handled un-der sterile conditions and maintained under microisolators in theanimal facility located at the Tokai University School of Medicine.Human hematopoietic cells at the indicated doses were transplantedby tail–vein injection into sublethally irradiated mice (350 cGy us-ing a 4 3 106 V linear accelerator). Cells were co-transplanted withirradiated (15 Gy using a 137Cs g-irradiator) nonrepopulating CD34dim

cells as accessory cells. Mice were killed 7 weeks after transplanta-tion, and the bone marrow (from the femurs and tibiae), spleen, andperipheral blood cells (from the retro-orbital venous plexus usingheparin-coated micropipettes) were harvested. The presence of hu-man hematopoietic cells was determined by detection of cells posi-tively stained with FITC-conjugated antihuman CD45 using flowcytometry. Southern blot analysis using a human chromosome 17-specific a-satellite probe was also performed to confirm flow cyto-metric results, as described previously [27,28].

Scanning electron microscopyCB CD34bright cells were cocultured with HESS-5 cells in the LPDculture system. On Day 5 of coculture, the membrane to whichboth cultured cells were adherent was removed from the cell cul-

Figure 3. Effects of several culture systems on ex vivo expansion of hematopoietic progenitors. CD34bright cells were cocultured with HESS-5 cells in serum-containing medium supplemented with TPO and FL-2 using several culture systems. The results represent the mean fold increase 6 SEM of three differentexperiments on Days 7 and 14 of culture. *No significant differences between different culture systems (p . 0.05); **p , 0.05 as compared with low poredensity (LPD) culture or noncontact culture. j 5 high pore density (HPD) culture; d 5 LPD culture; m5 noncontact culture; r 5 classical culture.

H. Kawada et al./Experimental Hematology 27 (1999) 904–915 909

ture insert; the membrane was fixed in 2.5% glutaraldehyde at 48Cfor 1 hour, stained with 1% OsO4/2% tannic acid, then freeze-driedwith t-butyl alcohol. The specimen was coated with gold by ionsputtering and examined with a scanning electron microscope(JSM 840A, JEOL, Tokyo, Japan).

Statistical analysisData were compared using analysis of variance. Where significantdifferences were inferred, sample means were compared using theStudent’s t-test.

ResultsEvaluation of supportive effects of HESS-5 cells on ex vivoexpansion of CB-PPC in synergy with TPO/FL-2. We firstassessed the effects of TPO and FL-2 on ex vivo expansionof CB-PPC in the stroma-free serum-containing culture. Assingle agents, neither TPO nor FL-2 could effectively sup-port proliferation of CD34bright cells (data not shown). How-

ever, the combination of factors (TPO/FL-2) enhanced pro-duction of progenitors; the number of CD34bright cells wasapproximately four times the input number after 14 days ofculture (Table 1). The supportive effects of HESS-5 cells onproliferation of CB-PPC were then studied in LPD culture.Electron microscopic examination confirmed direct contactbetween cultured human hematopoietic cells and cytoplas-mic villi of HESS-5 cells, which had passed through theetched 0.45-mm pores of the membrane in the cell cultureinsert (Fig. 2). Under cytokine-free conditions, althoughHESS-5 cells alone weakly supported production of nucle-ated cells, the number of CD34bright cells was decreased.However, in the presence of TPO/FL-2, HESS-5 cells couldeffectively support proliferation of progenitor cells, espe-cially in the CD34bright/CD38dim cell subpopulation; themean number of CD34bright/ CD38dim cells was approxi-mately 150 times the input number after 14 days of culture.The output of CFU-C and CFU-GEMM also was enhanced.

Figure 4. Comparison of CD34 and CD38 expression among hematopoietic cells in different culture systems. CD34bright cells derived from a single deliverywere cocultured with HESS-5 cells in serum-containing medium supplemented with TPO and FL-2 in different culture systems. On Day 7 of culture, aliquotsof harvested cells were subjected to flow cytometric analysis. While there were no significant differences in the number of total nucleated cells (p . 0.05), dif-ferences in CD34bright/CD38dim cells were noted between different culture systems. Data represent three different experiments. Results of this trial are dis-played quantitatively in Fig. 3. LPD 5 low pore density culture; HPD 5 high pore density culture.


Assessment of the supportive effects of HESS-5 cells on proliferation of CB-PPC in the presence of TPO/FL-2 using several culture systemsNext, we determined suitable culture conditions in whichHESS-5 cells could most effectively support proliferation ofCB-PPC in synergy with TPO/FL-2. CD34bright cells werecocultured with HESS-5 cells in several culture systems(Fig. 1) in serum-containing medium supplemented withTPO and FL-2. During culture, there were no significantdifferences in the number of total nucleated cells betweenthe different culture systems (Fig. 3). However, CD34bright

cells, especially CD34bright/CD38dim cells, were generatedmore by the HPD culture and the classical coculture than bythe LPD culture or the noncontact culture; the mean numberof CD34bright/CD38dim cells was over 100 times the initial in-put number in HPD culture within 7 days (Figs. 3 and 4). Bycontrast, synergistic effects of HESS-5 cells with TPO/FL-2were not seen in the noncontact culture (Fig. 3). Effects ofTPO/FL-2 alone or HESS-5 cells alone were identical tothose described in Table 1 (data not shown).

Ex vivo expansion of CB-PPC in a very-short-term (5 days) serum-free cultureThe synergistic effects of HESS-5 cells and TPO/FL-2 werealso studied in serum-free HPD culture for a very short du-ration. After 5 days of culture, HESS-5 cells alone could notsignificantly stimulate production of nucleated cells or pro-genitors in cytokine-free conditions (Table 2). However, inthe presence of TPO/FL-2, the number of progenitor cells,especially CD34bright/ CD38dim cells, was remarkably in-creased. Moreover, by addition of SCF or IL-3, those effectswere further enhanced. As a result, the mean number ofCD34bright/CD38dim cells was approximately 50 to 100 timesthe initial input number. The outputs of CFU-C and CFU-GEMM were increased 10- to 30-fold and 10- to 20-fold, re-spectively. The combination of TPO/FL-2 and IL-3 was themost effective cytokine combination to obtain expansion ofCD34bright/CD38dim cells in this system. Three-color flowcytometry revealed that most of the CD34bright/CD38dim pop-

ulation amplified by this system was negative for mostlineage-committed markers but was positive for HLA-DR(Table 3).

LTC-IC assay using the CD34bright population isolated from cells cultured in the very short-term xenogeneic coculture systemTo determine whether cells generated in the 5-day serum-free stroma-contact culture system could preserve the abilityto sustain long-term hematopoiesis, the LTC-IC frequency incells cultured by this system was quantified. Initially, iso-lated CD34bright CB cells were cultured in the presence ofTPO/FL-2, IL-3, and HESS-5 cells under serum-free HPDculture conditions. Cultured cells were harvested and sub-jected to a second CD34bright cell purification by sorting onDay 5 of culture. LTC-IC assay was performed using sortedCD34bright cell subpopulations (CD34bright/CD38bright andCD34bright/ CD38dim cells), as well as those initially preparedfrom CB (control samples). The LTC-IC frequency was de-termined as previously described [26]. Although the numberof LTC-IC in CD34bright/CD38bright cells was decreased afterculture, that in CD34bright/ CD38dim cells was markedly am-plified despite the short duration of culture (Table 4).

Table 2. Evaluation of synergistic effects between HESS-5 cells and TPO/FL-2 containing cytokines on ex vivo expansion of hematopoietic progenitors in very short-term serum-free culture

Cell population or colony Cytokine-free TPO 1 FL-2 TPO 1 FL-2 1 SCF TPO 1 FL-2 1 IL-3

Total number of cells 1.1 6 0.0 5.3 6 0.6 14.3 6 1.2* 17.2 6 3.9**CD34bright cells 1.1 6 0.0 2.3 6 0.4 4.8 6 0.1* 4.9 6 1.3*CD34bright/CD38bright cells 1.1 6 0.0 1.4 6 0.2 3.0 6 0.4* 2.9 6 0.8CD34bright/CD38dim cells 1.1 6 0.4 48.4 6 7.6 103.1 6 10.3* 113.9 6 21.7**CFU-C 1.5 6 0.1 12.4 6 2.3 34.2 6 6.7** 33.2 6 5.3*CFU-GEMM 0.5 6 0.1 9.4 6 2.5 18.3 6 5.3 11.5 6 1.2

In the presence or absence of the above-mentioned cytokines, cord blood (CB) CD34 bright cells were cultured with HESS-5 cells under serum-free conditions.HESS-5 cells directly adhered to the CB cells through 0.45-mm pores of high pore-density membranes in the cell culture insert system. Each number for thecell populations indicates the mean fold increase 6 SEM of three different experiments on Day 5 of culture. Suitable aliquots of cultured cells were assayedfor CFU-C and CFU-GEMM. After 2 weeks of clonal cell culture, colonies were scored and the results represent the fold increase (mean 6 SEM of three dif-ferent experiments). *p < 0.05; **p < 0.01 as compared with TPO 1 FL-2; SCF 5 stem cell factor.

Table 3. Immunophenotypes of hematopoietic progenitor cells expanded by the very-short-term serum-free culture

Marker CD34bright/CD38bright cells CD34bright/CD38dim cells

CD15 29.6 6 8.2 3.2 6 1.1CD19 3.9 6 2.6 0.0 6 0.0CD2 4.8 6 2.3 0.7 6 0.7CD41 4.7 6 3.3 6.4 6 2.4Gly A 4.5 6 3.8 2.5 6 1.6HLA-DR 88.7 6 3.8 90.9 6 3.7

In the presence of TPO, FL-2, and IL-3, cord blood CD34bright cells werecocultured with HESS-5 cells under serum-free conditions. After 5 days ofculture, cells were harvested and immunophenotypes of expanded CD34bright

cells were assessed using three-color flow cytometry. Each number indicatesthe mean percentage of positive cells 6 SEM of three different experiments.


Effects of the 5-day serum-free stroma-contact culture system on human reconstituting hematopoietic progenitorsAccordingly, we studied the SRC assay to determinewhether cells cultured in the very short-term xenogeneiccoculture system are capable of long-term multilineage re-constitution in vivo. Purified 1 3 105 CB CD34bright cellswere initially cocultured with HESS-5 cells in the presenceof TPO, FL-2, and IL-3 in serum-free media for 5 days; har-vested cells were then transplanted into NOD/SCID mice.As controls, uncultured 1 3 105 CB CD34bright cells ob-tained from the same sources were also transplanted intoother mice. Seven weeks after transplantation, humanCD451 cells were found in the bone marrow, spleen, andperipheral blood cells of mice transplanted with the culturedcells (Fig. 5A, and data not shown). There were marked dif-ferences in the percentage of chimerism between bone mar-row cells in mice transplanted with cultured cells and thosetransplanted with control samples. The proportion of humanhematopoietic cells in the murine bone marrow was alsoquantitatively confirmed by Southern blot analysis using ahuman chromosome 17-specific a-satellite probe (data notshown). Human CD451 cells in the murine bone marrowwere further subjected to flow cytometric analysis to deter-mine multilineage reconstitution. As a result, human CD451

cells were positive for CD33, CD14, CD41, glycophorin A,or CD19 (Fig. 5B). CD34-positive cells were also deter-mined. However, CD3 was considered to be negative (datanot shown).

DiscussionIn this study we demonstrated that the murine stromal cellline HESS-5 could effectively support rapid expansion ofcryopreserved CB-PPC in synergy with human cytokines.We first assessed the supportive effects of two early-actingcytokines, TPO and FL-2, on proliferation of PPC; syner-gistic effects of these factors were confirmed [8]. We thenassessed the supportive effects of HESS-5 cells on prolifer-ation of human PPC. While HESS-5 cells alone did not ef-fectively support proliferation, HESS-5 cells could dramati-cally enhance generation of progenitors, especially CD34bright/CD38dim cells, in synergy with TPO/FL-2 over a short pe-riod. The addition of SCF or IL-3 further enhanced production.

In ex vivo expansion of human PPC, there are severalbenefits to using a xenogeneic stromal cell line as the feeder:(1) HESS-5 cells can be maintained easily; (2) consistenthematopoietic-supportive effects are repeatedly obtained;and (3) cell differentiation due to exposure to various fac-tors secreted by stromal cells can be avoided. AlthoughHESS-5 cells produced high GM-CSF activity, murine GM-CSF does not exhibit activity on cells of human origin[16,29]. We previously studied the supportive effects ofnormal human bone marrow fibroblasts on proliferation ofCB-PPC, but variable results were noted because of theirmarked differentiation-supportive potential (data not shown).However, in stroma-contact culture, contamination of stro-mal cells into cultured cells is a problem. Furthermore, it isvery difficult to harvest cultured hematopoietic cells com-pletely, since a number of cultured cells migrate under stro-mal layers. Therefore, we established a novel culture systemin which HESS-5 cells were easily seeded and grew on thereverse side of the membrane of the cell culture insert; cul-tured human cells were simply harvested without contami-nation by HESS-5 cells. Evaluation of the hematopoietic-supportive effects of HESS-5 cells using several differentculture systems suggested that proliferation of CB-PPCenhanced by HESS-5 cells was not due to soluble factorsproduced by HESS-5 cells but was mediated by direct cell-to-cell interaction. Therefore, the HPD culture was more ef-fective for expansion of PPC than LPD culture, indicatingthat progenitor-stroma-contact density is important for ef-fective expansion. However, the effects of HPD culturewere comparable with those obtained by classical coculture.Hence, the HPD culture was considered to be the most suit-able system for ex vivo manipulation of PPC under stroma-contact culture conditions.

On the basis of these observations, we then assessed theHPD culture system in serum-free conditions. BecauseHESS-5 cells usually require horse serum for growth andactivity [16], we attempted ex vivo manipulation for a veryshort duration; adequate expansion of CB-PPC was ob-tained. Furthermore, compared with serum-containing cul-ture in cytokine-free conditions, the number of CD34bright

cells was not decreased and the total nucleated cells werenot significantly increased during culture, suggesting thatserum-free conditions prevent primitive-cell differentiation.

Table 4. Results of LTC-IC assay using CB CD34bright cells or those generated by very-short-term serum-free culture

LTC-IC frequency

Cell population Pre-expansion Post-expansion Fold LTC-IC amplification

CD34bright/CD38bright cells 1/215.0 6 85.0 1/2833.3 6 1593.3 0.2 6 0.1CD34bright/CD38dim cells 1/87.5 6 32.5 1/134.0 6 42.1 25.3 6 16.5

Long-term culture–initiating cells (LTC-IC) frequency was compared between cryopreserved cord blood (CB) CD34bright cells (pre-expansion) and those gen-erated by very short-term (5 days) serum-free coculture with HESS-5 in the presence of TPO, FL-2, and IL-3 (post-expansion). The fold increase in LTC-ICwas determined by the LTC-IC frequency and the fold increase in CD34bright/CD38bright or CD34bright/CD38dim cells during culture. Results represent mean 6SEM of three different experiments.


Most of the expanded CD34bright/CD38dim cell populationdid not express various lineage-committed markers, butwere positive for HLA-DR. In contrast to previous findingsin bone marrow, CB LTC-IC were shown to be present

among the CD34bright/HLA-DRbright cell fraction [30,31].Furthermore, Bhatia et al. [13] recently identified the SRCthat were capable of multilineage reconstitution of humanhematopoiesis in the bone marrow of NOD/SCID mice; the

Figure 5A. Determination of human hematopoietic reconstitution in NOD/SCID mice 7 weeks after transplantation. Expression of human CD45 on bonemarrow cells (BMC) collected from NOD/SCID mice 7 weeks after transplantation. (a) Cells in the gated area were analyzed by flow cytometry. (b) Bonemarrow cells (BMC) from an untransplanted mouse (negative control). (c) BMC from a mouse transplanted with 1 3 105 human CD34bright cells (Sample 1)before culture. (d) BMC from a mouse transplanted with cells obtained after cultivation of those 1 3 105 human CD34bright cells (Sample 1) in the very-short-term xenogeneic coculture system. Additional results using another cord blood sample are also shown [Sample 2, (e) before and (f) after culture, respectively].


CD34-positive SRC were found exclusively in the CD34bright/CD38dim cell fraction [13]. Therefore, the expanded hemato-poietic progenitors were expected to sustain long-term he-matopoiesis. As a result, 25-fold LTC-IC amplification wasobserved in CD34bright/CD38dim cells expanded by the very

short-term xenogeneic coculture, although the number ofLTC-IC in the more mature population of CD34bright cells,i.e., CD34bright/CD38bright cells, was decreased during cul-ture. The SRC assay indicated the reconstituting ability ofthese cultured human PPC. Although we could not perform

Figure 5B. Determination of human hematopoietic reconstitution in NOD/SCID mice 7 weeks after transplantation. Specific subsets of human CD451 cellsin bone marrow cells of the NOD/SCID mouse transplanted with the human cord blood cells [Sample 1, Fig. 5(A)] after the xenogeneic coculture. Harvestedcells were stained with PE-conjugated CD45 and various FITC-conjugated monoclonal antibodies.


a quantitative SRC assay, the difference in the percentage ofchimerism of human CD451 cells between bone marrowcells of mice transplanted with cultured cells and thosetransplanted with control samples strongly suggests the ex-tensive ability of these ex vivo-generated PPC to sustainand reconstitute long-term human hematopoiesis in vivo.

The mechanism of hematopoietic-supportive effects ofHESS-5 cells, especially in the CD34bright/CD38dim cell frac-tion, remains unknown. Recently, a cell surface moleculeidentified as delta-like/preadipocyte factor-1 (dlk) on a mu-rine stromal cell line (AFT024) was reported to show activ-ity for murine stem cells by promoting the formation of cob-blestone areas that contain both high-proliferative potentialprogenitors and in vivo repopulating cells [32]. Althoughexpression of dlk in HESS-5 cells could not be detected byNorthern blot analysis (in preparation), a novel molecularpathway should play an important role in stem cell regula-tion by HESS-5 cells.

AcknowledgmentsThe authors thank Hiroko Miyatake and Ayako Kanemura fortechnical assistance; Masayoshi Tokunaga and Akira Akatsuka forelectron microscopic examination; Tadayuki Sato for Southernblot analysis; Hideyuki Matsuzawa for Northern blot analysis; andthe members of the Tokai CBSC Study Group for providing hu-man cord blood. This study was supported by The Japan Societyfor the Promotion of Science (JSPS) grant no. JSPS-RFTF97 |00201; a Grant-in-Aid for Scientific Research and a ResearchGrant of The Science Frontier Program from the Ministry of Edu-cation, Science, Sports and Culture of Japan; and grants from 1998Tokai University School of Medicine Research Aid and the JapanResearch Foundation for Clinical Pharmacology.

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