1. No category

From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
1995 85: 997-1005
Functionally distinct human marrow stromal cell lines immortalized
by transduction with the human papilloma virus E6/E7 genes
BA Roecklein and B Torok-Storb
Updated information and services can be found at:
http://www.bloodjournal.org/content/85/4/997.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
Functionally Distinct Human Marrow Stromal Cell Lines Immortalized by
Transduction With the Human Papilloma Virus E6/E7 Genes
By Bryan A. Roecklein and Beverly Torok-Storb
A replication-defective recombinant retrovirus containing
the human papilloma virus E6/E7 genes (LXSN-16 E6E7)
was
used t o immortalize stromal cells from human marrow. The
E6/E7 gene products interfere with the function of tumorsuppressor proteins p53 and Rb, respectively, thereby preventing cell cycle arrestwithout causing significant transformation. Twenty-seven immortalized clones designated HS1t o HS-27 were isolated, four of which are characterizedin
this report. Two cell lines,HS-5 and HS-21,appear t o be
fibroblastoid and secrete significant levels of granulocyte
colony-stimulating factor (G-CSF), granulocyte-macrophageKit ligand (KL),
CSF(GM-CSF),macrophage-CSF(M-CSF),
macrophage-inhibitory protein-la, interleukin-6 (IL-6). 11-8.
and IL-11. However, only HS-5 supports proliferation of hematopoietic progenitor cells when cocultured in serum-deprived media with noexogenous factors.Conditioned media
(CM) from HS-5 promotes growth of myeloid colonies t o
significantly greater extent than a cocktail of recombinant
factors containing 10 ng/mL of IL-l, IL-3, IL-6, G-CSF, GMCSF, and KL and 3U of erythropoietin (Epo). Two additional
clones, HS-23 and HS-27, resemble "blanket" cells, with an
epithelioid morphology, and are much larger, broader, and
flatter when compared with HS-5 and HS-21.These lines
secrete low levels of growth factors and do not support
proliferation of isolated progenitor cells in cocultures. CM
from HS-23 and HS-27 also fail t o support growth of myeloid
colonies. Both HS-23 and HS-27 expressrelatively high levels
of VCAM-1, yet HS-27 is theonly line that supports the formation of "cobblestone" areas by isolated CD34+38'" cells.
We hypothesize that HS-5, HS-21, HS-23, and HS-27 represent functionally distinct components ofthe marrowmicroenvironment.
0 1995 by The American Societyof Hematology.
D
In this report, a different procedure was used to establish
cell lines representative of the heterogeneous ME. Primary
LTC were exposed to an amphotropic retrovirus which carries the E6 and E7 open reading frames of the human papilloma virus type 16 (HPV16). The E6 and E7 gene products
have been shown in other systems to immortalize differentiated cells without significant
This report
provides the initial characterization of WV16 E6/E7 immortalized human marrow stromal cell clones which support
the proliferation of hematopoietic progenitors and maintain
colony-forming cells (CFC) for up to 8 weeks in culture.
EFINING THE functional components of the marrow
microenvironment (ME) is a prerequisite to understanding how the proliferation and differentiation of progenitor cells is coordinately regulated. The cellular complexity
of the ME has been shown both in situ and in vitro using a
variety of histochemical techniques.'*2It has been established
that both hematopoietic and stromal or mesenchymal-derived
cells comprise the ME. The stromal cells include endothelial
cells that form the sinuses and adventitial reticular cells that
have characteristics consistent with adipocytes, fibroblasts,
and smooth muscle cell^.^.^ Numerous advances in recent
years have provided considerable information on the ontogeny and development of hematopoietic cells5'"; however,
ontogeny of the stromal components and their precise role
in controlling hematopoiesis has proven e l ~ s i v e . ' ~ " ~
Long-term cultures (LTCs) are an in vitro approximation
of the in vivo ME and have been informative with respect
to the identification of growth factors, adhesion proteins,
and extracellular matrix proteins that mediate the interaction
between the hematopoietic cells and the stromal element~.'~"'One improvement of this system was the use of
stromal precursors, positive for the STRO-1 antigen, to establish LTCs that are devoid of myeloid components and
less heterogeneous than primary cultures, but still capable
of supporting hematopoiesis.20However, both the STRO-1initiated cultures and the primary LTCs are too complex to
delineate contributions from individual cell types. Moreover,
primary cultures can be highly variable and change with
time, further complicating the issue.
Immortalized stromal cell lines have been used to circumvent some of these problems. Numerous spontaneous murine
cell lines have been
however, unlike mouse
lines, human cell lines undergo senescence unless first immortalized with a r e t r o v i r ~ sThe
. ~ ~ few human bone marrow
stromal cell lines that are available were established using
the SV40 virus large T an tiger^.'^.^' Some of these lines are
promising with respect to the maintenance of hematopoietic
cells; unfortunately, some also display transformed phenotypes, which limits their usefulness for extrapolation to the
normal ME."
Blood, Vol 85, No 4 (February 15). 1995: pp 997-1005
MATERIALS AND METHODS
Long-term bone marrow cultures (LTBMCs). Adult BM was
obtained from normal donors after informed consent as defined by
the Internal Review Board (I-)
at the Fred Hutchinson Cancer
Research Center (FHCRC; Seattle, WA) and LTBMCs were established as described by Gartner and K a ~ l a nBriefly,
. ~ ~ buffy coat cells
from marrow aspirates were plated in plastic tissue culture dishes
at 1 to 2 X lo6cellslmL. Adherent cells were grown in LTC medium
containing Iscove's, 12.5% horse serum, 12.5% fetal calf serum
(FCS), L-glutamine (0.4 mg/mL), sodium pyruvate (1 mmoVL), penicillin (100 U/mL), streptomycin sulfate (100 pg/mL), hydrocortisone sodium succinate
mom), and P-mercaptoethanol
mom).
From the Program in Transplantation Biology, Clinical Research
Division, Fred Hutchinson Cancer Research Center, Seattle, WA.
Submitted August 8, 1994; accepted October 19, 1994.
Supported in part by Grants No. DK34431, HL.36444, CA18029,
and CA18221 from the National Institutes of Health, Department of
Health and Human Services.
Address reprint requests to Bryan A. Roecklein, PhD, Clinical
Research Division, Fred Hutchinson Cancer Research Center, 1124
Columbia St, M318, Seattle, WA 98104.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006~4971/95/8.504-0017$3.00/0
997
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
998
Transduction of LTBMC. The amphotrophic retrovirus LXSN16 E6E7 was packaged in the PA317 murine cell line in the laboratory of Denise Galloway (FHCRC) as previously de~cribed.’~
Primary LTCs were exposed to virus in the presence of 4 pg/mL
polybrene (Aldrich Chemical CO Inc, Milwaukee, WI) for 2 hours
at 37°C. The virus-containing media was removed and the cells were
incubated for an additional 5 hours with media-containing polybrene.
Cells were then washed and fed with LTC media and incubated for
an additional 48 hours. Cell cultures were then trypsinized and replated at limiting dilution. Transduced clones were selected with 50
pg/mL G418, and resistant colonies were picked and grown in LTC
using standard tissue culture techniques. After expansion, most
clones were switched to RPMI containing 10% serum andHS-5
was switched to serum-deprived medium containing 1% Nutridoma
(Boehringer Mannheim, Indianapolis, IN), 100 mmoK glutamine,
100 mmoVL sodium pyruvate, and 100 U/mL penicillin-streptomycin in Iscove’s media.
Indirect-immunojuorescencestaining. The antigenic phenotype
of the stromal cell lines was determined by standard immunochemistry using IA4-IgG2a (Sigma, St Louis, MO) for smooth muscle
actin; CD14 (leuM3-IgG2b; Becton Dickinson, San Jose, CA) for
macrophages; anti-FVLII antigen (human type I; Calbiochem, La
Jolla, CA) for endothelial cells; 6.19-IgG2a for fibroblasts, endothelial cells, and adipocytes (C. Frantz, University of Rochester School
of Medicine and Dentistry, Rochester, NY); CD34 (12.8, IgM; courtesyof I. Bernstein, FHCRC); PIHI1 (fibronectin) and PlHl-C9
(vimentin) from Bill Carter (FHCRC); 60.5 (class I major histocompatibility complex [MHC]; from Dr P. Martin, FHCRC); anti-VLA4 and anti-VCAM-1 (4B9 ascites) from John Harlan (University of
Washington, Seattle, WA); anti-collagen type I (MAB1340, IgG),
type I11 (MAB1343, IgGl), and type IV (MAB 1910, IgG1) were
obtained from Chemicon (Temecula, CA). For immunofluorescence
staining, semiconfluent cells were rinsed withwarm Hanks’ Balanced Salt Solution (HBSS) and fixed for IO minutes with l% formaldehyde in phosphate-buffered saline (PBS) at 25°C.The cells were
washed with PBS and treated with 0.2 m o m glycine in PBS for 5
minutes at 25°C. One additional washwas performed with PBS
before incubation with a specific antibody or irrelevant nonspecific
isotype control antibody for 1 hour at 25°C. After incubation with
the primary antibody, the cells were washed three times and incubated with a secondary antibody (goat antimouse IgG/IgM fluorescein isothiocyanate [FITCI-conjugated antibody; Tago, Burlingame,
CA) for 1 hour at 25°C and washed with PBS before viewing with a
Nikon Diaphot fluorescent microscope (Chiyoda-Ku, Tokyo, Japan).
Primary LTCs and foreskin fibroblasts (FSFs) were used as controls
for antibody staining.
Cytochemical analysis. Alkaline phosphatase activity was determined using cells fixed with 2% formaldehyde in absolute methanol
for 30 seconds at 4OC” and then washed with distilled water and air
dried. After drying, the cells were incubated at 37°C for 30 minutes
in filtered reaction buffer containing 0.2 mol/L Tris HCI (pH 9.1)
and 1.0 mg/mL Fastblue BB with or without 0.2 m o m napthal AS
phosphate in N,N-dimethylformamide. After incubation, the cells
were washed with distilled water, overlaid with Aqua Mount (Lerner
Laboratories, Pittsburgh, PA), and photographed with a Nikon Diaphot microscope.
For analysis of acid phosphatase activity, the cells were fixed with
60% acetone in 0.04 m o m citrate buffer (pH 5.4) for 30 seconds
at 25°C,26 rinsed with distilled water, air dried, and incubated with
the reaction buffer. The reaction buffer was made up in 24 mL of
0.1 moVL acetate buffer with or without 12.5 mg Napthal AS-B1
phosphate as substrate; 7.5 mg of Fast Garnet GBC dye was added
as counterstain. This solution was filtered through a Whatman #4
filter (Whatman Inc, Clifton, NJ) and then incubated with cells for
1 hour at 25°C protected from light. After staining, the cells were
ROECKLEIN AND TOROK-STORB
washed with distilled water, overlaid with Aqua-mount, photographed (Nikon Diaphot microscope), and scored for the presence
of acid phosphatase.
Adipogenesis. Confluent stromal lines were incubated with corticoid steroids for 4 weeks and stained with oil red 0 to determine if
these lines contain adipogenic cells.37Cultures were fed weekly with
either dexamethasone (lo” mom), hydrocortisone (IO-‘ mom),
insulin ( I O mg/mL), or dexamethasone combined with insulin in
RPMI containing 10% FCS. After the incubation period, the cells
were washed extensively with PBS and then fixed with 10%formalin
in PBS for 30 minutes. The excess formalin was washed off with
PBS and the cells were stained for 15 minutes with filtered oil red
0 (0.3% wt/vol in isopropanol). The stain was then differentiated
with 60% isopropanol and washed and the cells were counterstained
with Mayers haematoxylin for 30 seconds.
Southern analysis. Genomic DNA was isolated from I X 10’
stromal cells using a modification of the technique described in
Ausebel et al.38Before Southern hybridization, IO yg of genomic
DNA was digested with excess EcoRI overnight at 37°C. The DNA
was extracted with pheno1:chloroform and precipitated. Ten micrograms of the digested genomic DNA was separated on a 0.5% agarose gel in TBE and then transferred to a nylon membrane according
to manufacturer’s specifications (Hybond; Amersham, Arlington
Heights, IL). The membrane was hybridized withrandom primed
probes generated against the E6E7 insert. A total of 50,000 cpm
was hybridized overnight at 42°C and washed twice with 2 x SSPE
at 25°C and then washed two more times with 0.2X SSPE containing
0.1% sodium dodecyl sulfate (SDS) at 60°C before autoradiography.
Isolation of CD34+/38’, 38‘”cells. Adult marrow from cadaveric
donors was obtained from the Northwest Tissue Center (Seattle,
WA). The mononuclear cells were isolated by Ficoll density centrifugation and red blood cells (RBCs) were removed by hemolysis with
150 mmom NH4CL, at 37°C.Marrow mononuclear cells were stored
frozen in RPMI, 36% FCS, 10% dimethylsulfoxide (DMSO), 90 U
penicillin, 90 mg/mL streptomycin sulfate, and 0.36 mg/mL glutamine. The stored cells were thawed at 37°C and slowly diluted on
ice to a final DMSO concentration less than 1 %. After washing, the
CD34’ cells were labeled with anti-CD34 conjugated to fluorescein
isothiocyanate (FITC; HPCA-2 [IgG,]; Becton Dickinson, San Jose,
CA) for 20 minutes on ice, washed with PBS containing I % bovine
serum albumin (BSA), and then labeled withrat antimouse IgGl
conjugated to superparamagnetic microbeads (Miltenyi Biotec
GmbH, Bergisch-Gladbach, Germany).” The CD34’ cells were positively selected using High-Gradient Magnetic Cell Sorting (Miltenyi
Biotec GmbH). The CD34’-enriched population was then incubated
with anti-CD38 conjugated to phycoerythrin (PE; leu-17; Becton
Dickinson) for 20 minutes on ice, washed, and sorted using a FACStar plus (Becton Dickinson). Cells with medium to high forward
light scatter and low side scatter were selected and both the 38’ and
the 38’” populations of CD34’ cells were collected.
Stromal cell functional analysis. Screening was initiated by plating stromal cells at a density of 600 to 1,000 per well in terasaki
96-well plates (Nunc, Naperville, IL) 2 days before the addition of
approximately 200 hematopoietic cells. 3gh’and 38’”cells were added
to the cultures in serum-deprived medium (Nutridoma-HU; Boehringer Mannheim, Indianapolis, IN) and incubated at 37°C in a 5%
CO, humidified incubator for 5 days. The viability and proliferation
of progenitors was scored after the addition of 5 pL of a staining
mixture that contained 2.5% India ink, 250 yg/mL ethdium bromide,
and 75 yg/mL acridine orange in HBSS. The number of viable cells
was determined for each well by inverted fluorescence microscopy
(Nikon Diaphot microscope).
Long-term support of CFCs. Two- to four-week-old primary
LTCs. established and maintained according to previously published
and stromal cell lines were irradiated at 2,000 cGy
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
999
IMMORTALIZED HUMAN STROMAL CELLLINES
and plated into 24-well plates at least 24 hours before the addition
of hematopoietic cells. Irradiated stromal cells were plated at sufficient cell densities to insure formation of monolayers. The stromal
cultures were seeded with1 , 0 0 0 to 3,000 38’”cells and were demidepletedweekly for 5 or 8 weeks. The nonadherentandadherent
cellswereharvestedandanalyzed
for CFCs using colony assays
(described below).
Conditioned media (CM). Media were conditioned by exposure
to semiconfluent cultures for 1 week.BothRPM1 containing 10%
FCS and serum-deprived ( 1 % Nutridoma-HU) media were used.The
culture debris was pelleted by centrifugation at 2 , 0 0 0 ~for 10 minutes
and the supernatant was then aliquoted and frozen at -20°C.
CM
were thawed only once before use. Concentrated CM was obtained
usingAmicon centriprep I O concentrator (Amicon, Beverly,MA)
according to the manufacturer’s specifications, and protein content
assay (BioRad LaborawasdeterminedusingtheBioRadprotein
tories, Richmond,CA). The same batchofCMwas
assayed for
colony-stimulating activity in standard colony assays and for cytokine content with enzyme-linked immunosorbent assays (ELISAs;
see below). In addition to these assays, 38‘ cells wereincubated
withCM or GF mix for 5 days andthenstained for viability as
described above.
Colony assavs. Colony assays were performed using a stock of
1.2%methylcellulose, 2.5%BSA,25%FCS
(Hyclone796). 100
U penicillin, 1 0 0 pglmL streptomycin sulfate, and 0.1 mol/L pmercaptoethanol. Colony-stimulating activity was provided
either by
a growth factor mix containing I O ng/mL interleukin-l (IL-l), IL3, IL-6. granulocytecolony-stimulatingfactor (G-CSF), granulocyteand 3 U/mL erythromacrophage-CSF (GM-CSF), Kit ligand (U),
poietin (Epo) or by 10% CM. Hematopoietic cells (38’ or 38“’) plus
1 0 0 pL growth factor mix or CMwasaddedto0.9mLofthe
methylcellulose stock. Colony formation was scored at day 14 and
designations were periodically confirmedby Wright-Giemsa staining
of colony cytospins.
ELISAs. Cytokine content of the CM was determined with Quantikinekits(R & D Systems, Minneapolis, MN) according to the
manufacturer’sspecifications.Supernatantswereanalyzedneat
at
1:2 and at 1 5 dilutions.
RESULTS
Immortalization of BM cell lines. LTBMCs were infected with the LXSN-16 E6E7 retrovirus that was packaged
in thePA317 cell line, and infected cells were plated at
limiting dilutions. Twenty-seven foci were identified and
isolated using cloning rings to establish stromal cell lines
(HS-I to HS-27). of which 24 were retained and proved to
be resistant to G418 at 50 pg/mL. All lines were initially
characterized morphologically and histochemically, screened
for maintenance andor proliferation of hematopoietic progenitors (HPs; see below), and then frozen. Several clones,
designated HS-5, HS-21, HS-23, and HS-27, were selected
for more detailed analysis and have been maintained in continuous culture for up to 20 months with periodic analysis
of their phenotypes.
Based on morphology, two distinct cell types were observed, small fibroblastic (HS-5 and HS-21) and large flattened epithelioid-like (HS-23 andHS-27). The two fibroblastic lines, although similar in morphology, differed in
regards to growth patterns. HS-5 forms a reticulum of overlapping cells, reminiscent of astrocytes, whereas HS-21 cells
are well separated and line up in parallel arrays. At higher
densities, HS-5 forms a dense “net” of cells, whereas HS-
- 9.4
”6.6
-4.4
-2.3
2.0
LXSN16 E6E7
->.,m
Fig 1. Southern hybridization of genomic DNA from stromal cell
lines using a radiolabeled probe directed against the E6E7genes.
Bands represent products from EcoRl digestion at a site within the
retroviral insert and a site provided by the flanking genomic DNA.
The lane labeled E6E7 is an EcoRl digest of the pLXSN16E6E7 construct that generates a 6.8-kb band and FSF is genomic DNA from a
nontransformed foreskin fibroblast line. The molecular weight markers (M) are ethidium bromide-stained A Hindlll DNA fragments.
21 forms a contiguous monolayer with discernible cell
boundaries. HS-23 and HS-27 formlarge flattened polygonal
shaped cells that exemplify “blanket” cells andmaintain
numerous intercellular contacts with neighboring cells. HS23 and HS-27 will also form monolayers; however, because
of their flattened morphology, it is difficult to identify distinct cell boundaries.
Clonal analysis of stromal cell lines. Southern hybridization on genomic DNA from these four cell lines was used
first to confirmthat LXSN-16 E6E7 hadintegratedand
second to establish clonality. Figure 1 indicates that all cell
lines contained a retroviral insert(s) with only a single band
present in HS-5 and HS-23. However, HS-21and HS-27
have two bands, indicating either that they contain two inserts or that two clones contribute to the line. Analysis of
foreskin fibroblasts andplasmidDNA
indicated that the
probe was specific for the LXSN-I6 E6E7 integrant.
Indirect immunofluorescence and cytochemistry. All cell
lines were negative for major histocompatibility complex
(MHC) class I1 (DR) and CD14, a macrophage-specific
marker (Table l ) , and positive for antigens normally associated with nonhematopoietic stromal cells. All lines expressed
collagen 111 and IV, with low levels of collagen I detected
on HS-5 and HS-27. Analysis of VCAM-I showed that HS-5
and HS-21 expressed low levels, HS-23 was heterogeneously
positive, and HS-27 was homogeneously strongly positive.
All lines were positive for acid phosphatase, with some differences in the degree of staining. In contrast, HS-5 was
negative for alkaline phosphatase staining, whereas all others
were heterogeneously positive.
These cell lines were also tested for their ability to undergo
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
1000
ROECKLEIN AND TOROK-STORB
Table 1. Indirect Immunofluorescentand Cytochemical
Analysis of the Stromal Cell Lines
Stromal Cell Lines
MoAb
Markers
Smooth
muscle
IA4
actin
classMHC
I
class MHC
II
Fibroblasts, adipocytes,
endothelial
cells
antigen
FVlll
Macrophage (CD14)
Endopeptidase
(CDIO)
Fibronectin
12.8
CD34
endothelial
Stromal,
Mesenchymal
(vimentin)
PlH1-C9
Collagen I
Collagen 111
Collagen IV
489 VCAM-1
phosphatase
Alkaline
phosphatase
Acid
HS-5
HS-21
+++
++t
++t
HS-23
HS-27
+++
60.5
P4.1
+++
++
++
-
-
-
-
6.19
++
++
++
++
-
-
-
-
-
-
-
-
+++
+
leuM3
J5
P l H l++
l
STRO-1
MoAb1340
MoAb1343
MoAbl9lO
++
+
++
+
++
++
-
-
-
-
-
-
-
-
+
+
+
+
-
-
+
+++
++++
+
+
++
+++
+++
+
+l-
+++
+
+
+l-
+
c+
+ +l- +++
+l-
+++
++
Abbreviations: +/-, the cell lines were heterogeneously positive;
-, lack of detectable antigen; ++, good staining; +++,the cells were
strongly positive.
lipogenesis in response to corticosteroids. Dexamethasone,
hydrocortisone, insulin, and a combination of dexamethasone and insulin were added to confluent cell layers for 4
weeks, at which point the cultures were stained for lipid
vacuoles with oil red 0. HS-5 and HS-21 did not accumulate
lipids, whereas a few cells (approximately 1% to 2%) from
HS-27 formed lipid vacuoles in the presence of all steroids
tested. HS-23 formed lipid vacuoles in the presence of dexamethasone only. However, none of the lines acquired the
large multilocular vacuoles commonly observed in adipocytes that are present in LTCS.'~.~'
Short-term hematopoiesis. A rapid screening assay was
developed to assess the viability and expansion of hematopoietic cells when cocultured with the cell lines. Both 38'"
and 38+ cells were cocultured on stromal cell lines in serumdeprived medium (Nutridoma-HU) for 5 days with andwithout IL-3 (10 ng/mL) and then stained to differentiate stromal
from hematopoietic cells and viable cells from dead cells.
All 24 cell lines maintained the viability of both 38' and
38'" subpopulations of CD34' cells for 5 days. When IL-3
was added to the cocultures, the 38' cells increased in number in all cases. However, one cell line, HS-5, was able to
induce the 38' cells to proliferate without exogenous IL-3,
and the addition of IL-3 to HS-5 coculture did not increase
the extent of proliferation beyond that observed with HS-5
alone. Figure 2 shows the difference between the maintenance of 38' cells on HS-21 and the proliferation of these
cells on HS-5. No proliferation of 38" cells was observed
within the 5-day time span of this experiment.
Long-term hematopoiesis. To determine whether these
lines could support less mature hematopoietic cells, we monitored their ability to maintain or produce CFCs from the
38"' population after 5 and 8 weeks (Fig 3). The two repre-
sentative experiments are indicative of the range of CFC
production andshowthat
the stromal lines canmaintain
CFCs at levels comparable to primary LTCs for upto 8
weeks. Interestingly, HS-27, which expresses the highest
levels of VCAM-1, was the only cell line to establish cobblestone regions when incubated with 38'" cells (Fig 4).
Biologic activiv of CM. Only CM from HS-5 induced
proliferation of38' cells in the absence of stromal cells.
Figure 5 shows the extent of proliferation induced byCM
from HS-5 compared with that induced byCM from HS21 and a recombinant growth factor mix. Additionally, we
determined whether CM from HS-5, HS-21, HS-23, and HS27 could support colony formation using colony assays. Consistent with the results of the shorter assay, only CM from
HS-5 supported the growth of colonies from the 38' population and the 38" population. Figure 6 is a comparative analysis between the activity of HS-5 CM, HS-21 CM (with and
without serum), and growth factor mix (GF mix). HS-5 CM,
independent of serum content, generated an equivalent number of G/GM colonies from 38" cells as the GF mix; however, HS-5 CM generated significantly more colonies from
38+ cells (Fig 6A). In contrast, CM from HS-21 supported
significantly fewer G E M from both CD34' subpopulations
compared with HS-5 or GF mix. The relative numbers of
burst-forming units-erythroid (BFU-E) generated by these
CMs from 38" cells were also significantly different and
paralleled the observations with the G E M colonies (Fig 6B).
However, the GF mix generated significantly more BFU-E
from 38+ cells than any CM.
Cytokine analysis in CM. The CMs from four cell lines
were assayed for G-CSF, GM-CSF, KL, leukemia-inhibitory
factor (LIF), IL-6, IL-la, L-3, and IL-11. Only HS-5 and
HS-21 CM contained significant amounts of these cytokines
and were additionally tested for the presence of IL-Ip, ILIRA, IL-2, IL-7, IL-8, epidermal growth factor (EGF), tumor
necrosis factor a (TNFa), transforming growth factor a
(TGFa), and macrophage-inhibitory protein-la (MIP- la).
Figure 7 shows that the majority of the cytokines are present
in HS-5 and HS-21 supernatants at similar levels. However,
HS-5 additionally secretes IL-la, IL-lp, IL-lRA, and LIF.
IL-3 was not found in any supernatant and wasnot detectable
using reverse-transcriptase polymerase chain reaction for IL3 message in mRNA from the HS-5 or HS-21 cell lines (data
not shown). Although it has been reported that IL-3 mRNA
can be detected in primary LTCs, it is not clear what the
cellular source is.'6 Given that the low amounts detected
may be produced by lymphocytes present in LTCs, it is not
surprising that HS-5 does not produce IL-3.
DISCUSSION
The hematopoietic ME is essential for controlling differentiation and proliferation of progenitor cells. The ME is
approximated in vitro by the LTC system, which generates
complex stromal cell layers that are composed of multiple
cell types reflecting the in vivo heterogeneity.',' Unfortunately this complexity complicates efforts to define specific
cellular components that are required for regulation of hematopoiesis. However, investigators have used this system to
define cytokines, adhesion molecules, andmatrix compo-
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
IMMORTALIZED HUMAN STROMALCELLLINES
1001
HS-5
Fig 2. Coculture of 38+ cells with the HS-5 and
HS-21 stromal cell lines for5 days in the absence of
serum or additional cytokines. Small round cells are
viable hematopoietic cells
that have accumulated acridine orange and fluoresce green, whereas
the large
flat cells are stromal cells. Nonviable cells have incorporated ethidium bromide and fluoresce orange. The
same number of 38+ cells were plated into each culture at the initiation of the experiment. The microof a terasakiwell by inverted
graph shows a portion
fluorescencemicroscopy(originalmagnification
x
401.
HS - 21
nents that contribute to hematopoietic reg~lation.’”’~
A furthe metallothionein-regulated promoter system23 that allows
ther dissection of this system into functional components,
cells to return to their “normal” phenotype; however, the
such as the cell lines described here,is needed to determine
residual effects of transformed growth cannot be assessed.
what each component contributes to the overall
ME and how
Previous reports indicated that epithelial cells immortalized by the HPV 16 E6and E7 genes can retain their differenthese activities are regulated.
Human BM stromal cell lines have been generated with
tiated
although
aneuploidy
is observed
late
in
passage cells.“ At the molecular level, E6 binds to and dewhereas attempts for transforthe SV40 large T a~~tigen;~-~’
S
gradesp53,whichfunctionstopreventcellentryinto
mation with c-myc, N-rus, and v-ras have not proven suc~ e s s f u l .Several
~ ~ ~ ~ ’labs using different
SV40 constructs have phase in the event
of DNA dan1age.4~
E7 alone has the ability
to immortalize cells; however, when combined with E6 in
generated cell lines that differ in their morphology and funcLXSN-16 E6E7, they immortalize epithelial cells at a higher
tional activity in ways that appear to be influenced by the
SV40 construct?’ Although these lines display proteins that frequency.” E7 functions by inhibiting the hypophosphoryE2F, thuspreventingcell
are indicativeof nontransformed cell lines, they also exhibit latedformofRb,whichbinds
cycle progression.” Rb, by virtue of binding E2F, is central
properties of transformed cells and some are not
To eliminate or reduce these effects, investigators have usedto cell cycle regulation and, more specifically, entry into S
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
ROECKLEIN AND TOROK-STORB
1002
A
80 -
l
-
T
60 -
K
U
3
G
40
T
20 -
1
OJ-
LTC
B
60
-
50
-
40
-
g
U
3
HS5
HS23
HS27
T
theymay alter cellular phenotypes sufficiently to prevent
characterization of the specific cell types and an understanding of their normal function.
Consistent with the E6E7 immortalization of epithelial
cells, the stromal cell lines reportedherehaveincreased
growth rates, do not undergo senescence (some have been
in continuous culture for 2 years), and retain characteristics
of normal differentiated BM stromal cells. Positive staining
with antibody 6.19 (specific for fibroblasts, endothelial, and
adipocytes), P4.1 (CD10, endopeptidase), and PIHI1 (vimentin) and the absence of a macrophage marker (CD14)
indicate that the cells are mesenchymal in origin. The lack
of W111 antigen indicates that they are not endothelial; however, all lines express collagen type IV, which is consistent
with the endothelial nature of BM ~troma.'~.~''
Only the HS23 cell line responds to dexamethasone, suggesting that it
may be preadip~cytic.~~
The cell lines display a normal staining pattern for smooth muscle actin, vimentin, and cell-associated fibronectin
and
growth is inhibited
confluency.
at
CD34 and STRO-I are absent, which is consistent with the
loss of these markers in normal BM cultures after several
weeks of growth?"' All 24 lines, except HS-5, were heterogeneously positive for both alkaline phosphatase and acid
phosphatase. Similar to HS-5, alkaline phosphatase-negative
cell lines were identified by Lanotte et aIZ4in their attempt
to generate spontaneous human lines.
Overall, the morphologic and phenotypic characteristics
of these cell lines are similar to those of murine BM stromal
T
30-
G
20 10
LTC
HS5 HS27
HS21
Fig 3. Two experiments that represent the range of CFC production by primary LTCs and the stromal cell lines HS-5, -23, and -27
in (A) and HS-5, -21, and -27 in (B). N = 3 for experiment no. 1 and
N = 4 for experiment no. 2. Data are reported as the mean of the
absolute number of CFC produced from adherent and nonadherent
layers in replicate cultures that were initiated with 1,500 38" cells.
Error bars represent SEM. 1.
5 weeks; (0)
8 weeks.
phase by controlling the expression of genes such as crny~.''''.~~
In the LXSN-16 E6E7 retroviral construct, E7 is
downstream of E6, resulting in lower expression of E7. This
level of expression is still sufficient to facilitate cell cycle
progression without significant transformation of the cell.
The SV40 large T antigen also binds Rb, thereby facilitating
entry into S phase; however, numerous additional activities
have been attributed to the large T antigen. Although these
activities are not intrinsically involved in transformation,
Fig 4. Phase contrast micrograph of a cobblestone region from
an 8-week LTC with theHS-27 stromal cell line and 38'" cells cultured
in LTC media (original maginification x 100).
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
IMMORTALIZED HUMAN STROMAL CELL LINES
1003
lines.*'-" However, onlyHS-5can
support CFU growth,
whereas HS-2 1 CM did not support CFU even when concentrated eightfold. There are several ways to interpret this observation: (1) unidentified growth-promoting activity may
be present in HS-5 CM; or (2) the cytokines present in both
HS-5 andHS-21may have more favorable concentrations
in HS-5, or HS-21 may contain an inhibitor. The last possibility was ruledout by mixing experiments (data not shown),
making it reasonable to suggest that HS-5 either has additional unique activities or more favorable combination of
known activities. Either interpretation would support the hypothesis that HS-5 CM would be useful for expanding committed cells ex vivo to facilitate peripheral recovery after
myeloablative therapy. To this end, we have initiated expansion of CD34' cells with HS-5 CM in the absence of serum.
Preliminary data indicates that a 12- to 15-fold expansion
can be achieved within 10 days with a fourfold to sixfold
30
1
CD34+38 lo
f
CD34+38+
T
Fig 5. Small-scale expansion of 38' cells
with GF mix (A), HS-5
CM (B), and HS-21 CM (C). The same number of cellswere added per
well at time0, expanded with different media for 5 days, and stained
with ethidium bromide and acridine orange.
cell
SV40-transformed human cell line^,*^-^' and
transient nontransformed human lines.'" However, no spindle shaped cells were observed, as others have previously
rep~rted,?~
and the HPV-immortalized lines remain MHC
class 11 (DR) negative, as observed with normal marrow.27
Consistent with the results of Novotny et al,27we did not
obtain cell lines with small round hematopoietic cells that
have been observed after SV40 virus transformation of marrow stroma."
HS-5 andHS-21 secrete significant levels of numerous
cytokines, which is consistent with other BM stromal cell
Fig 6. Number of hematopoietic colonies grown from 38' or 38'"
cells in thepresence of GFmix, HS-5 CM, and HS-21 CM with serum
(5) or serum deprived (sd). (A) Granulocyticlmonocyticcolony numbers (GIGM); (B) erythroid bursts (BFU-E). RPMls represents RPM1
media supplemented with 10% FCS. Results significantly different
from HS-5 are designated 1. Results significantly different from GF
mix are designated 2. Error bars represent SEM. * P < .Ol; **P < .05.
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
ROECKLEIN AND TOROK-STORB
1004
IL-8
> std
IL-7
> std
IL-6
1L-4
<std
IL-3
estd
IL-2
<std
IGlRA
ILlg
IL-la
4
500
0
IO00
1500
m
(P%mL)
IL-I1
GM-CSF
tenance factors at levels that are sufficient to support immature pluripotent progenitors. Previous investigations have
also indicated a lack of correlation between cytokine secretion and support of hematopoiesis by stromal cell^.'^.^^ Also
consistent with these observations is the ability of a murine
cell line (MS-5) to maintain immature human hematopoietic
cells:* which accounts for the ability of fixed 3T3 cells to
support murine hematopoietic ~ e l l s . The
4 ~ support of cobblestone regions by the HS-27 cell line indicates that it provides
additional signals for the burrowing of precursors and their
subsequent proliferation beneath the cell layer. This line
should be useful for the identification and isolation of the in
vivo equivalent of the blanket cells0and the signals required
for cobblestone formation.
Together, these observations suggest that the factors responsible for differentiation and proliferation of committed
progenitors are distinct from those required for maintenance
of the immature pre-CFC pool. Moreover, it suggests that
these maintenance factors are ECM or membrane associated.
Continued analysis of the differences between these diverse
cell lines will be useful in determining the role of different
stromal elements in the regulation of hematopoiesis.
ACKNOWLEDGMENT
-I
G-CSF
M-CSF
The authors thank Gretchen Johnson, Laurie Pfister, and Ludmila
Golubev for excellent technical assistance and Bonnie Larson for
preparing the manuscript.
KL
REFERENCES
“la
LIF
TGFa
<std
TNFa
estd
%F
estd
0
i
1 o O 0 m 3 o O 0 4 o O 0 5 o o o
(*L)
Fig 7. ELISA results showing the similarity in cytokines secreted
by HS-5 and HS-21. (M) HS-5; ( 0 )HS-21. <std indicates that the
cytokine level was less than thedetectable limits of the ELISA. >std
indicates that the level was greater than the standard curve. The
supernatantswere analyzed neat at 1:2 and 1:5 dilutions. Data represent the concentrationfrom one or more dilutions that were within
the standard curve.
increase in clonogenic cells (data not shown). Additional
experiments are necessary to evaluate the retention or expansion of LTCIC with this CM. In contrast to HS-5 and HS21, the “blanket” cell lines HS-23 and HS-27 only secrete
small quantities of cytokines and, as expected, CM from
these lines do not support colony growth (data not shown).
Both the fibroblastic and “blanket” cell lines supported
CFC for 5 to 8 weeks at levels comparable to those of
primary LTC. These results indicate that, independent of
phenotypic differences and detectable cytokine secretion,
these diverse stromal cell lines produce hematopoietic main-
1. Lichtman MA: The ultrastructure of the hematopoietic environment of the marrow: A review. Exp Hematol 9:391, 1981
2. Allen TD, Dexter TM: The essential cells of the hematopoietic
microenvironment. Exp Hematol 12517, 1984
3. Charbord P, Gown AM, Keating A, Singer J W : CGA-7 and
HHF, two monoclonal antibodies that recognize muscle actin and
react with adherent cells in human long-term bone marrow cultures.
Blood 66:1138, 1985
4. Charbord P, Lerat H, Newton I, Tamayo E, Gown AM, Singer
JW, Herve P The cytoskeleton of stromal cells from human bone
marrow cultures resembles that of cultured smooth muscle cells.
Exp Hematol 18:276, 1990
5. Galmiche MC, Koteliansky VE, Briere H, Herve P, Charbord
P: Stromal cells from human long-term marrow cultures are mesenchymal cells that differentiate following a vascular smooth muscle
differentiation pathway. Blood 82:66, 1993
6. Sutherland HJ, Lansdorp PM, Henkelman DH, Eaves AC,
Eaves CJ: Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow
stromal layers. Proc Natl Acad Sci USA 87:3584, 1990
7. Simmons PJ: CD34 expression by stromal precursors in normal
human adult bone marrow. Blood 78:2248, 1991
8. Sutherland HJ, Eaves CJ, Eaves AC, Dragowska W, Lansdorp
PM: Characterization and partial purification of human marrow cells
capable of initiating long-term hematopoiesis in vitro. Blood
74:1563, 1989
9. Visser JW, Van Bekkum DW: Purification of pluripotent hemopoietic stem cells: Past and present. Exp Hematol 18:248, 1990
10. Terstappen L W ” , Huang S, Safford M, Lansdorp PM, COken MR: Sequential generations of hematopoietic colonies derived
from a single nonlineage committed CD34+/CD38-progenitor cells.
Blood 77:1218, 1991
1 1. Eaves CJ, Sutherland HJ, Udomsakdi C, Lansdorp PM, Szil-
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
IMMORTALIZED HUMAN STROMAL CELL LINES
vassy SJ, Fraser CC, Humphries RK, Bamett MJ, Phillips GL, Eaves
AC: The human hematopoietic stem cell in vitro and in vivo. Blood
Cells 18:301, 1992
12. Ogawa M: Differentiation and proliferation of hematopoietic
stem cells. Blood 81:2844, 1993
13. Muller-Sieburg CE, Deryugina EI: Stromal cells in long term
cultures: Keys to theelucidation of hematopoietic development? Crit
Rev Immunol 13:115, 1993
14. Dorshkind K: Regulation of hemopoiesis by bone marrow
stromal cells and their products. Annu Rev Immunol 8:lll. 1990
15. Liesveld JL, Winslow JM, Kempski MC, Ryan DH, Brennan
JK, Abboud CN: Adhesive interactions of normal and leukemic
human CD34+ myeloid progenitors: Role of marrow stromal, fibroblast, and cytomahix components. Exp Hematol9:391, 1981
16. Kittler ELW, McGrath H, Temeles D, Crittenden RB, Kister
VK, Quesenberry PJ: Biological significance of constitutive and subliminal growth factor production by bone marrow stroma. Blood
79:3168, 1992
17. Eaves CJ, Cashman JD, Kay RI, Dougherty GJ, Otsuka T,
Baboury LA, Hogge DE, Lansdorp PM, Eaves AC, Humphries RK:
Mechanisms that regulate cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. 11. Analysis of
positive and negative regulators produced by stromal cells within
the adherent layer. Blood 78:110, 1991
18. Clark BR, Gallagher JT, Dexter TM: Cell adhesion inthe
stromal regulation of hemopoiesis. Clin Haematol 5:619, 1992
19. Heinrich MC, Dooley DC, Freed AC, Band L, Hoatlin ME,
Keeble WW, Peters ST, Silvey KV, Ey FS, Kabat D, Maziarz RT,
Bagby GC Jr: Constitutive expression of steel factor gene by human
stromal cells. Blood 82:771, 1993
20. Simmons P, Torok-Storb B: Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody,
STRO-l. Blood 7855, 1991
21. Zipori D, Friedman A, Tamir M, Silverberg D, Malik Z:
Cultured mouse cell lines: Interactions between fibroblastoid cells
and monocytes. J Cell Physiol 118:143, 1984
22. Zipori D, Duskin D, Tamir M, Argman A, Toledo J, Malik
Z: Cultured mouse marrow stromal cell lines. 11. Distinct subtypes
differing in morphology, collagen types, myelopoietic factors, and
leukemic cell growth modulating activities. J Cell Physiol 122:81,
1985
23. Song ZX, Quesenbeny PJ: Radioresistant murine marrow
stromal cells: A morphologic and functional characterization. Exp
Hematol 12523, 1984
24. Lanotte M, Allen TD, Dexter TM: Histochemical and ultrastructural characteristics of a cell line from human bone-marrow
stroma. J Cell Sci 50:281, 1981
25. Harigaya K, Handa H: Generation of functional clonal cell
lines from human bone marrow stroma. Proc Natl Acad Sci USA
82:3477, 1985
26. Tsai S, Emerson SG, Sieff CA, Nathan DG: Isolation of human stromal cell strain secreting hemopoietic growth factors. J Cell
Physiol 127:137, 1986
27. Novotny JR, Duehrsen U, Welch K, Layton JE, Cebon JS,
Boyd AW: Cloned stromal cell lines derived from human WhitlocW
Witte-type long-term bone marrow cultures. Exp Hematol 18:775,
1990
28. Slack JL, Nemunaitis H, Andrews DF 111, Singer JW: Regulation of cytokine and growth factor gene expression in human bone
marrow stromal cells transformed with simian virus 40. Blood
75:2319, 1990
29. Singer J W , Charboard P, Keating A, Nemunaitis J, Raugi G ,
Wight TN, Lopez JA, Roth GJ, Dow LW, Fialkow PJ: Simian virus
40-transformed adherent cells from human long-term marrow cultures: Cloned cell lines produce cells with stromal and hematopoietic
characteristics. Blood 70:464, 1987
1005
30. Cicutinni FM, Martin M, Salvaris E, Asnman L, Begley CG,
Novotny J, Maher D, Boyd AW: Support ofhuman cord blood
progenitor cells on human stromal cell lines transformed by SV40
large T antigen under the influence of an inducible (metallothionein)
promoter. Blood 80:102, 1992
31. Thalmeir K, Meibner P, Reisbach G , Falk M, Brechtel A,
Dormer P: Establishment of two permanent human bone marrow
stromal cell lines with long-term post irradiation feeder capacity.
Blood 83:1799, 1994
32. Halbert CL, Demers GW, Galloway DA: The E7 gene of
human papillomavirus type 16 is sufficient for immortalization of
human epithelial cells. J Virol 65:473, 1991
33. Ryan MJ, Johnson G , Kirk J, Fuerstenberg SM, Zager RA,
Torok-Storb B: HK-2: An immortalized proximal tubule epithelial
cell line from normal adult human kidney. Kidney Int 45:48, 1994
34. Gartner S, Kaplan HS: Long-term culture of human bone
marrow cultures. Blood 56:117, 1980
35. Kaplow LS: Cytochemistry of leukocyte alkaline phosphatase. Use of complex napthol AS phosphates in azo dye-coupling
technics. Am J Clin Path01 39:439, 1963
36. Burstone MS: Enzyme Histochemistry and Its Application in
the Study of Neoplasms. New York, NY, Academic, 1962
37. Kodama HA, Amagai Y, Koyama H, Kasai S: Hormonal
responsiveness of a preadipose cell line derived from newborn mouse
calvaria. J Cell Physiol 112233, 1982
38. Ausebel F, Brent R, Kingston RE, Moore DD, Seidman JG,
Smith JA, Struhl K (eds): Current Protocols in Molecular Biology.
New York, NY, Wiley Interscience, 1987
39. Miltenyi S, Muller W, Weichel W, Radbruch A: High gradient
magnetic cell separation with MACS. Cytometry 11:231, 1990
40. Eaves CJ, Cashman JD, Eaves AC: Methodology of longterm culture of human hematopoietic cells. J Cult Meth 1355, 1991
41. Rothstein L, Pierce JH, Klasser V, Greenberger JS: Amphotrophic retrovirus vector transfer of the V-ras oncogene to human
hematopoietic and stromal cells in continuous bone marrow cultures.
Blood 65:744, 1985
42. White AE, Livanos EM, Tlsty TD: Differential disruption of
genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins. Genes Dev 8:666, 1994
43. Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM: The E6oncoprotein encoded by human papillomavirus types
16 and 18 promotes degradation of p53. Cell 63:1129, 1990
44. Hamel PA, Gill RM, Phillips RA, Gallie BL: Transcriptional
repression of the E2-containing promoters EIIa, c-myc, and RBI by
the products of the RB1 gene. Mol Cell Biol 12:3431, 1992
45. Weintraub SJ, Prater CA, Dean DC: Retinoblastoma protein
switches the E2F site from a positive to a negative element. Nature
358:259, 1992
46. Zipori D: Cultured stromal cell lines from hematopoietic tissues, in Tavassoli M (ed): Handbook of the Hematopoietic Microenvironment. Clifton, NJ, Humana, 1989, p 287
47. Kittler EL, McGrath H, Temeles D, Crittenden RB, Kister
VK, Quesenheny PJ: Biologic significance of constitutive and subliminal growth factor production hy hone marrow stroma. Blood
79:3168, 1992
48. Issaad C, Croisille L, Katz A, Vainchenker W, Coulombel L:
A murine stromal cell line allows the proliferation of very primitive
human CD34"/CD38- progenitor cells in long-term cultures and
semisolid assays. Blood 81:2916, 1993
49. Roberts RA, Spooncer E, Parkinson EK, Lord BI, Allen TD,
Dexter TM: Metabolically inactive 3T3 cells can substitute for marrow stromal cells to promote the proliferation and development of
multipotent haemopoietic stem cells. J Cell Physiol 132:203, 1987
50. Clark BR, Gallagher JT, Dexter TM: Cell adhesion inthe
stromal regulation of haemopoiesis. Clin Haematol 5:619, 1992