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Connexin-43-Type Gap Junctions Mediate Communication Between
Bone Marrow Stromal Cells
By Kenneth Dorshkind, Lora Green, Angela Godwin, and William H. Fletcher
Several morphologic studies have suggested that gap
junctions exist between bone marrow stromal cells. This
possibility was examined by analysis of stromal cells present in the adherent layer of primary long-term lymphoid
bone marrow cultures and in additional studies using a
stromal cell line. Results showing that the fluorescent dye
lucifer yellow, when microinjected into a single stromal
cell, transferred between most other contacting stroma
and that stromal cells were electrotonically coupled provided support that cell-cell communication occurs between these microenvironmental elements. Additional
studies showed that transcripts for connexin (Cx) 43, but
not for Cx26 or Cx32, were present in a stromal cell line.
To examine the potentialfor regulatedcell-cellcommunication between the stroma, cells were treated with interleukin-1 (IL-1), a cytokine known to affect stromal cell function, and the effects on dye transfer were examined. IL-1
treatment resulted in a reversible decrease in the ability of
dye to transfer between stromal cells in contact. Taken
together, these studies show that gap junctions exist between stromal cells and that their permeability can be regulated. However, gap junction-mediated cell-cell communication could not be shown between the stroma and
developing lymphoid cells.
0 1993 by The American Society of Hematology.
H
whether their expression is sensitive to physiologic mediators known to affect stromal cell function. Expression of Cx
protein and the permeability of gap junctions between cells
in many tissues is a highly regulated process, and hormones
and/or growth factors can affect the degree to which intercellular communication occurs.
In addition to associations between the stroma, considerable heterocellular contacts occur between stromal cells and
hematopoietic cells in the medullary cavity. This is particularly true for developing cells of the B-lymphocyte lineage,
which intimately associate with stromal cell membrane processes.1,2,22
Because gap junctions occur between heterologous cell types,I3the possibility that the stroma can transmit
developmental signals to immature lymphocytes via gap
junctions must also be considered.
To address these issues, experiments were conducted to
establish if gap junctions and cell-cell communication are
present between stromal cells in lymphoid long-term bone
marrow cultures and to investigate the potential for gap
junction-mediated heterocellular contacts between stromal
cells and developing B-lineage cells. Taken together, the
data indicate that gap junctions exist between stromal cells
and suggest that cell-cell communication in the bone
marrow is a dynamic process affected by mediators known
to regulate stromal cell function. The results also indicate
that gap junction-mediated cell-cell communication could
not be shown between the stroma and developing lymphocytes.
EMATOPOIESIS occurs in the intersinusoidal spaces
of the medullary cavity in association with a three-dimensional framework of supporting stromal cells.'-4 The
stroma are considered to form a hematopoietic microenvironment that supports blood cell de~elopment,~
and this is
mediated in part via the regulated secretion of soluble factors and through direct contacts with developing blood
cells."* A characteristic property of stromal cells is that they
interact with each other via numerous filopodia3x4;several
morphologic studies have concluded that gap junction-like
structures exist at these site^.^-^ Gap junctions have been
implicated to be important in the control of normal function between ~ e l l s . ' If~ they
~ ' ~ exist between the stroma, this
could have considerable consequences for the coordination
of stromal cell function and for defining the structural organization of the hematopoietic microenvironment.
Gap junctions are formed by a family of proteins referred
to as connexins (Cx).I5-l9However, the Cx phenotype of
stromal cells has not been established and sensitive measures of cell-cell communication, such as electrotonic coupling, have not been performed. If bona fide gap junctions
are present between stromal cells, an additional issue is
From the Division of Biomedical Sciences, University of California, Riverside, CA; and the Department of Anatomy. Loma Linda
University School of Medicine and Molecular Cytology, Veterans
Administration Medical Center, Loma Linda. CA.
Submitted August 27, 1992; accepted February 8, 1993.
Supported by National Institutes of Health (NIH) Grants No.
HL36591 (K.D.) andHD21318 (W.H.F.), the VeteransAdministration Research Service, and Loma Linda University School ofMedicine. K.D. is the recipient of Research Career Development Award
AI00843 fvom the NIH. W.H.F. is a Research Career Scientist
Awardee of the Veterans Administration.
Address reprint requests to Kenneth Dorshkind, PhD, Division of
Biomedical Sciences, University of California, Riverside, CA
92521-0121.
The publication costs of this article were defvayed 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 1993 by The American Society of Hematology.
0006-4971/93/8201-0012$3.00/0
38
'3320,21
MATERIALS AND METHODS
Mice and cell preparation. Male or female BALB/cAn mice, 4
to 8 weeks old, were bred and maintained in the vivarium of the
Division of Biomedical Sciences, University of California (Riverside, CA). Mice were killed by cervical dislocation and femurs and
tibiae were removed and placed in a-minimal essential medium
(a-MEM; GIBCO, Grand Island, NY).
Long-term bone marrow cultures. For the studies described
herein, the long-term lymphoid bone marrow culture system described by Whitlock and WitteZ3was used because of the relative
ease in distinguishing hematopoietic and stromal cell populations
from one another. Cultures were initiated by plating 13.5 mL of a 1
X IO6 cells/mL suspension of bone marrow into IO-cm diameter
tissue culture plates in RPMI- 1640 (GIBCO) supplemented with
Blood, VOI 82,NO 1 (July 1). 1993:pp 38-45
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39
Cx43 GAP JUNCTIONS BETWEEN STROMAL CELLS
Fig 1. Dye transfer between
primary stromal cells. (A) Phase
contrast photomicrograph showing stromal cells in primary
long-term lymphoid bone marrow culture conditions and (E)
dye transfer between stromal
cells in contact. (*) The injected
stromal cell. The circular areas
to which dye did not transfer
are lymphocytes (see Fig 7 and
text).
5% fetal calf serum (FCS: JR Scientific, Woodland. CA) and 5 X
IO-' mol/L 2-mercaptoethanol(2-ME: Sigma, St Louis. MO). By 2
weeks postinitiation, a confluent adherent layer had established: by
4 weeks. foci of lymphoid cells were apparent. Cultures were fed
twice weekly.
Cell lines. The generation of the S I7 stromal cell line has been
described in detaiLz4The line was isolated from the adherent layer
of an established myeloid long-term bone marrow culture and
forms confluent, contact-inhibited monolayers. This line was chosen because it supports myelopoiesis and lymphopoiesis by direct
cell-cell interactions and via secretion of soluble mediators that affect the proliferation and/or differentiation of hematopoietic
celIs.2'26 The line was maintained in a-MEM supplemented with
5% FCS and incubated in a 5% CO, and air incubator at 37°C.
Electrotonic coupling and dye
Preparation ~~/e~~si~r.slipcirltrrrc.s.
injection studies were performed in cells grown on precleaned. 25mm diameter glass coverslips. Coverslip cultures were initiated
with SI7 stromal cells alone or with SI7 cells mixed with B-lineage
cells harvested from 4- to 6-week postinitiation primary lymphoid
bone marrow cultures. Alternatively, the adherent layer of a primary long-term culture was harvested with collagenase treatment
and replated onto coverslips.
In some experiments 50 U/mL of interleukin-I (IL-I; specific
activity, 2 X IO8 U/mg: Hoffmann-LaRoche, Nutley. NJ) were
added to stromal cell cultures grown on coverslips for different periods of time. The amount of RPMI-1640 medium in which IL-I
was added did not exceed I % of the total volume of the cultures.
This procedure eliminated any effects on intercellular communication attributable to replacement of larger volumes of medium.
Microinjwtion und c4ectrotonic coirpling stirdies. Microinjection was performed by standard iontophoretic techniques and digital images were obtained using a microcomputer-based image processor (Universal Imaging. Media. PA) with input from a
silicon-intensified target camera (Dage, Model 66) sideport
mounted to a Zeiss IM 405 inverted phase/fluoresence microscope
(Zeiss Inc, Thornwood. NY). Electrotonic coupling studies were
performed on stromal cell cultures without lymphocytes. Stromal
cell pairs were impaled with microelectrodes and the amplitude of
ionic coupling was measured by described methods?'.28
f.solution ( ? / R N A . Total cellular RNA from stromal cells was
prepared by solubilizing cell pellets in TSM (IO mmol/L Tris. pH
7.5. 150 mmol/L NaCI, 2 mmol/L MgCI,. 0.5% NP40) and Vanadium ribonucleoside complex (BRL. Grand Island. NY) on ice for
3 minutes with occasional vortexing. After 1 minute ofcentnfuga-
Table 1. Cell-Cell Communication Between Stromal Cells
No. of
Cultures
Attempts'
Primary cultures
Stromal cell line
21
23
No. of Stroma in
Contact With
Dye-Injected
Cellt
91
119
No. of Contacting
Cells to Which
Dye Transferred
Frequency
Frequency
of 1st Order
of2nd Order
Transfert
Transfer5
60
65.9
87
73.0
47.6
43.0
Number of individual cells injected with dye.
t Total number, from all experiments, of stromal cells in contact with dye-injected cell.
*
$
Frequency =
No. of Contacting Cells to Which Dye Transferred
x 100
No. of Cells in Contact With Injected Cell
5 These data refer to the frequency with which transfer to at least one second order cell was observed. Thus, of t h e 21 attempts with primary
cultures, dye transfer was observed in second order cells in 10 instances. Of the 23 attempts with the stromal cell line, dye transfer to at least one
second order cell occurred in 10 instances.
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DORSHKIND ET AL
40
Fig 2. Dye transfer between
cloned stromal cells. (A) shows
a photomicrograph of the S17
stromal cell line grown under
lymphoid culture conditions and
(E) demonstrates that, after injection of lucifer yellow into a
single stromal cell, dye transfer
to second order neighbors can
be observed. (*) The microinjected cell.
tion. supernatants were mixed with TSE ( 10 mmol/L Tris. pH 7.5.
I 50 mmol/L NaCI. 5 mmol/L EDTA. and I r i , sodium dodeoylsulfate [SDS]). After two extractions for 30 seconds on ice with an
q u a l volume o f pheno1:chloroform:isoamylalcohol (PCI: 2424: I ),
I 5 pL of 500 mmol/L EDTA was addcd to the aqueous phasc.
followed by one sequential extraction i n PCI followed by chloroform:isoamyl alcohol. One-tenth volume o f 3 mol/L sodium acetate plus 2 vol of IOO''; ethanol was added to the aqueous phase. and
after I hour at -70°C. the pellet obtained was rcsuspendcd in TE
( I O mmol/L Tris. pH 7.5. I mmol/l. EDTA. and 0. If';,SDS) and
1/10 vol 3 mol/L sodium acetate plus 2 vol 100r;8
ethanol. Afier.an
additional I hour of incubation at -70°C. the pellet was redissolved
in TE plus 0.1% SDS and stored at -70°C. Alternatively. total
cellular RNA was prepared from cells by extraction with guanidinium thiocyanate followed by centrifugation in a CsCl gradient.
Liver and heart poly(/\+) RNA was further purified from total cytoplasmic RNA by chromatography on oligddT)-ccllulose columns.
.Vor//wrfr /)/o//itt,q. RNA WJS electrophoresed under denaturing
conditions i n a 1.5T agarose gel containing 0.66 mol/L formaldchydc and IX MOPS buffer. The gels were capillap blotted with
high salt onto Hybond-N nylon membranes (Amersham. Arlington
Heights. IL).Transcripts were visualized by autoradiography o f "PCx43. CxX. or Cx26 CDNAS" (generous gifts o f Drs D. Cioodcnough. D. Paul. R. Nicholson. and E. Rcycr) or n-actin cDNA (a
generous gift o f S . Spindler) labeled by random priming to a specific
activity o f IOR cpm/pg or more. I-lybridizations were performed at
42°C for I 8 to 24 hours i n buffer consistingof 50%deionized formamide. 5 X Dcnhardt's. SX SSPE. pH 7.4. O.lrk SDS. 100 to 200
pg/mL sheared sperm DNA. and I to 2 X IO6 cpm/mL o f labeled
Cx cDNA. ARer hybridiiation. blots were washcd twice with 6X
+.
A
4
do
18s28s
I,
Cx32
Cx43
18s28s18s
~
Cx26
-
19
ACTIN
Fig 4. Expression of connexin mRNA by bone marrow stromal
cells. (A) RNA from heart or S17 stromal cells was probed with a
Fig 3. Stromal cells are electrotanically coupled. Each S17 stromal cell of a pair was impaled with a high resistance microelectrode. When 2 nA current was injected (V,) into one cell, it was
detected (V,) in the partner cell at a ratio of about 0.7 (V2/Vl).
Cx43 cDNA. Heart tissue was used as a positive control. because
Cx43 represents the major component of the heart gap j~nction.'~
(E) No Cx26 or Cx32 message can be detected in stromal cells.
Liver was used as a positive control, because Cx26 and Cx32 are
major components of the liver gap juncti~n.'~
In this experiment,
the same filter was stripped and reprobedwith the various cDNAs.
Note the presence of actin transcripts in the stromal cell lane.
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41
3
78.3f
100
1 7
02.7f
86.6f
4 0
6 A
65.8f
--
80
-
60
-
..
11
-2
C
0
0
c
0
8
a
W
LL
(I)
z
<
a 40
e
-
w
>
n
19.0 f
4.9
20 -
L
NO
IL-1
1L-1
16hr
IL-1
20hr
IL-1
24hr
IL-1
30hr
I
Fig 5. Effects of IL-1 on stromal cells at various times after
treatment. The frequency of dye transfer in stromal cells at 16,20,
24, and 30 hours after IL-1 exposure relative to that occurring in
stromal cells not exposed to IL-I is plotted. The numbers over each
bar indicate the frequency f standard error in which dye transferred to first order neighbors. The data are compiled from one of
two independent experiments and represent at least three independent microinjections per time point.
SSPE, 0.1% SDS at 42°C for 15 minutes; twice with 1X SSPE, 0.1%
SDS at 42°C for 15 minutes: and then once with 0.5X SSPE, 0.1%
SDS at 65°C for 30 minutes. Autoradiography was performed with
X-Omat (Eastman Kodak, Rochester, NY) film and Dupont
Cronex screens (Dupont, Wilmington, DE).
In some experiments, blots were rehybridized after the removal
of radioactive probes by incubating membranes in 1 mmol/L TrisC1 (pH 8.0), 1 mmol/L EDTA (pH 8.0), and 0.1X Denhardt's reagent for 2 hours at 75°C.
RESULTS
Gap junctions are present between stromal cells. The
contacts observed between stromal cell processes in vivo are
present under long-term lymphoid bone marrow culture
conditions. Therefore, primary cultures were used initially
to test the potential for cell-cell communication by injecting
the fluorescent dye lucifer yellow (molecular weight, 443
Kd) into a single stromal cell. As shown in Fig I , after the
introduction of dye to the interior of one stromal cell, appreciable dye transferred to contacting stromal cells. In 9 1 in-
stances in which distinct contacts between the primary
neighbors and the injected stromal cell could be confirmed
by phase contrast microscopy, dye transferred to the first
order cell with a frequency of 65.9%.In addition, transfer to
at least one second order cell was observed in 47% of cases
(Table 1 and Fig 2). In cultures initiated with a stromal cell
line that supports B lymphopoiesis, the frequency of dye
transfer to contacting first order stromal cells was 73%; in
43% of cases, transfer to at least one second order neighbor
was observed (Table 1).
In the above experiments, dye transfer between the injected stromal cell and its neighbors occurred in 10 seconds
or less (data not shown). However, in approximately 30% of
attempts using either primary or cloned stromal cells, dye
did not transfer between all cells in first order contact with
the injected cell. In some instances, dye stayed in the injected cell even though it contacted one to three neighbors.
In other cases, dye moved out of the injected cell to some
but not all contacting cells.
In addition to dye transfer, the degree of ionic coupling
between stromal cells was tested using the cell line to provide a more sensitive measure of cell-cell communicaIn the example shown, stromal cell pairs were
found to be well-coupled electrotonically, with a bidirectional V2/V, ratio of 0.7 or higher (Fig 3).
Stromal cells express Cx43 trunscripts. To identify the
gap junction phenotype of stromal cells, stromal cell
mRNA, prepared from the stromal cell line, was probed
with cDNAs specific for Cx26, 32, and 43. As shown in Fig
4, only Cx43 mRNA could be identified in stromal cells.
Cell-cellcommunication between stromal cells is sensitive
to extracellular mediators. The expression of gap junctions between cells is known to be sensitive to a variety of
regulatory signals that include growth factor^.'^,^',^' To determine if this effect potentially applies to the stroma, 50
U/mL of IL- 1a, a mediator known to bind to receptors on
stromal cells and affect the type and concentration of factors
~ e c r e t e d , ' .was
~,~~
added to cultures initiated with the stromal cell line and dye transfer between cells analyzed at
various intervals thereafter.
IL- I treatment resulted in a decrease in dye transfer between stromal cells that was first observed at 20 hours postIL-I treatment (Fig 5); at the 24-hour time point, there was
an 80% reduction in transfer relative to the control cultures
(Figs 5 and 6). Phase contrast microscopy indicated that this
inhibition of intercellular communication was not due to
observable effects of IL-I on cell adhesion or numbers of
intercellular contacts. The degree of dye transfer between
cells had returned to steady-statelevels by 30 hours post-ILI treatment (Fig 5).
Gapjunctions cannot be shown between lymphocytes and
stromal cells. It has been suggested that gap junctions occur between stromal cells and hematopoietic cell^.^,^^ To
determine the potential for such communication between
the stroma and B-lineage cells, the DNA-binding fluorescent dye lucifer yellow was injected into an S 17 stromal cell
with which numerous lymphoid cells were associated. Although dye often transferred to contacting stromal cells (Fig
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DORSHKIND ET AL
42
Fig 6. 11-1 treatment of
stromal cells results in an inhibition of dye transfer. (A) shows
the phase contrast photomicrograph of cloned stromal cells
and dye transfer between them
(B)in cultures at 16 hours posttreatment with IL-1. (C) shows
the phase contrast photomicrograph of cloned stromal cells at
24 hours post-IL-1 treatment.
(D) shows that dye transfer between cells was inhibited at
this time.
7). none was ever observed to transfer to associated lymphoid cells (Table 2).
DISCUSSION
Numerous studies have concluded that gap junction-like
structures exist at sites of stromal-stromal cell contact^.^^.^*
However. no study has established whether these populations are electronically coupled, a more sensitive indicator
of cell-cell communication. Also. no study has defined the
Cx phenotype of gap junctions that potentially unite the
stroma. Accordingly, experiments were designed to molecularly characterize these gap junctions. Additional studies
investigated the potential for gap junction-mediated communication between stromal cells and developing B
lymphocytes.
It has been suggested that hematopoietic cells can form
gap junctions with the ~troma.~.’”’~
To investigate whether
these structures play a role in stromal cell-dependent B lymphopoiesis, a long-term bone marrow culture system optimal for that process was used. Dye transfer between stromal
cells and contacting lymphocytes was never observed under
these lymphoid permissive culture conditions, a result consistent with our previous ultrastructural analysis that failed
to detect junctional complexes between lymphocytes and
stromal cells.34Therefore. if heterocellular gap junction-mediated contacts occur between lymphoid and stromal cells
in these cultures. they exist at a frequency below the ability
to be detected by the approaches used herein. The Whitlock-Witte culture conditions were used because the aim of
this study was to analyze heterocellular contacts between
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Cx43 GAP JUNCTIONS BETWEEN STROMAL CELLS
43
Fig 7. No dye transfer occurs between stromal cells and
lymphocytes. (A) Phase contrast photomicrograph showing
t w o foci of lymphocytes associated with S17 stromal cells
and (B) lackof dye transfer from
the injected stromal cell into
contacting lymphoid cells,
which appear as black holes.
However, note that dye has
transferred to the adjacent
stromal cell. The nuclei of two
stromal cells (*) and lymphoid
cell clusters (?) are indicated.
stromal cells and lymphocytes. Ncvcrthclcss. thc prcsent rcsults do not exclude the possibility that hctcroccllular gap
junctions may exist hctwccn stromal cells and hematopoietic cells under different culture conditions. although it has
k e n noted that gap junction-mcdiatcd intcrccllular contacts do not play a major role in mycloid long-tcrm bonc
marrow
Several indcpcndcnt mcasurcmcnts indicatc that gap
junctions exist bctwccn the stroma. First. transfcr of the low
molecular weight dye lucifcr ycllow could hc shown hctween cells. Examination of cloned and primary stroma
showed that in more than 200 instanccs in which direct
contacts bctween the injected cells and first order neighbors
could he observed. dye transfcr occurrcd in approximatcly
70rh of instances. In agreement with thcsc observations arc
the findings that show that at lcast somc stromal cells are
Table 2. Lack of Dye Transfer Between Stromal Cells and
Developing Lymphocytes
No. of Stromal
Cell-Associated
Lvmphocvtes'
Experiment
No.
Frequency
of Dye
Transfer (%)
~~
0
0
3
4
5
6
7
8
9
10
3
2
2
1
9
17
19
15
26
0
11
19
1
2
23
0
0
0
0
0
0
0
0
In most instances. multiple lymphoidcells were observedin association with a single stromal cell.
clcctrotonically couplcd. Finally. Northcrn blot analysis
showcd that transcripts for Cx43 wcrc prcscnt in stromal
cclls.
Gap junctions arc formcd by protein subunits known as
Cxs. and molccular cloning techniques indicatc that at least
eight distinct spccics may exist.36Of thcsc spccics. thc oncs
most thoroughly studicd are CxX. 3 2 . and 43. Thc data
prcscntcd hcrc indicatc that Cs43. but not Cx26 or 32.
mRNA is prcscnt in stromal cclls. a finding consistcnt with
thc observation that many mesodcrmally dcrivcd tissucs.
including capillary cndothclium and fibroblasts. also cspress that spccics.3" Howcvcr. until a thorough suncy of
additional Cxs has hccn madc. it is prcmaturc to concludc
that additional typcs arc not prcscnt in stromal cclls.
The data indicatc that thcrc arc somc stromal cells that do
not communicate with contacting neighbors. hccause in a p
proximately 30"; of cascs dyc did not transfcr hctwccn an
injcctcd stromal ccll and a contacting neighbor. Umczawa
et alzl rcportcd that prcadipocytcs lose their ability to
transfcr dvc upon convcrsion to adipocytes. so one cxplanation for this occurrcncc is that the cclls to which dye did not
transfcr havc a diffcrent developmental status. Another possibility is that thcrc isconstant formation and dissolution of
gap junctions hctwccn stromal cclls in response to clcmcnts
in thcir milieu. This latter premise is consistcnt with data
showing that hormones and growth factors can affcct cclland with findings in this study
cell comm~nication'~.''.~~.~'
showing the potential for cell-ccll communication in the
marrow to bc rcgulatcd by cytokines such as I L - 1 .
Whether the I L - I effccts arc direct or mediated by other
cytokincs whose cxprcssion is induced in stromal cclls by
IL-l is unknown. I t is also not clear at what lcvcl these
cffccts on ccll-ccll communication are mcdiatcd. Cx cxprcssion can he rcgulatcd at thc transcriptional. translational.
and posttranslational I C V C I S . ~ ~ and
~ ~ ~ further studics arc re-
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44
DORSHKIND ET AL
quired to determine which if any of these processes are affected by IL- 1, However, in preliminary experiments,
changes in stromal cell Cx43 mRNA levels after IL-1 treatment have not been observed (data not shown).
There is considerable evidence that gap junctions allow
hormone-secreting cells to coordinately regulate this activity36and that cells in communication via gap junctions can
exchange signal molecules that activate CAMP-dependent
protein kinases.39In view of these observations and data in
this report indicating that gap junctions exist between stromal cells, it may be appropriate to consider hematopoietic
microenvironments in terms ofstromal cell assemblies composed of cells in functional communication with one another. However, results indicating that exogenous mediators can influence intercellular communication also suggest
that such communication-competent microenvironments
may be dynamic rather than fixed. Accordingly, it will be
important to investigate further how the expression of gap
junctions between the stroma and the potential for cell-cell
communication to be modulated by growth factors may
influence stromal cell function and the resultant pattern of
hematopoiesis.
ACKNOWLEDGMENT
The authors thank Drs Dan Goodenough, David Paul, and Eric
Beyer for Cx32 and Cx43 cDNAs; Dr Bruce Nicholson for Cx26
cDNA; Dr Stepen Spindler for actin cDNA; and Linda Collins for
expert technical assistance.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1993 82: 38-45
Connexin-43-type gap junctions mediate communication between
bone marrow stromal cells
K Dorshkind, L Green, A Godwin and WH Fletcher
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