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Department of Pathology and Laboratory Medicine and the Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los Angeles, California
Correspondence: Dr. Kenneth Dorshkind, Department of Pathology and Laboratory Medicine, 173216 UCLA School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095-1732. E-mail: kdorshki{at}mednet.ucla.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONGAP JUNCTION STRUCTURE AND...STROMAL CELLS CAN FORM...GAP JUNCTIONS IN HEMATOPOIETIC...REGULATION OF Cx EXPRESSION...HEMATOPOIETIC DEFECTS IN Cx43...EXPRESSION OF Cx43 IS...IMPLICATIONS AND FUTURE...REFERENCES |
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Key Words: bone marrow • thymus • stromal cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONGAP JUNCTION STRUCTURE AND...STROMAL CELLS CAN FORM...GAP JUNCTIONS IN HEMATOPOIETIC...REGULATION OF Cx EXPRESSION...HEMATOPOIETIC DEFECTS IN Cx43...EXPRESSION OF Cx43 IS...IMPLICATIONS AND FUTURE...REFERENCES |
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These in vitro analyses have led to the realization that, in addition to interacting with hematopoietic cells via direct cell-to-cell contacts, stromal cells are an important source of soluble mediators that affect blood cell growth, maturation, and survival [1 2 3 4 5 6 7 ]. However, it has also become evident that stromal cells do not produce all such factors constitutively. Instead, they express cell surface receptors that allow them to respond to signals in their external milieu, and this in turn can affect the concentration and type of growth factors they produce. For example, binding of cytokines such as interleukin (IL)-1 to stromal-cell receptors can result in increased production of myeloid growth factors such as the colony-stimulating factors [1 , 7 , 8 ].
This functional plasticity provides stromal cells a means to modify their hematopoietic support capacity in response to changing demands for production of particular blood cells. It is possible that stromal cells function as independent entities in mediating these effects. However, the observations that stromal-cell processes are frequently in contact [7 , 9 , 10 ] and gap junction-like structures can be detected morphologically at these sites [11 12 13 14 15 16 ] has fueled speculation that groups of stromal cells might form functional domains in which their responses to environmental stimuli are synchronized. As reviewed below, gap junctions are intercellular channels that allow direct exchange of small molecules between the cytoplasm of coupled cells [17 18 19 20 21 22 23 24 ].
These observations prompted studies which confirmed that genes encoding gap junction structural proteins are expressed in hematopoietic tissues and functional gap junction-mediated communication occurs between stromal cells [25 26 27 28 ]. As reviewed below, there are indications that hematopoietic cells might also have the potential to form gap junctions. However, definitive evidence that gap junctions play a critical role during blood cell production has been obtained only recently through the demonstration of hematopoietic defects in a strain of mice in which the gene encoding connexin (Cx) 43, the principal gap junction protein expressed in hematopoietic tissues, has been disrupted [27 , 29 , 30 ].
This review summarizes these results and highlights their clinical implications for procedures such as bone marrow transplantation.
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GAP JUNCTION STRUCTURE AND FUNCTION |
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TOPABSTRACTINTRODUCTIONGAP JUNCTION STRUCTURE AND...STROMAL CELLS CAN FORM...GAP JUNCTIONS IN HEMATOPOIETIC...REGULATION OF Cx EXPRESSION...HEMATOPOIETIC DEFECTS IN Cx43...EXPRESSION OF Cx43 IS...IMPLICATIONS AND FUTURE...REFERENCES |
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1,200 Da)
between cells. Gap junction-mediated intercellular communication allows
groups of cells in contact to respond to environmental stimuli in a
coordinated manner [17
18
19
20
21
22
23
24
]. For example, the rapid
cell-to-cell transfer of action potentials between cardiac muscle cells
in contact allows contraction to be synchronized, whereas in
nonexcitable cells, gap junctions are thought to take part in the
coordination of metabolic responses [24
]. Gap junctions are found at sites where the plasma membranes of two adjacent cells interact, and these junctions consist of multiple channels that allow intercytoplasmic passage of small molecules. Each of these channels is formed when a connexon, i.e., a multimeric transmembrane structure composed of six Cx subunits (Fig. 1 ) in one cell, aligns with its counterpart in an adjoining cell. At least 14 mouse Cx genes exist, and their protein products are named according to their molecular size. All of the Cx proteins within a connexon can be the same, or different types of Cx proteins can associate to form a connexon.
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The remaining sections of this review synthesize the literature indicating that Cx gene expression occurs in hematopoietic tissues and focus on studies that have demonstrated hematopoietic defects in the Cx43-deficient mouse [29 ].
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STROMAL CELLS CAN FORM FUNCTIONAL GAP JUNCTIONS |
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TOPABSTRACTINTRODUCTIONGAP JUNCTION STRUCTURE AND...STROMAL CELLS CAN FORM...GAP JUNCTIONS IN HEMATOPOIETIC...REGULATION OF Cx EXPRESSION...HEMATOPOIETIC DEFECTS IN Cx43...EXPRESSION OF Cx43 IS...IMPLICATIONS AND FUTURE...REFERENCES |
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The ability to grow stromal cells in vitro [36 , 37 ] has made it possible to perform detailed analyses of Cx gene expression by phenotypic analysis, Northern blotting, and/or reverse transcriptase (RT)-PCR [26 27 28 , 38 39 40 41 42 ]. These studies revealed that the principal Cx expressed by bone marrow stromal cells is Cx43. Cx43 was originally described in the myocardium and is detectable in almost all vertebrate organs in various amounts. Cx31 and Cx45 have been detected in some stromal-cell lines by RT-PCR, but the expression of two other widely expressed Cxs—Cx26 and Cx32—has not been detected in bone marrow stroma [26 , 27 ].
Dye transfer experiments have confirmed that functional gap junctions
exist between stromal cells [25
26
27
, 35
,
39
, 40
41
42
43
]. This technique relies on the
fact that when a low-molecular-mass fluorescent dye such as lucifer
yellow (400 daltons) is microinjected into a single cell, it passes
to all other cells connected through gap junctions [44
].
In fact, second- and third-order dye transfer to cells not directly in
contact with the dye-injected cells is also observed
[26
] (Fig. 2
). The results of the dye transfer studies have been corroborated
by studies showing that electronic current can pass between stromal
cells via gap junctions [26
].
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Taken together, these results provide strong support for the hypothesis that stromal cells in the bone marrow [26 , 38 , 39 , 45 ] and thymus [46 ] form a network of cells that communicate via gap junctions.
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GAP JUNCTIONS IN HEMATOPOIETIC CELLS |
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TOPABSTRACTINTRODUCTIONGAP JUNCTION STRUCTURE AND...STROMAL CELLS CAN FORM...GAP JUNCTIONS IN HEMATOPOIETIC...REGULATION OF Cx EXPRESSION...HEMATOPOIETIC DEFECTS IN Cx43...EXPRESSION OF Cx43 IS...IMPLICATIONS AND FUTURE...REFERENCES |
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There are also indications that gap junction-mediated communication between hematopoietic cells can occur. Numerous studies have concluded that lymphocytes are electrophysiologically or metabolically coupled, that lymphoid cells in secondary lymphoid organs express Cx proteins, and that dye or electronic current can transfer between them [48 49 50 51 52 53 ]. It has also been reported that macrophages can form gap junctions, some of which contain Cx43 [54 55 56 57 58 ], although not all reports indicate that this occurs [59 , 60 ].
At least within the bone marrow, the degree to which gap junctions between stromal and hematopoietic cells occur is unclear, and this issue remains controversial. It has been reported that dye transfer from a cultured stromal-cell line to blood cells occurs at a frequency of about 10% [25 ]. Studies in our own laboratory have been unable to observe dye transfer between stromal and hematopoietic cells in cultures of stromal and B-lineage cells [26 ] (Fig. 2) or to detect Cx expression in hematopoietic cells by RT-PCR. For instance, Montecino-Rodriguez et al. [30 ] found that the pluripotent hematopoietic stem cell-enriched population of lineage-negative, c-kit+, Sca-1+ cells or B-cell precursors do not express Cx43 mRNA. Similarly, Cancellas et al. reported that bone marrow cells enriched for lymphoid-, myeloid- (monocytes, macrophage, and granulocytes), and erythroid-lineage cells do not express Cx43, Cx45, or Cx31 [27 ].
Thus, although gap junctions might form between stromal cells and hematopoietic cells in some instances, the functional significance of such couplings is unresolved.
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REGULATION OF Cx EXPRESSION IN STROMAL CELLS |
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TOPABSTRACTINTRODUCTIONGAP JUNCTION STRUCTURE AND...STROMAL CELLS CAN FORM...GAP JUNCTIONS IN HEMATOPOIETIC...REGULATION OF Cx EXPRESSION...HEMATOPOIETIC DEFECTS IN Cx43...EXPRESSION OF Cx43 IS...IMPLICATIONS AND FUTURE...REFERENCES |
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Although these data indicate that the high frequency of dye transfer observed in vitro is a culture-induced phenomenon, the observations indicate that gap junction communication between the stroma can be regulated. This conclusion is consistent with the fact that numerous hormones, cytokines, and pharmacologic agents can affect the expression of Cx and coupling of cells [26 , 41 , 61 62 63 ]. Another clear example that the level of gap junction communication is dynamic was provided by comparison of Cx43 protein expression in neonatal versus adult bone marrow.
There is minimal staining of marrow sections from adult mice with anti-Cx43 antibodies, a finding consistent with the low degree of dye transfer in freshly isolated adult bone marrow. The Cx43 expression that is observed is located primarily between the osteoblasts that line the endosteal-hematopoietic margin [38 ]. Hematopoietic stem cells and early progenitors are thought to be located primarily in this area of the bone marrow [8 , 64 ]. In contrast, Cx43 expression in the bone marrow of neonatal mice is localized in this same area, but the area of bone marrow labeled with the anti-Cx43 antibody is 80-fold greater than in adults. This result suggests that Cx43 expression is highest when vigorous blood cell production is required to meet the needs of a growing organism [25 , 38 , 39 ]. Consistent with this premise is the finding that bone marrow of 5-fluorouracil (5-FU)-treated mice exhibits Cx43 labeling that is 100-fold higher than in control animals [38 ]. 5-FU eliminates cycling hematopoietic cells, and the surviving, immature hematopoietic progenitors are driven into cell cycle to replenish the lymphoid and myeloid compartments [65 , 66 ]. A final piece of data that supports the hypothesis that gap junctions are required during periods of active hematopoiesis is that their numbers are decreased when stromal cells differentiate into adipocytes, which results in formation of yellow, nonhematopoietic bone marrow [40 , 42 , 43 ].
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HEMATOPOIETIC DEFECTS IN Cx43 KNOCKOUT MICE |
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TOPABSTRACTINTRODUCTIONGAP JUNCTION STRUCTURE AND...STROMAL CELLS CAN FORM...GAP JUNCTIONS IN HEMATOPOIETIC...REGULATION OF Cx EXPRESSION...HEMATOPOIETIC DEFECTS IN Cx43...EXPRESSION OF Cx43 IS...IMPLICATIONS AND FUTURE...REFERENCES |
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Cx43-/- mice are viable throughout embryonic development but die within hours of delivery. Examination of these mice revealed a malformation in which internal septa block the conus arteriosus region of the developing heart, resulting in an occlusion of the pulmonary artery and decreased cardiac output from the right ventricle. As a result, Cx43-/- mutants fail to oxygenate their blood, and they die of hypoxia [29 ]. It is interesting that mutations in the gene encoding Cx43 have been shown to correlate with a number of cardiac malformations in children [67 ].
Because Cx43-/- mice die perinatally, initial studies
focused on blood cell development in embryos and newborns
[27
, 30
]. During the embryonic and neonatal
periods of development, the liver, spleen, and thymus are hematopoietic
tissues [68
69
70
] and are easily harvested. The study of
these hematopoietic organs has shown that Cx43-/- mice
have reduced numbers of granulocyte-macrophage colony-forming units
(GM-CFUs) and erythroid bursts (BFU-Es) in fetal liver from 14- to
15-day-old embryos [27
]. In addition, a defect in
terminal lymphocyte maturation in Cx43-/- mice, which
correlates with a reduced frequency of surface immunoglobulin (Ig) M+ B
cells and 
ß+ T cells, was observed [30
].
Similar hematopoietic defects were also present in Cx43+/- heterozygote mice. The frequency of CD4+, T-helper cell, and surface IgM+ B-lineage cells was lower than in wild-type mice. However, these defects were not as severe as in the Cx43-/- littermates, suggesting that a gene dosage effect was operative. This finding is not unprecedented. Reaume and coworkers noted that the frequency of lucifer yellow dye transfer between Cx43+/- fibroblasts was intermediate between values observed in Cx43+/+ and Cx43-/- cells [29 ]. Additional studies have demonstrated that precise levels of Cx43-mediated gap junction communication are necessary for processes such as neural crest migration [71 ] and cardiac development [72 ], and other analyses have reported that ventricular contraction in Cx43+/- mice is 30% slower than in wild-type littermates [73 , 74 ].
Nevertheless, the observation that Cx43+/- mice have hematopoietic defects during the embryonic/neonatal period of their lives was quite surprising, because young adult Cx43+/- mice do not exhibit any hematopoietic abnormalities in the bone marrow or spleen. These observations can be reconciled if, as noted earlier, gap junctions are most critical during times when the hematopoietic system is establishing or regenerating [38 , 39 ]. To test this hypothesis, studies in our laboratory assessed the kinetics of hematopoietic recovery in young adult Cx43+/- mice after 5-FU treatment. The results were striking. Nine days after administration of 5-FU, bone marrow cellularity in Cx43+/- mice was only 20% of that in untreated animals, while 5-FU-treated Cx43+/+ mice had recovered to 70% of levels in untreated wild-type mice. This delayed recovery was due to effects in multiple hematopoietic lineages that included the B, erythroid, and macrophage series. Similarly, thymic recovery in Cx43+/- mice was only 30% of that in untreated animals, whereas Cx43+/+ mice exhibited no thymic abnormalities at this time. It is interesting that, although thymus cellularity was depressed in the Cx43+/- mice after 5-FU treatment, no effect on the frequency of thymocyte subpopulations was observed. Yet, in embryos and neonates, failure to express Cx43 resulted in a diminution of the frequency of CD4- and CD8-expressing cells. The reasons are not clear, but these findings might indicate that the manner in which gap junctions regulate hematopoiesis during embryonic development is not comparable with what occurs during regeneration of blood cell formation in the adult.
These in vivo data represent the first evidence that Cx43-type gap junctions play a physiologic role in the regulation of hematopoiesis. However, the question still remained as to whether Cx43 expression in stromal cells, hematopoietic cells, or both was most critical.
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EXPRESSION OF Cx43 IS MOST CRITICAL IN NONHEMATOPOIETIC CELLS |
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TOPABSTRACTINTRODUCTIONGAP JUNCTION STRUCTURE AND...STROMAL CELLS CAN FORM...GAP JUNCTIONS IN HEMATOPOIETIC...REGULATION OF Cx EXPRESSION...HEMATOPOIETIC DEFECTS IN Cx43...EXPRESSION OF Cx43 IS...IMPLICATIONS AND FUTURE...REFERENCES |
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To identify the compartment in which the requirement for Cx43 was manifest in vivo, Montecino-Rodriguez et al. [30 ] constructed bone marrow chimeras by reciprocal transplantation of bone marrow cells between Cx43+/- and Cx43+/+ mice. The recipient mice were preconditioned with 900 Rad of irradiation prior to transplantation, a procedure that in addition to being necessary for donor cell engraftment, also results in an up-regulation of Cx43 expression in the bone marrow [38 ]. In these experiments, thymic cellularity in Cx43+/- recipients was significantly reduced, regardless of the genotype of the bone marrow donor, whereas no significant difference was observed in the thymus of Cx43+/+ mice that had received either Cx43+/- or Cx43+/+ bone marrow cells. These data strongly suggest that the effects of Cx43 are manifest at a nonhematopoietic level.
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IMPLICATIONS AND FUTURE DIRECTIONS |
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TOPABSTRACTINTRODUCTIONGAP JUNCTION STRUCTURE AND...STROMAL CELLS CAN FORM...GAP JUNCTIONS IN HEMATOPOIETIC...REGULATION OF Cx EXPRESSION...HEMATOPOIETIC DEFECTS IN Cx43...EXPRESSION OF Cx43 IS...IMPLICATIONS AND FUTURE...REFERENCES |
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What stages of hematopoiesis are dependent on Cx43 expression?
Experiments involving cytoablative treatments have shown that Cx43
expression is overwhelmingly up-regulated at the endosteal surface of
the bone marrow, with minimal expression in deeper areas of the
medullary cavity towards the central sinus. It has been suggested that
the most immature hematopoietic cells, including pluripotent
hematopoietic stem cells and immature progenitors, are located in the
endosteal area [8
, 64
]. These observations
raise the possibility that the most primitive hematopoietic cells are
dependent on gap junction expression by their supporting stroma. This
premise is consistent with data showing that the number of progenitors
that form colonies in response to IL-3, IL-6, and c-kit ligand, a
cytokine combination that targets developmentally immature precursors,
was lower in Cx43+/- mice treated with 5-FU than in their
Cx43+/+ littermates (E. Montecino-Rodriguez and K.
Dorshkind, unpublished results).
Intercellular communication between bone marrow stromal cells and hematopoieitc progenitor cells has been suggested to occur [47 , 75 ], but the inability to detect expression of Cx43 in stem cell-enriched bone marrow cells [27 , 30 ] is not consistent with this conclusion. However, until a thorough examination of other Cx genes in stem cells from normal and regenerating tissues has been made, it is not possible to exclude the possibility that such heterotypic junctions might exist. Furthermore, because Cx expression is up-regulated during active hematopoiesis, it might be more informative to analyze cells harvested from embryos/neonates or from adult Cx43+/- mice at various times after 5-FU treatment.
Are all stromal cells coupled into a single syncytium or do
stromal-cell domains exist?
As development of the embryo proceeds, gap junction communication
becomes restricted at distinct boundaries, and the embryo is
compartmentalized into communication-competent domains
[76
]. This raises the question of whether all stromal
cells in the bone marrow and thymus that express Cx43 are coupled in a
single syncytium. In this case, responses in one cell could be rapidly
transmitted to all others in contact (Fig. 3
A). Alternatively, as depicted in Figure 3B
, distinct stromal-cell
domains might exist. In this case, one possibility is that niches for
individual stem cells and their progeny exist. Finally, as shown in
Figure 3C
, functional domains might also overlap.
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What signal(s) is being coordinated by an increase in the number of
Cx43-type gap junctions?
The question of what signal is coordinated by increasing the
number of Cx43-type gap junctions is not only an issue in the
hematopoietic system but one that remains a significant challenge for
cell biologists who study the function of gap junctions. Although it is
clear that small signaling molecules can pass through gap junction
channels, the precise nature of those involved in the regulation of
stromal- (or possibly hematopoietic)-cell function remains enigmatic.
It is tempting to speculate that stromal cells coupled via gap junctions can coordinate their secretory activity [77 , 78 ]. In this regard, it has been noted that normal insulin secretion requires the coordinated function of the numerous ß cells that form pancreatic islets. Inhibition of gap junction communication in these cells results in reduced insulin secretion in response to physiologic levels of glucose. By analogy, the coordinated secretion of one or more cytokines required for the growth, differentiation, and/or survival of immature hematopoietic cells might be regulated most efficiently when stromal cells that are in contact with one another express an optimal number of gap junctions.
A major objective is to determine whether this is the case and to identify relevant cytokines whose coordinated production is dependent on gap junction expression. A recent observation that production of the chemokine stromal-derived factor-1 is up-regulated after conditioning with DNA-damaging agents that include 5-FU may be of relevance [79 ]. Stromal-derived factor-1 is produced by bone marrow stromal cells, osteoblasts, and endothelial cells in the bone marrow and affects many aspects of stem cell function, including growth and differentiation.
What role is played by gap junctions in secondary lymphoid
tissues?
The focus of the present review has been on the expression of gap
junctions in the bone marrow and thymus. However, there is now evidence
for the existence of gap junctions in secondary lymphoid tissues such
as spleen and lymph nodes [80
81
82
], The function of gap
junctions in those organs is in need of further investigation.
Is the requirement for Cx43 expression clinically relevant?
One complication after autologous or allogeneic bone marrow
transplantation is failure to establish hematopoietic engraftment. This
risk in turn can exaggerate and prolong the susceptibility to infection
and increase mortality. Most investigations of engraftment failure have
focused on the pool of transplanted cells. For example, it is now
appreciated that it is important to transplant sufficient numbers of
donor cells [83
].
Comparatively less attention has been focused on the integrity of the hematopoietic microenvironment of the recipient or on how pre- and posttransplant conditioning regimens might affect its ability to support engraftment. The fact that Cx43 is expressed in human bone marrow makes it all the more relevant [25 ] and necessary to consider that damage to the hematopoietic microenvironment might directly compromise stem cell self-renewal and differentiation. In this regard, it is important to appreciate that gap junction expression between cells is sensitive to a number of pharmacologic agents. If one or more chemotherapeutic agents used in various pretransplant conditioning regimens also interfere with gap junction expression or function, this could have significant effects on the recovery of blood cell production in transplant patients.
With an increasing number of laboratories interested in gap junctions in general and their role in hematopoiesis in particular, at least some of these questions will hopefully be answered in the not too distant future.
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ACKNOWLEDGEMENTS |
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Received April 24, 2001; revised May 3, 2001; accepted May 7, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONGAP JUNCTION STRUCTURE AND...STROMAL CELLS CAN FORM...GAP JUNCTIONS IN HEMATOPOIETIC...REGULATION OF Cx EXPRESSION...HEMATOPOIETIC DEFECTS IN Cx43...EXPRESSION OF Cx43 IS...IMPLICATIONS AND FUTURE...REFERENCES |
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1 gap junction) is down-regulated at the transcriptional level during adipocyte differentiation of H-1/A marrow stromal cells Cell Struct. Function. 17,177-184[Medline]
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