scholarly journals Connexin-43 gap junctions are involved in multiconnexin-expressing stromal support of hemopoietic progenitors and stem cells

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 498-505 ◽  
Author(s):  
Jose A. Cancelas ◽  
Wendy L. M. Koevoet ◽  
Alexandra E. de Koning ◽  
Angelique E. M. Mayen ◽  
Elwin J. C. Rombouts ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gap Junctions ◽  
Stromal Cells ◽  
Connexin 43 ◽  
Fetal Liver ◽  
Large Family ◽  
Wild Type ◽  
Murine Bone Marrow ◽  
Stromal Support

Abstract Gap junctions (GJs) provide for a unique system of intercellular communication (IC) allowing rapid transport of small molecules from cell to cell. GJs are formed by a large family of proteins named connexins (Cxs). Cx43 has been considered as the predominantly expressed Cx by hematopoietic-supporting stroma. To investigate the role of the Cx family in hemopoiesis, we analyzed the expression of 11 different Cx species in different stromal cell lines derived from murine bone marrow (BM) or fetal liver (FL). We found that up to 5 Cxs are expressed in FL stromal cells (Cx43, Cx45, Cx30.3, Cx31, and Cx31.1), whereas only Cx43, Cx45, and Cx31 were clearly detectable in BM stromal cells. In vivo, the Cx43-deficient 14.5- to 15-day FL cobblestone area–forming cells (CAFC)-week 1-4 and colony-forming unit contents were 26%-38% and 39%-47% lower than in their wild-type counterparts, respectively. The reintroduction of the Cx43 gene into Cx43-deficient FL stromal cells was able to restore their diminished IC to the level of the wild-type FL stromal cells. In addition, these Cx43-reintroduced stromal cells showed an increased support ability (3.7-fold) for CAFC-week 1 in normal mouse BM and 5-fold higher supportive ability for CAFC-week 4 in 5-fluorouracil-treated BM cells as compared with Cx43-deficient FL stromal cells. These findings suggest that stromal Cx43-mediated IC, although not responsible for all GJ-mediated IC of stromal cells, plays a role in the supportive ability for hemopoietic progenitors and stem cells.

scholarly journals Connexin-43 gap junctions are involved in multiconnexin-expressing stromal support of hemopoietic progenitors and stem cells

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 498-505 ◽  
Author(s):  
Jose A. Cancelas ◽  
Wendy L. M. Koevoet ◽  
Alexandra E. de Koning ◽  
Angelique E. M. Mayen ◽  
Elwin J. C. Rombouts ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gap Junctions ◽  
Stromal Cells ◽  
Connexin 43 ◽  
Fetal Liver ◽  
Large Family ◽  
Wild Type ◽  
Murine Bone Marrow ◽  
Stromal Support

Gap junctions (GJs) provide for a unique system of intercellular communication (IC) allowing rapid transport of small molecules from cell to cell. GJs are formed by a large family of proteins named connexins (Cxs). Cx43 has been considered as the predominantly expressed Cx by hematopoietic-supporting stroma. To investigate the role of the Cx family in hemopoiesis, we analyzed the expression of 11 different Cx species in different stromal cell lines derived from murine bone marrow (BM) or fetal liver (FL). We found that up to 5 Cxs are expressed in FL stromal cells (Cx43, Cx45, Cx30.3, Cx31, and Cx31.1), whereas only Cx43, Cx45, and Cx31 were clearly detectable in BM stromal cells. In vivo, the Cx43-deficient 14.5- to 15-day FL cobblestone area–forming cells (CAFC)-week 1-4 and colony-forming unit contents were 26%-38% and 39%-47% lower than in their wild-type counterparts, respectively. The reintroduction of the Cx43 gene into Cx43-deficient FL stromal cells was able to restore their diminished IC to the level of the wild-type FL stromal cells. In addition, these Cx43-reintroduced stromal cells showed an increased support ability (3.7-fold) for CAFC-week 1 in normal mouse BM and 5-fold higher supportive ability for CAFC-week 4 in 5-fluorouracil-treated BM cells as compared with Cx43-deficient FL stromal cells. These findings suggest that stromal Cx43-mediated IC, although not responsible for all GJ-mediated IC of stromal cells, plays a role in the supportive ability for hemopoietic progenitors and stem cells.

High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 855-862 ◽  
Author(s):  
Robert A. J. Oostendorp ◽  
Julie Audet ◽  
Connie J. Eaves
Keyword(s):  
Stem Cells ◽  
Fetal Liver ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Flow Cytometric ◽  
Differentiation Status ◽  
Kinetics Of

The kinetics of proliferation of primitive murine bone marrow (BM) cells stimulated either in vitro with growth factors (fetal liver tyrosine kinase ligand 3 [FL], Steel factor [SF], and interleukin-11 [IL-11], or hyper–IL-6) or in vivo by factors active in myeloablated recipients were examined. Cells were first labeled with 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and then incubated overnight prior to isolating CFSE+ cells. After 2 more days in culture, more than 90% of the in vivo lymphomyeloid repopulating activity was associated with the most fluorescent CFSE+ cells (ie, cells that had not yet divided), although this accounted for only 25% of the repopulating stem cells measured in the CFSE+ “start” population. After a total of 4 days in culture (1 day later), 15-fold more stem cells were detected (ie, 4-fold more than the day 1 input number), and these had become (and thereafter remained) exclusively associated with cells that had divided at least once in vitro. Flow cytometric analysis of CFSE+ cells recovered from the BM of transplanted mice indicated that these cells proliferated slightly faster (up to 5 divisions completed within 2 days and up to 8 divisions completed within 3 days in vivo versus 5 and 7 divisions, respectively, in vitro). FL, SF, and ligands which activate gp130 are thus efficient stimulators of transplantable stem cell self-renewal divisions in vitro. The accompanying failure of these cells to accumulate rapidly indicates important changes in their engraftment potential independent of accompanying changes in their differentiation status.

Enhancement of repopulation haemopoiesis by heterozygous connexin 43 stem cells seeded on wild-type connexin 43 stroma

2003 ◽  
Vol 105 (5) ◽  
pp. 561-568 ◽  
Author(s):  
Martin ROSENDAAL ◽  
Mervyn STONE
Keyword(s):  
Stem Cells ◽  
Connexin 43 ◽  
Fetal Liver ◽  
Wild Type ◽  
Knock Out ◽  
Influence Model ◽  
Glucose Phosphate ◽  
Blood Formation ◽  
Cell Repopulation ◽  
Proliferation Potential

The present paper analyses the results of competitive blood-cell repopulation experiments in which Cx43-WT (connexin 43 wild-type) host mice, whose own HSCs (haemopoietic stem cells) were deleted, were grafted with fetal liver cells: 50% Gpi-1a (glucose phosphate isomerase-1a)/Cx43-WT cells competing with 50% Gpi-1b/Cx43-WT, 50% Gpi-1b/Cx43-HZ (heterozygous) or 50% Gpi-1b/Cx43-KO (knock-out) cells. The percentages of platelets, granulocytes, red cells, B-cells and T-cells containing Gpi-1b in blood samples obtained from 22 to 186 days after grafting, and the percentages of high-proliferation-potential colony-forming cells containing Gpi-1b at 255 days after grafting, were measured. The results show that, if we wait 4 months so that we measure the percentages of Gpi-1b end-cells formed by initially resting stem cells in the graft, values in HZ mice are greater than those in WT and KO mice by 10% or more. We propose a bipolar influence model for blood formation by grafted HSCs to explain this difference and other features of the data. Influence A is a direct one: for individual HSCs, the combined effect on HSC niching and HSC proliferation of Cx43 is superior to that of the KO allele. Influence B is a demographic one: HZ foundation mice compensate by having more HSCs than WT mice. The net outcome of influences A and B is that HZ is the winner.

Hematopoietic Stem Cell Engraftment in Bone Marrow Is Dependent upon Gsα.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 857-857
Author(s):  
Gregor B. Adams ◽  
Ian R. Alley ◽  
Karissa T. Chabner ◽  
Ung-il Chung ◽  
Emily S. Marsters ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Conditional Knockout ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Engraftment

Abstract During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, which remains the site of hematopoiesis throughout adulthood. In the bone marrow the HSCs are located at the endosteal surface, where the osteoblasts are a key component of the stem cell niche. The exogenous signals that specifically direct HSCs to the bone marrow have been thought to include stimulation of the chemokine receptor CXCR4 by its cognate ligand stromal derived factor-1α (SDF-1α or CXCL12). However, experiments in which CXCR4−/− fetal liver hematopoietic cells were transplanted into wild-type hosts demonstrated efficient engraftment of the HSCs in the bone marrow. In addition, treatment of HSCs with inhibitors of Gαi-coupled signaling, which blocks transmigration towards SDF-1αin vitro, does not affect bone marrow homing and engraftment in vivo. Therefore, we examined whether Gsα-coupled mechanisms play a key role in the engraftment of the HSCs in the bone marrow environment. Utilizing an inducible-conditional knockout of Gsα, we found that deletion of the gene in hematopoietic bone marrow cells did not affect their ability to perform in the in vitro primitive CFU-C or LTC-IC assay systems. However, Gsα−/− cells were unable to establish effective hematopoiesis in the bone marrow microenvironment in vivo in a competitive repopulation assay (41.1% contribution from wild-type cells versus 1.4% from knockout cells). These effects were not due to an inability of the cells to function in the bone marrow in vivo as deletion of Gsα following establishment of hematopoiesis had no effects on the HSCs. Examining the ability of the HSCs to home to the bone marrow, though, demonstrated that deletion of Gsα resulted in a marked impairment of the ability of the stem cells to localize to the marrow space (approximately 9-fold reduction in the level of primitive cell homing). Furthermore, treatment of BM MNCs with an activator of Gsα augmented the cells homing and thus engraftment potential. These studies demonstrate that Gsα is critical to the localization of HSCs to the bone marrow. Which receptors utilize this pathway in this context remains unknown. However, Gsα represents a previously unrecognized signaling pathway for homing and engraftment of HSCs to bone marrow. Pharmacologic activation of Gsα in HSC ex vivo prior to transplantation offers a potential method for enhancing stem cell engraftment efficiency.

Murine Bone Marrow Stromal Cells Sustain In Vivo the Survival of Hematopoietic Stem Cells and the Granulopoietic Differentiation of More Mature Progenitors

Stem Cells ◽  
2005 ◽  
Vol 23 (10) ◽  
pp. 1626-1633 ◽  
Author(s):  
Frédérique Hubin ◽  
Chantal Humblet ◽  
Zakia Belaid ◽  
Charles Lambert ◽  
Jacques Boniver ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow

Dissection of the Role of Connexin-43 in Hematopoietic Progenitors and Stromal Cells Unveils Distinct Hematopoietic Intrinsic and Extrinsic Regulatory Functions.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 607-607
Author(s):  
Lina Li ◽  
Bhuvana Murali ◽  
Dealma N. Worsham ◽  
Susan K. Dunn ◽  
Jose A. Cancelas
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Connexin 43 ◽  
Chimeric Mice ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Cx43 Expression

Abstract Bone marrow (BM) stromal cells seem to be crucial in the establishment of the hematopoietic niches in bone marrow. BM stromal cells can communicate through gap junctions, which consist of narrow channels between contacting cells and are composed by connexins. Connexin 43 (Cx43) is expressed by BM stromal cells and upon adhesion to stroma, by hematopoietic stem cells and progenitors (HSC/P). Cx43 has been shown to be essential in controlling osteoblast and fibroblast function. We have previously reported that Cx43 is critical for the interaction between stroma and HSC/P in CAFC assays (Cancelas J.A. et al., Blood 2000) and in adult hematopoiesis after 5-fluorouracil (5-FU) administration in Mx1-Cre-Tg;Cx43KO mice (Presley C, et al., Cell Comm. Adh., 2005). We have also previously shown that after 5-FU administration, Cx43 is predominantly expressed in the endosteum and the deficiency of Cx43 in stroma of Collagen I (ColI)-Cre;Cx43KO and chimeric mice impairs their hematopoiesis by impairing the homing of wild-type (WT) hematopoietic progenitors and after 5-FU administration, the hematopoietic progenitor cycling inducing a ∼30% expansion of the long-term stem cell compartment in BM (Li L et al., ASH 2006). Interestingly, stromal Cx43-deficient mice contain around twice as many CFU-F as wild-type (WT) mice. Now, we have further investigated the role of stromal Cx43 expression in the regulation of hematopoietic progenitor adhesion to stroma, trans-stromal migration and mobilization. Cx43-deficient stromal cells display complete absence of intercellular communication as assayed by calcein dye transfer which can be reverted by retroviral transduction of Cx43. Trans-stromal migration of hematopoietic progenitors through Cx43-deficient irradiated stroma is impaired (7.8% vs 13.8% in WT stroma, p=0.015) but primary adhesion to Cx43-deficient irradiated stroma and in vivo mobilization response to G-CSF in ColI-Cre;Cx43KO mice were similar to WT controls, suggesting that stromal Cx43 plays a role in the regulation of the post-adhesion migration of HSC/P. On the other hand, Cx43-deficient HSC/P from Vav1-Cre;Cx43KO primary and chimeric mice show severe impairment of blood cell formation during the recovery phase after 5-FU administration (day +14) compared to wild-type controls (ANC: 0.23±0.12 vs 1.40±1.25 x 109 neutrophils/L; Platelet count: 135±91 vs 572±205 x 109 platelets/L; p < 0.05). Cx43 deficiency in hematopoietic progenitors did not significantly impair their homing ability in wild-type mice. Taken together, these studies indicate that Cx43 expression plays distinct roles in the regulation of hematopoietic intrinsic and extrinsic mechanisms. While Cx43 expression in stroma seems to be crucial in the regulation of the stromal progenitor and HSC pool content as well as HSC/P trans-stromal migration and homing, deficiency of Cx43 in either hematopoietic cells or stromal cells independently induce a significant impairment in the post-chemotherapy blood formation in vivo, suggesting that, under stress, hematopoietic regeneration depends on complete Cx43 channels communicating HSC/P and stromal cells.

Hematopoietic capacity of connexin43 wild-type and knock-out fetal liver cells not different on wild-type stroma

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 2996-2998 ◽  
Author(s):  
Martin Rosendaal ◽  
Chris Jopling
Keyword(s):  
Gap Junctions ◽  
Stromal Cells ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Wild Type ◽  
Knock Out ◽  
Critical Gap ◽  
Glucose Phosphate ◽  
Fetal Liver Cells

Abstract In 1995 and 1997 we proposed that gap junctions between stromal and hematopoietic cells formed by connexin43 (Cx43) determine hematopoiesis. If this were the case, are the critical gap junctions in this regard those between hematopoietic and stromal cells, or those between stromal cells alone? To test the first possibility, we compared hematopoietic repopulating capacity between fetal liver hematopoietic cells expressing the different mouse Cx43 genotypes, wild type (WT), hemizygous, or knock-out (KO) on WT host mice stroma. We deleted host glucose phosphate isomerase 1a (Gpi-1a) stems and then raced identifiable Cx43 WT host fetal liver against congenic donor Cx43 WT, hemizygous, or KO cells in sets, comparing their capacity to form 5 end cells. Hematopoietic capacity did not differ between the Cx43 WT and KO genotypes. The role of Cx43 gap junctions in hematopoiesis remains uncertain.

Fetal liver pro-B and pre-B lymphocyte clones: expression of lymphoid-specific genes, surface markers, growth requirements, colonization of the bone marrow, and generation of B lymphocytes in vivo and in vitro

1992 ◽  
Vol 12 (2) ◽  
pp. 518-530
Author(s):  
R Palacios ◽  
J Samaridis
Keyword(s):  
B Cell ◽  
B Lymphocytes ◽  
Stromal Cells ◽  
B Lymphocyte ◽  
Germ Line ◽  
Fetal Liver ◽  
Rag 1 ◽  
B Cell Precursors

We describe here the development and characterization of the FLS4.1 stromal line derived from 15-day fetal liver of BALB/c embryos and defined culture conditions that efficiently support the cloning and long-term growth of nontransformed B-220+ 14-day fetal liver cells at two stages of B-cell development, namely, pro-B lymphocytes (immunoglobulin [Ig] genes in germ line configuration) and pre-B cells (JH-rearranged genes with both light-chain Ig genes in the germ line state). All B-cell precursor clones require recombinant interleukin-7 (rIL-7) and FLS4.1 stromal cells for continuous growth in culture, but pro-B lymphocyte clones can also proliferate in rIL-3. None proliferate in rIL-1, rIL-2, rIL-4, rIL-5, rIL-6, or leukemia inhibitory factor. FLS4.1 stromal cells synthesize mRNA for Steel factor but not for IL-1 to IL-7; all pro-B and pre-B clones express c-Kit, the receptor for Steel factor, and a c-Kit-specific antibody inhibits the enhanced proliferative response of fetal liver B-220+ B-cell precursors supported by FLS4.1 stromal cells and exogenous rIL-7 but does not affect that promoted by rIL-7 alone. Northern (RNA) blot analysis of the expression of the MB-1, lambda 5, Vpre-B, c mu, RAG-1, and RAG-2 genes in pro-B and pre-B clones show that transcription of the MB-1 gene precedes IgH gene rearrangement and RNA synthesis from c mu, RAG-1, RAG-2, lambda 5, and Vpre-B genes. All clones at the pre-B-cell stage synthesize mRNA for c mu, RAG-1, and RAG-2 genes; transcription of the lambda 5 and Vpre-B genes seems to start after D-to-JH rearrangement in B-cell precursors, indicating that the proteins encoded by either gene are not required for B-cell progenitors to undergo D-to-JH gene rearrangement. These findings mark transcription of the MB-1 gene as one of the earliest molecular events in commitment to develop along the B-lymphocyte pathway. Indeed, both pro-B and pre-B clones can generate in vitro and in vivo B lymphocytes but not T lymphocytes; moreover, these clones do not express the CD3-gamma T-cell-specific gene, nor do they have rearranged gamma, delta, or beta T-cell antigen receptor genes.

scholarly journals An in vivo inflammatory loop potentiates KRAS blockade

2019 ◽  
Author(s):  
Kristina A.M. Arendt ◽  
Giannoula Ntaliarda ◽  
Vasileios Armenis ◽  
Danai Kati ◽  
Christin Henning ◽  
...  
Keyword(s):  
Interleukin 1Β ◽  
Wild Type ◽  
Poor Survival ◽  
Murine Bone Marrow ◽  
Chemokine Ligand ◽  
Isoprenylcysteine Carboxylmethyltransferase ◽  
Chemokine Ligand 2 ◽  
Deficient Mice

ABSTRACTKRAS inhibitors perform inferior to other targeted drugs. To investigate a possible reason for this, we treated cancer cells with KRAS inhibitors deltarasin (targeting phosphodiesterase-δ), cysmethynil (targeting isoprenylcysteine carboxylmethyltransferase), and AA12 (targeting KRASG12C), and silenced/overexpressed mutant KRAS using custom vectors. We show that KRAS-mutant tumor cells exclusively respond to KRAS blockade in vivo, because the oncogene co-opts host myeloid cells via a C-C-motif chemokine ligand 2/interleukin-1β signaling loop for sustained tumorigenicity. Indeed, KRAS-mutant tumors did not respond to deltarasin in Ccr2 and Il1b gene-deficient mice, but were deltarasin-sensitive in wild-type and Ccr2-deficient mice adoptively transplanted with wild-type murine bone marrow. A KRAS-dependent pro-inflammatory transcriptome was prominent in human cancers with high KRAS mutation prevalence and predicted poor survival. Hence the findings support that in vitro systems are suboptimal for anti-KRAS drug screens, and suggest that interleukin-1β blockade might be specific for KRAS-mutant cancers.

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