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

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.

Hypoxia is a major stimulator of osteoclast formation and bone resorption.

Hypoxia is known to act as a general stimulator of cells derived from marrow precursors. We investigated the effect of oxygen tension on the formation and function of osteoclasts, the cells responsible for bore resorption, which are of promonocytic origin. Using 7- and 13-day cultures of mouse marrow cells on ivory discs, we found that reducing oxygen tension from the ambient atmospheric level of 20% by increasing the proportion of nitrogen caused progressive increases in the formation of multinucleated osteoclasts and resorption pits. Peak effects occurred in 2% oxygen, where stimulations of resorption up to 21-fold were measured. Significant stimulations of osteoclast formation and resorption were observed even in severely hypoxic cultures gassed with 0.2% oxygen. Short-term cultures of cells disaggregated from rat bones indicated that hypoxia did not alter the resorptive activity of mature osteoclasts, but reduced their survival or adherence. In 3-day organ cultures of mouse calvarial bones, exposure to 2% oxygen resulted in maximal, fivefold stimulation of osteoclast-mediated calcium release, an effect equivalent to that of prostaglandin E(2) (PGE(2)), a reference osteolytic agent. Hypoxia also caused a moderate acidosis in calvarial cultures, presumably as a result of increased anaerobic metabolism; this observation is significant because osteoclast activation is dependent on extracellular acidification. Our experiments reveal a previously-overlooked mechanism of considerable potential importance for the regulation of bone destruction. These findings may help explain the bone loss associated with a wide range of pathological states involving local or systemic hypoxia, and emphasize the importance of the vasculature in bone.

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

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 1(a) (Gpi-1(a)) 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.

Hematopoiesis: Gap Junction Intercellular Communication is Likely to be Involved in Regulation of Stroma-dependent Proliferation of Hemopoietic Stem Cells.

The 80-100 fold increased immunohistological expression of the Gap Junction (GJ) protein Connexin-43 in murine bone marrow during the neonatal period and directly following cytoreductive treatment of adult mice suggests that the regulation of stem cell proliferation may involve GJ Intercellular Communication (GJIC). Using a series of stromal cell lines from foetal liver and neonatal bone marrow we observed that the percentage of cells with GJIC, as indicated by dye-coupling using microinjection of lucifer yellow, correlated with the stromal support for late appearing clones formed by primitive stem cells (CAFC week 3-5). In order to functionally block all GJIC between mutual stromal cells and stromal cells and hemopoietic cells, in long-term stroma-supported flask (LTC) and CAFC cultures, the lipophilic compounds amphotericin-B (AB), nystatin, alpha-glycyrrhetinic acid, tetraphenylboron, dipicrylamine and arachidonic acid were tested for their effect on GJIC and CAFC support. Only AB and nystatin, which induced complete and prolonged GJIC blockade, were able to dramatically inhibit cobblestone area (CA) formation and CFU-C generation in LTC. This inhibition could be fully abrogated by withdrawing AB within the first 2 weeks of culture. Low AB concentrations stimulated CA formation. The AB-mediated inhibition of hemopoiesis probably involved direct stromal contact with stem cells because a) AB did not inhibit CFU-C generation when stem cells were cultured in trans-well inserts above the stroma; b) conditioned media from AB-containing or normal LTC did not inhibit colony formation by normal cells in semi-solid, non-stromal cultures, and c) AB did not inhibit colony formation by bone marrow cells in semi-solid culture nor did it inhibit growth or maintenance of stromal cells. In addition, The inhibition of hemopoiesis by AB could also not be explained by changes in the amount of cytokine and chemokine transcripts, including TGF-b1, in AB-blocked stromal cells. Our findings support the involvement of GJIC in stroma-dependent regulation of hemopoietic stem cell proliferation.