Gap junction intercellular communication is likely to be involved in regulation of stroma-dependent proliferation of hemopoietic stem cells

R. E. Ploemacher, A. E M Mayen, A. E. De Koning, T. Krenács, M. Rosendaal

Research output: Contribution to journalArticle

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Abstract

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 hemopoeitic stem cell proliferation.

Original languageEnglish
Pages (from-to)133-147
Number of pages15
JournalHematology
Volume5
Issue number2
Publication statusPublished - 2000

Fingerprint

Gap Junctions
Amphotericin B
Stem Cells
Stromal Cells
Nystatin
Bone Marrow
Tetraphenylborate
Cell Proliferation
Glycyrrhetinic Acid
Connexin 43
Connexins
Microinjections
Conditioned Culture Medium
Chemokines
Arachidonic Acid
Bone Marrow Cells
Coloring Agents
Clone Cells
Maintenance
Cytokines

Keywords

  • Gap junction
  • Proliferation
  • Stem cells

ASJC Scopus subject areas

  • Hematology

Cite this

Gap junction intercellular communication is likely to be involved in regulation of stroma-dependent proliferation of hemopoietic stem cells. / Ploemacher, R. E.; Mayen, A. E M; De Koning, A. E.; Krenács, T.; Rosendaal, M.

In: Hematology, Vol. 5, No. 2, 2000, p. 133-147.

Research output: Contribution to journalArticle

Ploemacher, R. E. ; Mayen, A. E M ; De Koning, A. E. ; Krenács, T. ; Rosendaal, M. / Gap junction intercellular communication is likely to be involved in regulation of stroma-dependent proliferation of hemopoietic stem cells. In: Hematology. 2000 ; Vol. 5, No. 2. pp. 133-147.
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AU - Rosendaal, M.

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AB - 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 hemopoeitic stem cell proliferation.

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