Intercellular communication between bone marrow stromal cells and CD34+ haematopoietic progenitor cells is mediated by connexin 43-type gap junctions.
The existence of functional gap junctions between haematopoietic progenitor cells (HPCs) and stromal cells of the haematopoietic microenvironment in the human system is a controversial issue. Primary CD34+ HPCs isolated from leukapheresis products were co-incubated with the human fibroblastoid bone marrow stromal cell line L87/4 in short-term liquid culture. Using the highly sensitive double whole-cell patch-clamp technique, we found that the majority (91%) of CD34+ HPCs are electrically coupled to L87/4 cells. Importantly, efficient coupling was observed within 1 h of the attachment of CD34+ HPCs to plastic adherent L87/4 cells. By comparison, homologous cell pairs formed by L87/4 cells exhibited a significantly higher electric coupling. Analysis of single-channel conductances revealed an electric profile characteristic of connexin 43 (Cx43)-type gap junctions for both homologous and heterologous cell pairs. The Cx phenotype was confirmed using Cx43-specific monoclonal antibodies in a flow cytometric assay and reverse transcription polymerase chain reaction (RT-PCR) for the detection of Cx43 mRNA. Finally, the electrophysiological studies were complemented by dye-transfer experiments using the recently described 'parachute' technique that allows the monitoring of dye diffusion without disruption of the plasma membrane. Taken together, our data indicate that functional Cx43-type gap junctions exist between stromal cells and immature HPCs and, thus, may provide an important regulatory pathway in haematopoiesis.
Dürig
J
J
Department of Haematology, University Hospital Essen, and Institute of Physiology, University Essen, Germany.
Rosenthal
C
C
Halfmeyer
K
K
Wiemann
M
M
Novotny
J
J
Bingmann
D
D
Dührsen
U
U
Schirrmacher
K
K
D018952:Antigens, CD34; D001854:Bone Marrow Cells / Q000502:physiology*; D002450:Cell Communication / Q000502:physiology; D002460:Cell Line; D018920:Coculture Techniques; D018031:Connexin 43; D004058:Diffusion; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000502:physiology*; D019337:Hematologic Neoplasms / Q000473:pathology*; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000502:physiology*; D006801:Humans; D008228:Lymphoma, Non-Hodgkin / Q000473:pathology; D009101:Multiple Myeloma / Q000473:pathology; D018408:Patch-Clamp Techniques
D018952:Antigens, CD34; D018031:Connexin 43; D005456:Fluorescent Dyes