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a Institute of Pathology, University of Munich, Germany;
b Institute of Clinical Hematology, GSF, Munich, Germany;
c CellTec GmbH, Hamburg, Germany;
d Department of Hematology, University Hospital, Hannover, Germany;
e Department of Medicine III, Klinikum Grosshadern, Munich, Germany
Key Words. Hematopoiesis • Mesenchymal stem cell • CD34 • Fibroblast-like canine cells
Ralf Huss, M.D., Institute of Pathology, University of Munich, Thalkirchner Str. 36, D-80337 Munich, Germany; Telephone: 49-89-5160-4011; Fax: 49-89-5160-4043; e-mail: Ralf.Huss{at}lrz.uni-muenchen.de Received April 27, 2000; accepted for publication May 1, 2000.
The hematopoietic system of vertebrates can be completely reconstituted with hematopoietic stem cells derived from the bone marrow, fetal liver, or cord blood, or even from peripheral-blood-derived cells. A cellular marker to identify those cells is the proteoglycan CD34, although we have shown that the earliest identifiable hematopoietic stem cell is a CD34– fibroblast-like cell which can differentiate into CD34+ hematopoietic precursors. Peripheral blood mononuclear cells were isolated from the heparinized blood of a dog and incubated in tissue culture in the presence of interleukin 6. After 10-14 days, an adherent layer of fibroblast-like cells had developed and cells were immortalized using the SV-40 large T antigen. Cells were cloned and subcloned by measures of limiting dilution, and various fibroblast-like clones were established. These fibroblast-like cells either do not express the CD34 antigen or express CD34 on a low level, although transcribing CD34. The CD34–/low cells express osteocalcin as a mesenchymal cell marker. The fibroblast-like cells eventually differentiate spontaneously in vitro into CD34+ precursors and show colony formation. Prior to autologous stem cell transplantation, one clone of choice (IIIG7) was transfected with a retroviral construct containing the green-fluorescence protein (GFP). The recipient dog was totally irradiated with 300 cGy and received a stem cell transplant with GFP-containing, immortalized, fibroblast-like monoclonal autologous stem cells (0.5 x 108/kg dog). No additional growth factors were applied. The peripheral blood counts recovered after 23 days (WBC >500; platelets >10,000). A peripheral blood smear showed some dim but definite, although timely, limited expression of the GFP protein in nucleated peripheral blood cells just five weeks after transplantation. A bone marrow biopsy showed GFP-positive cells in the marrow cavity predominantly as "bone-lining cells."
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