THROMBOPOIETIN IS SYNTHESIZED BY BONE-MARROW STROMAL CELLS

We have previously characterized stromal progenitor cells contained in fetal bone marrow by fluorescence-activated cell sorting (FAGS) using the differential expression of CD34, CD38, and HLA-DR, and found that a small number were contained wit hin the CD34(+) cell fraction. In t he present study, the frequency of stromal progenitors in both the CD3 4(+) and CD34(-) subpopulations from samples of fetal and adult bone m arrow was approximately one in 5,000 of the mononuclear cell fraction. Using multiparameter single-cell sorting, one in 20 fetal bone marrow cells with the CD34(+), CD38(-), HLA-DR-, CDw90(+) phenotype were clo nogenic stromal progenitors, whereas greater than one in five single c ells with the CD34(-), CD38(-), HLA-DR-, CBw90(+) phenotype formed str omal cultures. We found that cultures initiated by hematopoietic and s tromal progenitors contained within the CD34(+) fraction oi: bone marr ow cells formed mixed hematopoietic/stromal cell cultures that maintai ned the viability of the hematopoietic progenitor cells for 3 weeks in the absence of added hematopoietic cytokines. We characterized some o f the hematopoietic cytokines synthesized by stromal cultures derived from either CD34(+) or CD34(-) bone marrow cells using reverse transcr iptase-polymerase chain reaction (RT-PCR) amplification of interleukin -3 (IL-3), stem cell factor (SCF), CD34, Flt3/Flk2 ligand (FL), and th rombopoietin (TPO) mRNA sequences. We found ubiquitous expression of T PO mRNA in greater than 90% of stromal cultures initiated by either CD 34(+) or CD34(-) cells, and variable expression of SCF, FL, and CD34 m RNA. in particular, SCF and CD34 mRNA were detected only in stromal cu ltures initiated by CD34(+) bone marrow cells, although the difference s between CD34(+) and CD34(-) stromal cells were not statistically sig nificant. IL-3 mRNA was not found in any stromal cultures. An enzyme-l inked immunosorbent assay (ELISA) of soluble SCF and TPO present in cu lture supernatants demonstrated that biologically significant amounts of protein were secreted by some cultured stromal cells: eight of 16 s amples of conditioned media from stromal cultures initiated by fetal a nd adult bone marrow contained more than 32 pg/ml SCF (in the linear r ange of the ELISA), with a median value of 32 pg/mL (range, 9 to 230), while 13 of 24 samples of conditioned media had more than 16 pg/mL TP O (in ?he linear range of the ELISA), with a median of 37 pg/mL (range , 16 to 106). Our data indicate that stromal cultures initiated by sin gle bone marrow cells can make FL, SCF, and TPO. Local production of e arly-acting cytokines and TPO by stromal cells may be relevant to the regulation of hematopoietic stem cell self-renewal and megakaryocytopo iesis in the bone marrow microenvironment. (C) 1997 by The American So ciety of Hematology.