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The growth factor requirements of STRO-1-positive human bone marrow stromal
precursors under serum-deprived conditions in vitro
S Gronthos and PJ Simmons
Matthew Roberts Laboratory, Leukaemia Research Unit, Hanson Centre for
Cancer Research, Institute of Medical and Veterinary Science, Adelaide,
Australia.
Factors that regulate the growth and development of primitive bone marrow
stromal cell precursors are not well defined. We have examined 25 purified
recombinant growth factors for their ability to initiate and support
clonogenic growth of fibroblast colony-forming cells (CFU- F) from adult
human bone marrow. Assays were performed using bone marrow mononuclear
cells (BMMNC) enriched in CFU-F by magnetic- activated cell sorting (MACS)
using the monoclonal antibody (MoAb) STRO- 1. A serum-deprived assay was
developed to avoid components of fetal calf serum (FCS) that may mask or
otherwise modify the response of CFU- F to exogenously added factors.
L-ascorbate and the glucocorticoid dexamethasone were found to be essential
for CFU-F colony development under serum-deprived conditions. Importantly,
clonogenic growth of CFU- F in this culture system was absolutely dependent
on an exogenous source of growth factor. Platelet-derived growth factor-BB
(PDGF) and epidermal growth factor (EGF) demonstrated the greatest ability
to support colony growth. Colony formation was dose-dependent, with half-
maximal colony numbers at approximately 0.2 ng/mL for either factor and
plateau numbers at concentrations in excess of 1.0 ng/mL. Simultaneous
addition of PDGF and EGF had no effect on the number of colonies initiated
but resulted in dose-dependent increases in mean colony diameter that were
significant (P < or = .05) when compared with the effect of either
factor alone or with the size of colonies elicited in control cultures by
20% FCS. Fluorescence-activated cell sorting (FACS) of BMMNC using MoAbs to
the alpha chain of the PDGF receptor and to the EGF receptor in combination
with the Moab STRO-1 demonstrated constitutive expression of both receptors
by greater than 90% on CFU-F. Receptors for insulin-like growth factor-1
(IGF-1) and nerve growth factor (NGF) were also detected on STRO-1+ CFU-F,
but in vitro both IGF- 1 and NGF did not support colony growth. This report
demonstrates the development of a simple, reproducible, and stringent
culture system for the growth and assay of stromal precursors under
serum-deprived conditions and represents an important prerequisite for
future studies of the role of growth factors in the regulation of stromal
cell proliferation, differentiation, and development.
Volume 85,
Issue 4,
pp. 929-940,
02/15/1995
Copyright © 1995 by The American Society of Hematology
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