Enumeration of the colony-forming units-fibroblast from mouse and human bone marrow in normal and pathological conditions

Abstract

Bone marrow stromal cell populations, containing a subset of multipotential skeletal stem cells, are increasingly contemplated for use in tissue engineering and stem cell therapy, whereas their involvement in the pathogenetic mechanisms of skeletal disorders is far less recognized. We compared the concentrations of stromal clonogenic cells, colony forming units-fibroblast (CFU-Fs), in norm and pathology. Initially, culture conditions were optimized by demonstrating that fetal bovine serum heat inactivation could significantly repress colony formation. Using non-heat-inactivated fetal bovine serum, the concentration of CFU-Fs (colony-forming efficiency, CFE) ranged from 3.5 +/- 1.0 to 11.5 +/- 4.0 per 1 x 10(5) nucleated cells in five inbred mouse strains. In four transgenic lines with profound bone involvement, CFE was either significantly reduced or increased compared to wild-type littermates. In normal human donors, CFE decreased slightly with age and averaged 52.2 +/- 4.1 for children and 32.3 +/- 3.0 for adults. CFE was significantly altered in patients with several skeletal, metabolic, and hematological disorders: reduced in congenital generalized lipodystrophy, achondroplasia (SADDAN), pseudoachondroplasia, and Paget disease of bone and elevated in alcaptonuria and sickle cell anemia. Our findings indicate that under appropriate culture conditions, CFE values may provide useful insights into bone/bone marrow pathophysiology.

Figure 1

Mouse and human CFE in medium with various FBS lots. Mouse and human bone marrow cells were plated in medium with FBS of 1 of 11 lots, either noninactivated or heat inactivated. (A) Mouse cultures. Mouse bone marrow cells from two FVB/N mice were plated in triplicate. Statistically significant differences between noninactivated lots: No. 1 from 5, P<0.05; 1 from 9, P<0.01; 2 from 5, P<0.01; 2 from 7, P<0.05; 2 from 9, P<0.001. (B) Human cultures. Human bone marrow cells from a single donor were plated in quadruplicate. Statistically significant differences between noninactivated lots: No. 3 from all other lots (except 5), P<0.001; 5 from all other lots (except 3), P<0.001. *P<0.05; **P<0.01; ***P<0.001.

Figure 2

Human CFE in cultures from several donors with noninactivated and heat-inactivated FBS. Bone marrow cells from five donors were plated in triplicate in medium containing FBS lot 1, either noninactivated or heat inactivated. The differences between the noninactivated and the heat-inactivated groups were statistically significant only for the first donor. *P<0.05.

Figure 3

CFE in mouse cultures. (A) CFE in cultures from various strains of inbred mice. Mouse bone marrow cells were plated in triplicate. Numbers of animals analyzed per strain were CBA, 11; Rosa, 4; FVB/N, 12; C57Bl, 5; NIH-bg-nu/nu-xid, 2. Only differences between Rosa and CBA and between Rosa and FVB/N were statistically significant. (B) CFE in cultures from transgenic mice and their wild-type littermates. Mouse bone marrow cells were plated in quadruplicate. Numbers of animals analyzed per line were MT1-MMP deficient, 2 wild type, 2 transgenic; IL-5 overproducing, 2 wild type, 2 transgenic; Col1-caPPR, 2 wild type, 4 transgenic (CFE previously reported in Kuznetsov et al., 2004); FGFR3 mutation, 3 wild type, 4 transgenic. *P<0.05; **P<0.01; ****P<0.0001.

Figure 4

Correlation between CFE and age in normal human donors.

Figure 5

CFE in cultures from normal and pathological human donors. (A) Pediatric patients <18 years of age. (B) Adult patients >18 years of age. Each black bar represents a single donor, an average of three or four cultures. All differences are in comparison with the CFE values of age-matched groups of normal donors (striped bars). *P<0.05; **P<0.01.