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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.

SUBMITTER: Kuznetsov SA

PROVIDER: S-EPMC2753860 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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

Kuznetsov Sergei A SA Mankani Mahesh H MH Bianco Paolo P Robey Pamela G PG

Stem cell research 20080812 1

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 i ...[more]

PMID: 19383412

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Project description:Measuring the abundance of microbes in a sample is a common procedure with a long history, but best practices are not well-conserved across microbiological ﬁelds. Serial dilution methods are commonly used to dilute bacterial cultures to produce countable numbers of colonies, and from these counts, to infer bacterial concentrations measured in colony-forming units (CFUs). The most common methods to generate data for CFU point estimates involve plating bacteria on (or in) a solid growth medium and counting their resulting colonies or counting the number of tubes at a given dilution that have growth. Traditionally, these types of data have been analyzed separately using different analytic methods. Here, we build a direct correspondence between these approaches, which allows one to extend the use of the most probable number method from the liquid tubes experiments, for which it was developed, to the growth plates by viewing colony-sized patches of a plate as equivalent to individual tubes. We also discuss how to combine measurements taken at different dilutions, and we review several ways of analyzing colony counts, including the Poisson and truncated Poisson methods. We test all point estimate methods computationally using simulated data. For all methods, we discuss their relevant error bounds, assumptions, strengths, and weaknesses. We provide an online calculator for these estimators.Estimation of the number of microbes in a sample is an important problem with a long history. Yet common practices, such as combining results from different measurements, remain sub-optimal. We provide a comparison of methods for estimating abundance of microbes and detail a mapping between different methods, which allows to extend their range of applicability. This mapping enables higher precision estimates of colony-forming units (CFUs) using the same data already collected for traditional CFU estimation methods. Furthermore, we provide recommendations for how to combine measurements of colony counts taken across dilutions, correcting several misconceptions in the literature.

Project description:Uncultured mesenchymal stromal cells (MSCs) are increasingly used in therapies; however, the effects of donor age on their biological characteristics and gene expression remain unclear. The aim of this study was to investigate age-related changes in bone marrow (BM) MSCs following minimal or no culture manipulation. Iliac crest BM was aspirated from 67 healthy donors (19-89 years old) and directly used for the colony-forming unit-fibroblast (CFU-F) assay or CD45lowCD271+ cell enumeration. The colonies were analysed for colony area and integrated density (ID) when grown in standard MSC media or media supplemented with human serum from young (YS) or old (OS) donors. There was a notable age-related decline in the number of MSCs per millilitre of BM aspirate revealed by the CFU-F assay (r = -0.527, p < 0.0001) or flow cytometry (r = -0.307, p = 0.0116). Compared to young donors (19-40 years old), colony IDs were significantly lower in older donors (61-89 years old), particularly for smaller-sized colonies (42% lower, p < 0.01). When cultured in media supplemented with OS, young and old donor MSCs formed colonies with lower IDs, by 21%, p < 0.0001, and 27%, p < 0.05, respectively, indicating the formation of smaller sparser colonies. No significant differences in the expression of selected adipogenic, osteogenic, stromal, and bone remodelling genes as well as CD295, CD146, CD106, and connexin 43 surface molecules were found in sorted CD45lowCD271+ MSCs from young and old donors (n = 8 donors each). Altogether, these results show similar trends for age-related decline in BM MSC numbers measured by the CFU-F assay and flow cytometry and reveal age-related effects of human serum on MSC colony formation. No significant differences in selected gene expression in uncultured CD45lowCD271+ MSCs suggest that old donor MSCs may not be inferior in regard to their multipotential functions. Due to large donor-to-donor variation in all donor groups, our data indicate that an individual's chronological age is not a reliable predictor of their MSC number or potency.

Dataset Information

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

Publications

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

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