Despite the increasing interest in the potential therapeutic applications of BMSCs, there are major obstacles to their processing. It has been demonstrated that the standard cultures of BMSCs contain a heterogeneous population of cells, varying in their capacity to differentiate.18
Currently, the most favored method for BMSC expansion is at low monolayer density, which reduces contact-induced differentiation.18
Irrespective of the care taken to expand BMSCs, the cells can dramatically lose their capacity to differentiate even after relatively short periods of expansion. This is probably due to dilution of the early progenitor cells with partially or fully committed precursors. There is therefore a need for an assay that rapidly and robustly predicts the potential efficacy of a given BMSC culture without the necessity for time-consuming and often subjective assays of differentiation. Equipped with such a tool, investigators have the ability to identify and quantify the early progenitor status in BMSC cultures and improve current BMSC culture strategies.
In an attempt to identify the most robust markers of differentiation potential, we carefully designed a comparative study consisting of early-passage BMSCs and a panel of negative controls. The controls consisted of the appropriately passaged donor-matched dermal fibroblasts, late-passage BMSCs, and ATCC-derived fibroblast cell lines. We applied in vitro
differentiation assays to osteoblasts, chondrocytes, and adipocytes to validate our sources of early progenitor BMSCs and negative controls. All of the controls had barely detectable or absent potential for transdifferentiation, with the exception of one of the donor-matched fibroblast preparations. The cells from this donor reproducibly differentiated into mineralizing osteoblasts but failed to elicit a positive response in the ALP assays that define early, premineralizing osteoblasts. The cells also failed to produce adipocytic cultures and chondrocyte pellets, suggesting that their transdifferentiation potential was confined to one particular osteogenic pathway that probably bypasses the immature osteogenic stage. Although the physiological relevance of such a phenomenon is therefore questionable, differentiation by fibroblasts has been documented in the literature,21,32,33
demonstrating the need for validation of fibroblast controls and alternative negative controls, such as late-passage BMSCs.
Initially, we set out to compose a detailed profile of surface epitopes currently favored for the identification and characterization of BMSCs. We found no differences in the expression of 24 of the most commonly used markers when fibroblasts and p2 BMSCs were compared. Not surprisingly, all cultures were negative for hematopoietic markers. Markers that are commonly used for proving the identity of BMSCs, especially CD105, CD49, and CD90, were expressed in fibroblasts, demonstrating that these markers are limited in utility.
Comparative transcriptome analysis using the Affymetrix microarray revealed that BMSCs and fibroblasts are strikingly similar, with only a handful of differentially expressed genes between them. The microarray data predicted that some surface epitopes may be useful for direct selection of early progenitor BMSCs, but when analyzed using fluorescence-activated cell sorting, levels of surface protein were variable (see supplemental data
). It was therefore decided to examine transcripts that encoded secreted proteins. One of the most prominent of this group of genes was LIF. LIF is a member of the interleukin (IL)6-type cytokine family comprising IL6, IL11, oncostatin M, ciliary neurotrophic factor, cardiotrophin-1, and cardiotrophin-like cytokine.34,35
In addition to their function in inflammation and immunity, these cytokines also play a crucial role in hematopoiesis, tissue regeneration, embryonic development, osteogenesis, and adipogenesis. LIF transduces its signal via sequestration of a monomer of the IL6 receptor (GP130) and the specific LIF receptor (LIFR).36
The resultant complex transmits conformational changes into the cell, leading to a sequence of phosphorylation events and the recruitment of Janus kinase type 3 (Jak3). Jak3 is transiently immobilized at the membrane, where it activates members 1, 3, and 5 of the signal transducer and activation of transcription (STAT) family of transcription factors.35,37
The presence of the LIFR transcript in BMSCs suggests that there is potential for LIF-induced autocrine modulation of the Jak/STAT pathway.
Although the role of LIF secretion by human BMSCs is poorly understood, LIF seems to have a negative effect on differentiation by stromal progenitors. Malaval and colleagues, who demonstrated that chronic LIF exposure could inhibit osteogenic differentiation by rat calvarial progenitor cells38–43
and that dexamethasone41,42
or a dominant negative form of LIF40,43
could antagonize this effect, first reported this. LIF has also been shown to inhibit adipogenesis by a multipotent murine bone marrow–derived cell line.44
Based on the available literature, the effects of LIF on adult stromal progenitor cells appear to mimic the effects of LIF on murine embryonic stem cells, inhibiting inappropriate differentiation while supporting proliferation.45
LIF has been reported to stimulate proliferation of human adipose–derived BMSCs and multipotential adult progenitor cells in vitro
Although our donor pool had a limited age range (24–46), we found that the proliferative and differentiation capacity of the BMSCs did not appreciably vary with age at early passage. Levels of LIF secretion, which was maintained between 86.4 and 166.16
ng/mL per 48
h, reflected this. Our results are similar to those of Igarashi et al.
who examined the effect of age on gene expression by iliac crest–derived bone marrow stromal cells. Samples were obtained from seven young donors (aged 18–39) and LIF secretion was compared with that of seven older donors (aged 53–81). No significant difference in the expression of LIF or differentiation potential was observed between these groups. Control fibroblasts in the study expressed negligible levels of LIF. Therefore, our observations and those of Igarashi et al.
strongly suggest that LIF could be of great importance in clinical studies for patients of various ages.
In contrast with some studies, we failed to detect any monopotent cells. In the two key studies that document their existence, their detection was probably dependent on the culture conditions. Muraglia et al.49
employed an insulin-mediated adipogenic protocol, whereas our experiments employed the protocol of Digirolamo et al.
which is based on pharmaceutical manipulation of peroxisome proliferator-activated receptors gamma activity and cyclic 3',5'-adenosine monophosphate levels.1
We propose that the adipogenic medium used in this study is more powerful in inducing the adipogenic phenotype, accounting for the detection of more adipogenic clones than in the Maraglia study.50
Indeed, Digirolamo demonstrated that all of their early-passage clones had adipogenic and osteogenic potential; only when the cells were cultured to senescence did they exhibit exclusively osteogenic potential. The BMSC cultures in this study were cultured and tested for differentiation potential within 28 days, a shorter duration than in the Muriglia and Digirolamo studies. It is likely that we would have detected clones with only osteogenic potential if we had increased the number of cell doublings.
In the future, LIF secretion may prove to have an essential role in the growth and maintenance of BMSCs, accounting for its presence in the conditioned medium. The data presented here demonstrate that detection of LIF secretion using ELISA assay represents a robust predictor and indicator of early progenitor status in adult bone marrow stromal cells. Measurement of this parameter is rapid and noninvasive to the culture, suggesting that it may be an invaluable future tool for the production of high-quality BMSCs for clinical trial purposes.