In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells

Abstract

One of the applications of bone marrow stromal cells (BMSCs) that are produced by ex vivo expansion is for use in in vivo bone tissue engineering. Cultured stromal cells are a mixture of cells at different stages of commitment and expansion capability, leading to a heterogeneous cell population that each time can differ in the potential to form in vivo bone. A parameter that predicts for in vivo bone forming capacity is thus far lacking. We employed single colony-derived BMSC cultures to identify such predictive parameters. Using limiting dilution, we have produced sixteen single CFU-F derived BMSC cultures from human bone marrow and found that only five of these formed bone in vivo. The single colony-derived BMSC strains were tested for proliferation, osteogenic-, adipogenic- and chondrogenic differentiation capacity and the expression of a variety of associated markers. The only robust predictors of in vivo bone forming capacity were the induction of alkaline phosphatase, (ALP) mRNA levels and ALP activity during in vitro osteogenic differentiation. The predictive value of in vitro ALP induction was confirmed by analyzing "bulk-cultured" BMSCs from various bone marrow biopsies. Our findings show that in BMSCs, the additional increase in ALP levels over basal levels during in vitro osteogenic differentiation is predictive of in vivo performance.

Characterization of Cellular and Molecular Heterogeneity of Bone Marrow Stromal Cells

“…Our data corroborate and extend these findings by reporting the effects of siRNA-mediated inhibition of ALP that resulted in an impaired hBMSC differentiation not only to osteoblasts, but also to adipocytes. Also, our results corroborate earlier studies that demonstrated in human bone biopsies the presence of ALP expression in bone marrow adipocytic cells [13, 17]. All these data suggest that ALP is a “stemness” marker of hBMSC and not just an indicator of osteoblastic lineage commitment.…”

“…Since ALP has been suggested as a marker for hBMSC progenitor cell lineage commitment [13] and was highly upregulated in CL1 cells, we tested its biological role in CL1 cells. ALP siRNA transfection decreased ALP protein level, ALP activity, and mRNA gene expression compared to control cells transfected with control siRNA ( p < 0.01) and this inhibition was detectable up to day 7 days after osteoblast differentiation induction (Figures 4(A) and 4(B)).…”

“…ALP is expressed in a wide variety of tissues, including kidneys, bone, and liver [36, 37], but tissue-nonspecific ALP (ALPL) is considered a commitment marker for osteoblastic lineage [13, 38]. However, in a recent study the authors examined the differentiation potential of a number of hMSC clones in vitro and in vivo and reported that the hMSC clones with high levels of ALP expression were committed to trilineage differentiation [13]. Our data corroborate and extend these findings by reporting the effects of siRNA-mediated inhibition of ALP that resulted in an impaired hBMSC differentiation not only to osteoblasts, but also to adipocytes.…”

Two-component thermosensitive hydrogels: Phase separation affecting rheological behavior

“…After dehydration using graded series of ethanol/water solutions and a final washing step with xylene, the dehydrated hydrogels were embedded in paraffin and finally sliced into 5 µm thick samples. Safranin-O/fast green assay was performed as previously described in literature [49], and images were collected using an Olympus microscope equipped with a digital camera (Olympus BX51 microscope, Olympus DP70 camera, Hamburg, Germany).…”

Glutamic acid ameliorates estrogen deficiency–induced menopausal-like symptoms in ovariectomized mice

“…Cell survival and proliferation are mediated principally through extracellular signal-regulated kinase (ERK) MAPK pathways [15]. Alkaline phosphatase (ALP) activity, which is a marker of osteoblast differentiation and bone formation [16], is up-regulated via the activation of ERK signaling [3]. The analysis of bone cell-specific markers, such as ALP, is frequently used to characterize osteoblasts [17].…”