Prins Henk-Jan, Braat A Koen, Gawlitta D, Dhert Wouter J A, Egan David A, Tijssen-Slump Estel, Yuan Huipin, Coffer Paul J, Rozemuller Henk, Martens Anton C
Department of Immunology, University Medical Center Utrecht, Lundlaan 6, 3584 EA Utrecht, The Netherlands.
Department of Cell Biology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
Stem Cell Res. 2014 Mar;12(2):428-40. doi: 10.1016/j.scr.2013.12.001. Epub 2013 Dec 12.
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.
体外扩增产生的骨髓基质细胞（BMSCs）的应用之一是用于体内骨组织工程。培养的基质细胞是处于不同分化阶段和具有不同扩增能力的细胞混合物，导致细胞群体异质性，每次形成体内骨的潜力可能不同。到目前为止，尚缺乏预测体内骨形成能力的参数。我们采用单克隆来源的BMSC培养物来确定此类预测参数。通过有限稀释法，我们从人骨髓中产生了16种单克隆集落形成单位成纤维细胞（CFU-F）来源的BMSC培养物，发现其中只有5种在体内形成了骨。对单克隆来源的BMSC菌株进行了增殖、成骨、成脂和成软骨分化能力以及多种相关标志物表达的检测。体内骨形成能力的唯一可靠预测指标是体外成骨分化过程中碱性磷酸酶（ALP）mRNA水平和ALP活性的诱导情况。通过分析来自各种骨髓活检的“批量培养”BMSCs，证实了体外ALP诱导的预测价值。我们的研究结果表明，在BMSCs中，体外成骨分化过程中ALP水平相对于基础水平的额外增加可预测其体内性能。
