Characterization of single cell derived cultures of periosteal progenitor cells to ensure the cell quality for clinical application

For clinical applications of cells and tissue engineering products it is of importance to characterize the quality of the used cells in detail. Progenitor cells from the periosteum are already routinely applied in the clinics for the regeneration of the maxillary bone. Periosteal cells have, in addition to their potential to differentiate into bone, the ability to develop into cartilage and fat. However, the question arises whether all cells isolated from periosteal biopsies are able to differentiate into all three tissue types, or whether there are subpopulations. For an efficient and approved application in bone or cartilage regeneration the clarification of this question is of interest. Therefore, 83 different clonal cultures of freshly isolated human periosteal cells derived from mastoid periosteum biopsies of 4 donors were generated and growth rates calculated. Differentiation capacities of 51 clonal cultures towards the osteogenic, the chondrogenic, and the adipogenic lineage were investigated. Histological and immunochemical stainings showed that 100% of the clonal cultures differentiated towards the osteogenic lineage, while 94.1% demonstrated chondrogenesis, and 52.9% could be stimulated to adipogenesis. For osteogenesis real-time polymerase chain reaction (PCR) of BGLAP and RUNX2 and for adipogenesis of FABP4 and PPARG confirmed the results. Overall, 49% of the cells exhibited a tripotent potential, 45.1% showed a bipotent potential (without adipogenic differentiation), 3.9% bipotent (without chondrogenic differentiation), and 2% possessed a unipotent osteogenic potential. In FACS analyses, no differences in the marker profile of undifferentiated clonal cultures with bi- and tripotent differentiation capacity were found. Genome-wide microarray analysis revealed 52 differentially expressed genes for clonal subpopulations with or without chondrogenic differentiation capacity, among them DCN, NEDD9, TGFBR3, and TSLP. For clinical applications of periosteal cells in bone regeneration all cells were inducible. For a chondrogenic application a fraction of 6% of the mixed population could not be induced.

对于细胞与组织工程产品的临床应用而言，详尽表征所用细胞的质量至关重要。骨膜（Periosteum）来源的祖细胞（Progenitor cells）已常规应用于临床上颌骨再生治疗。骨膜细胞除具备向骨组织分化的潜能外，还可向软骨与脂肪组织分化发育。然而，一个核心问题亟待解答：从骨膜活检样本中分离的全部细胞是否均具备向这三种组织分化的能力，抑或其中存在不同的细胞亚群？明确该问题对于骨或软骨再生的高效合规临床应用具有重要意义。因此，本研究从4名供者的乳突骨膜（Mastoid Periosteum）活检样本中分离得到新鲜人骨膜细胞，构建了83株独立克隆培养物，并测定了其生长速率。随后针对其中51株克隆培养物的成骨、成软骨及成脂分化潜能展开了研究。组织学与免疫化学染色结果显示，100%的克隆培养物可向成骨系分化，94.1%可发生成软骨分化，另有52.9%可被诱导完成成脂分化。针对成骨分化，通过实时聚合酶链式反应（Real-time Polymerase Chain Reaction，PCR）检测BGLAP与RUNX2的转录水平；针对成脂分化，则通过检测FABP4与PPARG的转录水平验证了上述染色结果。整体而言，49%的细胞具备三向分化潜能，45.1%具备双向分化潜能（仅可完成成骨与成软骨分化，无法发生成脂分化），3.9%为另一类双向潜能细胞（仅可完成成骨与成脂分化，无法发生成软骨分化），另有2%仅具备单向成骨分化潜能。流式细胞术（Fluorescence-Activated Cell Sorting，FACS）分析显示，具备双向与三向分化潜能的未分化克隆培养物，其表面标志物表达谱无显著性差异。全基因组微阵列分析显示，在具备与不具备成软骨分化能力的克隆亚群中，共鉴定出52个差异表达基因，其中包括DCN、NEDD9、TGFBR3与TSLP。若将骨膜细胞应用于骨再生的临床治疗，所有细胞均具备分化诱导可行性；但若用于成软骨再生的临床应用，则混合细胞群中约6%的细胞无法被诱导发生成软骨分化。