Tissue Engineering Research Group, Division of Molecular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.
Cytotherapy. 2012 Aug;14(7):791-801. doi: 10.3109/14653249.2012.674639. Epub 2012 Apr 12.
Although bone marrow (BM) stromal cells (SC; BMSC) isolated from adherent cultures of untreated BM are known to contain both committed and uncommitted osteogenic cells, it remains unknown whether BMSC isolated either by hemolysis or Ficoll centrifugation also contain both of these populations.
Differences in the osteogenic cell populations of rat BMSC isolated from untreated, hemolyzed or Ficoll-treated BM were analyzed by in vivo transplantation, flow cytometry, alkaline phosphatase (ALP) assay, real-time polymerase chain reaction (PCR) and alizarin red staining.
Transplantation of non-cultured samples indicated that the Ficolled BMSC contained the lowest number of committed osteogenic cells. Flow cytometric analysis of cultured, non-induced samples showed that the percentage of ALP-positive cells was significantly lower in Ficolled BMSC. Quantitative ALP assays confirmed that the lowest ALP activity was in the Ficolled BMSC. Hemolyzed BMSC also contained lower numbers of committed osteogenic cells than untreated BMSC, but still more than Ficolled BMSC. Interestingly, the Ficolled BMSC showed the greatest levels of osteogenic ability when cultured in osteogenic induction medium.
These findings suggest that, although Ficolled BMSC rarely contain committed osteogenic cells, they are able to show comparable or even greater levels of osteogenic ability after induction, possibly because they contain a greater proportion of uncommitted stem cells. In contrast, induction is optional but recommended for both untreated and hemolyzed BMSC before use, because both these groups contain both committed and uncommitted osteogenic cells. These findings are of significant importance when isolating BMSC for use in bone tissue engineering.
尽管从未经处理的骨髓（BM）贴壁培养物中分离的骨髓基质细胞（BMSC）已知含有已定向和未定向的成骨细胞，但仍不清楚通过溶血或菲可离心分离的 BMSC 是否也含有这两种细胞群体。
通过体内移植、流式细胞术、碱性磷酸酶（ALP）测定、实时聚合酶链反应（PCR）和茜素红染色分析了来自未经处理、溶血或菲可处理的 BM 的大鼠 BMSC 的成骨细胞群体差异。
未培养样本的移植表明，菲可分离的 BMSC 含有最少数量的已定向成骨细胞。培养、未诱导样本的流式细胞术分析表明，菲可分离的 BMSC 中 ALP 阳性细胞的百分比显著降低。定量 ALP 测定证实，菲可分离的 BMSC 的 ALP 活性最低。与未经处理的 BMSC 相比，溶血的 BMSC 也含有较少数量的已定向成骨细胞，但仍多于菲可分离的 BMSC。有趣的是，当在成骨诱导培养基中培养时，菲可分离的 BMSC 显示出最大的成骨能力。
这些发现表明，尽管菲可分离的 BMSC 很少含有已定向的成骨细胞，但在诱导后，它们能够表现出相当或甚至更高水平的成骨能力，可能是因为它们含有更大比例的未定向干细胞。相比之下，在用于骨组织工程之前，未经处理和溶血的 BMSC 都需要进行诱导，因为这两组细胞都含有已定向和未定向的成骨细胞。这些发现对于分离用于骨组织工程的 BMSC 具有重要意义。
