Objective. To compare the properties of human mesenchymal stem cells (MSCs) isolated from bone marrow, synovium, periosteum, skeletal muscle, and adipose tissue.Methods. Human mesenchymal tissues were obtained from 8 donors during knee surgery for ligament injury. After collagenase digestion or gradient-density separation, nucleated cells were plated at an appropriate density for expansion at the maximum rate without colony-to-colony contact. Yield, expandability, differentiation potential, and epitope profile were compared among MSCs from the 5 different tissue sources.Results. Colony number per 10 3 nucleated cells was lower, and cell number per colony was higher, in bone marrow than in other mesenchymal tissues. When the cells were replated at low density every 14 days, bone marrow-, synovium-, and periosteum-derived cells retained their proliferation ability even at passage 10. In chondrogenesis studies in which the cells were pelleted and cultured in vitro, pellets from bone marrow-, synovium-, and periosteum-derived cells were shown to be larger and stained more extensively for cartilage matrix. Synovium-derived cells, in particular, had the greatest ability for chondrogenesis. In adipogenesis experiments, the frequency of oil red O-positive colonies was highest in synovium-and adipose tissue-derived cells. In studies of osteogenesis, the rate of alizarin red-positive colonies was highest in bone marrow-, synovium-, and periosteum-derived cells. For epitope profiling, 15 surface antigens were measured. Most appeared to have similar epitope profiles irrespective of cell source.Conclusion. Our findings indicate that there are significant differences in MSC properties according to tissue source, beyond donor and experimental variation. Superiority of synovium as a potential source of MSCs for clinical applications was demonstrated.

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There are increasing reports that mesenchymal stem cells (MSCs) are present in various tissues other than bone marrow, including synovium. Here we investigated the optimal conditions for in vitro chondrogenesis of human synovium-derived MSCs and compared these cells with bone marrow-derived MSCs, especially in terms of their chondrogenesis potential. Synovium and bone marrow were harvested from six donors during knee operations for ligament injuries. Digested synovium cells or nucleated cells from bone marrow were expanded clonally. A pellet culture system was used for chondrogenesis, and the best combination of up to three cytokines of the seven assessed. Synovium-derived MSCs plated at a lower density expanded more rapidly. Contrary to previous reports, a combination of TGFbeta and dexamethasone was not sufficient to induce chondrogenesis. However, addition of BMP2 to TGFbeta and dexamethasone dramatically increased cartilage pellet size and the synthesis of cartilage matrix. The cartilage pellets were also analyzed by electron microscopy and immunohistology. DNA content per pellet decreased during chondrogenesis, indicating the pellet increased its size through the accumulation of newly synthesized extracellular matrix. Sequential chondrogenic gene expression was demonstrated by RT-PCR. Synovium-derived MSCs looked similar to the bone marrow-derived MSCs in their surface epitopes and proliferation potential; however, cartilage pellets from synovium were significantly larger than those from bone marrow in patient-matched comparisons. We demonstrated that the combination of TGFbeta, dexamethasone, and BMP2 was optimal for in vitro chondrogenesis of synovium-derived MSCs and that the synovium-derived MSCs have a greater chondrogenesis potential than bone marrow-derived MSCs.

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Several reports describe that the explant culture of the trabecular bone after collagenase treatment produces mesenchymal stem cells (MSCs). However, the suspended cells had not been intensively examined concerning MSCs. We hypothesized that the cells would acquire the properties of MSCs during their expansion and therefore compared them with marrow aspiratederived MSCs. Human trabecular bones were washed, digested, filtered, and expanded clonally for 14 days. Their proliferation ability (n ‫؍‬ 9) and differentiation potentials for chondrocyte, adipocyte, and osteoblast (n ‫؍‬ 6) were similar with those of marrow aspirate-derived MSCs. Epitope and mRNA analysis revealed some differences in both types of cells, which disappeared with expansion and subcultivation. A mixed population of collagenase-released (CR) cells had similar differentiation potentials with CR clone, CD31 ؉ fraction, and osteoblastic cells. For quantitative study, trabecular bone and bone marrow were harvested by single aspiration using a biopsy needle (n ‫؍‬ 16). Although the total nucleated cell number harvested was similar, the colony-forming efficiency of CR cells was approximately 100-fold higher than that of BM cells and more than 1 million CR cells could be obtained in 14 days from all donors. Enzymatically released cells from trabecular bone became virtually identical to marrow aspirate-derived MSCs, demonstrating that a trabecular bone fragment can be an alternative source of MSCs. IntroductionHuman cells derived from trabecular bones have been used for many years in the study of bone cell biology. [1][2][3][4][5] Recently, Tuli et al, 6,7 Noth et al, 8 and Sottile et al 9 reported that human trabecular bone is a good source of mesenchymal stem cells (MSCs) with the characteristics of self-renewal and multilineage differentiation potential. To isolate MSCs from trabecular bones, they digested human trabecular bone with collagenase to release cells, referred to as collagenase-released (CR) cells. Then, they did explant cultures of the remaining bare bone fragments and harvested MSCs that were derived from the trabecular bones. CR cells were first mentioned by Robey and Termine as part of a study of osteoblast metabolism in vitro. 1 However, the CR cells were not examined intensively because the CR cell cultures showed a high level of type III collagen, which the researchers simply regarded as fibroblastic contamination. 1 Also, Noth et al described contamination by foreign marrow cells, especially fibroblasts, often predominated in the CR cell cultures. 8 Furthermore, collagenase pretreatment followed by extensive washing resulted in trabecular bone fragments completely devoid of surface-adherent cells, with all soft-tissue elements removed. 8 Therefore, until now the CR cells have been thought to be a poor source of MSCs.A large body of literature has developed on MSCs derived from bone marrow aspirate, 10 referred to as bone marrow (BM) cells here. Bone marrow is the tissue filling the space between vascular sinuses and bone surfaces...

Cell-based or pharmacological approaches for promoting tendon repair are currently not available because the molecular mechanisms of tendon development and healing are not well understood. Although analysis of knockout mice provides many critical insights, small animals such as mice have some limitations. In particular, precise physiological examination for mechanical load and the ability to obtain a sufficient number of primary tendon cells for molecular biology studies are challenging using mice. Here, we generated Mohawk (Mkx) −/− rats by using CRISPR/Cas9, which showed not only systemic hypoplasia of tendons similar to Mkx −/− mice, but also earlier heterotopic ossification of the Achilles tendon compared with Mkx −/− mice. Analysis of tendon-derived cells (TDCs) revealed that Mkx deficiency accelerated chondrogenic and osteogenic differentiation, whereas Mkx overexpression suppressed chondrogenic, osteogenic, and adipogenic differentiation. Furthermore, mechanical stretch stimulation of Mkx −/− TDCs led to chondrogenic differentiation, whereas the same stimulation in Mkx +/+ TDCs led to formation of tenocytes. ChIP-seq of Mkx overexpressing TDCs revealed significant peaks in tenogenic-related genes, such as collagen type (Col)1a1 and Col3a1, and chondrogenic differentiation-related genes, such as SRY-box (Sox)5, Sox6, and Sox9. Our results demonstrate that Mkx has a dual role, including accelerating tendon differentiation and preventing chondrogenic/ osteogenic differentiation. This molecular network of Mkx provides a basis for tendon physiology and tissue engineering.Achilles tendon ossification T endons play a critical role in the musculoskeletal system by connecting muscle to bone to transmit mechanical loads and enable movement. Tendon injuries and damage are repaired slowly and incompletely because of poor intrinsic healing capacity, which in part results from tissue hypocellularity and hypovascularity (1). Even after surgical tendon repair, a standard treatment for tendon rupture, clinical outcomes are not satisfactory because of recurrent rupture or adhesions (2). To develop cell-based or pharmacological approaches for promoting tendon repair, the molecular mechanism of tendon development and regeneration must be determined; however, the key genome network for tendon differentiation and homeostasis has not been well characterized.We, along with other researchers, recently reported the tendon-specific expression and functions of the transcription factor Mohawk (Mkx), which regulates tendon-related gene expression (3, 4). Mkx knockout mice showed general tendon hypoplasia (5, 6), suggesting that Mkx plays an important role during tendon development. Moreover, overexpression of Mkx in mesenchymal stem cells (MSC) elevates tendon-related markers, and transplantation of these cells increases the diameter of collagen fibers in tendons (7,8), suggesting the potential application of Mkx in cell therapy for tendon injury.Although the results from analysis of Mkx knockout mice have provided critical informatio...

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