Adult stem-cell therapy holds promise for the treatment of gastrointestinal diseases. Here we describe methods for long-term expansion of colonic stem cells positive for leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5(+) cells) in culture. To test the transplantability of these cells, we reintroduced cultured GFP(+) colon organoids into superficially damaged mouse colon. The transplanted donor cells readily integrated into the mouse colon, covering the area that lacked epithelium as a result of the introduced damage in recipient mice. At 4 weeks after transplantation, the donor-derived cells constituted a single-layered epithelium, which formed self-renewing crypts that were functionally and histologically normal. Moreover, we observed long-term (>6 months) engraftment with transplantation of organoids derived from a single Lgr5(+) colon stem cell after extensive in vitro expansion. These data show the feasibility of colon stem-cell therapy based on the in vitro expansion of a single adult colonic stem cell.

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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.

Osteoarthritis in the knees, which can be caused by meniscal defect, constitutes an increasingly common medical problem. Repair for massive meniscal defect remains a challenge owing to a lack of cell kinetics for the menisci precursors in knee joint. The synovium plays pivotal roles during the natural course of meniscal healing and contains mesenchymal stem cells (MSCs) with high chondrogenic potential. Here, we investigated whether intra-articular injected synovium-MSCs enhanced meniscal regeneration in rat massive meniscal defect. To track the injected cells, we developed transgenic rats expressing dual luciferase (Luc) and LacZ. The cells derived from synovium of the rats demonstrated colony-forming ability and multipotentiality, both characteristics of MSCs. Hierarchical clustering analysis revealed that gene expression of meniscal cells was closer to that of synovium-MSCs than to that of bone marrow-MSCs. Two to 8 weeks after five million Luc/ LacZ1 synovium-MSCs were injected into massive meniscectomized knee of wild-type rat, macroscopically, the menisci regenerated much better than it did in the control group. After 12 weeks, the regenerated menisci were LacZ positive, produced type 2 collagen, and showed meniscal features by transmission electron microscopy. In in-vivo luminescence analysis, photons increased in the meniscusresected knee over a 3-day period, then decreased without detection in all other organs. LacZ gene derived from MSCs could not be detected in other organs except in synovium by real-time PCR. Synovium-MSCs injected into the massive meniscectomized knee adhered to the lesion, differentiated into meniscal cells directly, and promoted meniscal regeneration without mobilization to distant organs.

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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...

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