Proliferation and differentiation potential of chondrocytes from osteoarthritic patients

Autologous chondrocyte transplantation (ACT) has been shown, in long-term follow-up studies, to be a promising treatment for the repair of isolated cartilage lesions. The method is based on an implantation of in vitro expanded chondrocytes originating from a small cartilage biopsy harvested from a non-weight-bearing area within the joint. In patients with osteoarthritis (OA), there is a need for the resurfacing of large areas, which could potentially be made by using a scaffold in combination with culture-expanded cells. As a first step towards a cell-based therapy for OA, we therefore investigated the expansion and redifferentiation potential in vitro of chondrocytes isolated from patients undergoing total knee replacement. The results demonstrate that OA chondrocytes have a good proliferation potential and are able to redifferentiate in a three-dimensional pellet model. During the redifferentiation, the OA cells expressed increasing amounts of DNA and proteoglycans, and at day 14 the cells from all donors contained type II collagen-rich matrix. The accumulation of proteoglycans was in comparable amounts to those from ACT donors, whereas total collagen was significantly lower in all of the redifferentiated OA chondrocytes. When the OA chondrocytes were loaded into a scaffold based on hyaluronic acid, they bound to the scaffold and produced cartilage-specific matrix proteins. Thus, autologous chondrocytes are a potential source for the biological treatment of OA patients but the limited collagen synthesis of the OA chondrocytes needs to be further explained

In Situ Labeling and Magnetic Resonance Imaging of Transplanted Human Hepatic Stem Cells

The purpose is to address the problem in magnetic resonance imaging (MRI) of contrast agent dilution

Human hepatic stem cells from fetal and postnatal donors

Human hepatic stem cells (hHpSCs), which are pluripotent precursors of hepatoblasts and thence of hepatocytic and biliary epithelia, are located in ductal plates in fetal livers and in Canals of Hering in adult livers. They can be isolated by immunoselection for epithelial cell adhesion molecule–positive (EpCAM+) cells, and they constitute ∼0.5–2.5% of liver parenchyma of all donor ages. The self-renewal capacity of hHpSCs is indicated by phenotypic stability after expansion for >150 population doublings in a serum-free, defined medium and with a doubling time of ∼36 h. Survival and proliferation of hHpSCs require paracrine signaling by hepatic stellate cells and/or angioblasts that coisolate with them. The hHpSCs are ∼9 μm in diameter, express cytokeratins 8, 18, and 19, CD133/1, telomerase, CD44H, claudin 3, and albumin (weakly). They are negative for α-fetoprotein (AFP), intercellular adhesion molecule (ICAM) 1, and for markers of adult liver cells (cytochrome P450s), hemopoietic cells (CD45), and mesenchymal cells (vascular endothelial growth factor receptor and desmin). If transferred to STO feeders, hHpSCs give rise to hepatoblasts, which are recognizable by cordlike colony morphology and up-regulation of AFP, P4503A7, and ICAM1. Transplantation of freshly isolated EpCAM+ cells or of hHpSCs expanded in culture into NOD/SCID mice results in mature liver tissue expressing human-specific proteins. The hHpSCs are candidates for liver cell therapies

Human articular chondrocytes. Plasticity and differentiation potentials

Articular cartilage has no or very low ability for self-repair and untreated lesions may lead tothe development of Osteoarthritis (OA). One method, which has been proved to result in longterm repair of isolated lesions, is autologous chondrocyte transplantation (ACT). In this methodculture expanded chondrocytes isolated from full-thickness biopsies taken from the supromedialedge, a non-weight bearing area of the femoral condyle, are transplanted back to the patientunder a cover of periosteum. To be able to improve the method and widen the treatmentindication to patients with fully developed OA, the general aim of this thesis was to increasethe knowledge of the cellular and molecular mechanisms underlying the repair tissue generatedby culture expanded chondrocytes.Our initial hypothesis to be tested was whether chondrocytes have stem cell properties. Thisquestion was addressed by using the established differentiation protocols for mesenchymalstem cells (MSCs) on culture expanded dedifferentiated human articular chondrocytes isolatedfrom non OA patients. The chondrocytes were found to exhibit a level of phenotypic plasticitythat is comparable with that of MSCs. By utilizing a cartilage selective agarose culture system,the plasticity of the cartilage progenitor cells was further shown to originate from clonal true chondrocytes and not from the heterogenous population of cells generated from acartilage biopsy that could harbor contaminating bone marrow stromal cells.By supplementing monolayer cultured chondrocytes with human serum instead of foetal calfserum a higher proliferative rate was achieved without losing the ability for cartilageredifferentiation in a 3D environment under serum free culture conditions. With microarraytechnology, the dynamic process of chondrocyte redifferentiation was shown to involve genesknown to be expressed in early embryonic chondrogenesis.Chondrocytes from OA patients were shown to have a proliferation potential in monolayercultures and an ability to redifferentiate in a pellet model at the same level as chondrocytesfrom non-OA patients. The cells could also be loaded into a hyaluronic acid based scaffold inwhich they did bind and produce cartilage proteoglycans and collagen. However, even if theOA cells produced cartilage specific matrix proteins, the cells did not produce the sameamount of total collagen as cells from patients without OA. The OA cells also continued theirproliferation within the redifferentiation model, indicating a potentially disturbed control intheir cell cycle.These findings demonstrate that treatment with ACT utilizes chondroprogenitor cells whichhave the potential to recapitulate embryonic genes during redifferentiation. This knowledgeis important for ongoing research in identifying strategies for regenerative therapies of othertissues and organs

Topographic variation in redifferentiation capacity of chondrocytes in the adult human knee joint.

OBJECTIVES: The aim of this study was to investigate the topographic variation in matrix production and cell density in the adult human knee joint. Additionally, we have examined the redifferentiation potential of chondrocytes expanded in vitro from the different locations. METHOD: Full thickness cartilage-bone biopsies were harvested from seven separate anatomical locations of healthy knee joints from deceased adult human donors. Chondrocytes were isolated, expanded in vitro and redifferentiated in a pellet mass culture. Biochemical analysis of total collagen, proteoglycans and cellular content as well as histology and immunohistochemistry were performed on biopsies and pellets. RESULTS: In the biochemical analysis of the biopsies, we found lower proteoglycan to collagen (GAG/HP) ratio in the non-weight bearing (NWB) areas compared to the weight bearing (WB) areas. The chondrocytes harvested from different locations in femur showed a significantly better attachment and proliferation ability as well as good post-expansion chondrogenic capacity in pellet mass culture compared with the cells harvested from tibia. CONCLUSION: These results demonstrate that there are differences in extra cellular content within the adult human knee in respect to GAG/HP ratio. Additionally, the data show that clear differences between chondrocytes harvested from femur and tibia from healthy human knee joints exist and that the differences are not completely abolished during the process of de- and redifferentiation. These findings emphasize the importance of the understanding of topographic variation in articular cartilage biology when approaching new cartilage repair strategies

Human hepatic stem cells from fetal and postnatal donors

Human hepatic stem cells (hHpSCs), which are pluripotent precursors of hepatoblasts and thence of hepatocytic and biliary epithelia, are located in ductal plates in fetal livers and in Canals of Hering in adult livers. They can be isolated by immunoselection for epithelial cell adhesion molecule–positive (EpCAM+) cells, and they constitute ∼0.5–2.5% of liver parenchyma of all donor ages. The self-renewal capacity of hHpSCs is indicated by phenotypic stability after expansion for >150 population doublings in a serum-free, defined medium and with a doubling time of ∼36 h. Survival and proliferation of hHpSCs require paracrine signaling by hepatic stellate cells and/or angioblasts that coisolate with them. The hHpSCs are ∼9 μm in diameter, express cytokeratins 8, 18, and 19, CD133/1, telomerase, CD44H, claudin 3, and albumin (weakly). They are negative for α-fetoprotein (AFP), intercellular adhesion molecule (ICAM) 1, and for markers of adult liver cells (cytochrome P450s), hemopoietic cells (CD45), and mesenchymal cells (vascular endothelial growth factor receptor and desmin). If transferred to STO feeders, hHpSCs give rise to hepatoblasts, which are recognizable by cordlike colony morphology and up-regulation of AFP, P4503A7, and ICAM1. Transplantation of freshly isolated EpCAM+ cells or of hHpSCs expanded in culture into NOD/SCID mice results in mature liver tissue expressing human-specific proteins. The hHpSCs are candidates for liver cell therapies