Lisa M Grady

Chondrogenic Differentiation of Human Bone Marrow Stem Cells in Transwell Cultures: Generation of Scaffold-Free Cartilage

Human bone marrow stem cells (hMSCs) have been shown to differentiate in vitro into a number of cell lineages and are a potential autologous cell source for the repair and replacement of damaged and diseased musculoskeletal tissues. hMSC differentiation into chondrocytes has been described in high‐density cell pellets cultured with specific growth and differentiation factors. We now describe how culture of hMSCs as a shallow multicellular layer on a permeable membrane over 2–4 weeks resulted in a much more efficient formation of cartilaginous tissue than in established chondrogenic assays. In this format, the hMSCs differentiated in 14 days to produce translucent, flexible discs, 6 mm in diameter by 0.8–1 mm in thickness from 0.5 × 106 cells. The discs contained an extensive cartilage‐like extracellular matrix (ECM), with more than 50% greater proteoglycan content per cell than control hMSCs differentiated in standard cell pellet cultures. The disc constructs were also enriched in the cartilage‐specific collagen II, and this was more homogeneously distributed than in cell pellet cultures. The expression of cartilage matrix genes for collagen type II and aggrecan was enhanced in disc cultures, but improved matrix production was not accompanied by increased expression of the transcription factors SOX9, L‐SOX5, and SOX6. The fast continuous growth of cartilage ECM in these cultures up to 4 weeks appeared to result from the geometry of the construct and the efficient delivery of nutrients to the cells. Scaffold‐free growth of cartilage in this format will provide a valuable experimental system for both experimental and potential clinical studies.

Directed differentiation of human embryonic stem cells toward chondrocytes

We report a chemically defined, efficient, scalable and reproducible protocol for differentiation of human embryonic stem cells (hESCs) toward chondrocytes. HESCs are directed through intermediate developmental stages using substrates of known matrix proteins and chemically defined media supplemented with exogenous growth factors. Gene expression analysis suggests that the hESCs progress through primitive streak or mesendoderm to mesoderm, before differentiating into a chondrocytic culture comprising cell aggregates. At this final stage, 74% (HUES1 cells) and up to 95–97% (HUES7 and HUES8 cells) express the chondrogenic transcription factor SOX9. The cell aggregates also express cell surface CD44 and aggrecan and deposit a sulfated glycosaminoglycan and cartilage-specific collagen II matrix, but show very low or no expression of genes and proteins associated with nontarget cell types. Our protocol should facilitate studies of chondrocyte differentiation and of cell replacement therapies for cartilage repair.

The matrix-forming phenotype of cultured human meniscus cells is enhanced after culture with fibroblast growth factor 2 and is further stimulated by hypoxia

Human meniscus cells have a predominantly fibrogenic pattern of gene expression, but like chondrocytes they proliferate in monolayer culture and lose the expression of type II collagen. We have investigated the potential of human meniscus cells, which were expanded with or without fibroblast growth factor 2 (FGF2), to produce matrix in three-dimensional cell aggregate cultures with a chondrogenic medium at low (5%) and normal (20%) oxygen tension. The presence of FGF2 during the expansion of meniscus cells enhanced the re-expression of type II collagen 200-fold in subsequent three-dimensional cell aggregate cultures. This was increased further (400-fold) by culture in 5% oxygen. Cell aggregates of FGF2-expanded meniscus cells accumulated more proteoglycan (total glycosaminoglycan) over 14 days and deposited a collagen II-rich matrix. The gene expression of matrix-associated proteoglycans (biglycan and fibromodulin) was also increased by FGF2 and hypoxia. Meniscus cells after expansion in monolayer can therefore respond to chondrogenic signals, and this is enhanced by FGF2 during expansion and low oxygen tension during aggregate cultures.

Human meniscus cells express hypoxia inducible factor-1α and increased SOX9 in response to low oxygen tension in cell aggregate culture

In previous work we demonstrated that the matrix-forming phenotype of cultured human cells from whole meniscus was enhanced by hypoxia (5% oxygen). Because the meniscus contains an inner region that is devoid of vasculature and an outer vascular region, here we investigate, by gene expression analysis, the separate responses of cells isolated from the inner and outer meniscus to lowered oxygen, and compared it with the response of articular chondrocytes. In aggregate culture of outer meniscus cells, hypoxia (5% oxygen) increased the expression of type II collagen and SOX9 (Sry-related HMG box-9), and decreased the expression of type I collagen. In contrast, with inner meniscus cells, there was no increase in SOX9, but type II collagen and type I collagen increased. The articular chondrocytes exhibited little response to 5% oxygen in aggregate culture, with no significant differences in the expression of these matrix genes and SOX9. In both aggregate cultures of outer and inner meniscus cells, but not in chondrocytes, there was increased expression of collagen prolyl 4-hydroxylase (P4H)α(I) in response to 5% oxygen, and this hypoxia-induced expression of P4Hα(I) was blocked in monolayer cultures of meniscus cells by the hypoxia-inducible factor (HIF)-1α inhibitor (YC-1). In fresh tissue from the outer and inner meniscus, the levels of expression of the HIF-1α gene and downstream target genes (namely, those encoding P4Hα(I) and HIF prolyl 4-hydroxylase) were significantly higher in the inner meniscus than in the outer meniscus. Thus, this study revealed that inner meniscus cells were less responsive to 5% oxygen tension than were outer meniscus cells, and they were both more sensitive than articular chondrocytes from a similar joint. These results suggest that the vasculature and greater oxygen tension in the outer meniscus may help to suppress cartilage-like matrix formation.

IL-27 Receptor Signaling Regulates Memory CD4+ T Cell Populations and Suppresses Rapid Inflammatory Responses during Secondary Malaria Infection

ABSTRACT Interleukin-27 (IL-27) is known to control primary CD4+ T cell responses during a variety of different infections, but its role in regulating memory CD4+ T responses has not been investigated in any model. In this study, we have examined the functional importance of IL-27 receptor (IL-27R) signaling in regulating the formation and maintenance of memory CD4+ T cells following malaria infection and in controlling their subsequent reactivation during secondary parasite challenge. We demonstrate that although the primary effector/memory CD4+ T cell response was greater in IL-27R-deficient (WSX-1−/−) mice following Plasmodium berghei NK65 infection than in wild-type (WT) mice, there were no significant differences in the size of the maintained memory CD4+ T population(s) at 20 weeks postinfection in the spleen, liver, or bone marrow of WSX-1−/− mice compared with WT mice. However, the composition of the memory CD4+ T cell pool was slightly altered in WSX-1−/− mice following clearance of primary malaria infection, with elevated numbers of late effector memory CD4+ T cells in the spleen and liver and increased production of IL-2 in the spleen. Crucially, WSX-1−/− mice displayed significantly enhanced parasite control compared with WT mice following rechallenge with homologous malaria parasites. Improved parasite control in WSX-1−/− mice during secondary infection was associated with elevated systemic production of multiple inflammatory innate and adaptive cytokines and extremely rapid proliferation of antigen-experienced T cells in the liver. These data are the first to demonstrate that IL-27R signaling plays a role in regulating the magnitude and quality of secondary immune responses during rechallenge infections.

WSX-1 Signalling Inhibits CD4+ T Cell Migration to the Liver during Malaria Infection by Repressing Chemokine-Independent Pathways

IL-27 is an important and non-redundant regulator of effector T cell accumulation in non-lymphoid tissues during infection. Using malaria as a model systemic pro-inflammatory infection, we demonstrate that the aberrant accumulation of CD4+ T cells in the liver of infected IL27R−/− (WSX-1−/−) mice is a result of differences in cellular recruitment, rather than changes in T cell proliferation or cell death. We show that IL-27 both inhibits the migratory capacity of infection-derived CD4+ T cells towards infection-derived liver cells, but also suppresses the production of soluble liver-derived mediator(s) that direct CD4+ T cell movement towards the inflamed tissue. Although CCL4 and CCL5 expression was higher in livers of infected WSX-1−/− mice than infected WT mice, and hepatic CD4+ T cells from WSX-1−/− mice expressed higher levels of CCR5 than cells from WT mice, migration of CD4+ T cells to the liver of WSX-1−/− mice during infection was not controlled by chemokine (R) signalling. However, anti-IL-12p40 treatment reduced migration of CD4+ T cells towards infection-derived liver cells, primarily by abrogating the hepatotropic migratory capacity of T cells, rather than diminishing soluble tissue-derived migratory signals. These results indicate that IL-27R signalling restricts CD4+ T cell accumulation within the liver during infection primarily by suppressing T cell chemotaxis, which may be linked to its capacity to repress Th1 differentiation, as well as by inhibiting the production of soluble, tissue-derived chemotaxins.