Current therapies involving chondrocytes or mesenchymal stromal cells (MSCs) remain inefficient in restoring cartilage properties upon injury. The induced pluripotent stem-cell (iPSC)-derived mesenchymal progenitor cells (iMPCs) have been put forward as a promising alternative cell source due to their high proliferation and differentiation potential. However, the observed cell loss during in vitro chondrogenesis is currently a bottleneck in establishing articular chondrocyte generation from iPSCs. In a search for candidate mechanisms underlying the low iPSC-derived cartilage tissue yield, global transcriptomes were compared between iMPCs and MSCs and the cell properties were analyzed via a condensation assay. The iMPCs had a more juvenile mesenchymal gene signature than MSCs with less myofibroblast-like characteristics, including significantly lower ECM- and integrin-ligand-related as well as lower α-smooth-muscle-actin expression. This correlated with less substrate and more cell-cell adhesion, impaired aggregate formation and consequently inferior cohesive tissue properties of the iMPC-pellets. Along lower expression of pro-survival ECM molecules, like decorin, collagen VI, lumican and laminin, the iMPC populations had significantly less active ERK1/2 compared to MSCs. Overall, this study proposes that this ECM and integrin-ligand shortage, together with insufficient pro-survival ERK1/2-activity, explains the loss of a non-aggregating iMPC sub-fraction during pellet formation and reduced survival of cells in early pellets. Enhancing ECM production and related signaling in iMPCs may be a promising new means to enrich the instructive microenvironment with pro-survival cues allowing to improve the final cartilage tissue yield from iPSCs.
Mesodermal differentiation of induced pluripotent stem cells (iPSCs) in vitro and subsequent specification into mesodermal derivatives like chondrocytes is currently afflicted with a substantial cell loss that severely limits tissue yield. More knowledge on the key players regulating mesodermal differentiation of iPSCs is currently needed to drive all cells into the desired lineage and to overcome the current need for intermediate cell selection steps to remove misdifferentiated cells. Using two independent human iPSC lines, we here report that a short initial WNT/β-catenin pulse induced by the small molecule CHIR99021 (24 h) enhanced expression of mesodermal markers (PDGFRα, HAND1, KDR, and GATA4), supported the exit from pluripotency (decreased OCT4, SOX2, and LIN28A) and inhibited ectodermal misdifferentiation (reduced PAX6, TUBB3, and NES). Importantly, the initial CHIR pulse increased cell proliferation until day 14 (five-fold), adjusted expression of adhesion-related genes (CDH3 up, CDH6 down) and increased extracellular matrix (ECM)-related gene expression (COL6, COL1, COL3, COL5, DCN, NPNT, LUM, MGP, MATN2, and VTN), thus yielding more matrix-interacting progenitors with a high aggregation capability. Enhanced contribution to chondrogenic pellet formation increased the cell yield after eight weeks 200-fold compared to controls. The collagen type II and proteoglycan-positive area was enlarged in the CHIR group, indicating an increased number of cartilage-forming cells. Conclusively, short initial WNT activation improved mesoderm commitment and our data demonstrated for the first time to our knowledge that, acting via stimulation of cell proliferation, ECM expression and cell aggregation, WNT pulsing is a key step to make cell selection steps before chondrogenesis obsolete. This advanced understanding of the WNT/β-catenin function
The tumorigenicity of cancer cells is highly influenced by the extracellular matrix (ECM) through mechanisms that are poorly understood. Here it is reported that a variety of 3D ECM microenvironments strongly induce expression of Id1 and Id3 in melanoma cells. Genetic ablation of Id1/Id3 impairs melanoma cell outgrowth in 3D Matrigel culture and inhibits melanoma initiation in vivo. Mechanistically, 3D ECM microenvironments hinder diffusion of endogenously produced bone morphogenetic proteins, thereby fostering autocrine signaling and Id1/Id3 expression. A compound screen identifies new coumarin derivatives that potently inhibit both Id1/Id3 expression and melanoma initiation in vivo. Together, the findings reveal a novel mechanism through which the ECM increases tumorigenicity, identify Id1/Id3 as melanoma-relevant therapeutic targets, and characterize inhibitors of Id1/Id3 expression with therapeutic potential.
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