Effect of a retinoic acid analogue on BMP-driven pluripotent stem cell chondrogenesis.
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PROVIDER: PRJEB64544 | ENA |
REPOSITORIES: ENA
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Project description:Osteoarthritis is the most common degenerative joint condition, leading to articular cartilage (AC) degradation, chronic pain and immobility. The lack of appropriate therapies that provide tissue restoration combined with the limited lifespan of joint-replacement implants indicate the need for alternative AC regeneration strategies. Differentiation of human pluripotent stem cells (hPSCs) into AC progenitors may provide a long-term regenerative solution but are still limited due to the continued reliance upon growth factors to recapitulate developmental signalling processes. Recently, TTNPB, a small molecule activator of retinoic acid receptors (RARs), has been shown to be sufficient to guide mesodermal specification and early chondrogenesis of hPSCs. Here, we modified our previous differentiation protocol, by supplementing cells with TTNPB and administering BMP2 at specific times to enhance early development.
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Project description:Numerous studies have established a critical role for BMP signaling in skeletal development. In the developing axial skeleton, sequential SHH and BMP signals are required for specification of a chondrogenic fate in somitic tissue. A similar paradigm is thought to operate in the limb, but the signals involved are unclear. To investigate the nature of these signals we examined BMP action in mesenchymal populations derived from the early murine limb bud (~ E10.5). These populations exhibited a graded response to BMPs, in which early limb mesenchymal (EL) cells (from the distal hind limb) displayed an anti-chondrogenic response, whereas BMPs promoted chondrogenesis in older cell populations. To better understand the molecular basis of disparate BMP action in these various populations, gene expression profiling with Affymetrix microarrays was employed to identify BMP-regulated genes. These analyses showed that BMPs induced a distinct gene expression pattern in the EL cultures versus later mesenchymal limb populations (IM and LT). Mouse embryos at gestational age E10.5 were collected and various portions of the limb were micro-dissected. These led to the generation of three populations of cells, early (EL) limb mesenchymal cells from the distal half of the hind limb, an intermediate (IM) population derived from the distal 1/3 of the fore limb, and a later (LT) population from the proximal 2/3 of the fore limb. Mesenchymal cells were isolated and cultured with and without BMP4 treatment. RNA was extracted from cultures at either Day 0,1 or 2, labelled and hybridized to Affymetrix 430 2.0 microarrays. For each time point, RNA was collected from two biological replicates for each treatment condition.
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Project description:Retinoic acid (RA) is a standard-of-care neuroblastoma drug thought to act primarily via differentiation. However, this has never been conclusively demonstrated in vivo. Curiously, RA has little effect shrinking primary human tumors when applied in upfront treatment but can eliminate neuroblastoma cells from the bone marrow during consolidation therapy—a discrepancy that has never been explained. To investigate this, we treated a large cohort of neuroblastoma cell lines with RA and observed that the most RA-sensitive cells tended to undergo apoptosis or senescence, rather than differentiation. We conducted genome-wide CRISPR knockout screens under RA treatment, which identified BMP signaling as controlling the apoptosis/senescence vs differentiation cell fate decision and RA’s overall potency. We then conducted integrative analysis of high throughput ChIP-seq and RNA-seq data, which indicates that downstream BMP and RA transcription factors cooperate to determine cell fate, following exposure to RA.
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