Optogenetic manipulation of BMP signalling to drive chondrogenic differentiation of hPSCs
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ABSTRACT:
PROVIDER: PRJEB66461 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR12082309_1.fastq.gz | Fastqsanger.gz | |||
| ERR12082309_2.fastq.gz | Fastqsanger.gz | |||
| ERR12082310_1.fastq.gz | Fastqsanger.gz | |||
| ERR12082310_2.fastq.gz | Fastqsanger.gz | |||
| ERR12082311_1.fastq.gz | Fastqsanger.gz |
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Project description:Optogenetics is a rapidly advancing technology that combines photochemical, optical and synthetic biology techniques to control cellular behaviour and physiology. Combining sensitive light responsive optogenetic intervention tools with human pluripotent stem cell differentiation models has the potential to refine differentiation and unpick the processes by which morphogenetic signals direct tissue patterning, organisation and cell specification. Here, we utilised an optogenetic bone morphogenetic protein (BMP) signalling system (optoBMP) to drive chondrogenic differentiation of human embryonic stem cells (hESCs). We initially engineered light-sensitive hESCs through CRISPR/Cas9-mediated integration of the optoBMP system into the AAVS1 locus. Activation of optoBMP with blue light in lieu of BMP family growth factors during differentiation resulted in activation of BMP signalling pathway mechanisms and upregulation of a chondrogenic phenotype, with significant transcriptional differences compared to cells left in the dark. Furthermore, cells differentiated with light were capable of forming chondrogenic pellets consisting of a hyaline-like cartilaginous matrix.
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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).
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Project description:Differentiated chondrocytes from populations of human bone marrow stromal cells including skeletal stem cells (hBMSCs/SSCs) and induced pluripotent stem cells (hiPSCs) have the potential to restore damaged cartilage, yet chondrocyte hypertrophy is a barrier for translational therapy. However, hBMSCs/SSCs attached to a hyaluronic acid-coated fibrin microbead scaffold (HyA-FMBs) produce cartilage that resists hypertrophy. We show that HyA-FMBs initially suppress Bone Morphogenic Protein (BMP) signaling early in chondrogenic differentiation of hBMSCs/SSCs, then restore BMP signaling in a stable chondrogenic population enriched for Insulin-like Growth Factor Binding Protein 5 and Matrix Gla Protein. We subsequently developed a serumfree hiPSC differentiation strategy that inhibited, then activated BMP signaling in a purified subpopulation derived from ?chondrospheroids? that exhibited stable expression of Type II Collagen, Aggrecan (ACAN), and Lubricin (PRG4) and minimal e xpression of Type X Collagen. Overall, timed, and selective BMP signaling restrains chondrocyte hypertrophy, supporting the potential of chondrospheroid-derived cells for clinical utility in osteoarthritis.
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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:We report a bulk RNA sequencing analysis of a population containing different mesendoderm progenitor cells derived from hiPSCs in 3D environment. Specifically, 3D aggregates of hPSCs were differentiated into cardiac progenitor cells through WNT signaling activation from day 0 to day 1 and WNT signaling inhibition from day 3 to day 5 of differentiation, which allowed further efficient and reproducible specification into hiPSC-derived CMs. This represents the control protocol from which samples from D3 (D3N) and D5 (D5N) were collected. Additionally, adaptation of this control protocol by performing WNT and BMP signalling pathways activation from day 3 to day 5 of differentiation, allowed the modulation of the progenitor cells population to a more posterior splanchnic mesoderm which favours further specification of those progenitors into hiPSC-derived pro-epicardium/septum transversum cells. This represents the CB protocol from where samples from D3 (D3CB) and D5 (D5CB) were collected.
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Project description:Osteoarthritis (OA) is the most prevalent chronic joint disease that affects a majority of the elderly. Chondrogenic progenitor cells (CPCs) reside in late stage OA cartilage tissue, producing a fibrocartilagenous extra-cellular matrix and can be manipulated in-vitro, to deposit proteins of healthy articular cartilage. CPCs are under control of SOX9 and RUNX2 and in order to enhance their chondrogenic potential, we found that RUNX2 plays a pivotal role in chondrogenesis. In another approach, CPCs carrying a knockout of RAB5C, a protein involved in endosomal trafficking, demonstrated elevated expression of various chondrogenic markers including the SOX trio and displayed an increased COL2 deposition, whereas no changes of COL1 deposition was observed. We report RAB5C as an attractive target for future therapeutic approaches to increase the COL2 content in the diseased joint.
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