Biology subjects

Kimber, S. J.

Publications and source records attributed to Kimber, S. J..

18 recordsLinked to original sources

bioRxiv2025

Human pluripotent stem cell-derived macrophages modify development of human kidney organoids

IntroductionThe human fetal kidney contains macrophages, innate immune cells postulated to enhance its development. Macrophages have also been implicated in the pathobiology of human kidney malformations and Wilms tumour. Human pluripotent stem cell (hPSC)-derived kidney organoids contain nephrons differentiated from intermediate mesoderm-like precursors. Endothelia are present between organoid tubules but fail to efficiently populate glomeruli. These organoids lack macrophages, as expected because in vivo kidney macrophages invade from yolk sac and liver. MethodsWe hypothesised that combining hPSC-derived macrophages with hPSC-derived kidney precursors modifies nephrogenesis. Macrophages harvested at early or later maturation stages were added to kidney precursors in numbers of 1%, 5% or 20% compared with constant numbers of nephrogenic cells. ResultsNo macrophages, as assessed by CD68 immunostaining, were detected in organoids without added macrophages. In contrast, composite organoids contained macrophages located between tubules, mimicking native human fetal kidneys. Quantification of tissue macrophages at the end of 18-day organoid culture positively correlated with the numbers of macrophages added. Assessed by CD31/PECAM-1 and CD68 co-immunostaining, some macrophages were near vessels but added macrophages neither increased the proportion of vessels nor endothelial invasion of glomeruli. Early-stage macrophages significantly increased the percentage area occupied by glomeruli, as assessed by synaptopodin immunostaining. The highest macrophage numbers inhibited overall growth, assessed by organoid area, additionally generating dysmorphic tissue when later stage macrophages were added. ConclusionDepending on their maturation stage and quantity, macrophages have beneficial or harmful effects on organoids. This supports the proposition that macrophages play roles in normal and abnormal development of human kidneys.

developmental biology↗

bioRxiv2025

Human pluripotent stem cell model of multiple epiphyseal dysplasia with MATN3 mutation identifies altered Matrix organisation and upregulation of the cholesterol biosynthesis pathway

Multiple epiphyseal dysplasia (MED), caused by mutations in MATN3, is a chondrodysplasia affecting the cartilage growth plate and is characterised by delayed epiphyseal ossification, short stature, and early onset osteoarthritis. Here we generated an in vitro human pluripotent stem cell (hPSC) model of cartilage growth-plate development to identify pathogenic mechanisms underlying MED. hPSCs were differentiated to chondrocytes via a mesenchymal intermediate, followed by TGF{beta}3+BMP2 induced chondrogenic pellet culture. MATN3-mutant hPSCs were generated by reprogramming MED patient PBMCs or by CRISPR-Cas9 gene editing to introduce a MATN3 mutation in a hESC line. RNAseq was used to assess chondrogenesis and identify MED pathogenic mechanisms. Transmission electron microscopy (TEM) was used to assess extracellular matrix assembly. The resultant hPSC-derived cartilage pellets displayed a typical cartilage morphology and strongly expressed cartilage matrix markers, e.g., collagen II and matrilin-3. Matrilin-3 protein was detected within both the matrix and cells of heterozygous mutant hPSC-cartilage pellets. RNAseq of mutant hPSC-cartilage pellets revealed significant enrichment for ECM organisation and cholesterol biosynthesis pathway genes as well as sightly increased expression of some unfolded protein response (UPR) marker genes. MATN3 mutant hPSC-derived cartilage pellets displayed abnormal matrix assembly, distended ER, accumulation of lipid droplets, and increased cholesterol content. Our model revealed mutant matrilin-3 induces cholesterol biosynthesis pathway upregulation and abnormal matrix assembly during MED pathogenesis. This study provides new insights into the molecular mechanisms underlying MED and highlights potential therapeutic targets.

cell biology↗

bioRxiv2025

A Modular Bioinstructive Platform for Additive-Free, Topography-Driven Stem Cell Differentiation and Patterning

Recreating 3D bone formation in vitro without biochemical inducers remains a longstanding challenge in preclinical testing. We present a scalable, bioinstructive platform based on polylactic acid microparticles with controlled dimpled surface features that direct mesenchymal stem cell differentiation through endogenous topography-mediated mechanotransduction, establishing a mechanistically validated, additive-free platform. These 3D topographical cues drive cytoskeletal reorganisation and induce osteogenesis via canonical Hedgehog signalling. RNA-Seq revealed early significant upregulation of cytoskeletal components and osteochondral transcription factors, including runt-related transcription factor 2 (RUNX2) and SRY-box transcription factor 9 (SOX9), followed by activation of the insulin growth factor-II pathway and osteogenic commitment. To demonstrate translational potential, two-photon polymerisation lithography was employed to engineer precisely-patterned 3D topographies, inducing graded GLI1 expression without added soluble cues. This establishes a modular, versatile platform for stem cell engineering, offering a topography-driven, non-genetic analogue to mechanogenetics with broad utility for regenerative medicine and human-relevant development of bone models. O_FIG O_LINKSMALLFIG WIDTH=162 HEIGHT=200 SRC="FIGDIR/small/664383v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@1b06d7dorg.highwire.dtl.DTLVardef@1e464e1org.highwire.dtl.DTLVardef@19b1299org.highwire.dtl.DTLVardef@1e21f89_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

bioengineering↗

bioRxiv2025

Epigenome and transcriptome changes in KMT2D-related Kabuki syndrome Type 1 iPSCs, neuronal progenitors and cortical neurons

Kabuki syndrome type 1 (KS1) is a neurodevelopmental disorder caused by loss-of-function variants in KMT2D which encodes a H3K4 methyltransferase. The mechanisms underlying neurodevelopmental problems in KS1 are still largely unknown. Here, we track the epigenome and transcriptome across three stages of neuronal differentiation using patient-derived induced pluripotent stem cells (iPSCs) to gain insights into the disease mechanism of KS1. In KS1 iPSCs we detected significantly lower levels of functional KMT2D transcript and KMT2D protein, and lower global H3K4me1 and H3K4me2 levels. We identify loss of thousands of H3K4me1 peaks in iPSCs, neuronal progenitors (NPs) and early cortical neurons (CNs) in KS1. We show that the number of lost peaks increase as differentiation progresses. We also identify hundreds of differentially expressed genes (DEGs) in iPSCs, NPs and CNs in KS1. In contrast with the epigenomic changes, the number of DEGs decrease as differentiation progresses. Our analysis reveals significant enrichment of differentially downregulated genes in areas containing putative enhancer regions with H3K4me1 loss. We also identify a set of distinct transcription factor binding sites in differentially methylated regions and a set of DEGs related to KS1 phenotypes. We find that genes regulated by SUZ12, a subunit of Polycomb Repressive complex 2, are over-represented in KS1 DEGs at early stages of differentiation. In conclusion, we present a disease-relevant human cellular model for KS1 that provides mechanistic insights for the disorder and could be used for high throughput drug screening for KS1.

genetics↗

bioRxiv2024

Interaction between gene expression and morphokinetic parameters in undisturbed human embryo culture

The clinical in vitro fertilisation (IVF) need for optimal culture conditions and improved selection of human embryos for transfer led to the development of time-lapse systems built into incubators to allow a stable, well-defined and undisturbed culture environment with continual monitoring of embryo development. Clinical data suggest that both undisturbed culture of embryos and selection algorithms based on time-lapse morphometric parameters can potentially improve embryo development and live birth rates. However, there remains a need to validate and understand the mechanisms underlying the potential benefits of the time-lapse technology in IVF. In this study, we compared the gene expression of human embryos grown in an undisturbed time-lapse system with a conventional incubator and showed that there is no fundamental difference in the developmental program of the undisturbed embryos, which provides important reassurance concerning the time-lapse technology. We then performed a network-based integrative analysis based on the undisturbed blastocyst transcriptomes and identified time-lapse parameter correlated genes. These showed cross talk with identified embryo development gene functional modules, suggesting potential molecular mechanisms underlying the clinical predictive capabilities of embryo time-lapse morphokinetic parameters for subsequent live birth. This study suggests methodologies for assessing the impact of additional predictive correlates of value for optimal embryo development and understanding their mechanisms of action.

developmental biology↗

bioRxiv2024

Effect of glucose concentration in culture medium on the human preimplantation embryo methylome

Study questionDoes glucose concentration in culture medium have an impact on the DNA methylome of the early human embryo? Summary answerGlucose concentration is associated with changes in gene expression, global DNA methylation, methylation levels at CpG islands and at key histone modifications in human blastocysts. What is known alreadyPreimplantation human embryos are highly sensitive to their local environment, and this may have long term implications for the health of the developing embryo, fetus and offspring. Glucose is a standard component of human embryo culture media, due to its importance as a nutrient. However, concentrations of glucose differ widely between different commercially available types. The present study was designed to determine whether changes in glucose concentration could influence global methylation and gene expression in the human preimplantation embryo. Study design, size, durationHuman embryos were cultured in clinically relevant concentrations of glucose and global DNA methylation analysis was performed. The effect of glucose concentration on the embryo epigenome, specifically DNA methylation, was analysed. Participants/materials, setting, methodsHuman embryos surplus to treatment requirements were donated with informed consent from several ART centres. Embryos were cultured to the blastocyst stage in Vitrolife G-TL medium, either at 0.9 mM or 3.5 mM glucose, separated via immunosurgery into Inner Cell Mass (ICM) and trophectoderm (TE) samples, and compared for both DNA methylation and gene expression. This allowed us to evaluate the association between DNA methylation and previously importantly identified biological pathways. Main results and the role of chanceThe concentration of glucose in human embryo culture medium was associated with changes in gene expression and global DNA methylation in both ICM and TE, and methylation levels at CpG islands and key histone modifications. These results are significant because glucose is a major nutrient metabolised by human embryos in culture, and yet we know relatively little of its downstream effects on the genome and epigenome. Wider implications of the findingsCommercially available embryo culture media with varying glucose levels have also been associated with altered fetal growth, birthweight and postnatal development of IVF offspring. Our findings may have important ramifications for potential clinical markers of embryo quality and pregnancy initiation, and improve understanding of the mechanisms underlying the impact of the early environment on the long term health of ART offspring. Study funding/competing interest(s)This work was funded by the National Council for Science and Technology of Mexico (CONACyT), an NIHR pre-doctoral fellowship (PCAF) to MM, the NIHR Local Comprehensive Research Network and NIHR Manchester Clinical Research Facility, the University of Manchester and Manchester University NHS Foundation Trust. None of the authors has any conflict of interest to declare.

developmental biology↗

bioRxiv2024

Analysis of higher order interactions quantifies co-ordination in the epigenome and reveals novel biological relationships in Kabuki syndrome

Complex direct and indirect relationships between multiple variables are a characteristic of all natural systems and are defined as higher order interactions (HOIs). Traditional differential and network analyses fail to account for the richness of omic datasets and miss HOIs. We investigated genome-wide peripheral blood DNA methylation data from Kabuki syndrome type 1 (KS1) and control individuals, identified 2,002 differentially methylated points (DMPs), and inferred 17 differentially methylated regions, which represent only 189 DMPs. We followed these results with quantification of HOIs by applying hypergraph network models on all the CpGs in the two datasets and revealed differences in co-ordination of the DMPs along with lower entropy and higher co-ordination of the peripheral epigenome in KS1 implying reduced network complexity. We demonstrate that the hypergraph approach captures substantially more information, enables factoring trans-relationships, and identifies biologically relevant pathways that escape the standard analyses. These findings construct the basis of a suitable model that is not computationally intensive for the analysis of the organisation of the epigenome in rare diseases. This approach can be applied to other types of omic datasets, and to other fields of science and medicine to investigate mechanism in big data.

systems biology↗

bioRxiv2023

Single cell transcriptomics reveals chondrocyte differentiation dynamics in vivo and in vitro

The consistent production of in vitro chondrocytes that faithfully recapitulate in vivo development would be of great benefit for musculoskeletal disease modelling and regenerative medicine. Current efforts are often limited by off-target differentiation, resulting in a heterogeneous product. Furthermore, the lack of comparison to human embryonic tissue, precludes detailed evaluation of in vitro cells. Here, we perform single-cell RNA sequencing of embryonic long bones dissected from first trimester hind limbs from a range of gestational ages. We combine this with publicly available data to form a detailed atlas of endochondral ossification, which we then use to evaluate a series of published in vitro chondrogenesis protocols, finding substantial variability in cell states produced by each. We apply single-nuclear RNA sequencing to one protocol to enable direct comparison between in vitro and in vivo, and perform trajectory alignment between the two to reveal differentiation dynamics at the single-cell level, shedding new light on off-target differentiation in vitro. Using this information, we inhibit the activity of FOXO1, a transcription factor predicted to be active in embryonic bone development and in chondrogenic cells in vitro, and increase chondrocyte transcripts in vitro. This study therefore presents a new framework for evaluating tissue engineering protocols, using single-cell data from human development to drive improvement and bring the prospect of true engineered cartilage closer to reality.

bioengineering↗

bioRxiv2023

Effect of a retinoic acid analogue on BMP-driven pluripotent stem cell chondrogenesis

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. Transcriptomic analyses indicated that activation of RAR signalling significantly upregulated genes related to limb and embryonic skeletal development in the early stages of the protocol and upregulated genes related to AC development in later stages. Chondroprogenitors obtained from RAPID-E could generate cartilaginous pellets that expressed AC-related matrix proteins such as Lubricin, Aggrecan, and Collagen II. This protocol could lay the foundations for cell therapy strategies for osteoarthritis and improve the understanding of AC development in humans.

developmental biology↗

bioRxiv2023

Induced pluripotent stem cell model revealed impaired neurovascular interaction in genetic small vessel disease CADASIL

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common genetic small vessel disease caused by variants in the NOTCH3 gene. Patients with CADASIL experience recurrent strokes, developing into cognitive defect and vascular dementia. CADASIL is a late-onset vascular condition, but migraine and brain MRI lesions appear in CADASIL patients as early as their teens and twenties, suggesting an abnormal neurovascular interaction at the neurovascular unit (NVU) where microvessels meet the brain parenchyma. To understand the molecular mechanisms of CADASIL, we established induced pluripotent stem cell (iPSC) models from CADASIL patients and differentiated the iPSCs into the major NVU cell types including brain microvascular endothelial-like cells (BMECs), vascular mural cells (MCs), astrocytes and cortical projection neurons. We then built an in vitro NVU model by co-culturing different neurovascular cell types in Transwells and evaluated the blood brain barrier (BBB) function by measuring transendothelial electrical resistance (TEER). Results showed that, while the wild-type MCs, astrocytes and neurons could all independently and significantly enhance TEER values of the iPSC-BMECs, such capability of MCs from iPSCs of CADASIL patients was significantly impeded. Additionally, the barrier function of the BMECs from CADASIL iPSCs was significantly impaired, accompanied with disorganised tight junctions in iPSC-BMECs, which could not be effectively rescued by the wild-type MCs, astrocytes and neurons. Our findings provide new insight into early disease pathologies on the neurovascular interaction and BBB function at the molecular and cellular levels for CADASIL, which helps inform future therapeutic development.

cell biology↗

bioRxiv2023

Human pluripotent stem cell-derived kidney organoids reveal tubular epithelial pathobiology of heterozygous HNF1B-associated dysplastic kidney malformations

Hepatocyte nuclear factor 1B (HNF1B) encodes a transcription factor expressed in developing human kidney epithelia. Heterozygous HNF1B mutations are the commonest monogenic cause of dysplastic kidney malformations (DKMs). To understand their pathobiology, we generated heterozygous HNF1B mutant kidney organoids from CRISPR-Cas9 gene-edited human ESCs and iPSCs reprogrammed from a family with HNF1B-asscociated DKMs. Mutant organoids contained enlarged malformed tubules and displayed deregulated cell turnover. Numerous genes implicated in Mendelian kidney tubulopathies were downregulated, and mutant tubules resisted the cAMP-mediated dilatation seen in controls. Bioinformatic analyses indicated abnormal WNT, calcium, and glutamatergic pathways, the latter hitherto unstudied in developing kidneys. Glutamate ionotropic receptor kainate type subunit 3 was upregulated in mutant organoids and was detected in their tubules and in fetal human DKM dysplastic epithelia. These results reveal morphological, molecular, and physiological roles for HNF1B in human kidney tubule morphogenesis and functional differentiation. They additionally suggest druggable targets to ameliorate disease.

developmental biology↗

bioRxiv2021

KMT2D haploinsufficiency in Kabuki Syndrome disrupts neuronal function through transcriptional and chromatin rewiring independent of H3K4-monomethylation

Kabuki syndrome (KS) is a rare multisystem disorder, characterized by intellectual disability, growth delay, and distinctive craniofacial features. It is mostly caused by de novo mutations of KMT2D, which is responsible for histone H3lysine 4 mono-methylation (H3K4me1) that marks active and poised enhancers. We assessed the impact of KMT2D mutations on chromatin and transcriptional regulation in a cohort of multiple KS1 tissues, including primary patient samples and disease-relevant lineages, namely cortical neurons (iN), neural crest stem cells (NCSC), and mesenchymal cells (MC). In parallel, we generated an isogenic line derived from human embryonic stem cells (hESC) for the stepwise characterization of neural precursors and mature neurons. We found that transcriptional dysregulation was particularly pronounced in cortical neurons and widely affected synapse activity pathways. This was consistent with highly specific alterations of spontaneous network-bursts patterns evidenced by Micro-electrode-array (MEA)-based neural network. Profiling of H3K4me1 unveiled the almost complete uncoupling between this chromatin mark and the effects on transcription, which is instead reflected by defects in H3K27ac. Finally, we identified the direct targets of KMT2D in mature cortical neurons, uncovering TEAD2 as the main mediator of KMT2D haploinsufficiency. Our results uncover the multi-tissue architecture of KS1 dysregulation and define a unique electrical phenotype and its molecular underpinnings for the cortical neuronal lineage.

genetics↗

bioRxiv2021

A secreted proteomic footprint for stem cell pluripotency

With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the spent medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGF{beta} deficient media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of 4 E8- against 4 E6- enriched biomarkers provides 16 ratio abundances which are each robustly diagnostic for pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to recovery assays.

cell biology↗

bioRxiv2021

Directed differentiation of hPSCs through lateral plate mesoderm for generation of articular cartilage progenitors

Developmentally the articular joints are derived from lateral plate (LP) mesoderm. However, no study has produced LP derived prechondrocytes or preosteoblasts from human pluripotent stem cells (hPSC) in a chemically defined manner. Differentiation of hPSCs through the authentic route, via an LP-osteochondral progenitor (OCP), may aid understanding of human cartilage development and the generation of effective cell therapies for osteoarthritis. We refined our existing chondrogenic protocol, incorporating knowledge from development and other studies to produce a LP-OCP from which prechondrocytes- and preosteoblast-like cells can be produced. Results show the formation of an OCP, which can be further driven to prechondrocytes and preosteoblasts. Prechondrocytes cultured in pellets produced cartilage like matrix with lacunae and superficial flattened cells expressing lubricin. Additionally, preosteoblasts were able to generate a mineralised structure. This protocol can therefore be used to further investigate cartilage development and in the development of joint cartilage for potential treatments.

developmental biology↗

bioRxiv2020

Trophectoderm differentiation to invasive syncytiotrophoblast is induced by endometrial epithelial cells during human embryo implantation

At implantation, trophoblast derived from the trophectoderm of the blastocyst-stage embryo invades the endometrium to establish pregnancy. To understand how embryos breach the endometrial epithelium, we modelled human implantation using blastocysts or trophoblast stem cell spheroids cultured with endometrial epithelial cells (EEC). Blastocyst invasion of the EEC layer was initiated by multinuclear syncytiotrophoblast. Spheroids also invaded the epithelium with syncytiotrophoblast, and EEC induced upregulation of syncytiotrophoblast markers. Modelling implantation in silico using blastocyst and EEC transcriptomes revealed gene networks that exhibited greater connectivity and organisation in trophectoderm of the polar region of the embryonic axis. However, gene ontologies and machine learning suggested that EEC drives syncytiotrophoblast differentiation in polar and mural trophectoderm. This is the first evidence for endometrial epithelium-induced trophectoderm differentiation to invasive syncytiotrophoblast as the cellular mechanism of embryonic breaching of the endometrium in humans, with implications for reproductive medicine and our understanding of human embryonic development.

developmental biology↗

bioRxiv2020

Modelling the developmental spliceosomal craniofacial disorder Burn-McKeown syndrome using induced pluripotent stem cells

The craniofacial developmental disorder Burn-McKeown Syndrome (BMKS) is caused by biallelic variants in the pre-messenger RNA splicing factor gene TXNL4A/DIB1. The majority of affected individuals with BMKS have a 34 base pair deletion in the promoter region of one allele of TXNL4A combined with a loss-of-function variant on the other allele, resulting in reduced TXNL4A expression. However, it is unclear how reduced expression of this ubiquitously expressed spliceosome protein results in craniofacial defects during development. Here we reprogrammed peripheral mononuclear blood cells from a BMKS patient and her unaffected mother into induced pluripotent stem cells (iPSCs) and differentiated the iPSCs into induced neural crest cells (iNCCs), the key cell type required for correct craniofacial development. BMKS patient-derived iPSCs proliferated more slowly than both mother- and unrelated control-derived iPSCs, and RNA-Seq analysis revealed significant differences in gene expression and alternative splicing. Patient iPSCs displayed defective differentiation into iNCCs compared to maternal and unrelated control iPSCs, in particular a delay in undergoing an epithelial-to-mesenchymal transition (EMT). RNA-Seq analysis of differentiated iNCCs revealed widespread gene expression changes and mis-splicing in genes relevant to craniofacial and embryonic development that highlight a dampened response to WNT signalling, the key pathway activated during iNCC differentiation. Furthermore, we identified the mis-splicing of TCF7L2 exon 4, a key gene in the WNT pathway, as a potential cause of the downregulated WNT response in patient cells. Additionally, mis-spliced genes shared common sequence properties such as length, splice site strengths and sequence motifs, suggesting that splicing of particular subsets of genes is particularly sensitive to changes in TXNL4A expression. Together, these data provide the first insight into how reduced TXNL4A expression in BMKS patients might compromise splicing and NCC function, resulting in defective craniofacial development in the embryo.

genetics↗

bioRxiv2020

SIRT1 activity orchestrates ECM expression during hESC-chondrogenic differentiation through SOX5 and ARID5B

Epigenetic modification is a key driver of differentiation and the deacetylase Sirtuin1 (SIRT1) is an established regulator of cell function, ageing and articular cartilage homeostasis. Here we investigate the role of SIRT1 during development of chondrocytes by using human embryonic stem cells (hESCs). HESC-chondroprogenitors were treated with SIRT1 activator; SRT1720, or inhibitor; EX527, at different development stages. Activation of SIRT1 during 3D-pellet culture led to significant increases in expression of ECM genes for type-II collagen (COL2A1) and aggrecan (ACAN), and chondrogenic transcription factors SOX5 and ARID5B, with SOX5 ChIP analysis demonstrating enrichment on the ACAN -10 enhancer. Unexpectedly, while ACAN was enhanced, GAG retention in the matrix was reduced when SIRT1 was activated. Significantly, ARID5B and COL2A1 were positively correlated, with Co-IP indicating association of ARID5B with SIRT1 suggesting that COL2A1 expression is promoted by an ARID5B and SIRT1 interaction. In conclusion, SIRT1 activation positively impacts on the expression of the main ECM proteins, whilst altering ECM composition and suppressing GAG content during cartilage development. These results suggest that SIRT1 activity can be beneficial to cartilage development and matrix protein synthesis but tailored by addition of other positive GAG mediators.

developmental biology↗

bioRxiv2020

Optogenetic control of the Bone Morphogenetic Protein signalling pathway through engineered blue light-sensitive receptors

Bone Morphogenetic Proteins (BMPs) are members of the Transforming Growth Factor {beta} (TGF{beta}) superfamily and have crucial roles during development; including mesodermal patterning and specification of renal, hepatic and skeletal tissues. In vitro developmental models currently rely upon costly and unreliable recombinant BMP proteins that do not enable dynamic or precise perturbation of the BMP signalling pathway. Here, we develop a novel optogenetic BMP signalling system (optoBMP) that enables rapid induction of the canonical BMP signalling pathway through illumination with blue light. We demonstrate the utility of the optoBMP system in multiple human cell lines to initiate signal transduction through phosphorylation and nuclear translocation of SMAD1/5, leading to upregulation of BMP target genes including Inhibitors of DNA binding ID2 and ID4. Furthermore, we demonstrate how the optoBMP system can be used to fine-tune activation of the BMP signalling pathway through variable light stimulation. Optogenetic control of BMP signalling will enable dynamic and high-throughput intervention across a variety of applications in cellular and developmental systems.

molecular biology↗